The Interactive Fly

Genes involved in tissue and organ development

Testis and spermatogenesis

  • Development of the male germline stem cell niche in Drosophila
  • Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche
  • Hedgehog is required for cyst stem cell self-renewal but does not contribute to the GSC niche in the Drosophila testis
  • Nanotubes mediate niche-stem-cell signalling in the Drosophila testis
  • Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo
  • lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis
  • Occluding junctions maintain stem cell niche homeostasis in the fly testes
  • Steroid signaling promotes stem cell maintenance in the Drosophila testis
  • Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster
  • Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation
  • Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis
  • Socs36E controls niche competition by repressing MAPK signaling in the Drosophila testis
  • The novel tumour suppressor Madm regulates stem cell competition in the Drosophila testis
  • Conversion of quiescent niche cells to somatic stem cells causes ectopic niche formation in the Drosophila testis
  • Stage-specific control of niche positioning and integrity in the Drosophila testis
  • Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output
  • Survival motor neuron protein regulates stem cell division, proliferation, and differentiation in Drosophila
  • The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells
  • Somatic cell encystment promotes abscission in germline stem cells following a regulated block in cytokinesis
  • Phf7 controls male sex determination in the Drosophila germline
  • Escargot restricts niche cell to stem cell conversion in the Drosophila testis
  • Protein synthesis and degradation are critical to regulate germline stem cell homeostasis in Drosophila testes
  • Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila
  • Recruitment of Mediator complex by cell type and stage-specific factors required for tissue-specific TAF dependent gene activation in an adult stem cell lineage
  • Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues
  • A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage
  • Sequential changes at differentiation gene promoters as they become active in a stem cell lineage
  • Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells
  • RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila
  • tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation
  • Blocking promiscuous activation at cryptic promoters directs cell type-specific gene expression

  • Asymmetric inheritance of mother versus daughter centrosome in stem cell division
  • Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells
  • Centrosome misorientation reduces stem cell division during ageing
  • The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis
  • Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline
  • Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution

  • The regulated elimination of transit-amplifying cells preserves tissue homeostasis during protein starvation in Drosophila testis
  • Spermatid individualization is sensitive to temperature and fatty acid metabolism
  • A Krebs cycle component limits caspase activation rate through mitochondrial surface restriction of CRL activation
  • Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila
  • Reduced expression of CDP-DAG synthase changes lipid composition and leads to male sterility in Drosophila
  • Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis
  • Cell type-specific translational repression of Cyclin B during meiosis in males
  • Regulators of alternative polyadenylation operate at the transition from mitosis to meiosis
  • The piRNA pathway is developmentally regulated during spermatogenesis in Drosophila
  • Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes
  • Critical roles of long noncoding RNAs in Drosophila spermatogenesis

  • Cytoskeletal dynamics in male meiosis
  • A role for actin dynamics in individualization during spermatogenesis
  • tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility
  • Regulation of dynein localization and centrosome positioning by Lis-1 and asunder during Drosophila spermatogenesis
  • The cilium-like region of the Drosophila spermatocyte: an emerging flagellum?
  • A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids
  • Centriole remodeling during spermiogenesis in Drosophila
  • The THO complex is required for nucleolar integrity in Drosophila spermatocytes

  • Post-meiotic transcription in Drosophila testes
  • The search for Y-linked genes: Y chromosome fertility factors encode dynein heavy chain polypeptides
  • The B-type lamin is required for somatic repression of testis-specific gene clusters
  • The poly(A) polymerase GLD2 is required for spermatogenesis in Drosophila melanogaster
  • Replacement of histones by amines and Mst77F during chromatin condensation in late spermatids and role of Sesame in the removal of these proteins from the male pronucleus
  • Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila
  • The HMG-box-containing proteins tHMG-1 and tHMG-2 interact during the histone-to-protamine transition in Drosophila spermatogenesis
  • Prtl99C acts together with protamines and safeguards male fertility in Drosophila
  • Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche
  • tBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the Bromodomain and Extra-Terminal (BET) Family
  • Paternal diet defines offspring chromatin state and intergenerational obesity
  • Mitotic fidelity requires transgenerational action of a testis-restricted HP1
  • Bi-directional gap junction-mediated Soma-Germline communication is essential for spermatogenesis
  • Genomic and expression analysis of transition proteins in Drosophila
  • The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin
  • Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila

  • Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila accessory gland
  • Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects
  • BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior
  • Drosophila sperm surface α-l-fucosidase interacts with the egg coats through its core fucose residues
  • The fatty acid elongase Bond is essential for Drosophila sex pheromone synthesis and male fertility
  • Secondary cell expressed genes in the male accessory gland are needed for the female post-mating response in Drosophila melanogaster
  • Localized, reactive F-actin dynamics prevents abnormal somatic cell penetration by mature spermatids
    Genes Involved in Spermatogenesis

    Development of the male germline stem cell niche in Drosophila

    Stem cells are found in specialized microenvironments, or 'niches', which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. This study investigates the formation of the testis GSC niche, or 'hub', during the late stages of embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).

    The evidence indicates that an embryonic hub, which appears to give rise to the adult hub and create the male GSC niche, forms during the late stages of embryogenesis. A subset of anterior SGPs initiates expression of several molecular markers that are also expressed in the adult hub. These SGPs segregate into a tight cluster in a distinct region of the gonad, and a subset of germ cells organizes around these SGPs in a manner similar to the organization of GSCs around the adult hub. Since spermatogenesis begins by early larval stages, it is possible that the embryonic hub already forms a functional GSC niche. The formation of the hub, or indeed any stem cell niche, can be divided into the distinct issues of niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).

    The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression during stage 17 also express every other marker of adult hub identity tested, including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that these cells are specified as hub cells at this time. The fate of the anterior SGPs that lose esg expression and do not form part of the hub is unknown. An intriguing possibility is that these cells could form another important somatic cell type: the cyst progenitor cells (somatic stem cells) that associate with the hub along with the GSCs (Le Bras, 2006).

    Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to be regulated by esg in other tissues. It has been reported, however, that esg is required for hub maintenance, and that the hub is severely defective at later stages in esg mutants that survive embryogenesis. Thus, esg is critical for the male GSC niche, but is either not important for the initial formation of this structure, or acts redundantly with another factor (Le Bras, 2006).

    It has been possible to follow the morphogenesis of the hub from the time of gonad formation until the embryonic hub is fully formed. At the time of gonad coalescence, anterior SGPs interact with other SGPs, and with the germ cells, in a manner that is indistinguishable from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in their relationship to other SGPs and germ cells. Hub cells segregate away from other SGPs to one pole of the gonad, and coalesce tightly with one another. In addition, hub cells do not ensheath the germ cells at this stage. Instead, a defined interface between hub cells and germ cells forms which is labeled by DE- and DN-cadherin, but not Fasciclin 3. Thus, hub cells appear to maximize their interactions with one another, and minimize their interactions with other cells in the gonad, although they clearly still contact a subset of germ cells (Le Bras, 2006).

    It is apparent that the changes in cell–cell contact and morphology that occur during hub formation require changes in cell adhesion. Indeed, characteristic changes have been found in expression of the homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur during hub formation; all three are significantly upregulated in the embryonic and adult hub. Increased homophilic adhesion among hub cells could account for their ability to maximize their contacts with one another, and sort away from other SGPs. However, no changes were observed in embryonic hub formation in mutants for these cell adhesion molecules. Thus, these proteins, and possibly others, may act redundantly in this process (Le Bras, 2006).

    It is clear that a subset of germ cells organizes specifically with the developing hub as it forms. During the last stage of hub formation, germ cells become oriented in a rosette distribution around the developing hub in a manner characteristic of GSCs in the adult. These may represent the subset of germ cells that will become GSCs. The presence of DE- and DN-cadherin at sites of hub–germ cell contact suggests that cadherin-mediated adhesion may be important for niche–GSC interaction in the testis, as has been observed in the ovary. Interestingly, germ cells are not required for hub formation. Analysis of a number of hub identity markers indicates that these cell form normally from a subset of anterior SGPs in embryos that lack germ cells. The hub does not appear as well compacted in these embryos, consistent with observations of the adult hub, indicating that hub–germ cell contact (or hub–germ cell signaling) affects the final shape of the hub. Nevertheless, the GSC niche can form in the absence of one of its stem cell populations (somatic stem cells may still be present). It will be of great interest in the future to determine if the subset of germ cells organized around the male embryonic hub are, indeed, developing GSCs, and to study how their transition to stem cell identity might be regulated by the niche (Le Bras, 2006).

    The formation of the male GSC niche is a sex-specific characteristic of anterior SGPs. Male-specific expression of esg and hub formation both require the sex determination genes tra and dsx. In some tissues, DSXM is required to promote male development and repress female development, while the opposite is true for DSXF. Interestingly, it was found that embryonic hub development is entirely masculinized in dsx null mutants; XX and XY individuals appear identical when mutant for dsx and both resemble wild type males. Thus, no role is seen for DSXM in promoting embryonic hub formation, while DSXF is required in females to repress hub formation. Since esg is expressed male-specifically, it is one candidate for being directly regulated by DSX (Le Bras, 2006).

    We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the mechanism for how sexual dimorphism is created differs between the two cell types. msSGPs are present only in males because they have undergone sex-specific apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs. These cells appear to remain present in both sexes, but only form a hub in males. Thus, although the sex determination genes tra and dsx regulate sex-specific development of both cell types, the cellular mechanisms employed are different. Finally, as was observed for the hub, development of the msSGPs is completely masculinized in dsx mutant embryos. Thus, for both of these cell types, the male pattern of development in the embryonic gonad is the default state in the absence of dsx function, and it is the role of DSXF to repress male development in females. However, DSXM may well play a role in development of one or both of these gonad cell types at later stages, since proper testis development in males clearly requires dsx (Le Bras, 2006).

    The sex determination pathway must also ensure that GSC niches form in females and are different from those in males. Recently, it has been shown that germ cells populating the anterior of the gonad in female embryos are predisposed to become GSCs in the adult ovary, while germ cells populating the posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad may promote GSC identity, similar to what is proposed to happen in the male during hub formation. One possibility is that anterior SGPs give rise to GSC niches in both sexes, while genes such as tra and dsx control whether these niches will be male or female (Le Bras, 2006).

    In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).

    Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

    In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. This study shows that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. These observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated (Michel, 2012).

    Hh thus provides a niche signal for the maintenance and proliferation of the somatic stem cells of the testis. CySCs that are unable to transduce the Hh signal are lost through differentiation, whereas pathway overactivation causes overproliferation. Hh signalling thereby resembles Jak/Stat signalling via Upd. Partial redundancy between these pathways might explain why neither depletion of Stat activity nor loss of Hh signalling causes complete CySC loss (Michel, 2012).

    This study has shown that loss of Hh signalling in smo mutant cells blocks expression of the Jak/Stat target Zfh1, whereas mutation of ptc expands the Zfh1-positive pool. Overexpression of Zfh1 or another Jak/Stat target, Chinmo, is sufficient to induce CySC-like behaviour in somatic cells irrespective of their distance. By contrast, Hh overexpression in the hub using the hh::Gal4 driver only caused a moderate increase in the number of Zfh1-positive cells relative to a GFP control. Ectopic Hh overexpression in somatic cells under c587::Gal4 control increased this number further. However, unlike in somatic cells with constitutively active Jak/Stat signalling, the additional Zfh1-positive cells remained largely confined to the testis tip, although their average range was increased threefold. Thus, Hh appears to promote stem cell proliferation, in part, also independently of competition (Michel, 2012).

    It is tempting to speculate that further stem cell expansion is limited by Upd range. Consistently, cells with an ectopically activated Jak/Stat pathway remain undifferentiated, whereas ptc cells can still differentiate. Future experiments will need to formally address the epistasis between these pathways. However, the observations already show that Hh signalling influences expression of the bona fide Upd target gene zfh1, and therefore presumably acts upstream, or in parallel to, Upd in maintaining CySC fate (Michel, 2012).

    In addition, the reduction in GSC number following somatic stem cell loss implies cross-regulation between the different stem cell populations that presumably involves additional signalling cascades, such as the EGF pathway (Michel, 2012).

    In recent years, research has focused on the differences between the male and female gonadal niches. This paper instead emphasizes the similarities: in both cases, Jak/Stat signalling is responsible for the maintenance and activity of cells that contribute to the GSC niche, and Hh signalling promotes the proliferation of stem cells that provide somatic cells ensheathing germline cysts. In the testis, both functions are fulfilled by the CySCs, whereas in the ovary the former task is fulfilled by the postmitotic escort stem cells/escort cells and the latter by the FSCs. Finally, male desert hedgehog (Dhh) knockout mice are sterile. Dhh is expressed in the Sertoli cells and is thought to primarily act on the somatic Leydig cells. However, the signalling microenvironment of the vertebrate spermatogonial niche is, as yet, not fully defined. Future experiments will need to clarify whether these similarities reflect convergence or an ancestral Hh function in the metazoan gonad (Michel, 2012).

    Hedgehog is required for cyst stem cell self-renewal but does not contribute to the GSC niche in the Drosophila testis

    The Drosophila testis harbors two types of stem cells: germ line stem cells (GSCs) and cyst stem cells (CySCs). Both stem cell types share a physical niche called the hub, located at the apical tip of the testis. The niche produces the JAK/STAT ligand Unpaired (Upd) and BMPs to maintain CySCs and GSCs, respectively. However, GSCs also require BMPs produced by CySCs, and as such CySCs are part of the niche for GSCs. This study describes a role for another secreted ligand, Hedgehog (Hh), produced by niche cells, in the self-renewal of CySCs. Hh signaling cell-autonomously regulates CySC number and maintenance. The Hh and JAK/STAT pathways act independently and non-redundantly in CySC self-renewal. Finally, Hh signaling does not contribute to the niche function of CySCs, as Hh-sustained CySCs are unable to maintain GSCs in the absence of Stat92E. Therefore, the extended niche function of CySCs is solely attributable to JAK/STAT pathway function (Amoyel, 2013).

    This study has shown that Hh from the Drosophila testis niche is a self-renewal factor for CySCs and that Hh signaling does not contribute to the role of CySCs as a niche for GSCs. This supports the model that the Hh and JAK/STAT pathways act independently within CySCs. The results therefore confirm those recently reported by another group (Michel, 2012), who showed that Hh regulates CySC self-renewal, and extend their results by demonstrating the genetic independence of Hh and the other pathway (i.e. JAK/STAT) that is crucial in CySC function (Amoyel, 2013).

    It is notable that two signals regulate CySC self-renewal but only JAK/STAT signaling contributes to the GSC niche. Moreover, despite the drastic reduction in CySCs in hhts2 testes (from ~36 in controls to ~8), GSCs do remain in hh mutant animals albeit at reduced numbers. The reduction in GSCs in hh mutants is not due to changes in the size of the hub. These data suggest that most CySCs are dispensable for their niche function and that only a few BMP-producing CySCs are needed to maintain GSC self-renewal. This raises the question as to whether, in a wild-type animal, there are distinct populations of CySCs, some with activated Stat92E that produce BMPs and act as a niche for GSCs, and others with activated Hh signaling that participate only in self-renewal and the production of cyst progeny. This is consistent with the fact that, despite the presence of ~36 Zfh1-positive CySCs, elevated Stat92E is only seen in a few CySCs. However, it is also conceivable that all Zfh1-positive CySCs are equivalent and that high Stat92E correlates, for instance, with a specific phase of the cell cycle, such as the repositioning of the spindle during anaphase that brings the nucleus of the CySC closer to the hub interface and might expose that CySC to more Upd ligand. This possibility implies a much more dynamic stem cell niche for the GSCs than has been previously appreciated (Amoyel, 2013).

    The results indicate that the Hh and JAK/STAT pathways act mostly in parallel, although activating Hh may delay the differentiation of CySCs that are deficient for JAK/STAT pathway components. It is unclear why the CySC would require both signaling inputs to be maintained. However, it should be noted that these inputs contribute different information, as JAK/STAT signaling imparts niche potential, and Hh signaling additionally ensures that the right number of CySCs are present and provide cyst cells for normal spermatogonial development. Future work will establish whether self-renewal in CySCs depends on two sets of genes controlled separately by the Hh and JAK/STAT pathways or whether they converge on the same targets. The first possibility is supported by the fact that Hh does not contribute to the niche function of STAT in CySCs, indicating that different targets (presumably BMPs) are regulated differently (Amoyel, 2013).

    One consequence of this work is to lead to a reevaluation of the differences between male and female gonad development in Drosophila. Indeed, Hh signaling is an essential regulator of the self-renewal and the number of follicle stem cells, the offspring of which carry out a comparable function to cyst cells by ensheathing germ line cysts. In the ovary, as in the testis, JAK/STAT signaling in somatic cells is required for the maintenance of GSCs via BMP production. However, in the ovary, the escort cells and cap cells are the JAK/STAT-responsive niche cells, implying that CySCs in the male gonad fulfill the function of two cell types in the female gonad and require both the signals used in the female to do so. Finally, the data evoke the interesting possibility that Hh has a conserved ancestral role in male gonads. Mutation in one of the three mammalian hh homologs, desert hedgehog (Dhh), causes male sterility and a loss of somatic support cells called Leydig cells. However, the cellular niche for spermatogenesis in mammals is less well understood than in Drosophila and it remains to be established whether the Hh pathway orchestrates similar cellular functions (Amoyel, 2013).

    Nanotubes mediate niche-stem-cell signalling in the Drosophila testis

    Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. This study shows that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. The study proposes that microtubule-based nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem-cell signalling (Inaba, 2015).

    The Drosophila testis represents an excellent model system to study niche-stem-cell interactions because of its well-defined anatomy: eight to ten germline stem cells (GSCs) are attached to a cluster of somatic hub cells, which serve as a major component of the stem cell niche. The hub secretes at least two ligands: the cytokine-like ligand Unpaired (Upd), and a BMP ligand Decapentaplegic (Dpp), both of which regulate GSC maintenance. GSCs typically divide asymmetrically, so that one daughter of the stem cell division remains attached to the hub and retains stem cell identity, while the other daughter, called a gonialblast, is displaced away from the hub and initiates differentiatio. Given the close proximity of GSCs and gonialblasts, the ligands (Upd and Dpp) must act over a short range so that signalling is only active in stem cells, but not in differentiating germ cells. The basis for this sharp boundary of pathway activation remains poorly understood (Inaba, 2015).

    Using green fluorescent protein (GFP)-α1-tubulin84B expressed in germ cells (nos-gal4>UAS-GFP-αtub), this study found that GSCs form protrusions, referred to as microtubule-based (MT)-nanotubes hereafter, that extend into the hub. MT-nanotubes are sensitive to fixation similar to other thin protrusions reported so far, such as tunnelling nanotubes, explaining why they have escaped detection in previous studies. MT-nanotubes appear to be specific to GSCs: 6.67 MT-nanotubes were observed per testis in the GSC population (or 0.82 per cell). The average thickness and length of MT-nanotubes are 0.43 ± 0.29 µm (at the base of MT-nanotube) and 3.32 ± 1.6 µm, respectively. These GSC MT-nanotubes are uniformly oriented towards the hub area. By contrast, differentiating germ cells showed only 0.44 MT-nanotubes per testis (or <0.002 per cell), without any particular orientation when present. MT-nanotubes were sensitive to colcemid, the microtubule-depolymerizing drug, but not to the actin polymerization inhibitor cytochalasin B, suggesting that MT-nanotubes are microtubule-based structures. MT-nanotubes were not observed in mitotic GSCs, and GSCs form new MT-nanotubes as they exit from mitosis. By contrast, MT-nanotubes in interphase GSCs were stably maintained for up to 1 h of time-lapse live imaging. Although cell-cycle-dependent formation of MT-nanotube resembles that of primary cilia, MT-nanotubes are distinct structures, in that they lack acetylated microtubules and are sensitive to fixation. Furthermore, a considerable fraction of GSCs form multiple MT-nanotubes per cell (54% of GSCs with MT-nanotubes), and MT-nanotubes are not always associated with the centrosome/basal body, as is the case for the primary cilia (Inaba, 2015).

    To examine the geometric relationship between MT-nanotubes and hub cells further, MT-nanotubes were imaged in combination with various cell membrane markers, followed by three-dimensional rendering. Although the MT-nanotubes are best visualized in unfixed testes that express GFP-αTub in germ cells, adding a low concentration (1 μM) of taxol to the fixative preserves MT-nanotubes, allowing immunofluorescence staining. First, Armadillo (Arm, β-catenin) staining, which marks adherens junctions formed at hub cell/hub cell as well as hub cell/GSC boundaries, revealed that adherens junctions do not form on the surface of MT-nanotubes. Using FM4-64 styryl dye, it was found that the MT-nanotubes are ensheathed by membrane lipids. Furthermore, myristoylation/palmitoylation site GFP (myrGFP), a membrane marker, expressed in either the germline or hub cells illuminated MT-nanotubes, suggesting that the surface membrane of a MT-nanotube is juxtaposed to hub-cell plasma membrane (Inaba, 2015).

    Genes were examined that regulate primary cilia and cytonemes for their possible involvement in MT-nanotube formation. RNA interference (RNAi)-mediated knockdown of oseg2 (IFT172), osm6 (IFT52) and che-13 (IFT57), components of the intraflagellar transport (IFT)-B complex that are required for primary cilium anterograde transport and assembly, significantly reduced the length and the frequency of MT-nanotubes. Knockdown of Dlic, a dynein intermediate chain required for retrograde transport in primary cilia<, also reduced the MT-nanotube length and frequency. Knockdown of klp10A, a Drosophila homologue of mammalian kif24 (a MT-depolymerizing kinesin of the kinesin-13 family, which suppresses precocious cilia formation), resulted in abnormally thick/bulged MT-nanotubes. No significant changes were observed in MT-nanotube morphology upon knockdown of IFT-A retrograde transport genes, such as oseg1 and oseg3 (Inaba, 2015).

    Endogenous Klp10A localized to MT-nanotubes both in wild-type testes and in GFP-αTub-expressing testes. GFP-Oseg2 (IFT-B), GFP-Oseg1, GFP-Oseg3 (IFT-A) and Dlic also localized to the MT-nanotubes when expressed in germ cells. The localization of IFT-A components to MT-nanotubes, without detectable morphological abnormality upon mutation/knockdown, is reminiscent of the observation that most of the genes for IFT-A are not required for primary cilia assembly. Expression of a dominant negative form of Dia (DiaDN) or a temperature-sensitive form of Shi (Shits) in germ cells (nos-gal4>UAS-diaDN or UAS-shits), which perturb cytoneme formation, did not influence the morphology or frequency of MT-nanotubes in GSCs. Taken together, these results show that primary cilia proteins localize to MT-nanotubes and regulate their formation (Inaba, 2015).

    In search of the possible involvement of MT-nanotubes in hub-GSC signalling, it was found that the Dpp receptor, Thickveins (Tkv), expressed in germ cells (nos-gal4>tkv-GFP) was observed within the hub region, in contrast to GFP alone, which remained within the germ cells. A GFP protein trap of Tkv (in which GFP tags Tkv at the endogenous locus) also showed the same localization pattern as Tkv-GFP expressed by nos-gal4. By inducing GSC clones that co-express Tkv-mCherry and GFP-αTub, it was found that Tkv-mCherry localizes along the MT-nanotubes as puncta. Furthermore, using live observation, Tkv-mCherry puncta were observed to move along the MT-nanotubes marked with GFP-αTub, suggesting that Tkv is transported towards the hub along the MT-nanotubes. It should be noted that, in the course of this study, it was noticed that mCherry itself localized to the hub when expressed in germ cells, similar to Tkv-GFP and Tkv-mCherry. Importantly, the receptor for Upd, Domeless (Dome), predominantly stayed in the cell body of GSCs, demonstrating the specificity/selectivity of MT-nanotubes in trafficking specific components of the niche signalling pathways. A reporter of ligand-bound Tkv, TIPF localized to the hub region together with Tkv-mCherry, in addition to its reported localization at the hub-GSC interface. Furthermore, Dpp-GFP expressed by hub cells co-localized with Tkv-mCherry expressed in germline. These results suggest that ligand (Dpp)-receptor (Tkv) engagement and activation occurs at the interface of the MT-nanotube surface and the hub cell plasma membrane. Knockdown of IFT-B components (oseg2RNAi, che-13RNAi or osm6RNAi), which reduces MT-nanotube formation, resulted in reduction of the number of Tkv-GFP puncta in the hub area, concomitant with increased membrane localization of Tkv-GFP. A similar trend was observed upon treatment of the testes with colcemid, suggesting that MT-nanotubes are required for trafficking of Tkv into the hub area. By contrast, knockdown of Klp10A, which causes thickening of MT-nanotubes, led to an increase in the number of Tkv-GFP puncta in the hub area. Taken together, these data suggest that Tkv is trafficked into the hub via MT-nanotubes, where it interacts with Dpp secreted from the hub (Inaba, 2015).

    Knockdown of klp10A (klp10ARNAi) led to elevated phosphorylated Mad (pMad) levels, a readout of Dpp pathway activation, in GSCs. By contrast, RNAi-mediated knockdown of oseg2, osm6 and che-13 (IFT-B components), which causes shortening of MT-nanotubes, reduced the levels of pMad in GSCs. Dad-LacZ, another readout of Dpp signalling activation, exhibited clear upregulation upon knockdown of klp10A. GSC clones of che-13RNAi, osm6RNAi or oseg2452 were lost rapidly compared with control clones, consistent with the idea that MT-nanotubes help to promote Dpp signal transduction. Knockdown of oseg2, che-13 and osm6 did not visibly affect cytoplasmic microtubules, suggesting that GSC maintenance defects upon knockdown of these genes are probably mediated by their role in MT-nanotube formation. Global RNAi knockdown of these genes in all GSCs using nos-gal4 did not cause a significant decrease in GSC numbers , indicating that compromised Dpp signalling due to MT-nanotube reduction leads to a competitive disadvantage in regards to GSC maintenance only when surrounded by wild-type GSCs (Inaba, 2015).

    When klp10ARNAi GSC clones were induced, pMad levels specifically increased in those GSC clones, indicating that Klp10A acts cell-autonomously in GSCs to influence Dpp signal transduction. Importantly, klp10ARNAi spermatogonia did not show a significant elevation in pMad level compared with control spermatogonia, demonstrating that the role of Klp10A in regulation of Dpp pathway is specific to GSCs. pMad levels did not change in spermatogonia upon manipulation of MT-nanotube formation. GSC clones of klp10ARNAi or klp10A null mutant (klp10A24) did not dominate in the niche, despite upregulation of pMad, possibly because of its known role in mitosis. Importantly, these conditions did not significantly change STAT92E levels, which reflect Upd-JAK-STAT signalling in GSCs, revealing the selective requirement of MT-nanotubes in Dpp signalling. Together, these results demonstrate that MT-nanotubes specifically promote Dpp signalling and their role in enhancing the Dpp pathway is GSC specific (Inaba, 2015).

    Since cytonemes are induced/stabilized by the signalling molecules themselves, the possible involvement of Dpp in MT-nanotube formation was explored First, it was found that a temperature-sensitive dpp mutant (dpphr56/dpphr4) exhibited a dramatic decrease in the frequency of MT-nanotubes (0.067 MT-nanotubes per GSC) and the remaining MT-nanotubes were significantly thinner. Knockdown of tkv (tkvRNAi) in GSCs also resulted in reduced length and frequency of MT-nanotubes. Conversely, overexpression of Tkv (tkvOE) in germ cells led to significantly longer MT-nanotubes. Interestingly, expression of a dominant negative Tkv (tkvDN), which has intact ligand-binding domain but lacks its intracellular GS domain and kinase domain, resulted in thickening of MT-nanotubes, rather than reducing the thickness/length. This indicates that ligand-receptor interaction, but not downstream signalling events, is sufficient to induce MT-nanotube formation. Strikingly, upon ectopic expression of Dpp in somatic cyst cells (tj-lexA>dpp), spermatogonia/spermatocytes were observed to have numerous MT-nanotubes, suggesting that Dpp is necessary and sufficient to induce or stabilize MT-nanotubes in the neighbouring germ cells. In turn, MT-nanotubes may promote selective ligand-receptor interaction between hub and GSCs, leading to spatially confined self-renewal (Inaba, 2015).

    This study shows that previously unrecognized structures, MT-nanotubes, extend into the hub to mediate Dpp signalling. It is proposed that MT-nanotubes form a specialized cell surface area, where productive ligand-receptor interaction occurs. In this manner, only GSCs can access the source of highest ligand concentration in the niche via MT-nanotubes, whereas gonialblasts do not experience the threshold of signal transduction necessary for self-renewal, contributing to the short-range nature of niche signalling. In summary, the results reported here illuminate a novel mechanism by which the niche specifies stem cell identity in a highly selective manner (Inaba, 2015).

    Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo

    Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. This study shows that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. The driving force behind such behavior is accelerated proliferation. These results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process (Amoyel, 2014).

    lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis

    To function properly, tissue-specific stem cells must reside in a niche. The Drosophila testis niche is one of few niches studied in vivo. Here, a single niche, comprising ten hub cells, maintains both germline stem cells (GSC) and somatic stem cells (cyst stem cells, CySC). This study shows that lines is an essential CySC factor. Surprisingly, lines-depleted CySCs adopted several characteristics of hub cells, including the recruitment of new CySCs. This led to an examination of the developmental relationship between CySCs and hub cells. In contrast to a previous report, no significant conversion was seen of steady-state CySC progeny to hub fate. However, it was found that these two cell types derive from a common precursor pool during gonadogenesis. Furthermore, embryos mutant for lines, an obligate antagonist of bowl function (Hatini, 2005), exhibited gonads containing excess hub cells, indicating that lines represses hub cell fate during gonadogenesis. In many tissues, lines acts antagonistically to bowl, and it was found that this is true for hub specification, establishing bowl as a positively acting factor in the development of the testis niche (Dinardo, 2011).

    This analysis together with previous lineage-tracing shows that some hub cells and some CySCs are derived from the SGPs of PS11. The remaining CySCs could in principle derive from either PS10 or PS12. Currently, neither of those mesodermal parasegments can be uniquely lineage traced. However, the remaining hub cells probably derive from PS10 SGPs, as that would fit with the identification of receptor tyrosine kinase signaling as an antagonist of hub fate among posterior SGPs (Dinardo, 2011).

    Aside from pathways known to repress hub fate, work is also beginning to identify positive functions necessary to specify these cells. This study found that bowl is one factor, as mutants had fewer hub cells, and those present appeared compromised for hub cell function. Still, the existence of residual hub cells suggests that Bowl is not the only factor required for hub cell specification, and, indeed, Notch signaling is a second positively acting component (Dinardo, 2011).

    It is of interest that both Notch and bowl are positively required for hub cell specification, since these two genes act together in several other tissues. However, the exact epistatic relationship between bowl and the Notch pathway can be complex. There is some evidence that Notch activation leads to Bowl accumulation. Since it was found that Notch and also the relief-of-repression hierarchy consisting of drm/lines/bowl acts during hub cell specification, a simple model would be that Notch activation induces an antagonist of lines, for example, drm. This allows Bowl protein to accumulate in a subset of SGPs and to promote hub fate, while SGPs that retain functional Lines would adopt CySC fate. Attractive as this model is, testing some of its predictions was difficult. Attempts to visualize endogenous protein accumulation for Bowl and for Lines in the gonad has been frustrating. In addition, although drm mutants had reduced hub cell number, drm-expressing cells have not been identified within the forming gonad (Dinardo, 2011).

    Thus, the relationship between Notch and the drm/lines/bowl cassette may be indirect, an outcome of the fact that both systems use the co-repressor Groucho. It has been suggested that conditions which alter the levels of available Bowl, such as in drm (down) or lines (up) mutants, could reciprocally affect the amount of Groucho available to Suppressor of Hairless, which requires this co-repressor to maintain repression of Notch target genes. Whether or not the relationship between Notch and Bowl for hub cell specification is direct, loss of Notch has a stronger phenotype than loss of bowl. Thus, the Notch pathway must also engage a separate pathway that specifies some hub cells (Dinardo, 2011).

    During gonadogenesis, the current model suggests that Lines represses hub fate and promotes CySC fate. It is intriguing that a requirement for lines persists in CySCs during the steady-state operation of the testis. Analysis at this later stage suggests that lines plays a similar, though not identical, role. Although cells in gonads from lines mutant embryos fully adopt hub cell fate, in the testis the lines-depleted CySCs only partially adopt hub fate, as they do not recruit new GSCs. Thus, at steady-state, some additional regulation over the distinction between CySC and hub cell fate has been added on. Such a factor(s) remain to be identified (Dinardo, 2011).

    Even the partial conversion of lines mutant CySCs into hub cells is an intriguing phenotype. Recently, a lineage relationship has been described for several stem cell-niche pairs, where stem cells can generate cells of their niche. These include production of Paneth cells in the mammalian intestine, the production of transient niche cells in the fruitfly intestine, and the repair of ependymal cells by neural progenitors of the sub-ventricular zone. In the steady-state testis, it was recently suggested that CySCs can efficiently generate new hub cells. Thus, it is considered whether lines might be deployed at steady state to govern this transition, but no increase was detected in conversion in flies with decreased lines gene dose. In fact, in wild type it was found that the conversion of cells into hub fate was insignificant compared with what has been reported. As one method used in this study was essentially identical to one used in the original report, the reason for the discrepancy is uncertain. Lineage-marking was very efficient. For example, two days after delivery of FLP by one heat-shock, 85% of testes possessed a labeled CySCs, with an average of 1.5 CySCs per testis. In the previous report, a similar regimen produced only 13% of testes with labeled CySCs. Still, it is not clear how an increase in marking efficiency could account for a decrease in apparent frequency of conversion of CySC progeny into hub cells (Dinardo, 2011).

    Thus, since CySCs do not normally generate hub cells, why might lines function be maintained in CySCs so long after its embryonic requirement? The favored model is that lines is deployed during steady-state for a distinct purpose. For example, recent work on the lines/bowl cassette suggests that it assists in signal integration. This idea is appealing as the niche cells and their local environment are subjected to the action of a number of signaling pathways, such as Hh, Wnt, BMP, Jak/STAT and EGFR. Currently, it is not fully understood how these pathways function in the steady-state operation of the niche, nor how signals from distinct pathways integrate to produce a single outcome. Even the dogma of the heavily studied Jak/STAT pathway continues be challenged and refined by recent data. Perhaps as newer data uncovers the nuanced roles of several of these pathways, the lines/bowl cassette will figure into the integration of those signals (Dinardo, 2011).

    Finally, the fact that lines-depleted CySCs recruited neighboring wild-type somatic cells to adopt CySC fate is striking. Although the imaging tools necessary to reveal which somatic cells are recruited to CySC fate are unavailable, the fact of their recruitment suggests that under these mutant conditions cyst cells can de-differentiate into CySCs. It has been elegantly shown that maturing germ cells can de-differentiate, creating new GSCs. As those maturing germ cells are encysted by the somatic cyst cells, during de-differentiation this grouping must break apart to release individual germline cells that repopulate the niche. Whether cyst cells de-differentiate to CySCs in these cases has not been directly assessed. If this happens under physiological conditions, it would be of great interest to study how cyst cells de-differentiation occurs, and testes harboring lines-deficient clones might aid in such studies (Dinardo, 2011).

    Occluding junctions maintain stem cell niche homeostasis in the fly testes

    Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).

    The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).

    During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).

    Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).

    Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w1118 progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).

    Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).

    To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).

    Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).

    In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).

    One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).

    Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).

    Steroid signaling promotes stem cell maintenance in the Drosophila testis

    Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). This study shows that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which has been shown to maintain CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance (Li, 2014).

    This work shows that the steroid hormone 20E plays an important role in maintaining stem cells in theDrosophila testis: 20E, receptors of ecdysone signaling, and downstream targets are required directly in CySCs for their maintenance. When ecdysone signaling is lost in CySCs, GSCs are also lost, but it is unclear if their maintenance requires an ecdysone-dependent or independent signal from the CySCs. The requirement for EcR in the testis is isoform-specific: expression of EcR-B2 in the CySC lineage is sufficient to rescue loss of GSCs and CySCs and increased cell death in EcR mutant testes, suggesting that there might be a temporal and spatial control of ecdysone signaling in the adult testis. In addition, evidence is provided that ecdysone signaling, as in the ovary, is able to interact with an intrinsic chromatin-remodeling factor, Nurf301, to promote stem cell maintenance. Therefore, these studies have revealed a novel role for ecdysone signaling in Drosophila male reproduction (Li, 2014).

    Although ecdysone signaling is required in both ovaries and testes for stem cell maintenance, the responses in each tissue are likely to be sex-specific. In the ovary, 20E controls GSCs directly, by modulating their proliferation and self-renewal, and it acts predominantly through the downstream target gene E74. In contrast, male GSCs require ecdysone signaling only indirectly: ecdysone signaling was found to be required in the CySC lineage to maintain both CySCs and GSCs. In a previous study, RNAi-mediated knockdown of EcR, usp or E75 in the CySC lineage did not result in a significant loss of GSCs; however, the number of CySCs was not determined, and the phenotype was examined after 4 or 8 days, not 14 days as in this study. It is suspected that the earlier time points used in that study may not have allowed enough time for a significant number of GSCs to be lost (Li, 2014).

    During development, 20E is produced in the prothoracic gland (PG) and further metabolized to 20E in target tissues, but the PG does not persist into adulthood. In adult female Drosophila, the ovary is a source of 20E. In contrast, the identification of steroidogenic tissues in adult male Drosophila remains the subject of active investigation. The level of 20E in adult males is significantly lower than in adult females, but it can be detected in the testis. Furthermore, RNA-seq data show that shade, which encodes the enzyme that metabolizes the prohomone ecdysone to 20E, is expressed in the adult testis, suggesting that the adult testis may produce 20E. However, the sources of 20E production in adult Drosophila males remain to be determined experimentally (Li, 2014).

    20E, like other systemic hormones, can have tissue-specific effects or differential effects on the same cell type as development proceeds. These differences are mediated at least in part by the particular downstream target genes that are activated in each case. For example, in female 3rd instar larval ovaries, ecdysone signaling upregulates br expression to induce niche formation and PGC differentiation, but br is not required for GSC maintenance in the adult ovary; instead, E74 plays this role. Similarly, br is required for the establishment of intestinal stem cells (ISCs) in the larval and pupal stages but not for ISC function in adults. This study shows that ecdysone signaling in the adult testis is mediated by different target genes than in the ovary: E74, but not E75 or br, regulate stem cell function in the ovary, whereas E75 and ftz-f1 are important for stem cell maintenance in the testis. Since E75 is itself a nuclear hormone receptor that responds to the second messenger nitric oxide, it will be interesting to know whether E75's partner DHR3 also plays a role in CySCs. An intriguing question for future studies will be how different ecdysone target genes interact with the various signaling pathways that maintain stem cells in the ovary or testis (Li, 2014).

    Since 20E levels can actively respond to physiological changes induced by environmental cues, it is possible that the effect of 20E on testis stem cell maintenance might reflect changes in diet, stress, or other environmental cues. For example, in Aedes aegypti, ecdysteroid production in the ovary is stimulated by blood feeding and this is an insulin-dependent process. In Drosophila, ecdysone signaling is known to interact with the insulin pathway in a complex way. Ovaries from females with hypomorphic mutations in the insulin-like receptor have reduced levels of 20E. Furthermore, ecdysone signaling can directly inhibit insulin signaling and control larval growth in the fat body. Thus, ecdysone signaling may interact with insulin signaling during testis stem cell maintenance. Previously, it was shown that GSCs in the ovary and testis can respond to diet through insulin signaling, which is required to promote stem cell maintenance in both sexes. It is possible that diet can affect 20E levels and thus regulate stem cell maintenance. In addition to diet, stress can also affect 20E levels, as is the case in Drosophila virilis, where 20E levels increase significantly under high temperature stress. A similar effect has been found in mammals, where the steroid hormone cortisol is released in response to psychological stressor. Finally, 20E levels are also influenced by mating. In Anopheles gambiae, males transfer 20E to blood-fed females during copulation, which is important for egg production. In female Drosophila, whole body ecdysteroid levels also increase after mating. Studying the roles of hormonal signaling in mediating stem cell responses to stress and other environmental cues will be an exciting topic for future studies. From this work it is now clear that, as in mammals, steroid signaling plays critical roles in adult stem cell function during both male and female gametogenesis (Li, 2014).

    The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche

    Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). This study found that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (DsxM) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of DsxM. The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. This work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals (Ma, 2014).

    Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster

    The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, this study showed that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. It is proposed that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells (Qian, 2014).

    Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation

    Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. This study shows that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, TFIIH stability was not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients (Cruz-Becerra, 2016).

    Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche

    Adult stem cells reside in microenvironments called niches, where they are regulated by both extrinsic cues, such as signaling from neighboring cells, and intrinsic factors, such as chromatin structure. This study reports that in the Drosophila testis niche an H3K27me3-specific histone demethylase encoded by Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (dUTX) maintains active transcription of the Suppressor of cytokine signaling at 36E (Socs36E) gene by removing the repressive H3K27me3 modification near its transcription start site. Socs36E encodes an inhibitor of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Whereas much is known about niche-to-stem cell signaling, such as the JAK-STAT signaling that is crucial for stem cell identity and activity, comparatively little is known about signaling from stem cells to the niche. The results reveal that stem cells send feedback to niche cells to maintain the proper gene expression and architecture of the niche. dUTX acts in cyst stem cells (CySCs) to maintain gene expression in hub cells through activating Socs36E transcription and preventing hyperactivation of JAK-STAT signaling. dUTX also acts in germline stem cells to maintain hub structure through regulating DE-Cadherin levels. Therefore, these findings provide new insights into how an epigenetic factor regulates crosstalk among different cell types within an endogenous stem cell niche, and shed light on the biological functions of a histone demethylase in vivo (Tarayrah, 2013).

    This study identified a new epigenetic mechanism that negatively regulates the JAK-STAT signaling pathway in the Drosophila testis niche: the H3K27me3-specific demethylase dUTX acts in CySCs to remove the repressive H3K27me3 histone modification near the TSS of Socs36E to allow its active transcription. Socs36E acts upstream to suppress Stat92E activity and to restrict CySCs from overpopulating the testis niche. In addition, dUTX acts in CySCs to prevent hyperactivation of Stat92E in hub cells, which would otherwise ectopically turn on Zfh1 expression. When zfh1 cDNA was ectopically driven in hub cells using the upd-Gal4 driver, no obvious defect could be identified. Therefore, the biological consequence of ectopic Zfh1 expression in hub cells remains unclear. However, ectopic Zfh1 expression in hub cells and the overpopulation of Zfh1-expressing cells around the hub are two connected phenomena because both phenotypes are caused by loss of dUTX in CySCs (Tarayrah, 2013).

    UTX also acts in GSCs to regulate DE-Cadherin levels to maintain proper GSC-hub interaction and normal morphology of the hub. It has been reported that differential expression of different cadherins causes cells with similar cadherin types and levels to aggregate. In wt testes, hub cells express higher levels of DE-Cadherin and therefore tightly associate with each other. Loss of dUTX in germ cells leads to higher levels of DE-Cadherin in GSCs, which probably allows them to intermingle with hub cells and causes disrupted hub architecture. It has also been demonstrated that the major role of JAK-STAT in GSCs is for GSC-hub adhesion, suggesting that the expression and/or activity of cell-cell adhesion molecules, such as DE-Cadherin, depends on JAK-STAT signaling. Therefore, the abnormal DE-Cadherin activity in GSCs in dUTX testis could also result from misregulated JAK-STAT signaling in the testis niche. dUTX is a new negative epigenetic regulator of the JAK-STAT signaling pathway (Tarayrah, 2013).

    The JAK-STAT signaling pathway plays crucial roles in stem cell maintenance in many different stem cell types across a wide range of species. These studies identify the histone demethylase dUTX as a new upstream regulator of the JAK-STAT pathway, which directly controls the transcription of Socs36E. In addition to acting as an antagonist of JAK-STAT signaling, Socs36E has been reported to be a direct target gene of the Stat92E transcription factor (Terry, 2006). Therefore, increased Stat92E would be expected to upregulate Socs36E expression, but this was not observed in dUTX mutant testes. Instead, the data revealed that Socs36E expression decreased, whereas Stat92E expression increased, in dUTX testes, consistent with the hypothesis that Socs36E is a direct target gene of dUTX and acts upstream of Stat92E (Tarayrah, 2013).

    Sustained activity of the JAK-STAT pathway in cyst cells has been reported to activate BMP signaling, which leads to GSC self-renewal outside the niche and gives rise to a tumor-like phenotype in testis. To examine BMP pathway activity, immunostaining experiments were performed using antibodies against phospho-SMAD (pSMAD), a downstream target of BMP signaling. No obvious difference was detected in the pSMAD signal between the dUTX testes and wt control, nor were any germline tumors detected in dUTX testes. It is speculated that germline tumor formation upon activation of the JAK-STAT pathway is secondary to the overproliferation of Zfh1-expressing cells, which was not observed in dUTX testes (Tarayrah, 2013).

    This study also provides an example of the multidimensional cell-cell communication that takes place within a stem cell niche. Many studies of the stem cell niche have focused on understanding niche-to- stem cell signaling in controlling stem cell identity and activity. For example, in the Drosophila female GSC niche, Upd secreted from terminal filaments activates the JAK-STAT pathway in cap cells and escort cells, which subsequently produce the BMP pathway ligand Decapentaplegic (Dpp) to activate BMP signaling and prevent transcription of the differentiation factor bag-of-marbles (bam) in GSCs. In the Drosophila intestinal stem cell (ISC) niche, the visceral muscle cells underlying the intestine secrete Wingless to activate Wnt signaling and Upd to activate JAK-STAT signaling in ISCs, which are required to maintain ISC identity and activity (Tarayrah, 2013).

    More studies have now revealed the multidirectionality of signaling within the stem cell niche. For example, in the Drosophila female GSC niche, GSCs activate Epidermal growth factor receptor (Egfr) signaling in the neighboring somatic cells, which subsequently represses expression of the glypican Dally, a protein required for the stabilization and mobilization of the BMP pathway ligand Dpp. Through this communication between GSCs and the surrounding somatic cells, only GSCs maintain high BMP signaling. The current studies establish another example of the multidimensional cell-cell communications that occur within the testis stem cell niche, where CySCs and GSCs have distinct roles in regulating hub cell identity and morphology (Tarayrah, 2013).

    The data identified new roles of a histone demethylase in regulating endogenous stem cell niche architecture and proper gene expression. Previous studies have reported in vivo functions of histone demethylases in several model organisms. For example, mammalian UTX has been shown to associate with the H3K4me3 histone methyltransferase MLL2, suggesting its potential antagonistic role to the PcG proteins. The PcG proteins play a crucial role in Hox gene silencing in both Drosophila and mammals. Consistently, mammalian UTX has been reported to directly bind and activate the HOXB1 gene locus. In addition to antagonizing PcG function, H3K27me3 demethylases play crucial roles during development. For example, in zebrafish, inactivating the UTX homolog (kdm6al) using morpholino oligonucleotides leads to defects in posterior development, and in C. elegans the dUTX homolog (UTX-1) is required for embryonic and postembryonic development, including gonad development. Furthermore, loss of UTX function in embryonic stem cells leads to defects in mesoderm differentiation, and somatic cells derived from UTX loss-of-function human or mouse tissue fail to return to the ground state of pluripotency. These reports demonstrate that UTX is not only required for proper cellular differentiation but also for successful reprogramming. However, despite multiple reports on the in vivo roles of H3K27me3-specific demethylases, little is known about their functions in any endogenous adult stem cell system (Tarayrah, 2013).

    Whereas mammals have multiple H3K27me3 demethylases, dUTX is the sole H3K27me3-specific demethylase in Drosophila. This unique feature, plus the well-characterized nature of Drosophila adult stem cell systems, make interpretation of the endogenous functions of histone demethylases in Drosophila unambiguous. Because mammalian UTX has been reported as a tumor suppressor, understanding the endogenous functions of dUTX in an adult stem cell system might facilitate the use of histone demethylases for cancer treatment. In summary, these results demonstrate that stem cells send feedback to the niche cells to maintain their proper gene expression and morphology. Furthermore, this feedback is regulated through the JAK-STAT signaling pathway, the activity of which is controlled by a chromatin factor, providing an example of crosstalk between these two regulatory pathways (Tarayrah, 2013).

    Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis

    Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). This study shows a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. This work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche (Stine, 2014: PubMed).

    Socs36E controls niche competition by repressing MAPK signaling in the Drosophila testis

    Socs36E, which encodes a negative feedback inhibitor of the JAK/STAT pathway, is the first identified regulator of niche competition in the Drosophila testis. The competitive behavior of (Suppressor of cytokine signaling at 36E) (Socs36E) mutant cyst stem cells (CySCs) has been attributed to increased JAK/STAT signaling. This study shows that competitive behavior of Socs36E mutant CySCs is due in large part to unbridled Mitogen-Activated Protein Kinase (MAPK) signaling. In Socs36E mutant clones, MAPK activity is elevated. Furthermore, it was found that clonal upregulation of MAPK in CySCs leads to their outcompetition of wild type CySCs and of germ line stem cells, recapitulating the Socs36E mutant phenotype. Indeed, when MAPK activity is removed from Socs36E mutant clones, they lose their competitiveness but maintain self-renewal, presumably due to increased JAK/STAT signaling in these cells. Consistently, loss of JAK/STAT activity in Socs36E mutant clones severely impairs their self-renewal. Thus, these results enable the genetic separation of two essential processes that occur in stem cells. While some niche signals specify the intrinsic property of self-renewal, which is absolutely required in all stem cells for niche residence, additional signals control the ability of stem cells to compete with their neighbors. Socs36E is the node through which these processes are linked, demonstrating that negative feedback inhibition integrates multiple aspects of stem cell behavior (Amoyel, 2016a).

    Stem cell niches are complex environments that provide support for stem cells through molecular signals. Several well-characterized niches provide not just one but multiple signals which stem cells must integrate and interpret in order to remain at the niche and self-renew. How this integration is achieved is not well understood at present. Furthermore, in order to maintain the appropriate number of stem cells and the homeostatic balance between self-renewal and differentiation, it is necessary that self-renewal cues be present in limiting amounts or that their activity be dampened to prevent excessive accumulation of stem cells. One general feature of many signal transduction pathways is the presence of feedback inhibitors. These are dampeners of signaling, transcriptionally induced by the signaling itself, that prevent signal levels from being aberrantly high. One such family of feedback inhibitors is the Suppressor of Cytokine Signaling (SOCS) proteins, which were identified as inhibitors of JAK/STAT signal transduction, and are SH2- and E3-ligase domain-containing proteins. The SH2 domain binds phosphorylated (i.e., activated) signal transduction components and the E3-ligase targets them for degradation by Ubiquitin-dependent proteolysis. In mammals, SOCS proteins can thus inhibit several tyrosine kinase-dependent signaling pathways, including JAK/STAT and Mitogen-Activated Protein Kinase (MAPK) (Amoyel, 2016a).

    The Drosophila testis is an ideal model system to study questions of signal regulation and integration in stem cells. The testis niche, called the hub, supports two stem cell populations. The first, germ line stem cells (GSCs), gives rise to sperm after several transit-amplifying divisions leading up to meiosis. The second, somatic cyst stem cells (CySCs), gives rise to cyst cells, the essential support cells for germ line development. Many ligands for signaling pathways are produced by the hub, including the JAK/STAT pathway agonist, Unpaired (Upd), the Hedgehog (Hh) pathway ligand Hh and the Bone Morphogenetic Protein (BMP) homologs Decapentaplegic (Dpp) and Glass Bottom Boat (Gbb). The latter two signals are also produced by CySCs and are required in GSCs for self-renewal, indicating that CySCs constitute part of the niche for GSCs along with the hub. CySCs require JAK/STAT and Hh activity for self-renewal (Amoyel, 2016a).

    CySCs and GSCs compete for space at the niche, a phenomenon that was revealed by the analysis of testes lacking the JAK/STAT feedback inhibitor Socs36E. In these animals, excessive JAK/STAT activity was detected in CySCs, and Socs36E mutant CySCs displaced the resident wild type GSCs. Additionally, it has been shown that CySCs with sustained Hh signaling or sustained Yorkie (Yki) activity also outcompeted neighboring wild type GSCs, indicating that several signaling pathways can control niche competition. Moreover, prior to out-competing GSCs, mutant CySCs displaced neighboring wild type CySCs, indicating that both intra- (CySC-CySC) and inter-lineage (CySC-GSC) competition take place in the testis. While the two types of competition appear related, in that one precedes the other, there are instances in which only intra-lineage competition takes place. While the competitive phenotype of Socs36E mutant CySCs was ascribed to increased JAK/STAT signaling, it was surprising to find that clonal gain-of-function in JAK/STAT signaling in CySCs did not induce competitive behavior, and it was concluded that loss of Socs36E did not mimic increased JAK/STAT signaling in CySC (Amoyel, 2016a).

    This study addressed whether other mechanisms could account for the competitive behavior of Socs36E mutant CySCs. Because SOCS proteins can inhibit MAPK signaling in cultured cells and in Drosophila epithelial tissues, this study examined if Socs36E repression of MAPK signaling underlied the Socs36E competitive phenotype. Indeed, it was found that Socs36E inhibits MAPK signaling in CySCs during self-renewal, and that gain of MAPK activity induces CySCs to outcompete wild type CySCs and GSCs at the niche. This study dissected the genetic relationship between Socs36E and the MAPK and JAK/STAT pathways and shows that loss of Socs36E can compensate for decreased self-renewal signaling within CySCs. Thus, CySCs integrate multiple self-renewal signals through the use of a feedback inhibitor that controls at least two signaling pathways regulating stem cell maintenance at the niche (Amoyel, 2016a).

    The data presented in this study implicate MAPK signaling as a major regulator of CySC competition for niche access and establish that the competitiveness of CySCs lacking Socs36E is derived primarily from their increased MAPK activity. The ability of a stem cell to self-renew reflects not only intrinsic properties but also extrinsic relationships with its neighbors. For instance, if a cell is unable to compete for space at the niche then it will be no longer able to receive short-range niche signals and will be more likely to differentiate. Conversely, if a cell is more competitive for niche space, this cell and its offspring will replace wild type neighbors and colonize the entire niche. (Amoyel, 2016a).

    These data show that CySCs with increased MAPK signaling out-compete neighboring stem cells in CySC-CySC as well as CySC-GSC competition and that CySCs with reduced MAPK activity are themselves out-competed. The interpretation is favored that MAPK regulates primarily competitiveness rather than self-renewal because while MAPK mutant clones are lost from the niche, lineage-wide inhibition of the pathway does not result in a complete loss of stem cells. This contrasts with the role of JAK/STAT signaling in CySCs. Stat92E mutant CySCs are lost and lineage-wide pathway inhibition results in pronounced and rapid stem cell loss. Based on these results, it is argued that JAK/STAT signaling in CySCs primarily controls their intrinsic self-renewal capability while MAPK signaling regulates their competitiveness. Interestingly, there are important similarities between Hh and MAPK function in CySCs in that CySCs lacking Hh signal transduction are out-competed and those with sustained Hh activity out-compete wild type neighbors. Lastly, it is noted that CySCs mutant for the tumor suppressor Hippo (Hpo) (which leads to sustained Yki activation) or Abelson kinase (Abl) also have increased competitiveness, suggesting the existence of multiple inputs controlling the ability of stem cells to stay in the niche at the expense of their neighbors. In the future, it would be interesting to determine if genetic hierarchies exist between competitive pathways or if they independently converge on similar targets. One outstanding question is how altering the competitiveness of CySCs affects the maintenance of the germ line. In the case of Socs36E, MAPK, Hh and Hpo, the competitive CySC displaces not only wild type CySCs but also wild type GSCs. While these observations suggest that out-competition of CySCs and GSCs is linked, the result that Abl mutant CySCs only compete with CySCs and not with GSCs indicates that these two competitive processes are separable genetically (Amoyel, 2016a).

    It is well established that Egfr/MAPK signaling is required in somatic cells for their proper differentiation and for their encystment of the developing germ line. In this study, an additional function for Egfr/MAPK was identified in the somatic stem cells, specifically that this pathway regulates competitiveness of CySCs, with each other and with GSCs. Regarding the latter, it is possible that the loss of GSCs when somatic cells have high MAPK signaling is linked to their possibly increased encystment by these cells. Indeed, recent work has shown that Egfr activity in CySCs regulates cytokinesis and maintenance stem cell fate in GSCs. It is tempting to speculate that increased somatic Egfr activity leads to increased encystment of GSCs and loss of stem cell fate in GSCs (Amoyel, 2016a).

    MAPK may play a conserved role in niche competitiveness as mouse intestinal stem cells that acquire activating mutations in Ras bias normal stem cell replacement dynamics and colonize the niche. Interestingly, the activating ligand Spi is produced by germ cells, suggesting that the germ line coordinates multiple behaviors in the somatic cell lineage. In addition to transducing signals from the germ line, CySCs also receive ligands from hub cells (including Hh and the JAK/STAT ligand Upd) and they have to integrate these various stimuli. If unmitigated, the combined effect of all of these signals could produce highly competitive CySCs, with overall negative effects on niche homeostasis. The data are consistent with a model in which the induction of Socs36E by the primary self-renewal pathway (JAK/STAT) results in the restraint of a competitive trigger (MAPK) in CySCs. In this way, Socs36E acts to integrate signals from different sources and maintain homeostatic balance between resident cell populations that share a common niche (Amoyel, 2016a).

    The novel tumour suppressor Madm regulates stem cell competition in the Drosophila testis

    Stem cell competition has emerged as a mechanism for selecting fit stem cells/progenitors and controlling tumourigenesis. However, little is known about the underlying molecular mechanism. This study identified Mlf1-adaptor molecule (Madm), a novel tumour suppressor that regulates the competition between germline stem cells (GSCs) and somatic cyst stem cells (CySCs) for niche occupancy. Madm knockdown results in overexpression of the EGF receptor ligand vein (vn), which further activates EGF receptor signalling and integrin expression non-cell autonomously in CySCs to promote their overproliferation and ability to outcompete GSCs for niche occupancy. Conversely, expressing a constitutively activated form of the Drosophila JAK kinase (hop(Tum-l)) promotes Madm nuclear translocation, and suppresses vn and integrin expression in CySCs that allows GSCs to outcompete CySCs for niche occupancy and promotes GSC tumour formation. Tumour suppressor-mediated stem cell competition presented in this study could be a mechanism of tumour initiation in mammals.

    tBRD-1 selectively controls gene activity in the Drosophila testis and interacts with two new members of the Bromodomain and Extra-Terminal (BET) Family

    Multicellular organisms have evolved specialized mechanisms to control transcription in a spatial and temporal manner. Gene activation is tightly linked to histone acetylation on lysine residues that can be recognized by bromodomains. Previously, the testis-specifically expressed bromodomain protein tBRD-1 was identified in Drosophila. Expression of tBRD-1 is restricted to highly transcriptionally active primary spermatocytes. tBRD-1 is essential for male fertility and proposed to act as a co-factor of testis-specific TATA box binding protein-associated factors (tTAFs) for testis-specific transcription. This study performed microarray analyses to compare the transcriptomes of tbrd-1 mutant testes and wild-type testes. The data confirmed that tBRD-1 controls gene activity in male germ cells. Additionally, comparing the transcriptomes of tbrd-1 and tTAF mutant testes revealed a subset of common target genes. Two new members of the bromodomain and extra-terminal (BET) family, tBRD-2 and tBRD-3, were also characterized. In contrast to other members of the BET family in animals, both possess only a single bromodomain, a characteristic feature of plant BET family members. Immunohistology techniques not only revealed that tBRD-2 and tBRD-3 partially co-localize with tBRD-1 and tTAFs in primary spermatocytes, but also that their proper subcellular distribution was impaired in tbrd-1 and tTAF mutant testes. Treating cultured male germ cells with inhibitors showed that localization of tBRD-2 and tBRD-3 depends on the acetylation status within primary spermatocytes. Yeast two-hybrid assays and co-immunoprecipitations using fly testes protein extracts demonstrated that tBRD-1 is able to form homodimers as well as heterodimers with tBRD-2, tBRD-3, and tTAFs. These data reveal for the first time the existence of single bromodomain BET proteins in animals, as well as evidence for a complex containing tBRDs and tTAFs that regulates transcription of a subset of genes with relevance for spermiogenesis (Theofel, 2014: 25251222).

    Blocking promiscuous activation at cryptic promoters directs cell type-specific gene expression

    To selectively express cell type-specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. This study shows in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg; CG5204), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. These results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation (Kim, 2017).

    Highly specialized cell types such as red blood cells, intestinal epithelium, and spermatozoa are produced throughout life from adult stem cells. In such lineages, mitotically dividing precursors commonly stop proliferation and initiate a cell type-specific transcription program that sets up terminal differentiation of the specialized cell type. In the Drosophila male germ line, stem cells at the apical tip of the testis self-renew and produce daughter cells that each undergo four rounds of spermatogonial mitotic transit amplifying (TA) divisions, after which the germ cells execute a final round of DNA synthesis (premeiotic S-phase) and initiate terminal differentiation as spermatocytes. Transition to the spermatocyte state is accompanied by transcriptional activation of more than 1500 genes, many of which are expressed only in male germ cells. Expression of two-thirds of these depends both on a testis-specific version of the MMB (Myb-Muv B)/dREAM (Drosophila RBF, dE2F2, and dMyb-interacting proteins) complex termed the testis meiotic arrest complex (tMAC) and on testis-specific paralogs of TATA-binding protein-associated factors (tTAFs). Although this is one of the most dramatic changes in gene expression in Drosophila, it is not yet understood how the testis-specific transcripts are selectively activated during the 3-day spermatocyte period (Kim, 2017).

    To identify the first transcripts up-regulated at onset of spermatocyte differentiation, germ cells were genetically manipulated to synchronously differentiate from spermatogonia to spermatocytes in vivo using bam-/- testes, which contain large numbers of overproliferating spermatogonia. Brief restoration of Bam expression under heat shock control in hs-bam;bam-/- flies induced synchronous differentiation of bam-/- spermatogonia, resulting in completion of a final mitosis, premeiotic DNA synthesis, and onset of spermatocyte differentiation by 24 hours after Bam expression, eventually leading to production of functional sperm. Comparison by means of microarray of transcripts expressed before versus 24 hours after heat shock of hs-bam;bam-/- testes identified 27 early transcripts that were significantly up-regulated more than twofold in testes from hs-bam;bam-/- but not from bam-/- flies subjected to the same heat shock regime. Among these was the early spermatocyte marker RNA binding protein 4 (Rbp4). At this early time point, the transcript for CG5204 - now named kumgang (kmg), from the Korean name of mythological guardians at the gate of Buddhist temples - had the greatest increase among all 754 Drosophila predicted transcription factors (Kim, 2017).

    Kumgang (CG5204) encodes a 747-amino acid protein with six canonical C2H2-type zinc finger domains expressed in testes but not in ovary or carcass. Kmg protein was expressed independently from the tMAC component Always early (Aly) or the tTAF Spermatocyte Arrest (Sa), and both kmg mRNA and protein were up-regulated before Topi, another component of tMAC. Immunofluorescence staining of wild-type testes revealed Kmg protein expressed specifically in differentiating spermatocytes, where it was nuclear and enriched on the partially condensed bivalent chromosomes. Consistent with dramatic up-regulation of kmg mRNA after the switch from spermatogonia to spermatocyte, expression of Kmg was first detected with immunofluorescence staining after completion of premeiotic S-phase marked by down-regulation of Bam, coinciding with expression of Rbp4 protein (Kim, 2017).

    Function of Kmg in spermatocytes was required for male germ cell differentiation. Reducing function of Kmg in spermatocytes-either by means of cell type-specific RNA interference (RNAi) knockdown (KD) or in flies trans-heterozygous for a CRISPR (clustered regularly interspaced short palindromic repeats)-induced kmg frameshift mutant and a chromosomal deficiency (kmgΔ7/Df)-resulted in accumulation of mature primary spermatocytes arrested just before the G2/M transition for meiosis I and lack of spermatid differentiation. A 4.3-kb genomic rescue transgene containing the 2.3-kb kmg open reading frame fully rescued the differentiation defects and sterility of kmgΔ7/Df flies, confirming that the meiotic arrest phenotype was due to loss of function of Kmg. In both kmg KD and kmgΔ7/Df, Kmg protein levels were less than 5% that of wild type. kmgΔ7/Df mutant animals were adult-viable and female-fertile but male-sterile, which is consistent with the testis-specific expression (Kim, 2017).

    Function of Kmg was required in germ cells for repression of more than 400 genes not normally expressed in wild-type spermatocytes. Although the differentiation defects caused by loss of function of kmg appeared, by means of phase contrast microscopy, to be similar to the meiotic arrest phenotype of testis-specific tMAC component mutants, analysis of gene expression in kmg KD testes showed that many Aly (tMAC)-dependent spermatid differentiation genes were expressed, although some at a lower level than that in wild type. Among the 652 genes with more than 99% lower expression in aly-/- mutant as compared with wild-type testes, only four showed similar reduced expression in kmg KD as compared with that of sibling control (no Gal4 driver) testes. In contrast, transcripts from more than 500 genes were strongly up-regulated in kmg KD testes, with almost no detectable expression in testes from sibling control males. Hierarchical clustering identified 440 genes specifically up-regulated in kmg KD testes compared with testes from wild-type, bam-/-, aly-/-, or sa-/- mutant flies. These 440 genes were significantly associated with Gene Ontology terms such as 'substrate specific channel activity' or 'detection of visible light' that appeared more applicable to non-germ cell types, such as neurons. Analysis of published transcript expression data for a variety of Drosophila tissues revealed that the 440 were normally not expressed or extremely low in wild-type adult testes, but many were expressed in specific differentiated somatic tissues such as eye, brain, or gut. Confirming misexpression of neuronal genes at the protein level, immunofluorescence staining revealed that the neuronal transcription factor Prospero (Pros), normally not detected in male germ cells, was expressed in clones of spermatocytes that are homozygous mutant for kmg induced by Flp-FRT-mediated mitotic recombination. The misexpression of Pros was cell-autonomous, occurring only in mutant germ cells. Mid-stage to mature spermatocytes homozygous mutant for kmg misexpressed Pros, but mutant early spermatocytes did not, indicating that the abnormal up-regulation of Pros occurred only after spermatocytes had reached a specific stage in their differentiation program (Kim, 2017).

    A small-scale cell type-specific RNAi screen of chromatin regulators revealed that KD of dMi-2 in late TA cells and spermatocytes resulted in meiotic arrest, similar to loss of function of kmg. Immunofluorescence analysis of testes from a protein trap line in which an endogenous allele of dMi-2 was tagged by green fluorescent protein (GFP) revealed that dMi-2-GFP, like the untagged endogenous protein, was expressed and nuclear in progenitor cells and spermatocytes, as well as in somatic hub and cyst cells. dMi-2-GFP colocalized to chromatin with Kmg in spermatocytes, and the level of dMi-2 protein appeared lower and less concentrated on chromatin in nuclei of kmg-/- spermatocytes than in neighboring kmg+/+ or kmg+/- spermatocytes, suggesting that Kmg may at least partially help recruit dMi-2 to chromatin in spermatocytes. Furthermore, in testis extracts Kmg coimmunoprecipitated with dMi-2 and vice versa, suggesting that Kmg and dMi-2 form a protein complex in spermatocytes. Comparison of microarray data revealed that most of the 440 transcripts up-regulated in testes upon loss of function of kmg were also abnormally up-regulated in dMi-2 KD testes, suggesting that Kmg and dMi-2 may function together to repress expression of the same set of normally somatic transcripts in spermatocytes (Kim, 2017).

    Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that Kmg protein localized along the bodies of genes actively transcribed in the testis. ChIP-seq with antibody to Kmg identified 798 genomic regions strongly enriched by immunoprecipitation of Kmg from wild-type but not from kmg KD testes. Of the 798 robust Kmg ChIP-seq peaks, 698 overlapped with exonic regions of 680 different genes actively transcribed in testes. The enrichment was often strongest just downstream of the transcription start site (TSS), but with substantial enrichment along the gene body as well (Kim, 2017).

    ChIP-seq with antibody to dMi-2 also showed enrichment along the gene bodies of the same 680 genes bound by Kmg, with a similar bias just downstream of the TSS. The dMi-2 ChIP signal along these genes was partially reduced in kmg KD testes, suggesting that Kmg may recruit dMi-2 to the bodies of genes actively transcribed in the testis (Kim, 2017).

    RNA-seq analysis revealed that the 680 genes bound by Kmg were strongly expressed in testes and most strongly enriched in the GO term categories 'spermatogenesis' and 'male gamete generation'. One-third of the genes bound by Kmg were robustly activated as spermatogonia differentiate into spermatocytes and were much more highly expressed in the testes than in other tissues. The median levels of transcript expression of most of the 680 Kmg bound genes did not show appreciable change upon loss of Kmg (Kim, 2017).

    Genes that are normally transcribed in somatic cells that became up-regulated upon loss of Kmg function in spermatocytes for the most part did not appear to be bound by Kmg. Only 3 of the 440 genes up-regulated in kmg KD overlapped with the 680 genes with robust Kmg peaks, suggesting that Kmg may prevent misexpression of normally somatic transcripts either indirectly or by acting at a distance (Kim, 2017).

    Inspection of RNA-seq reads from kmg and dMi-2 KD testes mapped onto the genome showed that ~80% of the transcripts that were detected with microarray analysis as misexpressed in KD as compared with wild-type testes did not initiate from the promoters used in the somatic tissues in which the genes are normally expressed. Metagene analysis, as well as visualization of RNA expression centered on the TSSs annotated in the Ensembl database, showed that most of the 143 genes that are normally expressed in wild-type heads but not in wild-type testes were misexpressed in kmg or dMi-2 KD testes from a start site different from the annotated TSS used in heads. Transcript assembly from RNA-seq data by using Cufflinks for the 143 genes also showed that the transcripts that are misexpressed in kmg or dMi-2 KD testes most often initiate from different TSSs than the transcripts from the same gene assembled from wild-type heads (Kim, 2017).

    Of the 440 genes scored via microarray as derepressed in kmg KD testes, 346 could be assigned with TSSs in kmg KD testes based on visual inspection of the RNA-seq data mapped onto the genome browser. Of these, only 67 produced transcripts in kmg KD testes that started within 100 base pairs (bp) of the TSS annotated in the Ensembl database, based on the tissue(s) in which the gene was normally expressed. In contrast, for the rest of the 346 genes, the transcripts expressed in kmg KD testes started from either a TSS upstream (131 of 346) or downstream (148 of 346) of the annotated TSSs. Of the 346 genes, 262 were misexpressed starting from nearly identical positions in dMi-2 KD as in kmg KD testes, suggesting that Kmg and dMi-2 function together to prevent misexpression from cryptic promoters (Kim, 2017).

    Many of the ectopic promoters from which the misexpressed transcripts originated appeared to be bound by Aly, a component of tMAC, in kmg KD testes. ChIP for Aly was performed by using antibody to hemagglutinin (HA) on testis extracts from flies bearing an Aly-HA genomic transgene able to fully rescue the aly-/- phenotype. Of 346 genes with new TSSs assigned via visual inspection, 181 had a region of significant enrichment for Aly as detected with ChIP, with its peak summit located within 100 bp of the cryptic promoter. Motif analysis by means of MEME revealed that these regions were enriched for the DNA sequence motif (AGYWGGC). This motif was not significantly enriched in the set of 165 cryptic promoters at which Aly was not detected in kmg KD testes. Enrichment of Aly at the cryptic promoters was much stronger in kmg KD as compared with wild-type testes, suggesting that in the absence of Kmg, Aly may bind to and activate misexpression from cryptic promoters (Kim, 2017).

    Genetic tests revealed that the misexpression of somatic transcripts in kmg KD spermatocytes indeed required function of Aly. The neuronal transcription factor Pros, abnormally up-regulated in kmg KD or mutant spermatocytes, was no longer misexpressed if the kmg KD spermatocytes were also mutant for aly, even though germ cells in kmg KD;aly-/- testes appear to reach the differentiation stage at which Pros turned on in the kmg KD germ cells. Assessment by means of quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that misexpression of five out of five transcripts in kmg KD testes also required function of Aly. Global transcriptome analysis via microarray of kmg KD versus kmg KD;aly-/- testes showed that the majority of the 440 genes that were derepressed because of loss of function of kmg in spermatocytes were no longer abnormally up-regulated in kmg KD;aly-/- testes. Even genes without noticeable binding of Aly at their cryptic promoters were suppressed in kmg KD;aly-/-, suggesting that Aly may regulate this group of genes indirectly (Kim, 2017).

    Together, the ChIP and RNA-seq data show that Kmg and dMi-2 bind actively transcribed genes but are required to block expression of aberrant transcripts from other genes that are normally silent in testes. The mammalian ortholog of dMi-2, CHD4 (Mi-2β), has been shown to bind active genes in mouse embryonic stem cells or T lymphocyte precursors but also plays a role in ensuring lineage-specific gene expression in other contexts. It cannot be ruled out that Kmg and dMi-2 might also act directly at the cryptic promoter sites but that the ChIP conditions did not capture their transient or dynamic binding because several chromatin remodelers or transcription factors, such as the thyroid hormone receptor, have been difficult to detect with ChIP. Kmg and dMi-2 may repress misexpression from cryptic promoters indirectly by activating as-yet-unidentified repressor proteins. However, it is also possible that Kmg and dMi-2 act at a distance by modulating chromatin structure or confining transcriptional initiation or elongation licensing machinery to normally active genes (Kim, 2017).

    Changes in the genomic localization of Aly protein in wild-type versus kmg KD testes raised the possibility that Kmg may in part prevent misexpression from cryptic promoters by concentrating Aly at active genes. Of the 1903 Aly peaks identified with ChIP from wild-type testes, the 248 Aly peaks that overlapped with strong Kmg peaks showed via ChIP an overall reduction in enrichment of Aly from kmg KD testes as compared with wild type. In contrast, the Aly peaks at cryptic promoters were more robust in kmg KD testes than in wild type. In general, over the genome 4129 new Aly peaks were identified by means of ChIP from kmg KD testes that were absent or did not pass the statistical cutoff in wild-type testes. More than 30% of the genomic regions with new Aly peaks in kmg KD showed elevated levels of RNA expression starting at or near the Aly peak in kmg KD but not in wild-type testes, suggesting that misexpression of transcripts from normally silent promoters in kmg KD testes is more widespread than initially assessed with microarray. Together, these findings raise the possibility that Kmg may prevent misexpression of aberrant transcript by concentrating Aly to active target genes in wild-type testes, preventing binding and action of Aly at cryptic promoter sites (Kim, 2017).

    The results suggest that selective gene activation is not always mediated by a precise transcriptional activator but can instead be directed by combination of a promiscuous activator and a gene-selective licensing mechanism. Cryptic promoters may become accessible as chromatin organization is reshaped to allow expression of terminal differentiation transcripts that were tightly repressed in the progenitor state. It is posited that this chromatin organization makes a number of sites that are accessible for transcription dependent on the testis-specific tMAC complex component Aly. In this context, activity of Kmg and dMi-2 is required to prevent productive transcript formation from unwanted initiation sites, potentially by confining Aly to genes actively transcribed in the testis and limiting the amount of Aly protein acting at cryptic promoters (Kim, 2017).

    The initiation of transcripts from cryptic promoters is reminiscent of loss of function of Ikaros, a critical regulator of T and B cell differentiation and a tumor suppressor in the lymphocyte lineage. Like Kmg, Ikaros is a multiple-zinc finger protein associated with Mi-2β, which binds to active genes in T and B cell precursors. In T cell lineage acute lymphoblastic leukemia (T-ALL) associated with loss of function of Ikaros, cryptic intragenic promoters were activated, leading to expression of ligand-independent Notch1 protein, contributing to leukemogenesis. Thus, in addition to being detrimental for proper differentiation, firing of abnormal transcripts from normally cryptic promoters because of defects in chromatin regulators may contribute to tumorigenesis through generation of oncogenic proteins (Kim, 2017).

    Paternal diet defines offspring chromatin state and intergenerational obesity

    The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. This study presents a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identifies genes required for its encoding in offspring. Intriguingly, as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. Requirements were identified for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, evidence is found that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for understanding of phenotypic variation and evolution (Ost, 2014).

    This study shows that acute dietary interventions, as short as 24 hr, have the capacity to modify F1 offspring phenotype via the male germline. Reprogramming occurs in response to dietary manipulations over a physiological range, and phenotypic outcomes require polycomb- and H3K9me3-centric plasticity in spatially and chromatin-state-defined regions of the genome. The eye color shifts in wm4 h offspring and the reduced fat body H3K9me3 staining in adult IGMR offspring supports the conclusions, first, that there are chromatin state changes and, second, that these are stable lifelong. These data are corroborated by selective derepression of Su(var)3-9, SETBD1, Su(var)4-20, and polycomb-sensitive transcripts; chromatin-state-associated transcriptional rearrangements genome wide; selective reprogramming of highly dynamic histone-mark-defined regions; and the fact that intergenerational metabolic reprogramming (IGMR ) itself is sensitive to a string of distinct H3K9me3-centric and polycomb mutants. Although nontrivial, ChIP-seq comparisons of repressive chromatin architecture in mature sperm and multiple defined offspring tissues will be important to establishing the ubiquitousness of these regulatory events and the nature of intergenerational signal itself. These data highlight how acutely sensitive intergenerational control can be to even normal physiological changes, and they identify some of the first genes absolutely required for transmission evolution (Ost, 2014).

    First categorized simply as heterochromatin versus euchromatin, multiple empirical models now divide the genome into 5 to 51 chromatin states, depending on the analysis. Paternal high sugar increases gene expression preferentially of heterochromatic-embedded genes in embryos. Specifically, these genes are characterized by active deposition of H3K9me3 and H3K27me3, by long distance from class I insulators, and by sensitivity to fully intact expression of Su(var)3-9, Su(var)4-20, SetDB1, Pc, and E(z) . The data support a model where phenotype has been evolutionarily encoded directly into the chromatin state of relevant loci. Specifically, an abundance of genes important to both cytosolic and mitochondrial metabolism appear to be embedded into H3K9me3- and distinct polycomb-dependent control regions. Indeed, GO analysis of the five chromatin colors indicate a largely mutually exclusive picture, in which functional pathways are not randomly distributed across chromatin states. The paternal IGMR data set revealed clear and strong overlaps with pathways of black (lamin-associated) and blue (polycomb) chromatin and included many key metabolic pathways, including glycolysis, TCA cycle, mitochondrial OxPhos, chitin, and polysaccharide metabolism, changes that could well prime the system for altered functionality given the appropriate stimulus. Indeed, paternal IGMR phenotype is a susceptibility to diet-induced obesity and is most readily observable upon high-sugar diet challenge evolution (Ost, 2014).

    The data support a trans-acting mechanism. In the wm4h experiments, male offspring inherited their X chromosome and thus the reporter from their unchallenged mothers, i.e., the reporter allele never encounters the initial signal but is reproducibly reprogrammed. Further, the failure of Su(var) 4-20SP and SetDB11473 mutants to elicit IGMR responses in their wild-type offspring indicate that wild-type haploid sperm carry the same insufficient reprogramming template as their syncytial mutant counterparts. cis- and trans-acting mechanisms are not mutually exclusive though. Signals transmitted via paternal chromosomes, though likely transmitted in cis, may be manifest via expression of paternal transcripts, which then act in trans. Paternal reductions of Su(var)3-9, SetDB1, and Hp1, for instance, would affect the maternal genome in trans evolution (Ost, 2014).

    Despite their genetic similarity, isogenic or congenic animals reared under controlled conditions exhibit measurable variation in essentially all phenotypes. Such variability in genome output is thought to arise largely from probabilistic or chance developmental events in early. This study mapped a mechanism that couples acute paternal feeding and zygotic chromatin state integrity directly to phenotypic output of the next generation. These same signatures predict obesity susceptibility in isogenic mouse and human obesity cohorts. Because acute circadian fluctuations in feeding are essentially constant over evolutionary timescales, they are the perfect mechanistic input upon which a system could evolve to ensure defined phenotypic variation within a given population evolution (Ost, 2014).

    Mitotic fidelity requires transgenerational action of a testis-restricted HP1

    Sperm-packaged DNA must undergo extensive reorganization to ensure its timely participation in embryonic mitosis. Whereas maternal control over this remodeling is well described, paternal contributions are virtually unknown. This study shows that Drosophila melanogaster males lacking Heterochromatin Protein 1E (HP1E) sire inviable embryos that undergo catastrophic mitosis. In these embryos, the paternal genome fails to condense and resolve into sister chromatids in synchrony with the maternal genome. This delay leads to a failure of paternal chromosomes, particularly the heterochromatin-rich sex chromosomes, to separate on the first mitotic spindle. Remarkably, HP1E is not inherited on mature sperm chromatin. Instead, HP1E primes paternal chromosomes during spermatogenesis to ensure faithful segregation post-fertilization. This transgenerational effect suggests that maternal control is necessary but not sufficient for transforming sperm DNA into a mitotically competent pronucleus. Instead, paternal action during spermiogenesis exerts post-fertilization control to ensure faithful chromosome segregation in the embryo (Levine, 2015).

    Faithful chromosome segregation requires careful orchestration of chromosomal condensation, alignment, and movement of mitotic chromosomes during every eukaryotic cell division. The very first embryonic mitosis in animals requires additional synchronization. Paternally and maternally inherited genomes undergo independent chromatin reorganization and replication prior to mitotic entry. For instance, maternal chromosomes must complete meiosis and then transition from a meiotic conformation to an interphase-like state in preparation for replication. The sperm-deposited, paternal chromosomes must undergo an even more radical transition from a highly compact, protamine-rich state to a decondensed, histone-rich state before DNA replication. Despite these divergent requirements to achieve replication- and mitotic-competency, maternal and paternal genomes synchronously enter the first mitosis. Failure to carry out paternal chromosome remodeling in a timely fashion results in paternal genome loss and embryonic inviability (Levine, 2015).

    The transition from a protamine-rich sperm nucleus to a competent paternal pronucleus requires the action of numerous maternally deposited proteins in the egg. For instance, paternal genome decondensation post-fertilization requires the integration of histone H3.3, a histone variant deposited by the maternal proteins HIRA, CHD1, and Yemanuclein. Similarly, maternally-deposited MH/Spartan protein localizes exclusively to the replicating paternal genome and is required for faithful paternal chromosome segregation during the first embryonic division. These and other studies demonstrate the essential role of maternally-deposited machinery in rendering competent sperm-deposited DNA and ultimately, ensuring faithful paternal genome inheritance (Levine, 2015).

    Is paternal control also necessary for the extensive decondensation and re-condensation of the post-fertilization paternal genome? If so, disruption of such control would manifest as paternal effect lethality (PEL). Unlike male sterility mutants that lack motile sperm, PEL mutants make abundant motile sperm that fertilize eggs efficiently. However, embryos 'fathered' by PEL mutants are inviable. Only a handful of PEL genes have been characterized in animals. These encode proteins that mediate sperm release of paternal DNA, sperm centriole inheritance, and paternal chromosome segregation. Only one of these PEL proteins directly localizes to paternal chromosomes; the sperm-inherited K81 protein localizes exclusively to paternal chromosome termini and ensures telomere integrity. The maintenance of telomeric epigenetic identity joins a growing list of examples of sperm-to-embryo information transmission via protein or RNA inheritance (e.g., diet, stress, embryonic patterning, transcriptional competency). Despite a new appreciation of paternal control over epigenetic information transfer, there are no reports of paternal control over the global chromatin reorganization required for synchronous mitosis across paternally and maternally inherited genomes. Indeed, in the absence of any known paternal protein-directed genome remodeling, a model has emerged that maternal proteins might be sufficient for transforming tightly packaged sperm DNA into a fully competent paternal pronucleus (Levine, 2015).

    The notion that maternal control is sufficient to accomplish paternal genome remodeling is challenged by recent findings from the intracellular Wolbachia bacterium that infects more than 50% of insect species. Wolbachia-infected Drosophila males mated to uninfected females father embryos that arrest soon after the first zygotic mitosis. Embryonic arrest occurs because paternal genomes enter the first mitosis with unresolved sister chromatids that fail to separate on the mitotic spindle. Although the identity of the host factor(s) manipulated by Wolbachia to mediate this transgenerational effect is still unknown, what is clear is that pre-fertilization, Wolbachia subverts the paternal germline machinery that helps direct global genome remodeling of paternal chromosomes in the embryo. Wolbachia action during spermiogenesis leads to paternal-maternal genome asynchrony and ultimately, failure of paternal chromosomes to separate on the first mitotic spindle. Despite decades of interest, the molecular basis of paternal control has remained elusive (Levine, 2015).

    To investigate the potential for paternal control over sperm genome remodeling post-fertilization, a candidate gene approach was taken, focusing on the Heterochromatin Protein 1 (HP1) proteins that orchestrate genome-wide chromosomal organization in plants, animals, fungi, and some protists. HP1 proteins are defined as such by a combination of two domains - a chromodomain that mediates protein-histone interactions and a chromoshadow domain that mediates protein-protein interactions. The biochemical properties of HP1 members support a diversity of chromatin-dependent processes in the soma, including DNA replication, telomere integrity, and chromosome condensation (Levine, 2015).

    Recently, a detailed phylogenomic analysis of the HP1 gene family was carried out in Drosophila that revealed numerous testis-restricted HP1 proteins. Given the established roles of HP1 proteins, it was posited that these newly discovered male-specific HP1 genes might represent excellent candidates for encoding chromatin functions specialized for paternal genome organization and remodeling in the early embryo. Using detailed genetic and cytological analyses, this study shows that one of these testis-specific HP1 proteins, Heterochromatin Protein 1E (HP1E), is essential for priming the paternal genome to enter embryonic mitosis in synchrony with the maternal genome in D. melanogaster. Intriguingly, HP1E is able to mediate this priming function transgenerationally i.e., the HP1E protein itself is not epigenetically inherited. It was further shown that absence of HP1E especially imperils mitotic fidelity of the heterochromatin-rich, paternal sex chromosomes. Thus, this study firmly establishes that both maternal and paternal control are necessary for paternal genome remodeling in the early Drosophila embryo (Levine, 2015).

    Properly coordinated chromosome segregation during virtually all mitotic divisions relies on the function of multiple cell cycle checkpoint proteins. No such cell cycle checkpoint proteins have been identified to act in the very first embryonic mitotic cycle, which must nevertheless accomplish the difficult task of synchronizing maternal and paternal chromosomes that were inherited in very different chromatin states. To investigate the paternal contributions that ensure timely participation of the paternal genome in early embryogenesis, a detailed functional analysis was carried out of the testis-restricted HP1E gene in D. melanogaster. It was found that HP1E encodes a novel function that ensures paternal genome stability in the embryo. Cytological and transcriptome analysis revealed that HP1E is developmentally restricted within the male germline, where it contributes to heterochromatin integrity. HP1E depletion during sperm development results in a highly penetrant PEL phenotype in which paternal chromosomes, especially the paternal sex chromosomes, fail to condense in synchrony with the maternal chromosomes and ultimately cause mitotic catastrophe. It was further shown that the PEL embryonic phenotype could not be rescued by egg-supplied HP1E but could be rescued if the paternal DNA was excluded from participating in embryonic mitosis. These observations support a model under which HP1E acts pre-fertilization to ensure proper chromosome condensation and segregation of paternal chromosomes post-fertilization (Levine, 2015).

    The 'hit and run' priming function clearly distinguishes HP1E from all other previously characterized paternal effect lethal genes, which encode proteins that are transmitted to the embryo via sperm. These include the Drosophila paternal chromatin-associated PEL, k81, which encodes a protein that persists on paternal telomeres from late spermatogenesis to the first embryonic mitosis. The HP1E-depletion phenotype is instead reminiscent of Drosophila fathers infected with Wolbachia bacteria crossed to uninfected females. Embryonic lethality induced by Wolbachia testis infection is also caused by a pre-fertilization modification to the paternal genome that results in paternal-maternal chromatin asynchrony and mis-segregation at the very first zygotic mitosis. However, Wolbachia-associated PEL results in mis-segregation of the entire paternal genome rather than just the heterochromatin-rich chromosomes observed in HP1E PEL . Moreover, the HP1E PEL defect is completely independent of Wolbachia (PEL phenotype persists for Wolbachia-free males and females). It is therefore concluded that HP1E supports a novel chromatin requirement to prime paternally inherited genomes for synchronous and successful embryonic mitosis (Levine, 2015).

    How does HP1E ensure timely mitotic entry? It is formally possible that the PEL phenotype is the consequence of a dysregulated spermatid transcriptome that is, up- or down-regulation of a downstream gene. However, the finding that HP1E depletion results in the global up-regulation of heterochromatin-embedded genes, together with the observation that the heterochromatin-rich paternal sex chromosomes are most vulnerable to HP1E depletion, lead to favoring the alternate model that HP1E functions as a canonical HP1 protein during spermiogenesis. Based on antibody localization and chromatin bridge morphology, no evidence was found for defects in kinetochore assembly or replication machinery engagement in PEL embryos. Instead, the observation that the lethality phenotype first manifests as decondensed paternal chromosomes relative to maternal chromosomes implicates condensation delay of the heterochromatin-rich sex chromosomes. This delay could be the consequence of incomplete replication. Indeed, large stretches of uninterrupted heterochromatic DNA, as found on the Drosophila sex chromosomes, pose a unique challenge to replication. Alternatively, the mitotic delay may be the result of inadequate condensin protein recruitment, which is required for timely resolution of sister chromatids post-replication. Previous studies have shown that heterochromatin can also impair chromosome condensation. Timely completion of replication and condensation requires the action of HP1E's closest relative, HP1A, in somatic cells. However, in developing spermatids, HP1A localizes to telomeres rather than broadly to heterochromatin as observed in virtually all other cell types. It is posited that HP1E adopts a global, HP1A-like chromatin function during this highly specialized developmental stage and ensures the recruitment or retention of either replication or condensin proteins that are required post-fertilization (Levine, 2015).

    Previous studies have shown that HP1A is essential for embryo viability. This study shows that paternally-acting HP1E is also essential for embryogenesis. Both HP1A and HP1E evolve under purifying selection. However, unlike HP1A (encoded by Su(var)205), HP1E has an unusually dynamic evolutionary history. Despite ancient origins, HP1E has been recurrently lost over evolutionary time. HP1E has been apparently replaced by younger, testis restricted HP1 paralogs on at least two occasions during Drosophila evolution. Curiously, Drosophila pseudoobscura and related species encode neither HP1E nor a putative replacement testis-specific HP1 gene. How is the paradox of HP1E essentiality in D. melanogaster reconciled with its loss in D. pseudoobscura? It was previously found that HP1E loss along in D. pseudoobscura-related species occurred during the same 7-million evolutionary period as a major sex chromosome rearrangement event, in which the ancestral Y was lost, a neo-Y chromosome was born, and the ancestral X fused to an autosome. The finding that the D. melanogaster sex chromosomes are especially vulnerable to HP1E depletion, combined with the emergence of novel sex chromosome arrangements along the same narrow branch as HP1E pseudogenization, suggests a model under which rearrangements of heterochromatin-rich sex chromosomes in the obscuragroup rendered HP1E non-essential. Such karyotypic changes can bring distal heterochromatin into closer proximity to euchromatin and be sufficient to alter heterochromatin packaging, replication timing or even delete blocks of satellite repeats. Thus, heterochromatin evolution via chromosomal rearrangements may have obviated maintenance of HP1E's essential heterochromatin function, leading to its degeneration in D. pseudoobscura (Levine, 2015).

    The finding that HP1E is essential in D. melanogaster yet lost in the obscura group highlights the lineage-restricted essential requirements of chromatin genes. Intriguingly, the only other characterized PEL gene that supports paternal chromatin function in Drosophila embryos, k81, is similarly lineage-restricted despite being essential for paternal telomere function. In contrast, maternally deposited proteins required for paternal chromatin reorganization following fertilization are generally conserved from fly to human. This dichotomy is striking. It specifically suggests that even though the essential functions of paternal control of DNA deposition and chromatin remodeling for embryonic mitosis are likely to be conserved in most animals, whereas the identity of those genes is not. PEL chromatin genes like HP1E and k81 thus challenge the dogma that ancient, conserved genes always encode essential conserved functions. Not only can young genes rapidly acquire essential chromatin functions due to dynamic chromatin evolution, but chromatin changes, such as those driven by karyotype evolution, may also drive the extinction of ancient genes encoding once-essential functions (Levine, 2015).

    Asymmetric inheritance of mother versus daughter centrosome in stem cell division

    Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. This study shows developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation (Yamashita, 2007).

    Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell. The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation. Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Upd, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity. Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (Yamashita, 2007).

    The stereotyped orientation of the mitotic spindle is set up by the positioning of centrosomes during interphase. GSCs remain oriented toward the niche throughout the cell cycle. In G1 phase, the single centrosome is located near the interface with the hub. When the duplicated centrosomes separate in G2 phase, one stays next to the hub, whereas the other migrates to the opposite side of the cell. Centrosomes in the GSCs separate unusually early in interphase, rather than at the G2-prophase transition, so it is common to see GSCs with fully separated centrosomes without a spindle (Yamashita, 2007).

    Differences between the mother and daughter centrosomes underlie the stereotyped behavior of the centrosomes in Drosophila male GSCs. The mother centrosome normally remains anchored to the hub-GSC interface and is inherited by the GSC, whereas the daughter centrosome moves away from the hub and is inherited by the cell that commits to differentiation. Mother and daughter centrosomes were differentially labeled by transient expression of green fluorescent protein-pericentrin/AKAP450 C-terminus (GFP-PACT) from the Drosophila pericentrin-like protein under heat shock-Gal4 control. The PACT domain, which is necessary and sufficient for centriolar localization, is incorporated into centrioles only during centrosome duplication and does not exchange with the cytoplasmic pool. Both the mother and daughter centrosomes are labeled by GFP-PACT in the first cell cycle after heat shock. In the second cell cycle, the daughter centrosome retains GFP-PACT, whereas the mother centrosome is not labeled, thus distinguishing the mother and daughter centrosomes. After a short burst of GFP-PACT expression induced by a 2.5-hour heat shock, 20% - 30% of the GSCs had GFP-labeled centrosomes, indicating the duplication of centrosomes during the window of GFP-PACT expression. By 12 hours after heat shock, >90% of the labeled GSCs had two GFP-positive centrosomes, indicating that they had progressed to the G2 phase of the first cell cycle after GFP-PACT incorporation (Yamashita, 2007).

    By 18 to 24 hours after heat shock, the number of GSCs with two GFP-positive centrosomes had decreased, whereas the number of GSCs with one GFP-positive and one GFP-negative centrosome had increased, suggesting progression into the second cell cycle. Generally, the centrosome distal to the hub was labeled, whereas the centrosome proximal to the hub was GFP-negative, indicating that the daughter centrosomes migrate away from the hub-GSC interface during asymmetric GSC divisions (Yamashita, 2007).

    Labeling the mother rather than the daughter centrosomes confirmed that the male GSCs in the niche preferentially retain mother centrosomes over time. Centrioles assembled during early embryogenesis were labeled using the NGT40 Gal4 driver to drive the expression of GFP-PACT in blastoderm-stage embryos, shutting off after germband extension. In the first cell cycle after the depletion of the cytoplasmic pool of GFP-PACT in the GSCs, both the mother and daughter centrosomes should be labeled. In subsequent cell cycles, only the mother centrosomes should be labeled (Yamashita, 2007).

    In most GSCs in the second or later cell cycle after the depletion of cytoplasmic GFP-PACT, the labeled centrosome was positioned next to the hub-GSC interface, and the unlabeled centrosome had moved away from the hub. The frequency of GSCs that had the proximal, but not distal, centrosome labeled remained constant over time for 10 days (L3 larvae to day-3 adults), suggesting that the mother centrosomes are reliably retained by the GSCs, even through multiple rounds of GSC divisions. Some GSCs maintained cytoplasmic GFP-PACT, especially in L3 larvae, suggesting that the GFP-PACT had not yet been diluted out. Some GSCs with two labeled centrosomes were observed, suggesting that they are in the first cell cycle after the depletion of cytoplasmic GFP-PACT (Yamashita, 2007).

    The mother centrosomes in GSCs appeared to maintain robust interphase microtubule arrays. Ultrastructural analysis of the GSCs revealed that the centrosome proximal to the hub was commonly associated with many microtubules throughout the cell cycle. Nineteen centrosomes in GSCs were scored in serial sections of the apical tips of five wild-type testes. Eleven centrosomes were localized close to the adherens junctions between the hub and the GSCs. Nine of these proximal centrosomes appeared to be in interphase cells, based on nuclear morphology and microtubule arrangement. Typically, these interphase centrosomes proximal to the hub were associated with numerous microtubules. In some samples, microtubules appeared to extend from the centrosome toward the adherens junctions. The other two proximal centrosomes appeared to be in cells in mitotic prophase, based on their robust microtubule arrays containing bundled microtubules running parallel to or piercing the nuclear surface (Yamashita, 2007).

    In contrast, of the five distal centrosomes in the apparently interphase cells that were scored, four had few associated microtubules. The remaining three distal centrosomes appeared to be in cells in mitotic prophase, based on microtubule arrays containing bundled microtubules. Thus, the mother centrosomes may maintain interphase microtubule arrays that anchor them to the hub-GSC interface, whereas the daughter centrosomes may initially have few associated microtubules and be free to move, establishing a robust microtubule array only later in the cell cycle (Yamashita, 2007).

    Consistent with the idea that astral microtubules anchor the mother centrosomes to the hub-GSC interface, mother- versus daughter centrosome positioning was randomized in GSCs that were homozygous mutant for centrosomin (cnn), an integral centrosomal protein required to anchor astral microtubules to centrosomes. Analysis of mother and daughter centrosomes after transient expression of GFP-PACT revealed that, for cnn homozygous mutant GSCs where one of the two centrosomes was positioned next to the hub, it was essentially random whether the mother or the daughter centrosome stayed next to the hub. In addition, in >25% of total labeled GSCs, neither of the two centrosomes was next to the hub (Yamashita, 2007).

    These results indicate that the two centrosomes in Drosophila male GSCs have different characters and fates. The mother centrosome stays next to the junction with the niche and is inherited by the cell that self-renews stem cell fate. Thus, GSCs can maintain an old centriole assembled many cell generations earlier. In contrast, the daughter centrosome migrates away from the niche and is inherited by the cell that will initiate differentiation. It is postulated that the mother centrosomes in male GSCs may remain anchored to the GSC-niche interface throughout the cell cycle by attachment to astral microtubules connected to the adherens junction, whereas the daughter centrosomes may initially have few associated microtubules and thus can move away from the niche. Microtubule-dependent differential segregation of mother and daughter spindle-pole bodies (equivalent to centrosomes in higher organisms) is observed in budding yeast. In cultured vertebrate cells, the centrioles mature slowly over the cell cycle, and the mother centrosomes (containing a mature centriole) attach astral microtubules more effectively and are more stationary than daughter centrosomes in interphase. The unusually early separation of centrosomes in interphase male GSCs may provide a way to move the daughter centrosome out of range of the stabilizing influence of the adherens junction complex before it becomes competent to hold a robust microtubule array (Yamashita, 2007).

    Developmentally programmed anchoring of the mother centrosome may provide a key mechanism to ensure the stereotyped orientation of the mitotic spindle and thus the reliably asymmetric outcome of the male GSC divisions. Although it is tempting to speculate that determinants associated with the mother or daughter centrosome may play a role in specifying stem cell or differentiating-cell fates, such determinants are yet to be identified. Rather, the asymmetric inheritance of mother and daughter centrosomes in male GSCs may be a consequence of the cytoskeletal mechanisms that are imposed as part of the stem cell program to anchor one centrosome next to the niche throughout the interphase, ensuring a properly oriented spindle (Yamashita, 2007).

    Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells

    Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. This study shows that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the hub led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome anchoring machinery, at the hub-GSC interface. This perturbation of the centrosome anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control (Levings, 2016).

    Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline

    A long-standing question concerns how stem cells maintain their identity through multiple divisions. It has been reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. This study shows that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity (Xie, 2015).

    Epigenetic phenomena are heritable changes in gene expression or function that can persist throughout many cell divisions without alterations in primary DNA sequences. By regulating differential gene expression, epigenetic processes are able to direct cells with identical genomes to become distinct cell types in humans and other multicellular organisms. However, with the exception of DNA methylation, little is known about the molecular pathways leading to epigenetic inheritance (Xie, 2015).

    Prior research has shown that epigenetic events play particularly important roles in ensuring both proper maintenance and differentiation of several stem cell populations. Many types of adult stem cells undergo asymmetric cell division to generate a self-renewed stem cell and a daughter cell that will subsequently differentiate. Mis-regulation of this balance leads to many human diseases, ranging from cancer to tissue dystrophy to infertility. However, the mechanisms of stem cell epigenetic memory maintenance as well as how loss of this memory contributes to disease remain unknown (Xie, 2015).

    During the asymmetric division of the Drosophila male germline stem cell (GSC), the pre-existing histone 3 (H3) is selectively segregated to the self-renewed GSC daughter cell whereas newly synthesized H3 is enriched in the differentiating daughter cell known as a gonialblast (GB) (Tran, 2012). In contrast, the histone variant H3.3, which is incorporated in a replication-independent manner, does not exhibit such an asymmetric pattern. Furthermore, asymmetric H3 inheritance occurs specifically in asymmetrically dividing GSCs, but not in the symmetrically dividing progenitor cells. These findings demonstrate that global asymmetric H3 histone inheritance possesses both molecular and cellular specificity. The following model is proposed to explain these findings (Xie, 2015).

    First, the cellular specificity exhibited by the H3 histone suggests that global asymmetric histone inheritance occurs uniquely in a cell-type (GSC) where the mother cell must divide to produce two daughter cells each with a unique cell fate. Because this asymmetry is not observed in symmetrically dividing GB cells, asymmetric histone inheritance is proposed to be a phenomenon specifically employed by GSCs to establish unique epigenetic identities in each of the two daughter cells. Second, as stated previously, a major difference between H3 and H3.3 is that H3 is incorporated to chromatin during DNA replication, while H3.3 variant is incorporated in a replication-independent manner. Because this asymmetric inheritance mode is specific to H3, a two-step model is proposed to explain asymmetric H3 inheritance: (1) prior to mitosis, pre-existing and newly synthesized H3 are differentially distributed on the two sets of sister chromatids, and (2) during mitosis, the set of sister chromatids containing pre-existing H3 is segregated to GSCs, while the set of sister chromatids enriched with newly synthesized H3 is segregated to the GB that differentiates (Tran, 2012; Tran, 2013; Xie, 2015 and references therein)

    This study reports that a mitosis-enriched H3T3P mark acts as a transient landmark that distinguishes sister chromatids with identical genetic code but different epigenetic information, shown as pre-existing H3-GFP and newly synthesized H3-mKO. By distinguishing sister chromatids containing different epigenetic information, H3T3P functions to allow these molecularly distinct sisters to be segregated and inherited differentially to the two daughter cells derived from one asymmetric cell division. The selective segregation of different populations of histones likely allows these two cells to assume distinct fates: self-renewal versus differentiation. Consequently, loss of proper epigenetic inheritance might lead to defects in both GSC maintenance and GB differentiation, suggesting that both cells need this active partitioning process to either 'remember' or 'reset' their molecular properties (Xie, 2015).

    The temporal and spatial specificities of H3T3P make it a great candidate to regulate asymmetric sister chromatid segregation. First, H3T3P is only detectable from prophase to metaphase, the window of time during which the mitotic spindle actively tries to attach to chromatids through microtubule-kinetochore interactions. Second, the H3T3P signal is enriched at the peri-centromeric region, where kinetochore components robustly crosstalk with chromatin-associate factors. Third, H3T3 shows a sequential order of phosphorylation, first appearing primarily on sister chromatids enriched with pre-existing H3 and then subsequently appearing on sister chromatids enriched with newly synthesized H3 as the GSC nears metaphase. The distinct temporal patterns shown by H3T3P are unique to GSCs and would allow the mitotic machinery to differentially recognize sister chromatids bearing distinct epigenetic information; an essential step necessary for proper segregation during asymmetric GSC division. Furthermore, the tight temporal control of H3T3 phosphorylation suggests that rather than serving as an inherited epigenetic signature, H3T3P may act as transient signaling mark to allow for the proper partitioning of H3. It is hypothesized that H3T3P needs to be under tight temporal control in order to ensure proper H3 inheritance and germline activity (Xie, 2015).

    These studies have shown that H3T3P is indeed subject to stringent temporal controls during mitosis. The H3T3P mark is undetectable during G2 phase. Upon entry to mitosis, sister chromatids enriched with pre-existing H3-GFP histone begin to show H3T3 phosphorylation prior to sister chromatids enriched with newly synthesized H3-mKO. As the cell continues to progress toward metaphase, H3T3P signal begins to appear on sister chromatids enriched with newly synthesized H3-mKO. Such a tight regulation of H3T3P is compromised when levels of H3T3P are altered due to the incorporation of mutant H3T3A or H3T3D. Incorporation of the H3T3A mutant results in a significant decrease in the levels of H3T3P on sister chromatids throughout mitosis, such that neither sister becomes enriched with H3T3P as the GSC progresses toward metaphase. Conversely, incorporation of the H3T3D mutant would result in seemingly elevated levels of H3T3P early in mitosis. Although H3T3A and H3T3D act in different ways, both mutations significantly disrupt the highly regulated temporal patterns associated with H3T3 phosphorylation, the result of which is randomized H3 inheritance patterns and germ cell defects in testes expressing either H3T3A or H3T3D (Xie, 2015).

    To further evaluate the extent of H3T3A and H3T3D roles in the segregation of sister chromatids enriched with different populations of H3 during mitosis, all possible segregation patterns were modeled in male GSCs, and these estimates were compared to the experimental results. To simplify the calculations, two important assumptions were made: first, nucleosomal density was assumed to be even throughout the genome. This assumption allows the inference that the overall fluorescent signal contributed by each chromosome is proportional to their respective number of DNA base pairs. Second, by quantifying pre-existing H3-GFP asymmetry in anaphase and telophase GSCs, it was estimated that the establishment of H3-GFP asymmetry is ∼4-fold biased, i.e., 80% on one set of sister chromatids and 20% on the other set of sister chromatids, based on quantification of GFP signal in anaphase and telophase GSCs (Tran, 2012). With these two simplifying assumptions, both GFP and mKO ratios were caculated among all 64 possible combinations. If asymmetry is designed as a greater than 1.5-fold difference in fluorescence intensity, then based on a model of randomized sister chromatid segregation, it is estimated that a symmetric pattern should appear for 53.1% (34/64) of GSC-GB pairs whereas both conventional and inverted asymmetric patterns should occur with equal frequencies and account for 18.7% (12/64) of total GSC-GB pairs. The remaining 9.4% (6/64) of GSC-GB pairs should produce histone inheritance patterns with a 1.45- to 1.55-fold difference in signal intensity (Xie, 2015).

    This estimation is close to the experimental data in both H3T3A- and H3T3D-expressing testes. Of the 64 quantified post-mitotic GSC-GB pairs in nos>H3T3A testes, ∼71.9% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 9.4% and 12.5%, respectively, and 6.3% at the borderline. Similarly, of the 57 quantified post-mitotic GSC-GB pairs in nos>H3T3D testes, ∼79.0% showed symmetric inheritance pattern. Conventional and inverted asymmetric patterns were detected at 7.0% and 10.5%, respectively with 3.5% of pairs at the borderline. Some differences between predicted ratios and the experimental data could be due to the simplified assumptions, the limited sensitivity of the measurement, and/or some coordinated chromatid segregation modes that bias the eventual read-out. In summary, comparison between the modeling ratios and the experimental data suggest that loss of the tight control of H3T3 phosphorylation in GSCs randomizes segregation of sister chromatids enriched with different populations of H3 (Xie, 2015).

    If the temporal separation in the phosphorylation of H3T3 on epigenetically distinct sister chromatids facilitates their proper segregation and inheritance during asymmetric cell division, it is likely that mutations of the Haspin kinase will also affect the temporal control of H3T3 phosphorylation. In the context of H3T3A, where the levels of H3T3P are already reduced, a further decrease in H3T3P by reducing Haspin levels should limit the GSC's ability to distinguish between sister chromatids enriched with distinct H3. Indeed, haspin mutants enhance the phenotypes in nos>H3T3A testes. A different situation appears in the context of H3T3D where sister chromatids experience seemingly elevated levels of H3T3P at the start of mitosis. These elevated H3T3P levels may be exacerbated by the phosphorylation activity of the Haspin kinase. Therefore, it is conceivable that by halving the levels of the Haspin kinase, H3T3 phosphorylation should be reduced to a level more closely resembling wild-type. In this way, some of the temporal specificity that is lost in the H3T3D mutant is restored, resulting in suppression of the phenotypes observed in nos>H3T3D testes. An exciting topic for future study would be to further explore how exactly Haspin phosphorylates H3T3 in the context of chromatin and whether H3T3A and H3T3D mutations act synergistically or antagonistically in regulating asymmetric sister chromatids segregation through differential phosphorylation of a key histone residue (Xie, 2015).

    It would also be interesting to understand the potential connection between asymmetric histone inheritance and another phenomenon reported by several investigators: selective DNA strand segregation. Recent development of the chromosome orientation fluorescence in situ hybridization (CO-FISH) technique allows study of selective chromatid segregation at single-chromosome resolution. Using this technique in mouse satellite cells, it has been demonstrated that all chromosomes are segregated in a biased manner, such that pre-existing template DNA strands are preferentially retained in the daughter cell that retains stem cell identity. Interestingly, this biased segregation becomes randomized in progenitor non-stem cells. Using CO-FISH in Drosophila male GSCs, sex chromosomes have been shown to segregate in a biased manner. Remarkably, sister chromatids from homologous autosomes have been shown to co-segregate independent of any specific strand preference. Such findings hint at a possible epigenetic source guiding the coordinated inheritance of Drosophila homologous autosomes. In many cases of biased inheritance, researchers have speculated about the existence of a molecular signature that would allow the cell to recognize and segregate sister chromatids bearing differential epigenetic information. However, the identity of such a signature has remained elusive. The work represented in this paper provides experimental evidence demonstrating that a tightly-controlled histone modification, H3T3P, is able to distinguish sister chromatids and coordinate their segregation (Xie, 2015).

    Epigenetic processes play important roles in regulating stem cell identity and activity. Failure to appropriately regulate epigenetic information may lead to abnormalities in stem cell behaviors, which underlie early progress toward diseases such as cancer and tissue degeneration. Due to the crucial role that such processes play in regulating cell identity and behavior, the field has long sought to understand whether and how stem cells maintain their epigenetic memory through many cell divisions. Yhe results of this study suggest that the asymmetric segregation of pre-existing and newly synthesized H3-enriched chromosomes may function to determine distinct cell fates of GSCs versus differentiating daughter cells (Xie, 2015).

    Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

    Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). Extended live imaging of the Drosophila testis niche was developed, allowing the tracking of individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event termed 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. GSCs were captured undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements (Sheng, 2011).

    Live imaging of the Drosophila germline stem cell niche has directly demonstrated many aspects of GSC behavior that were impossible to observe in fixed tissues. Asymmetrically oriented divisions do not necessarily determine asymmetric cell fate, but can occasionally result in the production of two GSCs. This is the primary mechanism by which GSCs are replenished in healthy tissues to compensate for GSC loss. As GSC-daughter pairs are adjacent to the hub and are enriched in the maintenance factor STAT92E, the process of symmetric renewal is probably distinct from de-differentiation of spermatogonia (which are non hub-adherent and express the differentiation factor Bam). The frequency of symmetric renewal increases during GSC recovery after protein starvation, and during GSC regeneration after genetically induced stem cell depletion. In the latter case, where the rate of GSC regeneration is higher, GSCs are concurrently derived from de-differentiating spermatogonia, a process characterized by movement, fragmentation and adhesion to the hub by spermatogonial cells. Together, these data demonstrate that lost GSCs can be regenerated by multiple mechanisms, some or all of which may be similar to events occurring in other stem cell systems (Sheng, 2011).

    As changes in stem cell output are observed during regeneration, signaling from support cells or from systemic factors may underlie these effects. Niche-generating cells, transit amplifying daughter cells or even differentiated daughter cells may potentially signal to stem cells and modulate their division output. In the Drosophila testis, GSC maintenance depends on Jak-STAT signaling initiated from the hub, but it is not known whether this same pathway regulates division outcome. As STAT-null GSCs are rapidly lost from the niche, low levels of Jak-STAT signaling due to fluctuations in gene expression may be sufficient to cause GSC loss. In support of this hypothesis, three out of 556 GSCs examined for STAT92E expression had low levels of this protein. However, the mRNA expression pattern of the Jak-STAT pathway ligand Upd is unchanged during de-differentiation, suggesting that genes other than Upd may affect symmetric renewals. BMP signaling, which is required for GSC maintenance, is a good candidate. Combining live imaging with genetic tools for monitoring levels of signaling pathway activation in the Drosophila testis will provide a powerful platform for understanding how cell signaling affects the outcome of stem cell divisions in real time (Sheng, 2011).

    The observation that both symmetric renewal and GSC loss occur when the GSC is attached to a daughter cell suggests that there may be a cell cycle-specific gene expression profile that primes the cells for these events to occur during S or early G2 in the cell cycle. It is speculated that the abscission accompanying symmetric renewal is similar to that occurring in GSC-GB pairs, another G2 event. Cell cycle regulation, which is characterized by a short G1 phase and relatively long S phase, maintains pluripotency in many types of cultured stem cells. As GSCs in the Drosophila testis have short G1 phases, and Drosophila GSCs require distinct cell cycle regulators, investigation of cell cycle regulation of Drosophila GSC division outcome may be informative (Sheng, 2011).

    It was shown that GSCs in both centrosomin mutants and starved wild-type flies have increased frequencies of symmetric renewal, but surprisingly, there is no corresponding rise in GSC numbers. These results suggest that increased symmetric renewal is counterbalanced by increased GSC loss. Cnn mutant GSC are reported to have abnormal cell morphology and often appear to be detaching from the hub, suggesting an overall maintenance defect. During starvation, lowered insulin signaling results in GSCs loss, and this effect can be rescued by overactivation of insulin signaling. The results indicate that symmetric renewals of GSCs undergoing oriented divisions are the source of new GSCs. Starved flies initially have low insulin signaling, but when returned to normal food for a day have higher insulin signaling. However, this study found that both timepoints exhibited increased symmetric renewals, leading to the idea that activation of insulin signaling does not directly modulate division outcome. Perhaps during starvation, lowered insulin signaling causes GSC loss, which in turn triggers a compensatory increase in symmetric renewal. However, symmetric renewals are not able to fully compensate for the loss, yielding an overall decrease in GSC number. When flies are re-fed and insulin signaling returns to normal, GSCs are no longer rapidly lost, and the same rate of symmetric renewal is now able to increase overall GSC number. Together, these results suggest that the behavior of stem cells within the niche is much more dynamic than previously expected, and indicate that GSC number is controlled by the relative rates of symmetric renewal versus loss, not by the orientation of the division plane (Sheng, 2011).

    Why do the majority of Drosophila GSCs undergo asymmetric division if symmetric renewal plus symmetric differentiation produces the same output? As GSCs and CySCs function together within the niche during spermatogenesis, robust division orientation of both populations may enable differentiating germline cells to be generated at a rate that matches cyst cell production. Asymmetric divisions may also prevent clonal expansion of stem cells harboring harmful mutations within the niche, which can compete for niche occupancy. However, clonal expansion may not always be harmful; mammalian niches regularly progress towards mono-clonality with stem cells exhibiting neutral drift dynamics. Perhaps symmetrically renewing divisions are not detrimental to mammalian systems because mammalian niches are not as constrained spatially, and mammalian stem cells are often motile. So far, asymmetric division in Drosophila testes correlates with optimal GSC function, as it becomes less robust with aging. Whether symmetric divisions increase during aging has not been examined, but it might occur because GSCs are thought to be lost more frequently due to decreased maintenance cues. Interestingly, depleting STAT92E from GSCs displaces them from the hub, yet they are not lost from the tissue. Instead, they associate with BMP-producing CySCs, which probably promote GSC renewal. However, GSC division orientation is now randomized; suggesting that their output is composed of symmetric renewals and symmetric differentiation. Furthermore, APC2 mutants that affect centrosome position and E-cadherin mutants that have misoriented divisions still have wild-type GSC numbers. Together, these observations suggest that the Drosophila testis stem cell niche does not require invariant asymmetric GSC division outcomes (Sheng, 2011).

    As mammalian stem cells are thought to undergo symmetric renewal in combination with stochastic differentiation, rather than strict asymmetric divisions, GSCs in Drosophila may share more aspects of stem cell behavior with mammalian systems than has been previously assumed. Wild-type GSCs were observed losing niche attachment and directly differentiating, which is consistent with reports that subsets of undifferentiated spermatogonia in the mouse testes can directly differentiate. Although a lost GSC being replaced by a neighboring GSC undergoing symmetric renewal was observed, this was only a single example where these events are coupled together. Thus, stem cell loss and symmetric renewal may occur stochastically in Drosophila GSCs, as in the mouse testis. It was also shown that differentiating spermatogonia revert into GSCs, which is consistent with findings that differentiating spermatogonia can contribute to the stem cell pool during reconstitution of spermatogenesis in the mouse testes. Therefore, this system provides an ideal platform for determining regulators of stem cell loss and replacement in vivo that may also be conserved in mammalian tissues (Sheng, 2011).

    Conversion of quiescent niche cells to somatic stem cells causes ectopic niche formation in the Drosophila testis

    Adult stem cells reside in specialized regulatory microenvironments, or niches, where local signals ensure stem cell maintenance. The Drosophila testis contains a well-characterized niche wherein signals from postmitotic hub cells promote maintenance of adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells were considered to be terminally differentiated; this study shows that they can give rise to CySCs. Genetic ablation of CySCs triggers hub cells to transiently exit quiescence, delaminate from the hub, and convert into functional CySCs. Ectopic Cyclin D-Cdk4 expression in hub cells is also sufficient to trigger their conversion into CySCs. In both cases, this conversion causes the formation of multiple ectopic niches over time. Therefore, this work provides a model for understanding how oncogenic mutations in quiescent niche cells could promote loss of quiescence, changes in cell fate, and aberrant niche expansion (Hetie, 2014).

    Centrosome misorientation reduces stem cell division during ageing

    Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. This study shows that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. GSCs containing misoriented centrosomes accumulate with age, and these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, it is proposed that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. It was also shown that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia (Cheng, 2008).

    GSCs with misoriented centrosomes accumulate as flies age. Since such misoriented GSCs divide less frequently as compared to oriented GSCs, accumulation of misoriented GSCs contributes to the decline in spermatogenesis that occurs with age. Although misoriented GSCs rarely divide, they are not permanently arrested (or senescent) and are correctly oriented when they divide. Whether correction of GSC orientation is an active process that is part of the acquisition of stem cell identity remains to be determined. The low cell cycle activity of misoriented GSCs may also suggest that mechanisms are in place to detect misorientation and induce cell cycle arrest in response to this change, although the underlying mechanisms remain to be identified (Cheng, 2008).

    It was also demonstrated that misoriented GSCs originate, at least in part, from dedifferentiation of spermatogonia. Although dedifferentiated GSCs have high frequency (>40%) of centrosome misorientation, they can function as stem cells by resuming the cell cycle, with correctly oriented mitotic spindles just like as constitutive GSCs. GSC numbers do not decrease as quickly as expected from the calculated GSC half-life, suggesting that a mechanism to compensate for the loss of GSCs exists. Since misoriented spindles, or symmetric stem cell division, was rarely observed, it is speculated that dedifferentiation is the major mechanism to replace stem cells over time in the Drosophila male germ line (Cheng, 2008).

    A decline in GSC number in older males (day 50) was reported recently (Boyle, 2007) This decrease in stem cell number is likely due to failure of the niche function (via decreased signal from the niche as well as decreased E-cadherin-based attachment between the niche and GSCs. However, the decrease in the production of spermatogonia and testis involution precede the loss of GSCs such that decreasing GSC numbers cannot explain the testis involution that is observed at younger ages (Cheng, 2008).

    The present results provide a novel mechanistic link between the control of stem cell polarity and the age-related decline in tissue regenerative capacity. Mechanisms responsible for monitoring stem cell orientation with respect to the niche not only prevent overproliferation of stem cells by ensuring the asymmetric outcome of the stem cell division, but they contribute to the decline in tissue regenerative capacity during aging. Many of the misoriented GSCs originate from the dedifferentiation of spermatogonia, a mechanism thought to be responsible for maintaining the stem cell population over extended periods of time. Therefore, although GSCs produce less progeny over time, the system appears to maximize the number of progeny produced throughout life, while maintaining asymmetric stem cell division (Cheng, 2008).

    In summary, it is proposed that the GSCs with misoriented centrosome divide less frequently and that a combination of such a decreased stem cell division and a higher frequency of the GSC misorientation in aged testes leads to a decline in spermatogenesis with age (Cheng, 2008).

    Survival motor neuron protein regulates stem cell division, proliferation, and differentiation in Drosophila

    Spinal muscular atrophy is a severe neurogenic disease that is caused by mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein is required for the assembly of small nuclear ribonucleoproteins and a dramatic reduction of the protein leads to cell death. It is currently unknown how the reduction of this ubiquitously essential protein can lead to tissue-specific abnormalities. In addition, it is still not known whether the disease is caused by developmental or degenerative defects. Using the Drosophila system, this study shows that SMN is enriched in postembryonic neuroblasts and forms a concentration gradient in the differentiating progeny. In addition to the developing Drosophila larval CNS, Drosophila larval and adult testes have a striking SMN gradient. When SMN is reduced in postembryonic neuroblasts using MARCM clonal analysis, cell proliferation and clone formation defects occur. These SMN mutant neuroblasts fail to correctly localise Miranda and have reduced levels of snRNAs. When SMN is removed, germline stem cells are lost more frequently. It was also shown that changes in SMN levels can disrupt the correct timing of cell differentiation. It is concluded that highly regulated SMN levels are essential to drive timely cell proliferation and cell differentiation (Grice, 2011).

    This study shows a high demand for SMN in Drosophila stem cells. In addition, striking SMN concentration gradient, inversely proportional to the state of differentiation, has been identified in Drosophila larval CNS and testis. In Drosophila SMN mutant larvae, both the CNS and testis display growth defects which precede the previously reported motor defects and death. These larvae also fail to localise Miranda protein correctly at the basal membrane of the neuroblast. Clonal analysis indicates that SMN deficient stem cells have a reduced number of divisions and also generate cells with lower levels of U2 and U5 snRNPs. Overexpression of SMN alters the timing of CNS growth and disrupts the onset of pupariation and pupation. Using the male germline system, it was shown that prolonged SMN reduction leads to stem cell loss. Finally it was found that ectopic SMN expression in cells along the SMN gradient leads to changes in the timing of cell differentiation. It is therefore suggested that the fine-tuning of SMN levels throughout development can lead to complex developmental defects and reduce the capacity of stem cells to generate new cells in development (Grice, 2011).

    SMN levels have been reported to be extremely high in early development. This study shows that SMN up-regulation occurs in neuroblasts prior to the initiation of their cell division, suggesting a distinct increase of SMN levels is required for new rounds of neurogenesis and local proliferation. Fewer immature neurons are generated in the thoracic ganglion of smn mutant MARCM clones. Provisional data has suggested there may be proliferation defects in the spinal cord of severe mouse models. In addition, a recent study using the severe SMA mouse model has shown proliferation defects in the mouse hippocampus, a region associated with higher SMN levels (Wishart, 2010). Together these data suggest that, in part, the pathology observed in more severe forms of SMA may be caused by defects in tissue growth (Grice, 2011).

    Proteins involved in processes such as chromatin remodelling, histone generation and cell signalling have been identified as intrinsic factors for the maintenance of Drosophila stem cells. This is the first report of stem cell defects caused by the reduction of a protein involved in snRNP biogenesis. Although SMN is required in all cells, proper stem cell function requires a substantially higher level of SMN. This study also shows snRNP defects in Drosophila SMN mutant tissue. Previous studies in Drosophila have shown no gross changes in snRNP levels, including U2 and U5, in lysates from whole smnA and smnB mutant larvae. smnA MARCM neuroblast clones and male germline mitotic clones have reduced snRNP levels, suggesting snRNP assembly may be particularly sensitive to SMN reduction during CNS and germline development (Grice, 2011).

    SMN mutant neuroblasts have abnormal Miranda localisation. Miranda, an adaptor protein, forms a complex with the RNA binding protein Staufen which binds to prospero mRNA. In addition to snRNPs, SMN protein has been implicated in the biogenesis of numerous RNP subclasses, including proteins involved in the transport and localisation of β-actin mRNA at the synapse. Whether Miranda mislocalisation is due to direct or indirect associations with SMN should be addressed (Grice, 2011).

    SMN mutant larvae have been previously shown to have synaptic defects which include enlarged and fewer boutons and a reduction in the number of GluR-IIA clusters - the neurotransmitter receptor at the Drosophila neuromuscular junction. In addition, numerous developmental defects are observed including pupation and growth defects. Complementing this work, Drosophila Gemin5 a member of the Drosophila SMN-Gemin complex has been shown to interact with members of the ecdysone signalling pathway responsible for initiating pupation and growth. Drosophila Gemin5 is also enriched in pNBs, in a pattern comparable to SMN. There is increasing evidence that suggests the Drosophila SMN complex plays an important role in pupation. Ubiquitous overexpression of SMN using da-GAL4 advances CNS development and causes premature entry into pupation. The ecdysone pathway has been identified to play an important part in the regulation of neuroblast division and neuronal differentiation during development. How the Drosophila SMN complex plays a part in stem cell biology, and how the SMN complex interacts with specific signalling pathways should be the subject of further study (Grice, 2011).

    Larval and adult testes exhibit the most distinct SMN gradients in Drosophila tissues. Drosophila testes have a constant population of germline stem cells that start to divide in the late larval stages and produce sperm throughout life. The removal of SMN from male germline stem cells results in stem cell loss. In the smnB mutant testis, the reduction of SMN causes a contraction of the SMN gradient towards the apical stem cells. As SMN is lost from the primary spermatoctyes, more mature sperm are observed. Increasing SMN levels leads to an increase in primary spermatocytes and a reduction in mature sperm in the adult. This result is the first to demonstrate that high SMN levels in undifferentiated cells can repress differentiation in sperm development. Interestingly, along with the CNS, Drosophila testes have the highest number of alternative splicing events and the most differentially expressed splicing factors during development. Understanding if differential expression of SMN in specific cell types controls a shift in splicing factors as cells switch from proliferation to differentiation will be the target of future study. A recent study has identified defects in gametogenesis and testis growth in mice lacking the Cajal body marker coilin, a binding partner of SMN. The authors speculated that coilin may facilitate the fidelity and timing of RNP assembly in the cell and coilin loss may limit rapid and dynamic RNA processing. It will be important to understand how SMN and coilin genetically interact in stem cells and developing tissues (Grice, 2011).

    The Drosophila CNS and male germline offer two new tractable systems that can be used to study SMN biology in development and stem cells. It also offers a system to study how SMN, a protein associated with neuronal development, could cause SMA. Although SMA is classically a disease of the motor neuron, a severe reduction of SMN protein affects a wide spectrum of cells including stem cells. Consistent with this idea, symptoms in mild forms of SMA (type III or IV) are predominately limited to motor neurons. However, patients with the most severe type (type I), suffer from defects in multiple tissues including congenital heart defects, multiple contractures, bone fractures, respiratory insufficiency, or sensory neuronopathy. Elucidating the differential requirements of SMN in individual cell types, and how their sensitivity to SMN loss can mediate the disease, can contribute to the understanding of the selectivity of SMA (Grice, 2011).

    Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution

    Stem cells can self-renew and generate differentiating daughter cells. It is not known whether these cells maintain their epigenetic information during asymmetric division. Using a dual-color method to differentially label 'old' versus 'new' histones in Drosophila male germline stem cells (GSCs), it was shown that preexisting canonical H3, but not variant H3.3, histones are selectively segregated to the GSC, whereas newly synthesized histones incorporated during DNA replication are enriched in the differentiating daughter cell (see Experimental design and potential results). The asymmetric histone distribution occurs in GSCs but not in symmetrically dividing progenitor cells. Furthermore, if GSCs are genetically manipulated to divide symmetrically, this asymmetric mode is lost. This work suggests that stem cells retain preexisting canonical histones during asymmetric cell divisions, probably as a mechanism to maintain their unique molecular properties (Tran, 2013).

    Although all cells in an organism contain the same genetic material, different genes are expressed in specific cell types, allowing them to differentiate along distinct pathways. Epigenetic mechanisms regulate gene expression and maintain a specific cell fate through many cell divisions. Stem cells have the remarkable ability to both self-renew and generate daughter cells that enter differentiation. Epigenetic mechanisms have been reported to regulate stem cell activity in multiple lineages. However, there has been little direct in vivo evidence demonstrating whether stem cells retain their epigenetic information (Tran, 2013).

    The Drosophila male GSCs are well characterized in terms of their physiological location, microenvironment (i.e., niche), and cellular structures). Male GSCs can be identified precisely by their distinct anatomical positions and morphological features. A GSC usually divides asymmetrically to produce a self-renewed GSC and a daughter cell gonialblast (GB) that undergoes differentiation. Therefore, GSCs can be examined at single-cell resolution for a direct comparison (Tran, 2013).

    In eukaryotes, the basic unit of chromatin called nucleosome contains histone octamer [2×(H3, H4, H2A, H2B)] and DNA wrapping around them. Indeed, histones are one of the major carriers of epigenetic information. To address how histones are distributed during the GSC asymmetric division, a switchable dual-color method was developed to differentially label 'old' versus 'new' histones that uses both spatial (by Gal4; UAS system) and temporal (by heat shock induction) controls to switch labeled histones from green [green fluorescent protein (GFP)] to red [monomeric Kusabira-Orange (mKO)]. Heat shock treatment induces an irreversible DNA recombination to shut down expression of GFP-labeled old histones and initiate expression of mKO-labeled new histones. If the old histones are partitioned nonselectively, the GFP will initially exhibit equal distribution in the GSC and GB, and will be gradually replaced by the mKO. However, if the old histones are preferentially retained in the GSCs to constitute potentially GSC-specific chromatin structure, the GFP will be detected specifically in the GSCs. During DNA replication-dependent canonical histone deposition, histones H3 and H4 are incorporated as a tetramer, and histones H2A and H2B are incorporated as dimers. Therefore, independent transgenic strains were generated for H3 and H2B, respectively. On the other hand, histone variants are incorporated into chromatin in a transcription-coupled but DNA replication-independent manner. Therefore, the histone variant H3.3 was used as a control for canonical histones (Tran, 2013).

    To avoid potential complications caused by heat shock-induced DNA recombination on either one or both chromosomes in GSCs, each of the three transgenes (H3, H2B, and H3.3) was integrated as a single copy and analyzed in heterozygous flies. Examination of testes with the transgenes revealed nuclear GFP but little mKO signal before heat shock. After heat shock, mKO signals were detectable. Different GSCs undergo mitosis asynchronously, and an average cell cycle length of GSCs is approximately 12 to 16 hours. Among all GSCs, 75% to 77% are in G2 phase, 21% are in S phase, fewer than 2% are in mitosis, and G1-phase GSCs are almost negligible. Moreover, the GSC and GB arising from an asymmetric division remain connected after mitosis by a cellular structure known as the spectrosome, when they undergo the next G1 and S phases synchronously (Tran, 2013).

    To examine the distribution of old versus new histones in GSC and GB after a round of DNA replication-dependent histone deposition, testes were studied 16 to 20 hours after heat shock. In particular, GSC-GB pairs connected by spectrosomes were examined. On the basis of cell cycle length of GSCs, these GSC-GB pairs were from GSCs that switched from histone-GFP to histone-mKO genetic code during their G2 phase and then underwent the first mitosis followed by G1, S, and G2 phase and the second mitosis. Within this time frame, both old histones and new histones were detectable in GSCs at the second G2 phase because new histones had been synthesized and incorporated during the first S phase. For histone H3, the GFP signal was detected primarily in the GSC but not in the GB. By contrast, the mKO signals were present in both the GSC and the GB, with a relatively higher level in the GB. The asymmetric distribution of histone H3 was specific for GSC divisions, because both the GFP and mKO signals were equally distributed in spermatogonial cells derived from a symmetric division of the GB in the same testis samples. Quantification of fluorescence intensity revealed that the old H3 (GFP-labeled) signal was more enriched in the GSC than in the GB by a factor of ~5.7, whereas new H3 (mKO-labeled) signal was more enriched in the GB than in the GSC by a factor of ~1.6. By contrast, this differential distribution of old versus new histone was not detected for symmetrically dividing spermatogonial cells (Tran, 2013).

    In contrast to the asymmetric distribution pattern for the canonical histone H3, the histone variant H3.3 did not show this asymmetry during GSC divisions, by fluorescence images and by quantification. The symmetry of the histone variant H3.3 suggests that the asymmetric mode is specific for canonical histone H3 (Tran, 2013).

    Fewer than 2% of all GSCs are undergoing mitosis; thus, all analyses above were based on postmitotic GSC-GB pairs. To further examine the histone segregation pattern during mitosis, a screen was carried out for mitotic GSCs. Indeed, old histones were mainly associated with the chromatids segregated to the GSC side at metaphase, anaphase, and telophase. By contrast, new histones were more enriched at the chromatids segregated to GB side). These results suggest that the sister chromatids preloaded with old histones are preferentially retained in GSCs and that the ones enriched with new histones are partitioned to GBs during GSC mitosis (Tran, 2013).

    Next, the histone distribution pattern was examined during the first GSC division by recovering GSCs for 4 to 6 hours after heat shock. An asymmetric distribution pattern was also found in the GSC-GB pairs with the H3 transgene. By contrast, a symmetric distribution pattern was observed for both dividing spermatogonial cells with the H3 transgene and H3.3 during GSC division. Quantification of fluorescence intensity revealed that the old H3-GFP signal was enriched in the GSC by a factor of ~13 relative to the GB, whereas the new H3-mKO signal was enriched in the GB by a factor of ~2.4 relative to the GSC. By contrast, there was no differential distribution of the old versus new histone for the symmetrically dividing spermatogonial cells, or H3.3 during GSC division. Although an asymmetric histone distribution pattern was detected in postmitotic GSC-GB pairs, examination of the mitotic GSC at this stage did not show any asymmetry. These data suggest that the asymmetric segregation mode relies on replication-dependent histone incorporation prior to mitosis. However, the factor of >10 difference of GFP signal between GSC and GB could be contributed by faster turnover of old histones in GBs, probably as a mechanism to reset the chromatin for differentiation. By contrast, the difference of mKO in GSC and GB was less substantial, probably as a result of new histone synthesis in both cells. Furthermore, the H2B transgene showed a similar pattern to H3 after the first GSC division (Tran, 2013).

    The consistent asymmetric cell divisions of GSCs could be lost under certain conditions, such as ectopic activation of the key JAK-STAT signaling pathway in the niche. It has been shown that overexpression of the JAK-STAT ligand unpaired (OE-upd) induces overpopulation of GSCs. Consistent with the loss of asymmetry in expanded GSCs, the asymmetric distribution pattern of the histone H3 was not observed in OE-upd testes 16 to 20 hours after heat shock. These results demonstrate that the asymmetric histone distribution pattern is dependent on GSC asymmetric divisions. A two-step process is proposed as the favored explanation: Old and newly synthesized histones are incorporated to different sister chromatids during S phase; then, during mitosis, the sister chromatid preloaded with old histones is preferentially segregated to GSC (Tran, 2013).

    These data reveal that stem cells preserve preexisting histones through asymmetric cell divisions. The JAK-STAT signaling pathway required for the asymmetric GSC divisions contributes to the asymmetric histone distribution pattern. This work provides a critical first step toward identifying the detailed molecular mechanisms underlying old histone retention during GSC asymmetric division. These findings in the well-characterized GSC model system will facilitate understanding of how epigenetic information could be maintained by stem cells or reset in their sibling cells that undergo cellular differentiation (Tran, 2013).

    The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

    Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. This study demonstrates that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia (Shields, 2014).

    Chickadee, the only Drosophila profilin homolog, is required cell intrinsically for GSC maintenance in the testis. As profilin is a regulator of actin filament polymerization and filamentous actin (F-actin) plays a crucial role in the development and stabilization of cadherin-catenin-mediated cell-cell adhesion, profilin likely maintains attachment of Drosophila male GSCs to the hub through its effect on F-actin, which concentrates at the hub-GSC interface where localized adherens junctions anchor GSCs to hub cells. It is proposed that profilin-dependent stabilization of F-actin at the GSC cortex next to the hub may help localize E-cadherin and APC2 to the junctional region. E-cadherin and APC2 in turn may recruit β-catenin/Armadillo, stabilizing the adherens junctions that attach GSCs to the hub. Chickadee may thus facilitate maintenance of GSCs through a cascade of interactions leading to localization and/or retention of both E-cadherin and β-catenin at the hub-GSC interface (Shields, 2014).

    E-cadherin plays a crucial role in maintaining hub-GSC attachment. GSC clones mutant for E-cadherin are not maintained. In addition, germline overexpression of E-cadherin delayed GSC loss in stat-depleted GSCs. The results indicate that profilin function is required in GSCs for proper localization of E-cadherin to the hub-GSC interface. Several studies have shown that the actin cytoskeleton plays a crucial role in assembly and stability of adherens junctions. A favored model in the field is that actin filaments indirectly anchor and reinforce E-cadherin-mediated cell junctions by forming an intracellular scaffold for E-cadherin molecules. Indeed, binding to F-actin stabilized E-cadherin and promoted its clustering. Furthermore, the actin cytoskeleton participates in proper localization of E-cadherin molecules to cell-cell contacts. In chic/profilin mutant GSCs, disruption of actin polymerization at the cell cortex leading to local F-actin disorganization may destabilize E-cadherin and reduce its ability to localize to the GSC-hub junction, form clusters and build adequate adherens junctions (Shields, 2014).

    Destabilization of E-cadherin may contribute to the mislocalization of APC2 seen in chic mutant GSCs, as E-cadherin recruits APC2 to cortical sites in GSCs. Raising possibilities of a more direct link, actin filaments have been shown to be required for association of APC2 with adherens junctions in the Drosophila embryo and ovary. Treatment of embryos with actin-depolymerizing drugs resulted in complete delocalization of APC2 from adhesive zones and diffuse APC2 staining throughout the cell. Moreover, in ovaries of chic1320/chic221 females, APC2 was substantially delocalized from the plasma membranes of nurse cells and their ring canals, and increased levels of cytoplasmic APC2 staining were observed. Similarly, this study found that APC2 was delocalized from the hub-GSC interface in larval testes of chic11/chic1320 hypomorphs (Shields, 2014).

    In several studies, delocalization of APC2 from junctional membranes correlated with detachment of β-catenin/Armadillo from adherens junctions. APC2 co-localizes with Armadillo and E-cadherin at adherens junctions of Drosophila epithelial cells, nurse cells in Drosophila ovaries and at the hub-GSC interface in Drosophila testes. Disruption of APC2 function resulting in significant reduction of junctional APC2 was accompanied by delocalization of junctional Armadillo and increased levels of free cytoplasmic Armadillo in embryonic epithelial cells and ovaries. In a previous study, which used chic1320/chic221 strong loss-of-function mutants, the delocalizing effect on junctional Armadillo was variable, presumably due to incomplete penetrance of chic mutant effects. Although this study did not observe significant disruption in Armadillo staining along the hub-GSC interface of testes from chic hypomorphs, this may be due to incomplete penetrance. In addition, the Armadillo protein detected could be localized to the cortex of hub cells rather than GSCs (Shields, 2014).

    The finding that germline specific overexpression of APC2 in chic11/chic1320 hypomorphs partially rescued GSC loss is consistent with a previously proposed model that actin filaments shuttle APC2 to adherens junctions and APC2 in turn recruits cytoplasmic Armadillo to junctional membranes, reinforcing the adherens junctions. It is possible that in chic11/chic1320 hypomorphs, residual actin filaments associated with adherens junctions between the hub and GSCs are sufficient to shuttle the increased amounts of cytoplasmic APC2 to adherens junctions. This APC2 may in turn recruit free cytoplasmic Armadillo to the hub-GSC interface, locally stabilizing the adherens junctions and anchoring GSCs to their niche. Notably, however, germline specific overexpression of APC2 in testes of strong loss-of-function chic1320/chic221 mutants failed to rescue GSC loss. Thus either, adequate levels of actin filament polymerization may be required for the proposed translocation of junctional proteins to the plasma membrane, or APC2 function/localization may not be the only or even the major cell-autonomous target of profilin function important for maintaining GSCs. Indeed, loss of APC2 function did not lead to GSC loss. It is suggested that the localized cortical F-actin underlying adherens junctions at the GSC-hub interface, best candidate for the most direct target of chic function, strongly stabilizes adherens junctions between GSCs and the hub, with high levels of cortical APC2 able to in part make up for weak chic function by also stabilizing adherens junctions (Shields, 2014).

    Maintenance of hub-GSC attachment appears to be a key role of STAT in GSCs. The finding that STAT binds to a site near the upstream promoter of the chic gene raises the possibility that STAT might foster GSC attachment to the hub in part by ensuring high levels of transcription of profilin in GSCs. However, activation of STAT is clearly not the only regulatory influence on profilin expression as profilin is an essential gene expressed in many cell types, including those in which STAT is not active or detected. It is likely that transcription factors other than STAT turn on profilin expression in many cell types and that STAT acts along with other regulators to reinforce profilin expression in GSCs and CySCs. Conversely, overexpression of profilin was not sufficient to re-establish attachment of stat-depleted GSCs, suggesting that STAT probably regulates a number of genes to ensure that GSCs remain within the stem cell niche (Shields, 2014).

    Loss of chic function in somatic cyst cells impaired the ability of cyst cells to build and/or maintain the cytoplasmic extensions through which they embrace and enclose spermatogonial cysts. Two somatic cyst cells normally surround each gonialblast and enclose its mitotic and meiotic progeny throughout Drosophila spermatogenesis. The cyst cells co-differentiate with the germ cells they enclose. Several lines of evidence support the model that either the ability of somatic cyst cells to enclose germ cells or their ability to send signals to adjacent germ cells is important to restrict proliferation and promote differentiation of germ cells. In either case, activation of EGFR in cyst cells is required for cyst cells to enclose germ cells and/or send the signals for germ cells to differentiate. The similarities in phenotype between loss of chic function and loss of EGFR activation in somatic cyst cells raise the possibility that chic/profilin may act downstream of activated EGFR to modulate the actin cytoskeleton for the remodeling of cyst cells to form or maintain the cytoplasmic extensions that enclose germ cells. Indeed, activated EGFR is known in other systems to tyrosine phosphorylate phospholipase C-γ1 (PLC-γ1), a soluble enzyme in quiescent cells like daughter cyst cells, activating it to catalyze hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which binds profilin protein with high affinity, which inhibits the interaction between profilin and actin. The hydrolysis of PIP2 by activated PLC-γ1 results in localized release of profilin and other actin-binding proteins, enabling them to interact with actin and participate in cytoskeletal rearrangement and membrane protrusion. Thus, based on biochemical analysis in other systems, a link between EGFR activation and profilin leading to local remodeling of the actin cytoskeleton is plausible in somatic cyst cells, although it remains to be directly tested (Shields, 2014).

    Somatic cell encystment promotes abscission in germline stem cells following a regulated block in cytokinesis

    In many tissues, the stem cell niche must coordinate behavior across multiple stem cell lineages. How this is achieved is largely unknown. This study has identified delayed completion of cytokinesis in germline stem cells (GSCs) as a mechanism that regulates the production of stem cell daughters in the Drosophila testis. Through live imaging, a secondary F-actin ring was shown to form through regulation of Cofilin activity to block cytokinesis progress after contractile ring disassembly. The duration of this block is controlled by Aurora B kinase. Additionally, a requirement was identified for somatic cell encystment of the germline in promoting GSC abscission. It is suggested that this non-autonomous role promotes coordination between stem cell lineages. These findings reveal the mechanisms by which cytokinesis is inhibited and reinitiated in GSCs and why such complex regulation exists within the stem cell niche (Lenhart, 2015).

    This first real-time analysis of GSCs through abscission has revealed surprising complexities layered in cytokinesis. First, cytokinesis is blocked after central spindle and contractile ring disassembly and before entry to the abscission phase. This block is imposed by a secondary F-actin-ring. Second, AurB regulates the transition between phase one and phase two. That transition marks a vital step in the reinitiation of cytokinesis, permitting cytoplasmic isolation and recruitment of abscission machinery. Finally, somatic cell encystment is essential to abscission. Thus, three discrete nodes of regulation are layered on top of the canonical cytokinesis program to achieve tight temporal control over daughter cell production, and thus tissue maintenance by the resident stem cells (Lenhart, 2015).

    Incomplete cytokinesis is a deeply conserved feature of germ cells that establishes the syncytium necessary for robust germline development. Differentiating germ cells appear to arrest cytokinesis immediately following contractile ring ingression because the known components of stable ring canals are identical to those of the contractile ring. It was thought that delayed cytokinesis in GSCs was simply a remnant of this conserved program. In contrast, this study found that the delay is mechanistically distinct from that occurring in differentiating germ cells. GSCs complete ingression, disassemble their contractile ring F-actin, and dissolve central spindle microtubules before engaging a ROK-LimK-Cofilin pathway to regulate a secondary F-actin ring that blocks cytokinesis progression until its disassembly at the entry to phase two (Lenhart, 2015).

    Interestingly, the F-actin rings of gonial cells were not disrupted by manipulation of Cofilin activity, in contrast to their precocious disassembly in GSC-Gb pairs. This functional distinction is likely tied to the different biological goal of the stem cell versus the differentiating germ cell. One must release a differentiating daughter cell while the other must communicate syncitially for differentiation to progress normally. Ultimately, because the stem cell niche confers this functional distinction, future work will investigate whether it directly controls F-actin dynamics in the stem cell by possibly modulating Cofilin, or acts indirectly through other stem cell factors to do so (Lenhart, 2015).

    These data strongly indicate that the secondary F-actin ring must be disassembled for abscission to be reinitiated. This suggests that F-actin at the IC bridge inhibits abscission, and work in other cells supports this. Inhibition of the Cofilin phosphatase, activation of AurB, depletion of phosphoinositide 5-phosphatase, or of Rab35 all lead to retention of F-actin at the IC bridge and inhibit abscission. Importantly, abscission could be restored after Rab35 depletion by forcing F-actin disassembly (Lenhart, 2015).

    GSC-Gb pairs depleted for aurB fail to complete abscission prior to mitotic entry and form interconnected germ cells attached to the hub. This could suggest that AurB is normally required to promote abscission. However, expressing an activated form of Svn did not induce precocious abscission as would be expected in this model. Rather, SvnS125E expression advanced the transition from phase one to two, while aurB depletion delayed it. These reciprocal effects suggest instead that AurB times the phase one-phase two transition. In this model, the lack of abscission in aurB mutants is an indirect consequence of spending a shorter fraction of the total cycle in phase two. For example, this study has shown that ESCRTIII is localized during phase two and in the apparent absence of central spindle microtubules. In aurB-depleted cells, there simply may not be enough time during the shortened phase two for the already compromised recruitment of ESCRTIII machinery to promote abscission prior to mitotic entry. It is also noted that the lack of a central spindle raises the issue of how ESCRTIII components are delivered to the IC bridge. Perhaps the midbody performs this role, as has been suggested for the C. elegans first cell division (Lenhart, 2015).

    Recent studies have found that shrub is negatively regulated by AurB in female GSCs (Matias, 2015). Although the current results suggest that AurB activity should promote ESCRTIII function in the testis, it is compelling to speculate that AurB might control the phase one-phase two transition through shrub. Alternatively, AurB could directly control this transition by regulating disassembly of the secondary F-actin ring, as there is precedent for AurB controlling actin dynamics. For example, in the 'No Cut' pathway, maintenance of AurB activity late in cytokinesis is associated with persistence of F-actin at the IC bridge. Intriguingly, AurB can phosphorylate formin proteins and thereby regulate actin stress fiber formation. Although in this context AurB activity positively regulates actin polymerization, the interaction between AurB and formin suggests a direct link between CPC activity and actin dynamics. This connection is particularly compelling given that formins can also promote severing of actin filaments. Thus, it is intriguing to speculate that AurB phosphorylation of formins at the IC bridge in GSC-Gb pairs may promote severing of actin filaments in the secondary ring and thereby promote transition from phase one to phase two of delay (Lenhart, 2015).

    Perhaps most excitingly, this study has identified non-autonomous control over GSC-Gb abscission by somatic cell encystment. This sheds light on the functional relevance of abscission delay. Encystment of spermatogonia by two somatic cells is required for proper germ cell differentiation. However, GSCs and their flanking CySCs do not coordinate daughter cell production by synchronizing their cell cycles. Linking abscission to encystment is an elegant alternative for promoting coordinated release of stem cell daughters from the niche (Lenhart, 2015).

    Several questions are raised by the current observations, such as precisely when abscission is triggered relative to cyst cell engulfment of the Gb. It would be necessary to carry out live imaging simultaneously on germline and adjacent somatic cells to address this. However, imaging CySCs and cyst cells is fraught with difficulty due to their irregular morphology and small size. Thus, it has not yet been possible to image somatic cells with anywhere near the resolution achieved for GSC-Gb pairs (Lenhart, 2015).

    Encystment could promote abscission through contact-dependent signaling, where CySCs or cyst cells produce the ligand. Alternatively, the abscission trigger might be mechanical, because tension has been suggested to regulate abscission in cultured cells. Here, as daughter cells migrated apart in culture following mitosis, tension along the bridge connecting them increased and this lengthened the time to abscission. Experimentally decreasing bridge tension triggered earlier abscission. In the current system, most Gbs are displaced some distance from the hub during phase two, with a consequent elongation of the IC bridge connecting those cells to the GSC. Perhaps movement of the Gb away from its mother GSC generates increased tension along the bridge. Symmetric encystment might relieve that tension by providing equalizing forces on both sides of the IC bridge, inducing abscission while ensuring that the Gb is properly associated with two somatic cells. In culture, increased tension delayed abscission by disrupting assembly of functional ESCRTIII complexes at the IC bridge. Therefore, it will be interesting to address whether ESCRTIII complexes in GSCs are temporally regulated by encystment. Whatever the mechanism, the cyst cells are clearly poised for intimate contact at the appropriate time, because the midbody remnant is sometimes taken up byÊencysting somatic cells after abscission (Lenhart, 2015).

    This work has clarified the mechanism by which cytokinesis is delayed in GSCs, identifying three distinct regulatory events layered on top of the traditional program of cytokinesis. These events impose an appropriate delay, a timed reinitiation, and a regulated abscission in the GSCs. This stem cell-specific program assists in the coordinate release of differentiating daughter cells from the resident stem cell populations in this niche. Because similar requirements for synchronized daughter cell production between multiple stem cell populations exist in other tissues, it is enticing to speculate that regulated abscission might be used to promote coordination in other niches. Membrane scission is difficult to demonstrate in vivo in many systems, so it is not yet known if stem cells other than the germline exhibit abscission delay. As higher resolution methods are developed to visualize stem cell dynamics within endogenous niches, it will be interesting to see if abscission delay emerges as a conserved mechanism of niche-dependent control over stem cell proliferation (Lenhart, 2015).

    The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis

    Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. The COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. By establishing colcemid-induced microtubule depolymerization as a sensitive assay, this study examined the characteristics of COC activity and found that it functions uniquely in GSCs but not in their differentiating progeny. The COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types (Venkei, 2015).

    Stereotypical orientation of the mitotic spindle is a widely utilized mechanism to achieve asymmetric cell division. Although considerable knowledge has accumulated regarding how spindle orientation is established, little is known about whether cells possess a mechanism that monitors successful spindle orientation. In the present study, using colcemid treatment as a sensitive assay, the nature of the COC, which is the only known orientation/polarity checkpoint in multicellular organisms, was was investigated. It was established that: (1) the COC specifically operates in GSCs, but not differentiating germ cells (GBs and SGs); (2) the COC operates in G2 phase of the cell cycle to prevent precocious entry into mitosis upon centrosome misorientation; and (3) as a checkpoint mechanism, the COC is distinct from the SAC (Venkei, 2015).

    These results show that the COC is a GSC-specific checkpoint that monitors centrosome orientation and arrests cells in G2 phase when centrosomes are not correctly oriented. It remains unclear whether the COC-mediated G2 arrest might eventually undergo adaptation to allow mitotic entry, as is the case with mitotic slippage in the SAC. It is worth noting in this context that the GSC mitotic index never increased during 6 h of colcemid treatment, whereas SGs seem to undergo mitotic slippage by 6 h of colcemid treatment. This suggests that the COC-mediated G2 arrest is relatively strong. The findings that the mad2 mutation has no effect on G2 arrest of GSCs and that par-1 and cnn mutants have no effect on mitotic arrest in GBs/SGs upon colcemid treatment strongly support the notion that the COC and SAC constitute distinct checkpoint mechanisms. Although CySCs and SGs also orient their mitotic spindles, the present study shows that spindle orientation in these cells is not under the control of the COC. However, the lack of a COC in these cell types does not exclude the possibility that distinct polarity checkpoint mechanisms are in place to ensure correct spindle orientation. If the arrest points of such checkpoints are not prior to the arrest point of the SAC (i.e. metaphase), the assay using colcemid would not reveal their presence (Venkei, 2015).

    Mutant analysis of multiple centrosomal components in this study revealed a selective requirement for centrosomal components in the function of the COC. Sas-4 and Cnn are crucial for COC function, whereas Spd-2 and Apc1 are not. This indicates that not all of the centrosomal proteins are involved in COC function. Conversely, not all COC components are localized to the centrosome. As shown in a previous study, an essential component of the COC, Par-1, is localized to the spectrosome, where it regulates the localization of Cyclin A to regulate mitotic entry. How the information on centrosomal orientation is communicated to the spectrosome, where Par-1 and Cyclin A localize, remains to be determined. A major outstanding question in understanding the COC is how it senses the location of the centrosome with respect to the hub cells. Previous studies have shown that the mother centrosome is anchored to the adherens junctions formed at the hub-GSC interface via MTs. Therefore, it is plausible that the COC senses aspect(s) of these interactions. It awaits future investigation to understand how the association of the centrosome with the hub-GSC interface is mechanistically sensed, and how such information is integrated with the activity of COC component(s) on the spectrosome (Venkei, 2015).

    In summary, this present work establishes that the COC is a checkpoint mechanism that is distinct from the SAC and monitors correct centrosome orientation specifically in GSCs. It is speculated that a similar mechanism might be in place in other systems that rely on asymmetric cell division (Venkei, 2015).

    Phf7 controls male sex determination in the Drosophila germline

    Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. This study identified PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). It is proposed that PHF7 acts as a conserved epigenetic 'reader' that activates the male germline sexual program (Yang, 2012).

    Sex determination is key to sexual reproduction, and both somatic cells and germ cells need to establish sex-specific developmental fates. Germline sexual development is essential for the production of two distinct gametes, and underlies important differences in the regulation of male versus female fertility. In some species, germline stem cells are present in both males and females to sustain constant gamete production, but are regulated differently throughout development. In other species such as humans, sex-specific germ cell development produces a germline stem cell population only in males, whereas females have a much more limited capacity in making eggs. Defects in germline sexual development lead to a failure in gametogenesis, thus the study of germline sex determination is essential for understanding normal reproductive potential and treating infertility (Yang, 2012).

    In some animals, such as mammals and Drosophila, the sex chromosome compositions of the soma and germline are interpreted independently, and the 'sex' of the germline must match that of the soma for proper germ cell development to occur. For example, patients with Klinefelter's Syndrome have an XXY sex chromosome constitution and are almost always infertile. These individuals develop somatically as males due to the presence of a Y chromosome but the germline suffers from severe atrophy, including the loss of premeiotic germline and germline stem cells. This is due to the presence of two X chromosomes in the germ cells, as the limited spermatogenesis in these patients is from germ cells that have lost one of the X chromosomes. In Drosophila, XX females that are somatically transformed into males exhibit a similar germline loss due to a conflict in sexual identity between the masculinized soma and XX germline. Thus, fruit flies are a valuable model organism for studying how germ cells establish a proper sexual identity by coordinating intrinsic signals and those coming from the soma (Yang, 2012).

    In Drosophila, the presence of two X chromosomes promotes female somatic identity by activating an alternative splicing cascade that acts through Sex lethal (SXL) and Transformer (TRA), and ultimately leads to production of either the male or female forms of the transcription factors Doublesex (DSX) and Fruitless (FRU). DSX and FRU are responsible for virtually all sexually dimorphic somatic traits in Drosophila, with DSX being the key factor in the somatic gonad. In contrast, the germline does not determine its sex with this cascade and factors like TRA and DSX are not required in germ cells. Although SXL is required to promote female germ cell identity, its targets and mechanism of action in the germline are not known. The transcription factor OVO and the ubiquitin protease Ovarian Tumor (OTU) are also required in the female germline and thought to function upstream of SXL. Even less is known about how sexual identity is specified in male germ cells. Male germ cells receive a signal through the JAK/STAT pathway that promotes their sexual identity, but the downstream factors that are subsequently activated are not known. Similarly, how male germ cells respond to their own sex chromosome constitution is also not known (Yang, 2012).

    This study reports a histone code reader, Plant Homeodomain (PHD) Finger 7 (PHF7), that acts in the Drosophila germline to promote male sexual identity. PHF7 is specifically expressed in male germ cells from early stages of development and is restricted to male germline stem cells (GSCs) and spermatogonia. Phf7 is required for GSC maintenance and proper entry into spermatogenesis. Interestingly, expression of Phf7 in female germ cells causes ablation of the female germline. Moreover, Phf7 affects sexual compatibility between germline and soma. Loss of Phf7 in XY germ cells alleviates the germline loss typically observed when XY germ cells are surrounded by a female soma, and expression of Phf7 can induce spermatogenesis in XX germ cells nurtured by male soma. These findings indicate that Phf7 is an essential factor in determining sexual development in the Drosophila germline, and suggest that activation of the male identity occurs through interaction with the germline epigenome (Yang, 2012).

    The data indicate that Phf7 acts to promote a male identity in the germline. Loss of Phf7 function affected male GSC maintenance and spermatogenesis, but had no effect in females. Phf7-mutant GSCs exhibited a more female-like pattern of spectrosome localization, and male (XY) germ cells mutant for Phf7 were more compatible with a female soma than were wild-type male germ cells. Further, expression of PHF7 was able to masculinize the female germline: PHF7 expression induced apoptosis in developing XX germ cells and interacted with mutations in otu in a manner that indicates XX germ cells that express PHF7 are more male-like. Strikingly, PHF7 expression was able to induce spermatogenesis in XX germ cells when they are present in a male soma, something that XX germ cells are normally not able to do. Taken together, these results indicate that Phf7 promotes and is sufficient to induce male identity in the germline (Yang, 2012).

    Sex determination is thought to be initiated early during development, and sex-specific differences in the male and female germline are first observed during embryogenesis. The data indicate that Phf7 plays a role in early germline sexual development, rather than a late role to regulate germ cell differentiation and gametogenesis. First, PHF7 expression is observed in the embryonic gonad and, in the adult, PHF7 is found in the GSCs and early gonia and disappears dramatically as gonia transition to spermatocytes. Further, forced PHF7 expression disrupts early female germ cell development, around the time when they are first forming GSCs. Expression of PHF7 after the early cystoblast stage (Bam-Gal4, UAS-Gal4) had no effect on the female germline, indicating that it can only affect early stages of female germ cell development. Phf7 mutants show defects in male GSC behavior and maintenance, and in the initial progression to form spermatocytes, but it is possible that these defects are due to even earlier problems in male sexual identity (Yang, 2012).

    Germline sexual identity is determined by both the germ cell sex chromosome constitution and signals from the surrounding soma. Phf7 expression is activated in XX germ cells when in contact with a male soma and repressed in XY germ cells when contacting a female soma. However, in a female somatic environment, XY germ cells are somewhat more likely than XX germ cells to express Phf7, indicating that Phf7 may also respond to the sex chromosome constitution of the germ cells in addition to being regulated by the soma. Further, exogenous expression of Phf7 is required to promote spermatogenesis in XX germ cells when in a male soma. Thus, the Phf7 expression that is normally initiated in such germ cells by the male soma must either not be maintained, or may be insufficient to overcome the influence of the XX sex chromosome genotype (Yang, 2012).

    It is likely that Phf7 is not acting alone to control male sexual identity. Phf7 mutant males are still able to undergo spermatogenesis, but at a much reduced capacity. This appears to be the null phenotype for Phf7 as ther mutants have lost significant portions of the coding sequence. Further, when PHF7 is expressed in XX germ cells present in a male soma, these germ cells can undergo spermatogenesis, but the penetrance of this phenotype is low. Interestingly, the rescue of spermatogenesis in these XX germ cells follows an 'all or nothing' pattern; either the rescue is largely complete to give full testes and sperm production, or little rescue is observed. Therefore, there appears to be a threshold that must be crossed to promote male germline sexual identity, and that once this threshold is met, those germ cells either take over the testis, or induce other germ cells to also follow the male pathway. The simplest explanation for both the incomplete block to spermatogenesis in Phf7 mutants and the incomplete rescue of spermatogenesis by Phf7 in XX males is that an additional factor (or factors) exists that promotes male identity in addition to Phf7. Such a factor could function parallel to Phf7 in a single pathway, or represent independent input regarding germline sex determination (e.g., independent signals from the soma that influence germline sex) (Yang, 2012).

    PHD fingers, such as those found in PHF7, are best known for their ability to specifically bind histones that have been modified on their N-terminal tails, in particular methylated H3K4. This study shows that both Drosophila and human PHF7 can directly associate with dimethylated H3K4, indicating that PHF7 is indeed a histone code reader. It is uncommon for PHD domains to associate preferentially with H3K4me2 over H3K4me3, but this specificity has been observed previously, and is likely important for how PHF7 modulates expression of its targets. Both di- and trimethylated H3K4 are found at actively transcribed genes, but H3K4me2 is normally localized at the 5′ end of coding sequences, downstream of H3K4me3, which is near promoters. The two marks are also regulated by different demethylases. A few recent studies have started to dissect effects of H3K4me2 on gene transcription, but the exact mechanisms are not well understood. Some PHD finger proteins also contain other domains, such as those that modify histones enzymatically. This does not appear to be the case for PHF7, and the region of homology between PHF7 homologs of different species is restricted to the PHD domains. However, individual PHD fingers can bind modified histone tails independently, and it is yet unclear which PHD finger in PHF7 contacts H3K4me2 and what activities the others might have. The logic of how PHF7 is recruited to specific loci and affects chromatin structure and gene activity are interesting questions for future work (Yang, 2012).

    Another point of interest is how a reader of such a common epigenetic mark would have a sex-specific role in regulating male germline identity. It has been observed that mutation of an H3K4me2 demethylase in Caenorhabditis elegans, which leads to increased dimethylation at H3K4, results in ectopic activation of male-specific germline genes. A similar mutation in Drosophila causes female germline developmental defects, which may be related to the germline atrophy observed when PHF7 expression was upregulated in female germ cells. These data are consistent with the hypothesis that H3K4me2 has a role in regulating the male germline genome. Interestingly, another germline chromatin factor, No child left behind (NCLB), has been identifed that is expressed in germ cells of both sexes but required for GSC function only in males. Thus, NCLB may cooperate with PHF7 in regulating the male GSC transcriptional program (Yang, 2012).

    Based on sequence homology, orthologs of Phf7 are present in a wide range of mammalian species. Human and mouse PHF7 share extensive homology to Drosophila PHF7 throughout the N-terminus where the PHD fingers are present, and the results confirm that human PHF7 recognizes H3K4me2, similar to the fly protein. Interestingly, EST profiling indicates strong testis biases for Phf7 expression in many species, including humans, mice, rats, and dogs. Moreover, several studies that performed genome-wide RNA profiling from purified mouse germline populations indicate that mouse Phf7 expression is present in spermatogonia and is further induced in spermatocytes. Remarkably, human PHF7 was able to rescue fecundity defects in male flies mutant for Phf7. Thus, the sequence conservation observed between mammalian and Drosophila Phf7 represents true functional orthology (Yang, 2012).

    As in Drosophila, germline sex determination in mouse is regulated at an early stage and is controlled by important signals from the soma, which for the mouse include retinoic acid and FGF9. Such signals regulate the timing of meiotic entry, which is different between the sexes, and also influence sex-specific programs of germline gene expression, such as expression of the key male-specific factor nanos2. Significant changes in germ cell chromatin occur during this critical time in germ cell development, including changes in the H3K4 methylation state. Thus, Phf7 represents a prime candidate for interpreting these chromatin changes in a sex-specific manner to regulate male-specific gene expression. It will be of great interest to determine whether Phf7 plays a critical role in mouse and human male germ cell development, as is proposed for Drosophila (Yang, 2012).

    Escargot restricts niche cell to stem cell conversion in the Drosophila testis

    Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. Esg-interacting proteins were identified, and an interaction was demonstrated between Esg and the corepressor C-terminal binding protein (CtBP), which is also required for maintenance of hub cell fate. These results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo (Voog, 2014)

    Protein synthesis and degradation are critical to regulate germline stem cell homeostasis in Drosophila testes

    The homeostasis of self-renewal and differentiation in stem cells is strictly controlled by intrinsic signals and their niche. A large-scale RNA interference (RNAi) screen was conducted in Drosophila testes and 221 genes required for germline stem cell (GSC) maintenance or differentiation were identified. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in protein degradation pathway in cyst cells leads to testis tumor with overproliferated germ cells. Importantly, in the Cullin 4 - Ring E3 ubiquitin ligase (CRL4) complex, multiple proteins were identified that are critical to GSC self-renewal. pic/DDB1, the linker protein of CRL4, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice (Yu, 2016).

    Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, a genome-wide transgenic RNAi screen was performed through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). This study identified 530 genes that regulate GSC maintenance and differentiation. Of these, 113 selected genes were further knocked down using cell-type-specific Gal4s, and more than half were found to be external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a Myb/SANT-like DNA-binding domain-containing heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. It was also found that mitosis/cytokinesis proteins are especially important for male GSC maintenance. These findings provide valuable insights and resources for studying stem cell regulation at the organismal level (Liu, 2016).

    The regulated elimination of transit-amplifying cells preserves tissue homeostasis during protein starvation in Drosophila testis

    How tissues adapt to varying nutrient conditions is of fundamental importance for robust tissue homeostasis throughout the life of an organism, but the underlying mechanisms are poorly understood. This study shows that Drosophila testis responds to protein starvation by eliminating transit-amplifying spermatogonia (SG) while maintaining a reduced pool of actively proliferating germline stem cells (GSCs). During protein starvation, SG died in a manner that was mediated by the apoptosis of somatic cyst cells (CCs) that encapsulated SG and regulated their development. Strikingly, GSCs could not be maintained during protein starvation when CC-mediated SG death was inhibited, leading to an irreversible collapse of tissue homeostasis. The study proposes that the regulated elimination of transit-amplifying cells is essential to preserve stem cell function and tissue homeostasis during protein starvation (Yang, 2015).

    Spermatid individualization is sensitive to temperature and fatty acid metabolism

    Fatty acids are precursors of potent lipid signaling molecules. They are stored in membrane phospholipids and released by phospholipase A2 (PLA2). Lysophospholipid acyltransferases (ATs) oppose PLA2 by re-esterifying fatty acids into phospholipids, in a biochemical pathway known as the Lands Cycle. Drosophila Lands Cycle ATs oys and nes, as well as 7 predicted PLA2 genes, are expressed in the male reproductive tract. Oys and Nes are required for spermatid individualization. Individualization, which occurs after terminal differentiation, invests each spermatid in its own plasma membrane and removes the bulk of the cytoplasmic contents. This study developed a quantitative assay to measure individualization defects. Individualization is demonstrated to be sensitive to temperature and age but not to diet. Mutation of the cyclooxygenase Pxt, which metabolizes fatty acids to prostaglandins, also leads to individualization defects. In contrast, modulating phospholipid levels by mutation of the phosphatidylcholine lipase Swiss cheese (Sws) or the ethanolamine kinase Easily shocked (Eas) does not perturb individualization, nor does Sws overexpression. These results suggest that fatty acid derived signals such as prostaglandins, whose abundance is regulated by the Lands Cycle, are important regulators of spermatogenesis (Ben-David, 2015).

    A Krebs cycle component limits caspase activation rate through mitochondrial surface restriction of CRL activation

    How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. This study investigated caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. It was shown that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation (Aram, 2016). 

    Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila

    In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown mitochondrial shaping roles were uncovered for other testis-expressed ATP synthase subunits. This study demonstrates the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern (Sawyer, 2017).

    Reduced expression of CDP-DAG synthase changes lipid composition and leads to male sterility in Drosophila

    Drosophila spermatogenesis is an ideal system to study the effects of changes in lipid composition, because spermatid elongation and individualization requires extensive membrane biosynthesis and remodelling. The bulk of transcriptional activity is completed with the entry of cysts into meiotic division, which makes post-meiotic stages of spermatogenesis very sensitive to even a small reduction in gene products. In this study, the effect of are described changes in lipid composition during spermatogenesis using a hypomorphic male sterile allele of the Drosophila CDP-DAG synthase (CdsA) gene. The CdsA mutant shows defects in spermatid individualization and enlargement of mitochondria and the axonemal sheath of the spermatids. Furthermore, it was possible to genetically rescue the male sterile phenotype by overexpressing Phosphatidylinositol synthase (dPIS) in a CdsA mutant background. The results of lipidomic and genetic analyses of the CdsA mutant highlight the importance of correct lipid composition during sperm development and show that phosphatidic acid levels are crucial in late stages of spermatogenesis (Laurinyecz, 2016).

    Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis

    Mammalian DEAD box helicase DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. This study reports phenotypes associated with a null Ddx1 (Ddx1AX) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. This study demonstrates that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. These data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing (Germain, 2015).

    Recruitment of Mediator complex by cell type and stage-specific factors required for tissue-specific TAF dependent gene activation in an adult stem cell lineage

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. This study shows that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. These results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages (Lu, 2015).

    Developmental control of cell type specific gene expression programs is crucial to differentiation in embryonic and adult stem cell lineages. Developmental signaling pathways are ultimately interpreted in the context of cell type-specific chromatin states and by transcription machinery to establish the intricate patterns of gene expression unique to each differentiating cell type. Emerging evidence suggests that Mediator, a large, multiprotein complex that integrates transcriptional enhancing and repressing signals from transcription factors, chromatin modifiers, non-coding RNAs and elongation factors to deliver a calibrated output to the transcription machinery to modulate gene expression, plays critical roles in tissue and cell type specific gene expression programs in metazoans. For example, Mediator-enriched super enhancers contribute to regulation of key cell identity genes in ES cells and many differentiated cell types. Although Mediator was reported to be essential for ESC maintenance and embryonic development, and widely involved in human diseases and different types of cancer, the role(s) of Mediator in adult stem cell lineages are not well understood (Lu, 2015).

    This study investigated the function of Mediator in activating expression of a cell type specific transcription program for terminal differentiation in a model adult stem cell lineage, spermatogenesis in Drosophila. To initiate differentiation in this lineage, germ line stem cells divide asymmetrically, each producing one daughter that self-renews and one daughter that initiates a series of four spermatogonial mitotic transit amplifying divisions. The resulting 16 interconnected spermatogonia then undergo premeiotic S phase and become spermatocytes. One of the most dramatic cell type specific gene expression programs of the fly initiates at the spermatocyte stage, during which over 2000 genes are transcriptionally activated in meiotic prophase, many for the first time in development (Lu, 2015).

    Mutations in several genes cause failure to activate many genes in this transcription program and a meiotic arrest phenotype: mutant testes filled with mature primary spermatocytes that fail to enter the meiotic divisions or initiate spermatid differentiation. Molecular cloning and analysis revealed that proper activation of transcription of these terminal differentiation genes in spermatocytes depends on cooperative action of two classes of meiotic arrest genes, expressed specifically in spermatocytes, which encode homologs of either TBP-associated factors (tTAFs) or components of the testis meiotic arrest complex (tMAC), a testis-specific version of the mammalian MIP/dREAM and the C. elegans SynMuvB complexes. tMAC contains at least 3 potential DNA binding components, Comr, Topi and Tomb, as well as several subunits implicated in chromatin remodeling or performing structural roles within the complex. It has been suggested that the tMAC component aly may help remodel spermatocyte chromatin for global activation of the spermatocyte transcription program. Action of the tMAC complex is needed for transcription of the G2 cell cycle regulators Cyclin B, cdc25/twine and boule in spermatocytes and of a large set of spermatid differentiation genes, the tTAFs are required for full activation of the spermatid differentiation genes but are dispensable for expression of transcripts for the G2 cell cycle regulators (Lu, 2015).

    Expression of both the tTAFs and testis-specific components of the tMAC complex is turned on in early spermatocytes but the two classes of genes do not depend on each other to be transcribed. Recruitment of the tTAF protein Spermatocyte arrest (Sa) to promoters of target spermatid differentiation genes required function of the tMAC component, Aly. Several additional meiotic arrest genes not directly involved in regulation of the spermatocyte transcription program have also been discovered (Lu, 2015).

    This study shows that Mediator plays a key role in activating expression in spermatocytes of a large number of transcripts involved in meiotic cell cycle progression and spermatid differentiation. For several of the many Mediator subunits, spermatocyte specific RNAi knock down produced a range of meiosis I maturation arrest phenotypes in male germ cells. Knockdown of the mediator subunit Med22 by RNAi in spermatocytes resulted in a consistent meiotic arrest phenotype similar to the tTAF mutants, suggesting that Mediator may function with the tTAFs and tMAC to activate the transcription program for terminal differentiation. Expression of Drosophila Mediator complex components becomes upregulated in early spermatocytes just prior to expression of the tTAFs, and Mediator subunits colocalized with tTAFs in spermatocytes. Localization of Mediator subunits to chromatin in spermatocytes depended on tMAC but not tTAF function, while spermatocyte specific knockdown of Med22 by RNAi abolished localization of tTAFs to chromatin, suggesting that Med22 may recruit or stabilize tTAFs to chromatin for activation of transcription of differentiation genes. Consistent with this pathway, expression of most spermatid differentiation transcripts dependent on tMAC and the tTAFs also required function of Med22 in spermatocytes. Strikingly, expression of transcripts up regulated in early spermatocytes that did not depend on tMAC and the tTAFs remained largely unaffected in Med22 knockdown testes, suggesting that Mediator is not required for all activated transcription in spermatocytes, but mainly for turning on the developmentally controlled transcription program that depends on tMAC and tTAF. The Zn finger protein Topi, a component of tMAC, interacts structurally with Med22 and may recruit Mediator to target genes. These results suggest that Mediator serves as a key component in a gene regulatory cascade of transcription activation that establishes the expression program for terminal differentiation in the male germ line adult stem cell lineage (Lu, 2015).

    Activation of cell-type-specific gene expression profiles underlies terminal differentiation programs in both embryonic and adult stem cell lineages. In many cases such stage- and cell-type-specific gene expression programs require cooperative action of sequence-specific transcriptional activators and tissue-specific components of the basal transcription machinery. Previous studies have demonstrated that full activation in spermatocytes of the transcription program for terminal differentiation of male gametes requires sequential action of the testis specific tMAC complex and five testis specific homologues of components of the general transcription factor complex TFIID (the tTAFs). How tMAC and the tTAFs function at promoters of differentiation genes is not yet understood at the molecular level although function of tMAC was needed to recruit the tTAF protein Sa to promoters of spermatid differentiation genes. The new data presented in this study indicate that subunits of the Mediator coactivator complex mediate the regulatory function of tMAC on the tTAFs, acting in a pathway to turn on robust expression of terminal differentiation genes in spermatocytes. Potentially functioning as a testis specific TFIID complex, the tTAFs are needed for full activation of transcription of hundreds of spermatid differentiation genes in primary spermatocytes but are dispensable for transcription of meiotic cell cycle genes activated in the same cells. It is possible that the gene selectivity of the tTAFs is determined primarily by structures of the promoters of differentiation genes. The general TAFs and TFIID were shown to confer promoter selectivity and facilitate transcription activation at promoters with no or less stringent TATA boxes in Drosophila systems. Alternatively, as the current results suggest, the gene selectivity of the tTAFs may be partly achieved through DNA sequence specific components in the tMAC complex. tMAC contains at least two potential sequence-specific DNA-binding proteins, Tomb and Topi. This study found that Topi and MED22 physically interact when coexpressed in S2 cells. The tTAF protein Sa failed to localize to meiotic chromatin without Med22 function, suggesting the tTAFs may be recruited or stabilized by Mediator at promoters of the spermatid differentiation genes. Similar coactivator cross-talk between Mediator and canonical TAFs was observed during activation of the metal response gene, MtnA, in Drosophila cell culture. Although TFIID and Mediator were recruited separately to the MtnA promoter, TFIID was only functional in the presence of Mediator (Lu, 2015).

    Although expression and subcellular localization of Med22 protein proceeded protein accumulation and localization of Sa in spermatocytes, and the correct localization of Sa relied on function of Med22, the data do not prove direct recruitment of Sa by Med22. The tTAFs, including Sa, may be recruited by other mechanisms to promoters of differentiation genes, with function of Mediator needed to stabilize and facilitate assembly and function of the tTAF containing preinitiation complex. Despite that Sa and Mediator colocalized to both chromatin and the nucleolus in spermatocytes based on immunofluorescence staining, Sa protein was predominantly detected at the nucleolus, whereas the Mediator signal was more evenly distributed between chromatin and the nucleolus, again suggesting that Sa may not be directly recruited by Mediator, at least to the nucleolus. It is possible that once localized, Sa is stabilized through interaction with Mediator since both the nucleolar and chromatin localization of Sa was completely abolished in Med22RNAi. It was not possible to detect direct interaction between Mediator subunits and tTAF components in testis extracts. However, considering the limitation of the Drosophila in vivo system for such biochemical assays, the possibility cannot be ruled out that the two complexes physically interact in spermatocytes, since interactions between Mediator and TFIID have been demonstrated in vitro (Lu, 2015).

    Of all the Mediator subunits this study attempted to knock down in spermatocytes by RNAi, knock downs of only a few caused the meiotic arrest phenotype. Of these, Med22RNAi is the only knockdown for which the meiotic chromatin resembled tTAF mutant chromatin. Phenotypic variations among different Mediator subunits were also observed in a previous study in SL2 cells, in which each of mediator subunit was knocked down by RNAi. The heterogeneity of Mediator subunit knockdown phenotypes may be a result of variation in RNAi efficiency. Indeed, analysis of protein by immunofluorescence revealed that the RNAi hairpin against Med26 that was tested did not knock down expression of the Med26 protein. Since the Gal4-UAS system was used to drive RNA hairpin expression in spermatocytes, it is worth noting that Mediator had been shown to be required for Gal4 mediated transcription at UAS-containing promoters. Therefore, Mediator subunits needed for structural integrity of the complex or directly involved in interaction with Gal4-AD might not give strong phenotypes due to the potential negative feedback on the RNAi process. Alternatively, the differences in RNAi knockdown phenotypes of individual subunits could reflect functional diversity of particular subunits (Lu, 2015).

    Although previously thought to be a generally needed inert protein complex which passively bridge interactions between transcription activators and the general transcription machinery, including TFIID and PolII, recent studies have revealed a more active and even gene selective role for Mediator in transcription activation. Many Mediator subunits were found to selectively modulate and integrate regulatory signals of specific cellular and metabolic pathways. Previous studies also showed several Mediator subunits functioned as adaptors to bridge interactions of particular transcription factor(s) to the Mediator complex or subcomplexes. For example, the C-terminal domain and the activation domain of p53 specifically bind to the MED1 and MED17 subunits of Mediator, respectively. MED15 binds strongly to the activation domain of SREBP-1α to regulate lipid homeostasis, and MED23 binding to the ELK-1 activation domain is required specifically for adipogenesis. More interestingly, MED14 and MED25 mutants had opposite effects on cell size control in Arabidopsis, suggesting distinct Mediator subunits coexisting in the same cells can have distinct mechanistic roles in transcription regulation. The current work suggests Mediator is recruited in primary spermatocytes to target genes by the tissue- and cell type-specific transcription factor Topi through its physical interaction with MED22. Importantly, consistent with being recruited by sequence specific activators in the tMAC, Mediator is not generally required in spermatocytes for activating transcription, as most genes which did not require function of tMAC (as seen with aly-/-) or tTAFs (as seen with sa-/-), were expressed at relatively normal levels in Med22RNAi testes (Lu, 2015).

    This study detected physical interaction only between MED22 and Topi, it is certainly possible that other putative sequence specific DNA binding factors in tMAC, such as Tomb, or peripheral to tMAC, such as Achi/Vis also recruit Mediator through specific interactions with other Mediator subunits. Topi and Achi/Vis were suggested to be both needed at promoters of most genes controlled by the aly class of meiotic arrest genes. In spermatocytes, signals from different components of tMAC instructing activation of transcription may be integrated by Mediator through interactions between tMAC subunits and specific Mediator subunits, before being transduced to the general transcription machinery containing the tTAFs. This study on how transcriptional activation signals maybe routed through Mediator to the general machinery in differentiating spermatocytes will also shed light on how master transcription factors and Mediator-enriched super-enhancers may interact with the basal machinery in a gene selective fashion (Lu, 2015).

    Cell type-specific translational repression of Cyclin B during meiosis in males

    The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. This study shows that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, it was shown that Fest is required for proper execution of meiosis I (Baker, 2015).

    This study shows that the developmental program of male gametogenesis imposes several levels of cell type- and stage-specific post-transcriptional control on expression of the key G2/M cell cycle regulatory component CycB during meiotic prophase and identifies two key developmentally regulated trans-acting factors involved. First, the cycB RNA expressed in spermatocytes has a short 3' UTR, only 130 nt long and missing previously identified translational regulatory sequences used in other cell types. Second, the RNA-binding protein Rbp4, expressed starting early in meiotic prophase soon after completion of pre-meiotic DNA synthesis, binds the short 3' UTR and blocks translation of cycB in immature spermatocytes. Third, the Rbp4-interacting protein Fest, also upregulated early in the spermatocyte period, is also required for blocking CycB expression in immature spermatocytes (Baker, 2015).

    Rbp4 and Fest RNA and protein are expressed in very early spermatocytes prior to onset of transcription of cycB, which depends on action of the tMAC complex (White-Cooper, 1998; Beall 2007). As a result, when the cycB RNA is expressed, it arrives in a cytoplasm already primed for its proper cell type- and stage-specific translational repression. Expression of Cyclin B3 (Clb3) protein in budding yeast has been shown to be restricted to meiosis II via sequences in the CLB3 3' UTR that block translation during meiosis I. Although translational repression of CLB3 in meiosis I was important to prevent premature separation of sister chromatids, an event appropriate for meiosis II rather than meiosis I, trans-acting factors responsible for the stage-specific translational repression have yet to be identified. The current data reveal that translational repression of a cyclin (in this case CycB) is also a key feature of meiotic prophase during spermatogenesis in a metazoan animal. Surprisingly, expression of CycB in immature spermatocytes -- either in rbp4 or fest mutants or by a mutated CycB-eYFP reporter -- was insufficient to drive those cells immediately into meiotic division. This might be because action of the Cdc25 cell cycle phosphatase encoded by twine, which is also translationally repressed in immature spermatocytes and becomes translationally activated by the RNA-binding protein Boule only in mature spermatocytes, is also required to generate active Cdk1/CycB. (Baker, 2015)

    One general model for Fest function invokes the possibility that Rbp4 recruits Fest to the cycB 3' UTR, where Fest is able to interfere with cycB translation. However, no compelling evidence was found of specific binding of Fest to the cycB 3' UTR in biotin pull-down experiments from testis extracts from flies expressing eYFP-Fest either with or without functional Rbp4, which suggests that either Fest is not recruited to the cycB 3' UTR, or that the biotin pull-down assay has limitations in detecting indirect RNA-protein interactions. As a result, it is important to consider other mechanisms for Fest function, including the possibility that binding of Fest to Rbp4 is needed only briefly to enact a post-translational modification of or conformational change within Rbp4 to promote its ability to recruit partners and/or repress translation. It is also technically possible that Fest and Rbp4 act in parallel pathways to regulate CycB. Finally, as the fest germ cell phenotype is dramatically stronger than that of rbp4, it is likely that Fest regulates other proteins in addition to Rbp4 (Baker, 2015)

    It is not yet known how information about spermatocyte maturation is communicated to Rbp4 or Fest to allow translation of cycB in mature spermatocytes. One or more proteins could respond to input regarding cell size, given that spermatocytes grow 25-fold in volume during meiotic G2. Alternatively, given that translation of cycB in mature spermatocytes requires function of the testis TAF proteins, signals indicating the completion of the spermatocyte transcription program (not merely its onset) could trigger the reprieve from translational repression. Another possibility might be a meiotic arrest checkpoint mechanism triggered by transcriptional activity from unpaired chromatin, as seen in mammalian spermatocytes. Whatever the stimulus, it is clear that through stage-specific expression of the translational regulators Rbp4 and Fest in very early spermatocytes, the developmental program of male germ cell differentiation exerts additional layers of control over the core cell cycle machinery (Baker, 2015)

    Regulators of alternative polyadenylation operate at the transition from mitosis to meiosis

    In the sexually reproductive organisms, gametes are produced by meiosis following a limited mitotic amplification. However, the intrinsic program switching cells from mitotic to meiotic cycle is unclear. Alternative polyadenylation (APA) is a highly conserved means of gene regulation and is achieved by the RNA 3'-processing machinery to generate diverse 3'UTR profiles. In Drosophila spermatogenesis, this study observed distinct profiles of transcriptome-wide 3'UTR between mitotic and meiotic cells. In mutant germ cells stuck in mitosis, 3'UTRs of hundreds of genes were consistently shifted. Remarkably, altering the levels of multiple 3'-processing factors disrupted germline's progression to meiosis, indicative of APA's active role in this transition. An RNA-binding protein (RBP) Tut could directly bind 3'UTRs of 3'-processing factors whose expressions were repressed in the presence of Tut-containing complex. Further, this RBP complex could execute the repression post-transcriptionally by recruiting CCR4/Twin of deadenylation complex. Thus, it is proposed that an RBP complex regulates the dynamic APA profile to promote the mitosis-to-meiosis transition (Shan, 2017).

    Stage-specific control of niche positioning and integrity in the Drosophila testis

    This study addressed the complex question of how complex structures are maintained after their initial specification by studying the Drosophila male stem cell niche, called the hub. Once specified, the hub cells need to maintain their position and architectural integrity through embryonic, larval and pupal stages of testis organogenesis and during adult life. The Hox gene Abd-B, in addition to its described role in male embryonic gonads, maintains the architecture and positioning of the larval hub from the germline by affecting integrin localization in the neighboring somatic cyst cells. The AbdB-Boss/Sev cascade affects integrin independent of Talin, while genetic interactions depict integrin as the central downstream player in this system. Focal adhesion and integrin-adaptor proteins within the somatic stem cells and cyst cells, such as Paxillin, Pinch and Vav, also contribute to proper hub integrity and positioning. During adult stages, hub positioning is controlled by Abd-B activity in the outer acto-myosin sheath, while Abd-B expression in adult spermatocytes exerts no effect on hub positioning and integrin localization. The data point at a cell- and stage-specific function of Abd-B and suggest that the occurrence of new cell types and cell interactions in the course of testis organogenesis made it necessary to adapt the whole system by reusing the same players for male stem cell niche positioning and integrity in an alternative manner (Schardt, 2015).

    Cytoskeletal dynamics in male meiosis

    In animal cells, cytokinesis is accomplished by the contractile ring, a transient structure containing actin and myosin II filaments (Zipper) that is anchored to the equatorial cortex. Interactions between these filaments lead to the constriction of a ring that pinches the dividing cell in the middle like an ever tightening purse string until cleavage is completed. Male meiosis was examined in mutants of the chickadee (chic) locus, a Drosophila gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. Difficulties in meiotic cytokinesis are immediately obvious because of the characteristic appearance of spermatids directly after their formation at the so-called onion stage. Wild-type onion stage spermatids contain a single phase-light nucleus and a similarly sized phase-dark Nebenkern (a mitochondrial derivative). Failures in cytokinesis result in abnormally large Nebenkern associated with multiple normal-sized nuclei. The resulting phenotypes fall into multiple groups: in testes of males homozygous for chic a large fraction of onion-stage spermatids contain a single Nebenkern of larger than normal size, associated with two or more normal-sized nuclei. A substantial proportion have two nuclei with an intermediate-size Nebekern, but most frequently, these aberrant spermatids contain four nuclei and a very large Nebenkern. These phenotypes reflect failures of cytokinesis at either one or the other or both meiotic divisions, respectively, which would prevent proper subdivision of mitochondria and nuclei into daughter spermatids (Giansanti, 1998).

    In wild-type testes, phalloidin staining reveals an F-actin-enriched contractile ring that encircles the spindle midzone (the bundle of interdigitated microtubules between the separating chromosome complements during anaphase and telophase) from late anaphase through telophase of both meiotic divisions. In contrast, in many meiotic divisions of chic mutants, no actin staining is visible at the cell equator. In most mutant ana-telophases with equatorial actin staining, only irregular patches of F-actin are observed. Chic protein is found in clear concentrations near the cell cortex, particularly in the equatorial zone. In chic mutants abnormal aggregations of F-actin are observed primarily in premeiotic mature spermatocytes at the S5 stage. These aggregates are enriched in alpha-spectrin and are almost invariably associated with ring canals, suggesting that these actin aggregates are in fact relatively undegraded remnants of the male fusome (see Drosophila Spectrin for more information on fusomes). It is suggested that in the absence of chic the disintegration of the fusome is partially blocked (Giansanti, 1998).

    In addition to the absence of the contractile ring, the ana-telophases of all the chic mutants exhibit another obvious abnormality: a defect in central spindle morphology. In wild-type ana-telophases of both meiotic divisions, the two daughter nuclei are connected by a prominent bundle of interdigitating microtubules. In chic mutants, this central spindle structure is considerably less dense than in wild type, and microtubules show very little or no interdigitation. In wild type, the central spindle is already evident by mid-anaphase, before the actin ring constriction has caused substantial equatorial pinching. In the strongest chic mutants, these early stages of central spindle formation are never seen, indicating that the chic phenotype reflects a failure of central spindle assembly rather than a degradation of this structure in the absence of the actin ring. This result is surprising because the central spindle is a tubulin based cytoskeletal structure. There is substantial evidence that the central spindle is required for structuring of the actin based contractile ring but not similar evidence that the contractile ring is required for building the central spindle. Together, these observations indicate that chic mutations disrupt two major cytokinetic structures: the microtubule-based central spindle and the actomyosin contractile ring (Giansanti, 1998).

    In wild-type primary spermatocytes during the prophase-prometaphase transition of the first meiotic division (stage M1), centrosomes migrate from a position just under the plasma membrane to the nuclear envelope, where they nucleate prominent asters. The two asters then separate and move around the periphery of the nuclear membrane, so as to establish a bipolar spindle. Similarly, during late telophase of the first meiotic division and the short interphase between meiosis I and meiosis II, asters separate and migrate to the opposite poles of secondary spermatocytes. Although the asters in chic mutants are improperly positioned by the start of prometaphase, relatively normal-looking bipolar spindles eventually form by late metaphase. It is remarkable that the function of these spindles, with respect to chromosome segregation, appears to be largely unimpaired. Only a very low frequency of onion-stage spermatids contain irregularly sized nuclei, such as micronuclei (Giansanti, 1998).

    Lesions in twinstar (tsr), a gene encoding a Drosophila cofilin (an actin severing and depolymerizing protein), cause a syndrome of phenotypic effects that have both similarities and differences to those described above for chic mutations. In tsr, as in chic spermatocytes, centrosome separation and migration are defective, abnormal accumulations of F-actin are apparent, and cytokinesis often fails after one or both meiotic divisions. tsr and chic mutant phenotypes, however, can be easily distinguished on the basis of several criteria. In tsr mutants, the central spindle is normal, and the contractile ring still forms (though in misshapen and enlarged form), in contrast to the situation for chic. The types of F-actin aggregates formed in tsr spermatocytes are much different from those in chic. It is concluded that tsr and chic mutations differ in how they interfere with meiosis (Giansanti, 1998).

    To further investigate the relationships between the central spindle and the contractile ring, meiosis was examined in the cytokinesis-defective mutants KLP3A and diaphanous. The KLP3A gene encodes a kinesin-like protein that accumulates in the central spindle midzone during anaphase and telophase of both meiotic divisions. Accordingly, mutations in this gene disrupt central spindle formation and cause frequent failures in meiotic cytokinesis. To check whether the defect in central spindle integrity observed in KLP3A mutants also affects actin ring assembly, KLP3A mutant testes were stained with rhodamine-labeled phalloidin. The results of this experiment clearly show that most mutant ana-telophases (90%) are completely devoid of actin rings. The rare ana-telophases that exhibit thin and incomplete actin rings also contain more densely packed central spindles than those of cells completely lacking contractile rings. Despite the absence of the contractile ring, KLP3A mutants do not exhibit aberrant actin accumulations or problems in aster migration like those described above for chic and tsr mutants (Giansanti, 1998).

    The diaphanous gene encodes a protein that interacts with profilin through its proline-rich domain. All the ana-telophases present in testes homozygous for dia mutants are completely devoid of actin rings. It is of interest that these figures also show severe defects in the central spindle, similar to those observed in chic and KLP3A. The effects on the actomyosin contractile ring and the central spindle observed in chic and dia mutants could be specific consequences of lesions in the corresponding gene products. Alternatively, these effects could result from a more general disruption of the actin cytoskeleton. To discriminate between these possibilities, wild-type testes were treated with cytochalasin B prior to fixation and staining. Cytochalasin B binds the barbed ends of actin filaments and promotes the conversion of ATP-actin monomers to ADP-actin , preventing proper assembly of the contractile ring in most cell types. Remarkably, incubation with this drug produces an almost exact phenocopy of strong chic alleles. No F-actin staining is observed in any contractile ring-like structures at the equator of ana-telophase cells. (Giansanti, 1998).

    In all cases examined, the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: when one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, another consequence of chic mutations was observed: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration, and that the central spindle and the contractile ring are interdependent structures (Giansanti, 1998).

    The best candidate at present for mediating interactions between the central spindle and cortical actin, at least during male meiosis, is the KLP3A kinesin-like protein. This protein could interact directly with both the central spindle microtubules and components of the contractile ring. Alternatively, KLP3A could transport to the spindle midzone molecules that mediate F actin-microtubule interactions. At the moment, it is not possible to discriminate between these possibilities, nor is there any information on the proteins that bind to or might be transported by KLP3A. It is believed, however, that the isolation and characterization of additional mutations causing cytological phenotypes similar to those of KLP3A, chic, and dia, will eventually provide substantial insight into the mechanisms underlying microtubule-actin interaction during cytokinesis (Giansanti, 1998).

    A role for actin dynamics in individualization during spermatogenesis

    During late stages of spermatogenesis in Drosophila, a cyst of 64 syncytial spermatids elongates as the sperm axonemes are formed inside it. Then this elongated cyst is remodeled into individual sperm by a process called individualization. At the start of individualization actin cones assemble around the spermatid nuclei and then synchronously move from the heads to the tips of the tails. As the actin cones move, a large accumulation of cytoplasm and vesicles, called the cystic bulge, forms around them. In the cystic bulge, the membrane of the cyst is remodeled to enclose each sperm axoneme. Individualization is especially interesting as a cell biological process because it requires an unusual amount of membrane remodeling using a well-defined actin structure. The fully elongated cyst can be up to 1800 microm long; therefore, this process requires the actin structures important for the process to move unidirectionally over a significant length. During the process, the bulk of the cytoplasm is discarded from the cell body. However, there is little information about the mechanism of this process (Noguchi, 2003).

    In order to better understand the mechanism of sperm individualization, an in vitro culture system was developed in which live observation of individualization can be performed in isolated cysts. The whole process of individualization, during which a bundle of 64 syncytial spermatids is separated into individual sperm, takes place in these cultures. The speed of cystic bulge movement is fairly constant along the length of the cyst. Actin drugs, but not microtubule drugs inhibit cystic bulge movement, suggesting that the movement requires proper actin dynamics but not microtubules. GFP-tagged actin was expressed in the cyst and fluorescence recovery after photobleaching was monitored using confocal microscopy to analyze actin dynamics in cones. Actin turns over throughout the cone, with that at the leading edge of the cones turning over with slightly faster kinetics. Actin does not treadmill from the front to the back of the cone. Actin in moving actin cones turns over in about 12 minutes, although prior to onset of movement, turnover is much slower. Visualization of membrane using the dye FM1-43 reveals that the cystic bulge has an extremely complicated series of membrane invaginations and the transition from syncytial to individualized spermatids occurs at the front of the actin cones. It is also suggested that endocytosis and exocytosis might not be important for membrane remodeling. This system should be suitable for analysis of defects in male sterile mutants and for investigating other steps of spermatogenesis (Noguchi, 2003).

    These data are most consistent with the idea that actin cone movement is driven by actin polymerization, similar to lamellipodia extension and Listeria motility. The speed of the cystic bulge movement is similar to the speed of movement of the leading edge of lamellipodia. Cystic bulge movement is altered very quickly after inhibiting either assembly or disassembly of actin, consistent with the requirement for active actin assembly and disassembly for movement. In fluorescence recovery after photobleaching (FRAP) experiments, a slightly faster rate of turnover at the front was detected than in the rear of the cone. It is likely that the faster dynamics of actin at the front is important for movement. In addition, the observed acceleration of actin dynamics after the onset of movement supports this idea. Arp 2/3 complex, which is the key factor involved in promoting actin polymerization at the leading edge, is enriched at the front of actin cones, suggesting that this site is important for force generation. All these data support the idea that the driving force is actin polymerization (Noguchi, 2003).

    However, some puzzling differences in actin behavior in this structure when compared with leading edge protrusion make it difficult to explain how assembly drives movement in this case. (1) It had been expected that actin would treadmill through the actin cone from front to back, because of assembly at the front, i.e. in the direction of movement. This has been observed in other actin motility processes. However, this is not the case in actin cones. The filaments in the cone move forward relative to the substrate. (2) Actin turns over at a rate that is much slower than that of actin in lamellipodia and Listeria comet tails. In both of these structures, filaments turn over in 1-2 minutes, but in case of actin cones, turnover takes 12 minutes. (3). Another puzzle is the stability of actin cones to depolymerization by the actin depolymerizing drug latrunculin A (LTA). Actin completely turns over in 12 minutes in moving cones, so it might be expected that LTA would cause depolymerization in that time frame. However, even after 2 hours of LTA treatment, cones remain. It is likely that actin in cones is stabilized by binding of cross linkers or other proteins, but an understanding is not yet clear of the mechanism that regulates stability to permit turnover as the cones move, but prevents depolymerization when assembly stops (Noguchi, 2003).

    Despite these differences from other motility processes, the favored model involves only actin assembly as the driving force for motility. In order to explain the dynamics of actin in the cone and results of pharmacological experiments, it is suggested that there are two actin structural components in a moving actin cone. The first actin structure is the actin cone itself. Three characteristics (stability, filament translocation and slow turnover) suggest that the actin cone is a highly organized and stable structure compared to the actin network in lamellipodia. It is likely that each actin cone moves forward as one unit. The second actin structure component is an actin network near the membrane that pushes the actin cone forward by force of polymerization. Actin filaments elongate near the membrane, similar to the leading edge, but the membrane is held rigid, rather than protruding as it does at the leading edge. In this case, a photobleached GFP-actin in a filament would be pushed away from the membrane, i.e. `forward' relative to the membrane. Eventually, this filament would be crosslinked into the actin cone as new actin filaments assembled. This model is consistent with the data, no information is available about the orientation of actin filaments and the sites of actin monomer incorporation that would provide additional support for such a model. In addition, the molecules that might be important to keep the membrane rigid and prevent its protrusion are as yet unknown (Noguchi, 2003).

    An alternative model is that myosin based motility contributes to movement by generating force using cortical actin and/or actin cones as a substrate for movement. Using a motor protein to provide force is compatible with the idea that the actin cone moves forward as a unit. However, it is not obvious why motor-driven movement would be coupled with actin dynamics. Perhaps movement requires a dynamic cortical actin network around the actin cone, to provide tracks for myosin movement. This track might need to be continually assembled at new sites during movement. Since the actin cone is a very large and intensely labeled structure, it might be hard to detect the different dynamic behavior in a less prominent, thin cortical structure in the same region. Myosin VI is present on actin cones and essential for individualization, making it tempting to speculate that myosin VI might provide the driving force. However, cystic bulges of myosin VI mutants can move partway along the cyst, indicating its function is not required for cone movement. Instead, myosin VI is important for regulating actin dynamics during movement. Other myosins may be important for some aspects of cystic bulge movement, but this remains to be demonstrated. BDM, an inhibitor of myosin ATPases, did not block movement. This inhibitor has been demonstrated to block activity of myosin I, II and V, suggesting it is a general inhibitor. The lack of effect of BDM on cone movement makes a myosin-based motility model less likely: myosins cannot be ruled out as force generators in this process at this time. Mutant alleles of all the predicted myosins in the Drosophila are not available and the effect of BDM on myosins in many classes is unknown (Noguchi, 2003).

    Microtubule-based motility is not likely to be involved in cone movement. There are no cytoplasmic microtubules, which might participate in generating force in cooperation with microtubule motors and inhibitors of microtubule dynamics, and motors do not stop movement. In addition, when actin dynamics are altered, the cystic bulge stops immediately. If the movement was microtubule based, it is not clear why actin dynamics would be important (Noguchi, 2003).

    Further studies are required to provide support for this model of actin cone motility. Additional studies examining membrane dynamics, effect of disruption of actin polymerization regulators and ultrastructure of the actin cones will be needed for more insight into the similarities and differences in the mechanism of actin cone movement and lamellipodia extension (Noguchi, 2003).

    Although the mechanism of actin cone movement is not fully understood, it is possible to speculate about the role of actin cones during individualization. It is suggested that the actin cones have three roles: (1) the actin cones have the ability to push the cystic bulge forward, using actin polymerization; (2) the actin cones sweep the cytoplasm and organelles out of the sperm flagella, acting as a sieve; (3) the actin cones must bind the cell membrane around them and shape it into the observed thin tubular structure. Eventually, as the actin depolymerizes at the cone tip, the membrane must attach to the axoneme (Noguchi, 2003).

    There is an interesting transition that occurs as individualization begins. Microtubule staining disappears during a very short period around the onset of actin cone movement. The data suggests that this disappearance is due to tubulin degradation as movement begins. This idea is supported by observations that the amount of tubulin present in individualized spermatids is much less than in cysts prior to individualization, and that cytoplasmic microtubules disappear during individualization (Noguchi, 2003).

    This transition temporally coincides with the onset of actin cone movement, rather than sperm nucleus DNA condensation. FRAP experiments demonstrate that actin dynamics also accelerate after the onset of movement. Therefore, it is suspected that a global signal orchestrates these events to trigger the onset of individualization (Noguchi, 2003).

    Membrane remodeling does not require endocytosis or exocytosis. Conventional endocytosis may not be important for movement of the cystic bulge, because FM1-43 staining of cell membrane demonstrates that membrane uptake does not take place around the actin cones, and blocking endocytosis using temperature shift of the shibire (dynamin) mutant does not affect cystic bulge movement. In addition, no concentration of alpha-adaptin has been observed in the region around the actin cones, suggesting that no coated pit formation occurs there. Conversely, clathrin mutants have defects in individualization, but the reason that individualization fails has not been well studied. The discrepancies in these data will only be resolved by further analysis of the clathrin mutant phenotype and studies of the effects of loss of function in other proteins in the endocytosis pathway (Noguchi, 2003).

    Likewise, exocytosis may not play a major role in the membrane remodeling process, because membrane staining with the dye FM1-43 suggests that there is not a significant amount of membrane insertion at the sites around the actin cones, and treatment with the the exocytosis inhibitor BFA does not affect the movement of cystic bulge. These data do not completely exclude the possibility that exocytic events participate in remodeling, since the exocytosis could not be directly measured. However, it seems more likely that the large number of membrane invaginations that are present in the cystic bulge is a sufficient source of membrane to accomplish remodeling. The plasma membrane seems to be smoothly reorganized into thin tubular structures around the actin cones. Furthermore, ultrastructural observations have shown that the membrane around actin cones is flat, without any invaginating or docking membrane vesicles. These data support the idea that the cell membrane in the cystic bulge is directly deformed into a thin tubular structure (Noguchi, 2003).

    tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility

    Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. A tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin (CL) concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function has been generated earlier. This study shows that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A2, iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, it was shown that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA2 inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention (Malhotra, 2009).

    The cardiolipin metabolism defect associated with Barth syndrome is manifested by the triad of CL depletion, monolyso-CL accumulation, and CL species diversification, i.e., the generation of CL molecules with different fatty acyl compositions. It is not clear whether the abnormal CL homeostasis actually plays a role in the pathogenesis of Barth syndrome, and if so, which aspect is the key factor. This study addresses this issue in a Drosophila model of Barth syndrome (Malhotra, 2009).

    It was found that tafazzin deficiency in Drosophila, which alters CL homeostasis and reduces CL levels, also disrupts spermatid individualization during spermatogenesis, resulting in male sterility, and that this male-sterile phenotype can be suppressed by inactivation of the CL-degrading enzyme iPLA2-VIA, which partially restores CL homeostasis in double-mutant flies. These observations suggest that CL content, or at least the MLCL/CL ratio, plays a critical role in Drosophila spermatid individualization. It has been recently shown that the final stage of spermatid differentiation in Drosophila involves an apoptosis-like mechanism, in which the cytochrome c-dependent caspase activation is required for the elimination of excess cytoplasm. Cardiolipin has been shown to play important roles in mitochondria-dependent apoptosis and a recent report demonstrats that CL deficiency increases cells' resistance to apoptosis. Therefore, CL deficiency in Drosophila testes may prevent the syncytial spermatids from initiating the apoptosis-like mechanism required for normal spermatid individualization. (Malhotra, 2009).

    The cardinal characteristics of Barth syndrome are cardioskeletal myopathy, exercise intolerance, neutropenia, abnormal mitochondria, and altered CL metabolism. Because in eukaryotes CL is localized exclusively in mitochondria and is required for optimal mitochondrial function, it has been generally assumed that the defective CL metabolism causes the pathophysiology of Barth syndrome. This study tested this hypothesis by genetically manipulating CL metabolism in the Drosophila model. It was found that partial restoration of CL homeostasis through genetic inactivation of iPLA2-VIA suppresses the male-sterile phenotype of tafazzin-deficient flies; this provides the direct evidence that altered CL metabolism is a major contributing factor in Barth syndrome (Malhotra, 2009).

    The most abundant CL molecular species from various organisms and tissues contain only 1 or 2 types of fatty acids. In many mammalian tissues, the predominant fatty acyl moiety in CL is linoleic acid (C18:2). For example, 80% of CL molecules in heart and skeletal muscle are tetralinoleoyl CL. However, the role of CL molecular species in vivo remains speculative. The characteristic fatty acyl composition of CL in vivo is achieved through tafazzin-dependent remodeling of nascent CL. However, tafazzin deficiency, such as in Barth syndrome, results not only in abnormal CL acyl composition, but also in CL depletion and monolyso-CL accumulation. Thus, it is unclear which aspect of the CL metabolic disorder contributes to the pathogenesis of Barth syndrome. The finding in this study that the male-sterile phenotype of tafazzin-deficient flies can be suppressed by genetic inactivation of iPLA2-VIA, which prevents CL depletion and monolyso-CL accumulation without correcting the abnormal CL acyl composition, suggests that the abnormal levels of CL and/or monolyso-CL are important pathogenetic factors. Because a cardiolipin synthase mutant of yeast exhibits abnormal mitochondrial function, it is likely that the low CL content is critical in the molecular mechanism of Barth syndrome. Nevertheless, because Barth syndrome is a multisystem disorder, involvement of monolyso-CL accumulation and abnormal CL acyl composition may also play a role in certain tissues and organs (Malhotra, 2009).

    The mature acyl chain composition of CL is achieved through a remodeling process, which requires the action of tafazzin. It has been previously shown that tafazzin catalyzes phospholipid-lysophospholipid transacylation that involves both deacylation of a phospholipid such as CL and reacylation of a monolyso-phospholipid, such as monolyso-CL. Unlike the CoA-dependent deacylation-reacylation cycle (Lands cycle), in which a nascent phospholipid is deacylated by a phospholipase A to yield a free fatty acid and a lysophospholipid that is then reacylated by an acyl-CoA-dependent acyltransferase, transacylation does not require acyl-CoA, and proceeds directly by transferring a fatty acyl chain from a phospholipid to a lysophospholipid; no phospholipase is involved and no free fatty acid is generated in the process. It was found that although the calcium-independent phospholipase A2, iPLA2-VIA, is not required for CL remodeling, in the absence of tafazzin, i.e., in the Barth syndrome model, the enzyme plays a major role in the depletion of CL and the accumulation of monolyso-CL (Malhotra, 2009).

    The finding that the phenotypic features of tafazzin deficiency can be suppressed by inhibiting iPLA2-VIA activity identifies this enzyme as a potential target for therapeutic intervention in Barth syndrome. Indeed, it was found that treatment of cultured lymphoblasts from Barth patients with the iPLA2 inhibitor BEL partially restores CL homeostasis. The calcium-independent iPLA2-VIA has been implicated in a variety of biological processes, including phospholipid remodeling, arachidonic acid release, apoptosis, and store-operated calcium entry. In addition, iPLA2-VIA knockout mice develop age-dependent neurological impairment and mutations in the iPLA2-VIA gene have been identified in patients with infantile neuroaxonal dystrophy and neurodegeneration with iron accumulation in the brain. Therefore, a therapeutic approach to Barth syndrome based on the inhibition of iPLA2-VIA is likely to require either careful titration of the phospholipase inhibitor, or even its tissue-specific targeting (Malhotra, 2009).

    Regulation of dynein localization and centrosome positioning by Lis-1 and asunder during Drosophila spermatogenesis.

    Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, a male-sterile allele of the Drosophila homolog of human LIS1 was characterized. Centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, loss of attachments between the nucleus, basal body and mitochondria were observed. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. Dynein recruitment to the nuclear surface and spindle poles was shown to be severely reduced in Lis-1 male germ cells. It is proposed that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, since similar phenotypes were observed in flies null for Tctex-1, a dynein light chain. asunder (asun) was previously identified as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis (Anderson, 2009). It is now reported that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. A model is presented in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis (Sitaram, 2012).

    Analysis of a hypomorphic, male-sterile allele of Lis-1 revealed that Lis-1 plays essential roles during Drosophila spermatogenesis. The data suggest that loss of dynein function is the root cause of the defects that were observe in Lis-1k11702 testes, as mutation of the dynein light chain gene tctex-1 (Dynein light chain 90F) phenocopies mutation of Lis-1. Based on their overlapping phenotypes in male germ cells, genetic interaction, colocalization and co-immunoprecipitation, a model is presented in which Lis-1 and asun cooperate to regulate dynein localization during spermatogenesis (Sitaram, 2012).

    These observations suggest that centrosomes of Lis-1 spermatocytes remain attached to the cell cortex and fail to migrate to the nuclear surface at entry into meiotic prophase. The phenotype of persistent cortical centrosomes during meiotic divisions has been characterized in abnormal spindles and nudE testes, and the presence of cortical centrosomes has been noted in Lis-1k11702 metaphase spermatocytes in studies of nudE mutants. Dynein-dynactin and LIS-1 localize to the cell periphery in lower eukaryotes and cultured mammalian cells, as well as to the posterior cortex of Drosophila oocytes. However, no enrichment of dynein-dynactin or LIS-1 at the cortex of Drosophila spermatocytes has been detected. Cortical dynein has been implicated in regulation of mitotic spindle orientation in several systems, although the mechanism is not clear. These data suggest that dynein and LIS-1 are required in spermatocytes to release centrosomes from the cortex prior to meiotic entry (Sitaram, 2012).

    Lis-1 spermatocytes exhibit free centrosomes, albeit at a much lower frequency than the phenotype of cortical centrosomes. Detachment of centrosomes from the cortex of primary spermatocytes is an earlier step in male meiosis than reassociation of the centrosomes with the nuclear surface at G2/M; hence, a failure of centrosomes to detach from the cortex is likely to mask a subsequent failure of nucleus-centrosome coupling. LIS-1 colocalizes with dynein-dynactin at the nuclear surface, and localization of dynein-dynactin to this site is severely impaired in Lis-1 spermatocytes and spermatids. Dynein-dynactin anchored at the nuclear surface has previously been implicated in mediating interactions between the nucleus and centrosomes during both mitotic and meiotic cell cycles. It is proposed that defects in nucleus-centrosome coupling in Lis-1 spermatocytes stem from disruption in localization of dynein-dynactin to the nuclear surface (Sitaram, 2012).

    Previous studies in other systems concerning the role of LIS1 in dynein-dynactin recruitment to the nuclear surface have yielded conflicting results. In C. elegans embryos, dynein-dynactin was reported to localize normally to this site in the absence of Lis-1. In mammalian neural stem cells, however, Lis1 was shown to be required for recruitment of dynein to the nuclear surface at prophase entry. Similarly, it was observed that severe reduction of perinuclear dynein-dynactin in Drosophila Lis-1 spermatocytes at meiotic onset, suggesting that Lis-1 is required for this process. Conversely, normal levels of Drosophila LIS-1 were found at the nuclear surface of tctex-1 spermatocytes; thus, dynein-dynactin does not appear to be reciprocally required for LIS-1 recruitment to this site. This finding of reduced levels of dynein heavy chain on the nuclear surface of tctex-1 spermatocytes suggest that Tctex-1 light chain plays a specific role in localizing dynein complexes to the nuclear surface; alternatively, complex integrity may be compromised in tctex-1 mutants (Sitaram, 2012).

    Previously reported has been the finding that asun regulates dynein localization during Drosophila spermatogenesis (Anderson, 2009). The characterization of the hypomorphic Lis-1k11702 allele and the null asun93 allele during Drosophila male meiosis reveals overlapping but distinct phenotypes. Lis-1k11702 spermatocytes exhibit two classes of centrosome positioning defects: cortical (major phenotype) and free centrosomes (minor phenotype). By contrast, although most asun93 spermatocytes have free centrosomes, they do not share with Lis-1k11702 spermatocytes the phenotype of cortical centrosomes. These observations suggest that the role of asun in spermatocytes is limited to events at the nuclear surface, whereas Lis-1 additionally regulates cortical events. asun93 spermatocytes undergo severe prophase arrest, possibly owing to failure of astral microtubules of free centrosomes to promote nuclear envelope breakdown. In Lis-1k11702 spermatocytes, however, meiosis apparently progresses on schedule despite cortical positioning of centrosomes. The high percentage of asun93 spermatids with increased numbers of variably sized nuclei, probably a consequence of cytokinesis and chromosome segregation defects, are also absent in Lis-1k11702 testes. These observations suggest that spindle formation and normal progression through male meiosis require centrosomes to be anchored, either to the nuclear surface or the cortex (Sitaram, 2012).

    Hypomorphic Lis-1k11702 and null asun93 round spermatids also show similarities and differences in their phenotypes. Both genes are required for recruitment of dynein-dynactin to the nuclear surface; this pool of dynein probably mediates nucleus-basal body and nucleus-Nebenkern attachments, which are defective in both mutants. Genes encoding Spag4 (a SUN protein), Yuri Gagarin (a coiled-coil protein) and GLD2 [a poly(A) polymerase] are required for nucleus-basal body coupling in spermatids, although it is not known whether they interact with ASUN or LIS-1 in this process. The current studies suggest that Lis-1, but not asun, is required for proper Nebenkern shaping and Nebenkern-basal body association; these functions might be mediated by dynein/microtubules acting at the Nebenkern surface. Nebenkerne are generated through fusion of mitochondria following Drosophila male meiosis. Two Nebenkerne bodies are occasionally present in Lis-1 and tctex-1 spermatids, implicating dynein in regulation of mitochondrial aggregation at this stage. Together, these observations suggest that the role of asun in spermatids is limited to events at the nuclear surface, whereas Lis-1 plays additional roles in regulating Nebenkerne (Sitaram, 2012).

    Based on the studies of hypomorphic Lis-1k11702 and null asun93 mutant testes, a model is proposed in which LIS-1 is required for several dynein-mediated processes during Drosophila spermatogenesis, and ASUN is required for the subset of these processes that involve the nuclear surface. Both LIS-1 and ASUN promote recruitment of dynein-dynactin to the nuclear surface of spermatocytes and spermatids. The strong genetic interaction that was observe between Lis-1 and asun suggests that they cooperate in regulating dynein localization during spermatogenesis; the finding that LIS-1 accumulation on the nuclear surface is lost in asun male germ cells provides further support for this notion. The observed colocalization and coimmunoprecipitation of LIS-1 and ASUN suggest that they function within a shared complex to promote dynein-dynactin recruitment to the nuclear surface. Not interaction between Drosophila LIS-1 and ASUN proteins was detected by in vitro binding or yeast two-hybrid assays, suggesting that their association may be mediated by another protein(s) rather than being direct. Future studies on the nature of the ASUN-LIS-1 interaction should help elucidate the mechanism by which dynein-dynactin localizes to the nuclear surface during spermatogenesis (Sitaram, 2012).

    Several proteins that promote dynein recruitment and centrosomal tethering to the nuclear surface have been identified. In C. elegans embryos, the KASH-domain protein ZYG-12, which localizes to the outer nuclear membrane and binds the inner nuclear membrane protein SUN-1, is required for these events. Another KASH-domain protein, Syne/Nesprin-1/2 (see Drosophila Nesprin), works in concert with SUN-1/2 to mediate nucleus-centrosome interactions during mammalian neuronal migration. Two additional pathways required for dynein recruitment to the nuclear surface at prophase have recently been identified in cultured mammalian cells. BicD2 binds dynein and anchors it to the nuclear envelope via its interaction with a nuclear pore complex protein, RanBP2. Similarly, CENP-F and NudE/EL act as a bridge between dynein and Nup133. It has not yet been determined whether mammalian LIS1 and ASUN function within these pathways or whether they act via a parallel mechanism to promote dynein recruitment to the nuclear surface (Sitaram, 2012).

    The finding that a single copy of Lis-1k11702 can drastically decrease the size of asunf02815 testes suggests potential roles for Lis- 1 and asun in regulating division of male germline stem cells of Drosophila, as loss of cell proliferation can lead to reduction of testes size. Interestingly, Lis-1 has been reported to regulate germline stem cell renewal in Drosophila ovaries. Orientation of the cleavage plane during male germline stem cell division requires proper migration of centrosomes along the nuclear surface, and misorientation of the plane can lead to stem cell loss. Given the importance of Lis-1 and asun in mediating nucleus-centrosome coupling in Drosophila spermatocytes, it is possible that these genes also cooperate to regulate centrosomes during stem cell divisions in testes. In humans, the LIS1 gene is dose sensitive during brain development, as the disorder lissencephaly results from deletion or mutation of a single copy. Lis-1 spermatogenesis phenotypes reported in this study were observed in flies homozygous for a hypomorphic Lis-1 allele; flies carrying one copy of this allele displayed many of the same phenotypes but to a lesser degree. These findings suggest that precise regulation of LIS- 1 protein levels is essential for normal development in Drosophila. A requirement for Lis1 during spermatogenesis is conserved in mammals. Deletion of a testis-specific splicing variant of Lis1 in mice blocks spermiogenesis and prevents spermatid differentiation. LIS1 and dynein were shown to partially colocalize around wild-type spermatid nuclei, but dynein localization in Lis1 testes was not assessed. It remains to be determined if the functions of LIS1 in mammalian spermatogenesis are mediated through dynein and if the ASUN homolog regulates LIS1 localization in this system (Sitaram, 2012).

    The cilium-like region of the Drosophila spermatocyte: an emerging flagellum?

    Primary cilia and flagella are distinct structures with different functions in eukaryotic cells. Despite the fact that they share similar basic organization and architecture, a direct developmental continuity among them has not been reported until now. The primary cilium is a dynamic structure that typically assembles and disassembles during mitotic cell cycles, whereas the sperm axoneme is nucleated by the centriole inherited by the differentiating spermatid at the end of meiosis. Fruit flies display a remarkable exception to this general rule. Drosophila spermatocytes have an unusual axoneme-based structure reminiscent of primary cilia (the cilium-like region, or CLR). This structure persists through the meiotic divisions when it is internalized with the centriole to organize the centrosome and is finally inherited by young spermatids. Examination of elongating spermatids by transmission electron microscopy (EM) and cold regrowth experiments suggests that the motile axoneme derives directly from the elongation and remodelling of the immotile CLR. Both the CLR and elongating spermatid flagella have incomplete C-tubules that form longitudinal sheets associated with the B-tubule wall, unlike axonemes of other organisms in which C-tubules stop growing at the transition between the basal body and the axonemal doublets. Moreover, both the CLR and spermatid flagella lack a structured transition zone, a characteristic feature of ciliated cells. uncoordinated (unc) mutants that lack C-remnants have short centrioles, suggesting that the C-sheets play a role in the elongation of the centriole after it docks to the cell membrane. The structural similarities between CLR and sperm axoneme suggest that the CLR can be considered as the basal region of the future axoneme and could represent the start point for its elongation (Gottardo, 2013).

    The distal end of the Drosophila centriole carries thin projections that remember in their position the transition fibers found at the basis of the primary cilium of most animal cells. However, whereas the transition fibers usually emerge from the B-tubules, the projections associated to the Drosophila centriole originate from the C-tubules. Despite the apparent divergence between the two structural organizations, these projections may have a similar functional significance. They seem to tether the centriole to the cell surface, and might define the boundaries between the plasma and the ciliary membranes similarly to the distal appendages of the primary cilium in vertebrate cells. In veterbrate cells the transition fibers of primary cilia play a critical role as filter barrier to regulate the free diffusion of cytoplasmic components inside the ciliary compartment. A similar function for the projections associated to the distal end of the Drosophila centriole cannot be excluded. Interestingly, in Drosophila spermatocytes the membrane that surrounds the basal region of the CLR is particularly rich in vesicles that may represent a large stock of cytoplasmic components needed to refurnish the dynamic ciliary complex. Therefore, this membrane domain might be an important site for vesicular trafficking from and to the plasma membrane, as occurs in the ciliary pocket of some vertebrate cells (Gottardo, 2013).

    Two closely interconnected features usually identify the transition from the basal body to the axoneme: the end of the C-tubule, along with the extension of A- and B-tubules. Surprisingly, the outer C-tubule does not stop growing at the beginning of the axoneme in Drosophila CLRs. It loses some protofilaments reducing its size to form a continuous longitudinal sheet that runs along the whole axoneme in association with the B-tubule. The function of the C-remnant in the organization of the CLR is unclear, since this structure can assemble in unc mutants despite the C-tubule ends at the transition between the centriole and the ciliary axoneme. However, centrioles are shorter in unc mutants suggesting that C-remnants may play a role in the elongation of these organelles. Moreover, the lack of lateral projections points to the need of the C-remnants. The unc phenotype might explain how centrioles and associated ciliary axonemes increase their length at the same time in Drosophila primary spermatocytes. This is a unique process, since in other animal cells while the primary cilium elongates, the basal body does not change dimensions after its docking at the plasma membrane. It can be hypothesized that during the elongation of the A- and B-tubules, cytoplasmic precursors of the missing protofilaments join to the C-sheet at the distal end of the centriole, thus completing the tubule and allowing centriole to elongate concurrently (Gottardo, 2013).

    Thin radial projections emerge from the B-tubules at a right angle with the C-sheets. Projections associated to the axonemal doublets usually characterize the proximal region of the primary cilium of most organisms, the so-called 'transition zone'. These projections are typically Y-shaped, emerge at the A- and B-tubule junction, and directly contact the ciliary plasma membrane. The projections found in the CLR of Drosophila spermatocytes emerge from the B-tubule, are L-shaped in cross section and do not contact the ciliary membrane. It is important to remark that these projections were found along the whole CLR in Drosophila. This condition contrasts with findings in other eukaryotic cells where Y-links are restricted to the 'necklace', a distinct region at the proximal base of the primary cilium. The CLR of Drosophila spermatocytes somewhat remember the structure of the connecting cilium found in photoreceptors cells, where Y-shaped links extend along the cilium. Likewise, microtubule-membrane connections extending along much of the axoneme have been also described in Leismania amastigote flagella and in kidney primary cilia raising the possibility that both ciliary and flagellar axonemes may represent extended transition zones (Gottardo, 2013).

    It has been recently shown that in sensory neurons the coiled-coil protein Chibby (CBY) colocalizes at the distal end of the centriole with CG14870, the Drosophila orthologous of MKSR1, a component of the MKS module at the transition zone (Enjolaras et al., 2012). Similarly to typical transition zone components, CBY is required to regulate protein trafficking into and out sensory neuronal cilia. CBY was also found in Drosophila male germ cells to colocalize with Unc at the distal end of the centriole, and in later stages at the tip of the spermatid axoneme. It has been proposed that CBY is essential for proper centriole function and that, given its localization, both Drosophila CLR and sperm flagella may develop as elongated transition zones. Accordingly, there is evidence from different model systems that transition zone formation does not require intraflagellar transport (Gottardo, 2013).

    The unusual structural details found in the CLR of the Drosophila spermatocytes point to the lack of a conventional transition zone. This is consistent with the finding that the CG14870 gene product is associated with the distal end of the centriole in sensory neurons, but not in spermatocytes. However, it cannot be excluded that a structure molecularly similar to a transition zone, but morphologically different, may be assembled in Drosophila spermatocytes and maintained during spermatid elongation. Indeed, the appearance of the transition zone has been reported to vary between species and cell types, although the basic structural features appear to be conserved (Gottardo, 2013).

    In Drosophila primary spermatocytes the CLR elongates in the extracellular milieu and during the meiotic divisions internalizes into the cell by an invagination of the plasma membrane. A sheath with inner and outer membranes thus surrounds the ciliary axoneme inherited by young spermatids. This membrane invagination looks like the ciliary pocket that surrounds the proximal region of the primary cilium in several vertebrate cell types. However, despite these structures are morphologically related, they are not functionally equivalent. In fact, in Drosophila spermatids the marginal zone of the CPL does not correspond to the membrane domain found at the basis of the primary cilia. An important point is that the CPL of Drosophila spermatids is not correlated to the ciliogenesis pathway, but instead it is a consequence of the inward invagination of the CLR during the meiotic progression. During early spermatid elongation the Unc-GFP ring localized within the marginal zone of the CPL becomes free from the distal end of the centriole and was positioned toward the caudal end of the axoneme, tracking the leading edge of the CPL. Mutations in unc lead to the overgrowth of the CPL and the consequent reduction of the axoneme portion free in the cytoplasm. These data suggest that unc could be implicated in the complex dynamics of the CPL and in the maintenance of its shape (Gottardo, 2013).

    The current observations revealed a few vesicles at the basis of the CPL, suggesting that vesicular trafficking does not play an important role in the process of axoneme elongation in Drosophila spermatids. By contrast, vesicle docking is usually required in vertebrate cells to ensure membrane expansion and proper ciliogenesis. A ciliary pocket-like structure that does not support vesicular trafficking forms during vertebrate spermiogenesis as a consequence of the inward movements of the basal body toward the nucleus followed by the caudal extension of the cell body. However, in vertebrate spermatids the cell membrane surrounds the axoneme and growths in concert with the elongating microtubules. Thus in this case the ciliary pocket is a transitory structure that changes dimensions and then disappears. In Drosophila spermatids only the distal end of the elongating axoneme is surrounded by an inner membrane that remains contiguous with the outer plasma membrane throughout the assembly of the axoneme. Cold exposure revealed that the CPL held both stable and unstable axonemal microtubules. The latter population regrows quickly after recovery from cold treatment, suggesting that tubulin addition occurs in the distal-most end of the CPL. This agrees with previous findings showing that the elongation of flagellar axoneme occurs at its distal tips, with the addition of specific components that are translocate at the microtubule plus ends (Gottardo, 2013).

    The disorganized symmetry of the axoneme and the isolated A-tubules at the distal end of the CPL suggest that each microtubule doublet grows asynchronously. Longitudinal sections often revealed isolated A-tubules ending in a moderately dense material at the tip of the axoneme that might be implicated in microtubule nucleation. The wall of the A-tubules may act as template for the B-tubules that, in turn, may support the growth of the C-blade that give continuity to the longitudinal sheets emerging from the C-tubules of the centriole. Similar conclusions come from structural analysis of procentriole formation in human cultured cells. However, the polymerization of B- and C-tubules is bidirectional in cultured cells, whereas B-tubules and C-blades elongate at their distal/plus ends in the Drosophila spermatid axoneme (Gottardo, 2013).

    A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids

    In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. This study reports that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. Cep290 was shown to be essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity; MKS proteins also delimit a centriole-independent compartment in mouse spermatids. These findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly (Basiri, 2014).

    Centriole remodeling during spermiogenesis in Drosophila

    The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM). In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis. This study shows that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction. The ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and the PCL does not resemble a typical centriole. Additionally, Poc1 is enriched at the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, it was found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila (Khire, 2017).

    Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells

    The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. This study shows that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. These results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells (Vieillard, 2016).

    RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila

    This study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. Deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. This study found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins (Kotov, 2016).

    This study shows that RNA helicase Belle (DDX3) is required cell-autonomously for the survival and divisions of GSCs in Drosophila testes. In bel6/neo30 mutants as well as in the case of germline-specific RNAi belKD rapid elimination of germ cells via apoptosis occurred. But what events could trigger apoptosis? To address this issue, larval testes were analyzed. Testes of bel6/neo30 mutant larvae still contained all populations of early germ cells. This observation indicates that primordial germ cells (PGCs) correctly migrate into embryonic gonads during mid to late embryogenesis. In the mutant larval testes the wild-type hub and GSCs adjacent to the hub were clearly detected. It is known that the mechanism of capturing GSCs and CySCs to the hub involves a high level of adhesion molecule E-Cad on the hub/stem cells interface. In testes with STAT depletion the expression of E-Cad is severely disrupted accounting for the defects in hub-GSC adhesion and for the subsequent loss of GSCs. However, it was determined that STAT expression (in CySCs) and consequently upstream Upd signaling from the hub were not disrupted in the bel6/neo30 testes. Although the amount of Belle was strongly reduced in the bel6/neo30 testes, no reduction of E-Cad level was observed in CySCs. On the contrary, high ectopic expression of E-Cad was detected on the surface of CySCs surrounding the hub. The influence of Belle on the adherens junction formation in GSCs cannot be directly estimated. However, due to adhesion failures a loss of GSCs via premature differentiation could be expected followed by normal development of newly formed germline cysts. In contrast, this study detected a reduced germ cell content and morphological abnormalities of early germ cells including their giant nuclear and cellular sizes. It is assumed that these germ cells could not enter mitosis and are delayed in the G2 phase. Failure to enter mitosis after G2 delay appears to induce germ cell apoptosis in the bel testes, as previously has been shown for the how testes (Kotov, 2016).

    It is known that Drosophila mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, each form complexes with Cdc2, and they appear to function synergistically to provide a progression throughout mitosis. Sufficient levels of mitotic cyclins must be accumulated at the end of G2 to ensure the onset of mitosis. To date, cell cycle regulation of GSCs and their daughter gonial cells is still poorly understood. It is known that PGCs suppress mitotic activity during their migration to embryonic gonads due to translational repression of maternal cycB mRNA via its 3'UTR by Pumilio-Nanos complex and other unidentified factors. Pumilio and Nanos are also known to be expressed in GSCs of gonads of adult flies and are found to be essential for GSC maintenance. However, factors overriding the repressive Pumilio-Nanos-dependent signal and providing expression of zygotic Cyclin B protein during normal testis development are currently unknown (Kotov, 2016).

    It has been shown that Cyclin B and Cyclin A, but not Cyclin B3, are required in the gonad for the maintenance of early germ cells. A mutational depletion of Cyclin B leads to a complete missing of germ cells in the adult testes and their significant loss in the ovaries. However, the requirements for Cyclin A expression for the survival of early germ cells are currently obscure. It is known that overexpression of Cyclin A or expression of its nondegradable form leads to a rapid loss of GSCs in the ovaries (Kotov, 2016).

    This study found that the previously published cycB testis phenotype mimicked that in the case of bel6/neo30 mutants and germline-specific RNAi belKD. It was determined that both of the mitotic cyclins, but not Cyclin E and Cdc2, were significantly decreased in the belEY08943/neo30mutant testes. Furthermore, a considerable decrease was found of cycA and cycB mRNA levels. These results suggest a specific contribution of Belle to the transcriptional regulation of mitotic cyclins in the germline. It was also revealed that the constitutive level of Cyclin B expression in control testes was significantly lower than in control ovaries. Assuming that Belle regulates mitotic cyclins in a similar way in the germline of both sexes, it is believed that deficiency of Belle has a more severe effect on spermatogenesis, due to a sharply reduced level of Cyclin B protein below a threshold, whereas its dose in the bel6/neo30 ovaries is still sufficient to allow mitosis to occur. In support of this hypothesis a partial rescue was achieved of the RNAi belKD testis phenotype by transgenic germline-specific expression of Cyclin B, but not by Cyclin A overexpression (Kotov, 2016).

    The reduction of cycB transcription in belEY08943/neo30 testes places cycB downstream of bel. In rescue experiments a third copy of cycB was added to the system employing the germinal nos-Gal4 driver in combination with UAS-bel RNAi hairpin. It is assumed that the reduction of Belle in RNAi belKD testes would negatively affect the expression of both endogenous and transgenic cycB. In accordance with this assumption only a partial restoration of the Cyclin B protein level and only partial rescue of spermatogenesis was achieved. The data indicate that at least one crucial requirement for Belle in early germ cells is relevant to Cyclin B level maintenance for ensuring germ cell mitosis (Kotov, 2016).

    To date, evidences of participation of DDX3 proteins in cell cycle control both at the level of transcription and translations have been obtained. DDX3 specifically cooperates with transcription factor Sp1 to positively regulate the transcription of p21waf gene. A temperature-sensitive mutation of ddx3 gene in golden hamster cell culture at nonpermissive temperature leads to G1 arrest, which is accompanied by a decline of cycA mRNA and rather suggests the transcriptional level of regulation for cycA. It has been shown that in human HeLa cells DDX3 interacts with the GC-rich, highly structured 5'UTR of Cyclin E1 mRNA and regulates its translation initiation and a knockdown of DDX3 delays the entry to the S phase. DED1, a Schizosaccharomyces pombe homolog of DDX3, is involved in the translational control of B-type cyclin mRNAs (Cig2 and Cdc13), which have extended and expectedly highly structured 5'UTRs. It is known that only a single cyclin-dependent kinase and two B-type cyclins regulate both the S phase and mitosis in yeasts. Indeed, temperature-sensitive mutations of ded1 gene inhibit B-type cyclin translation and arrest cell cycle at both S phase and G2/M transition, whereas both cig2 and cdc13 mRNA levels remain unchanged (Kotov, 2016 and references therein).

    This study has presented experimental evidence that Belle has specific and essential functions in the male germline associated with proper transcriptional regulation of mitotic cyclin expression. The testis phenotype observed in Drosophila is similar to the SCOS phenotype in human testes, indicating a conserved function of DDX3 in spermatogenesis. Understanding the molecular basis for DBY (DDX3) functions in mammalian germ cell maintenance has proven to be challenging. The functions and regulation of A-type and B-type cyclins in mammalian spermatogenesis are not clearly understood. In this case, a study in the Drosophila model provides a useful insight into the mechanism of GSC maintenance in the male germline. The current findings support a mechanism according to which the determination of the fate of male GSCs is closely connected with the control of mitosis via the regulation of mitotic cyclin levels (Kotov, 2016).

    tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation

    Male germ cell differentiation proceeds to a large extent in the absence of active gene transcription. In Drosophila, hundreds of genes whose proteins are required during post-meiotic spermatid differentiation (spermiogenesis) are transcribed in primary spermatocytes. Transcription of these genes depends on the sequential action of the testis meiotic arrest complex (tMAC), Mediator complex, and testis-specific TFIID (tTFIID) complex. How the action of these protein complexes is coordinated and which other factors are involved in the regulation of transcription in spermatocytes is not well understood. This study shows that the bromodomain proteins tBRD-1 and tBRD-2 regulate gene expression in primary spermatocytes and share a subset of target genes. The function of tBRD-1 was essential for the sub-cellular localization of endogenous tBRD-2 but dispensable for its protein stability. Comparison of different microarray data sets showed that in primary spermatocytes, the expression of a defined number of genes depend on the function of the bromodomain proteins tBRD-1 and tBRD-2, the tMAC component Aly, the Mediator component Med22, and the tTAF Sa (Theofel, 2017).

    A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage

    In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. This study shows that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism (Insco, 2012).

    Adult stem cells act throughout life to replenish differentiated cells lost to turnover or injury. In many adult stem cell lineages, stem cell daughters destined for differentiation first undergo a limited number of mitotic divisions to amplify cell number prior to terminal differentiation. This transit amplifying (TA) division strategy may protect large long-lived animals from tumorigenesis by minimizing the number of stem cell divisions required for tissue homeostasis and preventing accumulation of oncogenic mutations in progenitor cells due to programmed differentiation. The mechanisms that limit the number of TA divisions and initiate terminal differentiation thus may provide tumor suppressor function, and defects may contribute to progression toward cancer in adult stem cell lineages (Insco, 2012).

    This study investigated the mechanisms that force TA cells to stop proliferating and initiate terminal differentiation in the Drosophila male germline adult stem cell lineage. Drosophila male germline stem cells (GSCs) reside in a niche at the tip of the testis, attached to somatic hub cells and flanked by somatic cyst stem cells (CySCs). When a GSC divides, one daughter remains in the niche and self-renews, while the other is displaced away and initiates differentiation. The resulting gonialblast, which is enveloped by a pair of CySCs, proceeds through four synchronous TA divisions with incomplete cytokinesis, producing a clone of 16 interconnected germ cells. These 16 mitotic sisters normally stop proliferating, undergo premeiotic DNA synthesis in synchrony, and switch to the spermatocyte program of cell growth, meiosis, and terminal differentiation. Because TA sister cells are contained within a common somatic cell envelope, are joined by cytoplasmic bridges, and divide in synchrony, mutations that cause overproliferation of TA cells can be easily identified (Insco, 2012).

    The bag of marbles (bam) gene is required cell autonomously for TA spermatogonia to stop proliferating and enter the spermatocyte differentiation program. Male germ cells mutant for bam undergo several extra rounds of mitotic TA division, fail to differentiate, and eventually die. The number of TA divisions appears to be set by the time required for Bam protein to accumulate to a critical threshold. Bam protein is normally first detected in 4-cell cysts, increases to a peak in 8-cell cysts, and is degraded in early 16-cell cysts immediately after premeiotic DNA replication. Lowering the bam dosage slowed Bam protein accumulation and delayed the transition to differentiation, whereas early accumulation of Bam protein caused a premature switch to differentiation (Insco, 2012).

    Bam, a protein with no recognizable domains, acts with a partner, benign gonial cell neoplasm (Bgcn), discovered in a genetic screen for Drosophila tumor suppressors. bam and bgcn have similar mutant phenotypes, and Bam protein directly interacts with Bgcn in Drosophila ovaries or when coexpressed in cultured cells or yeast. Bgcn is related to the DExH-box family of RNA-dependent helicases, indicating that Bgcn, and with it Bam, may regulate RNA (Insco, 2012).

    Consistent with a role in translational repression, Bam protein binds the translation initiation factor eIF4A. Furthermore, expression of Bam and Bgcn in Drosophila cultured cells resulted in a 4-fold reduction in expression of a luciferase reporter coupled to the 3′ untranslated region (UTR) of e-cadherin messenger RNA (mRNA), and tethering Bam to the 3′ UTR induced translational repression of the attached reporter. In female germ cells, Bam and Bgcn allow the onset of differentiation through translational repression of nanos (nos) via the nos 3' UTR. However, direct interaction of Bam or Bgcn protein with e-cadherin or nos mRNAs has not been demonstrated (Insco, 2012).

    This study identified the microRNA (miRNA) regulator and TRIM-NHL (tripartite motif and Ncl-1, HT2a, and Lin-41 domain) family member Mei-P26 both as a regulator of Bam protein accumulation and, subsequently, as a direct target of translational repression by Bam and Bgcn in male germ cells. Mei-P26 function facilitates both the switch from mitosis to meiosis and spermatocyte differentiation. In mei-P26 mutant males, Bam protein failed to accumulate to its normal peak levels. The overproliferation of TA cells in mei-P26 mutant testes was suppressed by expression of additional Bam, suggesting that the continued TA cell proliferation in mei-P26 mutant males is due to the failure of Bam protein to reach the threshold required for the switch to the spermatocyte state. In turn, Bam specifically binds the mei-P26 3' UTR, and Bam and Bgcn function are required for translational repression of mei-P26 via its 3' UTR in vivo. Mutating two potential let-7 target sites within the mei-P26 3' UTR derepressed reporter expression in vivo and disrupted Bam binding in vitro. These data suggest that a stepwise progression in regulatory states from [Mei-P26 on/Bam off] to [Mei-P26 on/Bam on] to [Bam on/Mei-P26 off], choreographed by translational regulation, accompanies the switch from TA cell proliferation to terminal differentiation in the Drosophila male GSC lineage (Insco, 2012).

    It is proposed that Mei-P26 and Bam act in a regulatory cascade based on translational control to affect the switch from TA cell proliferation to spermatocyte differentiation in the Drosophila male GSC lineage. First, wild-type function of Mei-P26 in TA cells facilitates accumulation of Bam protein. Consistent with this model, a mei-P26 hypomorphic allele enhanced the overproliferation of germ cell cysts in a bam/+ heterozygote. Furthermore, the finding that adding one extra copy of bam is sufficient to rescue the early germ cell overproliferation phenotype of mei-P26 mutant males indicates that allowing normal accumulation of Bam is the major role of Mei-P26 in regulating proliferation of early male germ cells. Second, as Bam protein levels rise, Bam and Bgcn repress translation of mei-P26 via its 3' UTR in late TA cell cysts. As a result, GSCs, gonialblasts, and two-cell cysts begin with Mei-P26 expressed and Bam off and transition to 4-cell and early 8-cell cysts wherein both Mei-P26 and Bam protein are expressed. In late 8-cell and early 16-cell cysts, Bam protein levels are high, causing Mei-P26 protein to drop to very low levels. Finally, in early spermatocytes, Mei-P26 levels rise again after Bam protein disappears to facilitate normal differentiation of spermatocytes and spermatids (Insco, 2012).

    Recent data suggest that Mei-P26 and related TRIM family proteins may function in the miRNA pathway. Mei-P26, two of its Drosophila homologs, and several mouse homologs have been shown to interact structurally with RISC effector proteins such as Ago-1. Mei-P26 protein localized to cytoplasmic puncta in early male and female germ cells, similar to the punctate distribution of mouse TRIM71. Many of the Mei-P26 puncta colocalized with the RISC component GW182, which accumulates in processing bodies that consist of enzymes involved in mRNA translational repression and degradation. The action of Mei-P26 in early male germ cells may facilitate accumulation of Bam protein by repressing an intermediate negative regulator of Bam. For example, Mei-P26 may function in TA cells to facilitate the accumulation of Bam protein through decreasing the function of the RNA-binding protein HOW. Previous studies suggest that HOW represses Bam expression in early male germ cells. In wild-type testes, HOW protein was expressed in early cells, including GSCs, gonialblasts, and two-cell cysts. However, in mei-P26 mutant testes, HOW protein perdured throughout the overproliferating cysts. Alternatively, given that Mei-P26 also contains a RING domain, it could facilitate degradation of an intermediate that normally degrades Bam. Bam has a C-terminal PEST sequence, a motif that targets proteins for ubiquitination and turnover by the proteasome, and expression of Bam lacking the PEST sequence resulted in early accumulation of high levels of Bam protein and a premature switch to the spermatocyte state (Insco, 2012).

    As Bam protein peaks in late TA cells, it acts with its binding partner Bgcn to repress translation of mei-P26 mRNA via sequences in the mei-P26 3' UTR. Bam protein specifically binds the mei-P26 3' UTR, suggesting that Bam and Bgcn act directly as translational repressors. Translational regulation via 3' UTR sequences frequently blocks formation of the translation initiation complex by inhibiting interactions between the cap binding protein eIF4E and the 5′ cap or the rest of the eIF4F complex. Bam protein physically interacts with eIF4A independent of RNA, and eIF4A/+ partially suppressed the phenotype of bam mutants in both the male and female germline systems, raising the possibility that Bam, recruited to a target 3' UTR as part of a translational repressor complex, may block translation initiation by antagonizing eIF4A. Mutating two potential let-7 binding sites within the mei-P26 3' UTR led to derepression of the in vivo reporter and disrupted binding of Bam to the mei-P26 3' UTR. These data raise the possibility that let-7 may work with Bam and Bgcn to translationally repress Mei-P26 in TA cells. In addition, introducing the let-7-CGK1 loss-of-function allele into a bamΔ86/+ mutant background enhanced the bam heterozygous mutant phenotype, suggesting that let-7 and Bam may share additional targets within the testes (Insco, 2012).

    Mei-P26 appears to play two distinct roles in the female germline as well: an early function in GSC maintenance and a later function required for cystocytes to switch to nurse cell and oocyte differentiation. However, there are also important differences between the male and female germline. Although Mei-P26 protein levels decreased when Bam was expressed in female germ cells, low levels of Mei-P26 were still detected. Bam and Bgcn may inhibit mei-P26 translation in female germ cells, although probably not to the same degree as in males. Notably, the mei-P26 3' UTR cloned from testes lacked the Vasa binding sites shown to be important for Mei-P26 expression in female germ cells. In addition, Bam is active at an earlier stage in the ovary, wherein the function of Bam is necessary for female GSCs to initiate the TA divisions rather than exit the TA divisions, as in males. Finally, Bam and Bgcn may have different mRNA targets in the female germline. In the female, Bam action directly or indirectly represses translation of the translational repressor Nanos, allowing the expression of proteins that initiate germ cell differentiation from the stem cell state. However, Nanos does not appear to play the same role in male as in female GSCs. Thus, the core machinery of Bam, Bgcn, and Mei-P26 probably acts through similar molecular mechanisms in female and male germ cell differentiation, but at a different point in the differentiation pathway, with different regulators and, most likely, on different targets (Insco, 2012).

    Strikingly, as is shown in this study for the Drosophila male germline, the switch from mitosis to meiosis is also controlled by a regulatory network based on translational control in the C. elegans germline. BLAST and ClustalW alignments revealed that Bgcn, a core component of the switch mechanism in the Drosophila germline, is a homolog of C. elegans proteins Mog1, Mog4, and Mog5, which are required for stopping mitosis and repressing target-mRNA translation via the 3' UTR (Insco, 2012).

    The requirement for TRIM-NHL proteins to facilitate the switch from proliferation to differentiation may be a widely conserved feature in many adult stem cell lineages. In Drosophila, loss of the Mei-P26 homolog dappled/wech causes large melanotic tumors, suggesting the continued proliferation of blood cells. Likewise, loss of the Drosophila Mei-P26 homolog brat in TA cells in certain neural lineages leads to brain tumors that are highly proliferative, invasive, transplantable, and lethal to the animal. In mammals, the mouse Mei-P26 homolog TRIM32 is necessary and sufficient for differentiation in neural lineages, and progenitor cells lacking TRIM32 retain proliferative status. Thus, elucidating the mechanisms by which Mei-P26 homologs and their interacting structural and regulatory partners control the switch from proliferation to differentiation in adult stem cell lineages may uncover a new class of tumor suppressors that act at the level of the developmental program rather than cell-cycle progression (Insco, 2012).

    The THO complex is required for nucleolar integrity in Drosophila spermatocytes

    The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. This study isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, this study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes (Moon, 2011).

    During spermatogenesis, the coordinated action of many gene products is essential for proper differentiation of sperm. In Drosophila, ∼25% of all genes expressed in the testis are testis-specific or testis-enriched, and most of these genes are transcribed in primary spermatocytes and stored until needed. Testis-specific gene regulation programs might ensure the coordinated expression of this large number of genes from transcription to translation. In Drosophila, two distinct classes of meiotic arrest genes, aly-class and can-class genes, represent testis-specific transcription regulation modules. The can-class genes, cannonball (can), spermatocyte arrest (sa), meiosis I arrest (mia) and no hitter (nht), encode the testis-specific TBP-associated factors (tTAFs), suggesting that their products form a testis-specific TFIID complex in primary spermatocytes. Interestingly, tTAFs, together with Polycomb group (PcG) proteins, mainly localize to a subcompartment of the nucleolus, rather than to euchromatin. Lack of tTAFs not only disrupts the nucleolar localization of PcG proteins but also causes PC to accumulate at tTAF target promoters. These findings suggested that tTAFs might also antagonize the Polycomb repressor complex (PRC1) to control the coordinated transcription of target genes (Moon, 2011).

    Most aly-class gene products form tMAC, a testis-specific meiotic arrest complex paralogous to Myb-MuvB. The aly-class gene products are mainly localized to euchromatin in primary spermatocytes, and this localization is essential for their function, suggesting that the major role of the tMAC complex is transcriptional activation of testis-specific genes. Thus, it now seems evident that the coordinated transcription of testis-specific genes is regulated by two specific complexes: tTAFs and tMAC. However, little is known about post-transcriptional regulation in the testis. This study shows that the THO complex, an evolutionarily conserved complex involved in the co-transcriptional formation of export-competent messenger ribonucleoproteins (mRNPs), is a novel regulator of Drosophila spermatogenesis (Moon, 2011).

    The THO complex was first found in budding yeast, Saccharomyces cerevisiae, as a multisubunit protein complex composed of Hpr1, Tho2, Mft1 and Thp2. Yeast cells that lack the THO complex show transcription impairment and transcription-dependent hyper-recombination phenotypes, implying that the THO complex connects transcription elongation to mitotic recombination. Subsequent studies showed that the THO complex, together with the mRNA export adaptor proteins Yra1 and Sub2, forms a larger complex called TREX (transcription-export complex), which is required for the co-transcriptional export of bulk mRNAs. Metazoans also have a functional homolog of the THO complex, but its subunit composition and function are slightly different from those of budding yeast. The metazoan THO complex lacks Mft1 and Thp2, but contains three other subunits, THOC5, THOC6 and THOC7, instead. Unlike budding yeast, metazoan cells require the THO complex for nuclear export of only a subset of transcripts, but almost nothing is known about the common features of the target transcripts. Although it is now clear that, at the single-cell level, a role of the THO complex in mRNP biosynthesis is conserved throughout evolution from yeast to human, its exact role in various types of cells at the multicellular organism level is still largely unknown. Recent studies using conditional knockout or a hypomorphic mutant mouse model have provided evidence that the THO complex has specific roles in cell differentiation during development. It has also recently been reported that the Drosophila THO complex is required for normal development through collaboration with E(Y)2 (or ENY2), a multifunctional protein important for transcription activation and mRNA export (Moon, 2011 and references therein).

    This study reports that a novel meiotic arrest gene, garmcho (garm), encodes the Drosophila THOC5 homolog. Flies lacking THOC5 showed complete male sterility with a meiotic arrest phenotype. Interestingly, unlike any other known meiotic arrest mutants, the nucleolar structure was severely disrupted in garm mutant primary spermatocytes. Both tTAF and PC proteins were abnormally distributed, whereas the expression and nuclear export of the three tTAF target mRNAs examined were mainly unaffected in mutant primary spermatocytes. Taken together, these data provide additional evidence that the THO complex is involved in a specific cell differentiation program, Drosophila spermatogenesis, probably by participating in the establishment or maintenance of nucleolar integrity in spermatocytes (Moon, 2011).

    The phenotypes of garm are different from those of other meiotic arrest mutants. First, the chromosomes were more condensed in arrested mutant spermatocytes than in wild-type spermatocytes. In other known meiotic arrest mutants, spermatocytes have chromosomes either similar to or less condensed than wild type. Second, unlike other mutants, the nucleoli were severely disrupted in garm mutant primary spermatocytes. Taken together, these findings suggest that garm represents a novel class of meiotic arrest gene, and that its gene product might play a role distinct from that of other meiotic arrest gene products, which are involved in the transcription of target genes. Indeed, THOC5 (encoded by garm) is a subunit of the THO complex, which is an evolutionarily conserved protein complex required for mRNP biosynthesis. So far, garm (thoc5) is the only meiotic arrest gene whose gene product is likely to be involved in a post-transcriptional step (Moon, 2011).

    THOC5 is likely to function in spermatocytes as a subunit of the THO complex. First, all subunits of the THO complex examined were not only colocalized in spermatocytes but also co-immunoprecipitated with each other, suggesting that they form a stable complex in spermatocytes. Second, in mutant spermatocytes lacking one of the THO subunits, not only was the localization of other THO subunits disrupted, but also their expression level was significantly reduce. Third, hypomorphic mutants of both thoc6 and thoc7 appeared to show mild disruption of the nucleoli, although they did not show the typical meiotic arrest phenotype (Moon, 2011).

    What is the specific role of the THO complex in Drosophila spermatogenesis? The finding that the nucleolar structure was severely disrupted in thoc5 mutants suggests that the THO complex might be required for the proper organization of the nucleolus. In accordance with this, all subunits of the THO complex accumulate at the peri-nucleolar region in pre-meiotic spermatocytes. Recent progress in understanding of the nucleolus suggests that not only does it function as the ribosome-producing factory, but it also regulates mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles. Consistent with these non-traditional functions, many proteins unrelated to ribosome assembly are found in the nucleolus. Moreover, the integration of many different sources of protein-protein interaction data showed that the spliceosomal complex is one of the major protein complexes in human nucleolus, suggesting that the spliceosomes are structural components of the nucleolus. Thus, it is speculated that the nucleolar disruption found in thoc5 spermatocytes might be caused by malformation of the spliceosomal complex, because the THO complex associates with splicesomal proteins independently of transcription. One of the common phenotypes caused by nucleolar disruption in mammalian cells is the p53-mediated cellular stress response, which includes cell-cycle arrest and apoptosis. To test whether the meiotic arrest phenotype seen in this study was also mediated by p53, genetic interactions were examined between thoc5 and p53. However, lack of p53 failed to suppress the meiotic arrest phenotype caused by thoc5 , suggesting that p53 is not required for the meiotic arrest phenotype seen in thoc5 mutant spermatocytes. This also suggested, if nucleolar disruption was the cause of the meiotic arrest, that some other unknown pathways are required for cell-cycle arrest in Drosophila spermatocyte. Alternatively, the THO complex might simply be required for the nuclear export of mRNAs for meiotic cell-cycle regulators, such as CycB or twine (Moon, 2011).

    The finding that PC localization to the nucleolus requires tTAFs raised a hypothesis that the nucleolus acts as a sequestering compartment for counteracting transcriptional silencing by PcG proteins in Drosophila spermatocytes. If this is the case, transcription of tTAF target genes might be affected in thoc5 mutant spermatocytes because the nucleolar localizations of both PC and the tTAF SA are abnormal in the thoc5 mutant. Surprisingly, however, tTAF target genes were transcribed; and even more surprisingly, their transcripts were still exported to cytoplasm in thoc5 mutant spermatocytes, suggesting that the nucleolar localization of tTAFs and PcG proteins is not essential for counteracting transcriptional silencing by PcG proteins. In addition to this, a male-sterile gene, bol, which is a target of another testis-specific meiotic arrest complex, tMAC, was also expressed and exported independently of THOC5. Taken together, these results suggest that the meiotic arrest phenotype in thoc5 might not be caused by the failure of the canonical THO function, mRNA export (Moon, 2011).

    The finding that don juan (dj) transcript normally appeared to be retained in the nucleus at the pre-meiotic stage of spermatogenesis is very interesting. The transcription of dj mRNA is known to be initiated in early spermatogenesis, but its translation is normally delayed until meiotic divisions are completed. Although it has been reported that translational repression of dj mRNA is mediated by the TRE (dj translational repression element) located at the 5'-UTR, it is speculated that nuclear retention of dj mRNA might be an additional mechanism by which dj mRNA is translationally suppressed. In mammalian cells, a novel regulation mechanism of gene expression through RNA nuclear retention has been recently proposed. In this model, certain mRNAs containing elements for adenosine-to-inosine editing within their 3'-UTR are retained in nuclear paraspeckles, and may be released when the demand for their protein products increases. Although it is unclear whether Drosophila has a similar regulatory mechanism, the nuclear retention of dj mRNA in pre-meiotic spermatocytes might be a sign of the existence of such a mechanism. Further studies are required to clarify this issue. From the finding that nuclear mRNA retention is not generally applicable to other tTAF target genes, the possibility that the dj probe might cross-hybridize with some other nuclear RNAs concentrated in a certain nuclear structure, such as the Y-loop, cannot be ruled out. However, this is unlikely because the signal was absent in mia mutant spermatocytes in which dj is not expressed; moreover, the nuclear dj message was still detectable in the testes of XO males, which lack the Y-loop. A previous report (Santel, 1997) that showed a strong cytoplasmic signal of dj mRNA by whole-mount in situ hybridization with colorimetric detection is also inconsistent with the current result. However, this study also failed to detect a clear signal of dj massages in the nucleus by a similar method. Thus, the discrepancy was probably due to methodological differences. Whether the signal detected by a dj anti-sense probe represents genuine dj message or not, nuclear retention of this RNA appears to be dependent on the THO complex (Moon, 2011).

    The THO complex is not a testis-specific protein complex. Why, then, might the testis be more sensitive to loss of THOC5 than other tissues are? There are possible reasons for this. First, THOC5 might not be essential for the function of the THO complex. In the thoc5 mutant, a significant amount of HPR1 was still detected in the nucleus, although its level was greatly reduced. Interestingly, the residual HPR1 was mainly located near the chromatins rather than the nucleolus. Thus, the residual subunits might still have some degree of activity, and this residual activity might be sufficient for most cells, but not enough for other cells, such as spermatocytes. Second, in addition to mRNP biogenesis, the THO complex might have a non-canonical function in the spermatocytes, such as the organization and/or maintenance of nucleolar structure, and possibly the localization of some nuclear proteins and RNAs. Third, spermatogenesis requires the coordinated expression of a large number of genes. To ensure that the spermatocytes regulate gene expression in a coordinated manner from transcription to nuclear export, the THO complex might still have a role, although its specific target mRNA was not detected (Moon, 2011).

    In summary, disruption of THOC5 caused severe defects in the primary spermatocyte nucleus, including nucleolar disruption, abnormal distribution of proteins (SA and PC) and an RNA transcript (dj), suggesting that it has a role in the establishment or maintenance of subnuclear structure in Drosophila primary spermatocytes. The main causative factor of meiotic arrest in thoc5 mutants might be the disruption of subnuclear structure rather than the defect in nuclear mRNA export (Moon, 2011).

    In addition to the meiotic arrest phenotype reported in this study, the thoc5 mutant also has other phenotypes, including wing bubble and uncoordinated behavior. Recent work also showed that longevity and tolerance to environmental stress were significantly reduced in the THO mutants. This suggests that the role of the THO complex is not limited to spermatogenesis, but is also important for other types of cells. When this study examined whether a similar nucleolar defect is seen in other cell types, no similar defect was found in other cell types except the salivary gland cells. This suggests that the specific role of the THO complex is different depending on the cell type. It will be interesting to clarify the specific roles of the THO complex in other types of cells (Moon, 2011).

    Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues

    Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on losing access to these signals. This study demonstrates that differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot properly differentiate. An insulin peptide produced by somatic cells immediately outside of the stem cell niche was found to act locally to promote somatic differentiation through Insulin receptor (InR) activation. These results indicate that there is a local 'differentiation' niche which upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, it was demonstrated that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, this study shows that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that local production of positive and negative InR signals regulate the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation is actively induced by signaling (Amoyel, 2016b).

    This study shows that PI3K/Tor activity is required for the differentiation of somatic stem cells in the Drosophila testis. Additionally, a 'differentiation' niche was identified immediately adjacent to the stem cell niche that, through the local production of Dilps, leads to the upregulation of PI3K/Tor activity in early CySC daughters and to their commitment to differentiation. The secretion of ImpL2 by CySCs antagonizes the initiation of differentiation in CySCs by blocking available Dilps in the stem cell niche. As a result, CySCs receive little free Dilp ligands. However, as their daughters move away from the hub, they encounter increasing levels of Dilps and decreasing levels of ImpL2, which leads to the upregulation of PI3K/Tor signaling and proper somatic cell differentiation. The fact that ImpL2 is upregulated by the main self-renewal signal (i.e., JAK/STAT) in CySCs leads to a model accounting for the spatial separation of the stem cell niche and the differentiation niche (Amoyel, 2016b).

    The results are consistent with a model in which autocrine or paracrine production of Dilp6by early cyst cells serves as a differentiation niche in the testis, defining where in the tissue upregulation PI3K/Tor signaling - a prerequisite for differentiation - occurs. This differentiation niche is critical for somatic development because stem cell markers like Zfh1 are maintained in the absence of signals like PI3K/Tor. Notably, JAK/STAT activity is not expanded outside of the niche upon somatic loss of PI3K/Tor signaling, suggesting that differentiation signals play a critical role in downregulating stem cell factors. Intriguingly, recent studies in the Drosophila ovary have identified a differentiation niche in this tissue: autocrine Wnt ligands produced by somatic support escort cells regulate escort cell function, proliferation and viability. Taken together, these studies reveal that at least in Drosophila gonads, there is a defined region immediate adjacent to the stem cell niche where autocrine production of secreted factors induces the differentiation of somatic cells, which in turn promote development of the germ line (Amoyel, 2016b).

    Several studies have examined the role of insulin signaling in gonadal stem cells. In both testes and ovaries, systemic Dilps have been shown to affect stem cell behavior. In both tissues, nutrition through regulation of systemic insulin controls the proliferation rate of GSCs. The current data showing that Akt1, Dp110 or Tor mutant CySC clones proliferate poorly are consistent with these findings and indicate that basal levels of insulin signaling are required for the proliferation and/or survival of both stem cell pools in the testis. This work also demonstrates that production of a secreted Insulin binding protein ImpL2 by CySCs reduces available Dilps in the stem cell niche, and ImpL2 in the niche milieu should reduce insulin signaling in GSCs and CySCs. While these data seemingly contradict the results that insulin is required for GSC maintenance, a model is suggested in which low constitutive levels of insulin signaling are required for stem cell proliferation and that higher levels are required to induce stem cell differentiation. (Amoyel, 2016b).

    Prior reports have found that both male and female flies with reduced Insulin or Tor activity are sterile, and the results presented in this study suggest that this is due at least in part to a lack of somatic cell differentiation. The results indicate that Dilp6, the IGF homolog, plays a local role in CySC differentiation, but acts redundantly with other presumably systemic factors, suggesting that both constitutive and nutrient-responsive inputs control CySC differentiation. Indeed, this study shows that in addition to controlling the proliferation of stem cells, systemic insulin is required for their differentiation, as the poorly proliferative Akt1, Dp110 or Tor mutant CySC clones do not differentiate and eventually die by apoptosis. This combination of reduced proliferation and increased apoptosis may explain why other studies suggest that Tor is required for self- renewal in GSCs; indeed prior reports indicate that while Tor mutant GSCs are lost, hyper-activation of Tor leads to faster loss of GSCs through differentiation and recent work indicates that lineage-wide Tor loss blocks the differentiation of GSCs. The use of hypomorphic alleles enabled a genetic separation of the proliferative effects and differentiation requirements of PI3K and Tor in CySCs. Finally, there is evidence that PI3K/Tor activity promotes differentiation of stem cells in gonads in mammals, suggesting that these findings may reflect a conserved role of Tor activity in promoting germ cell differentiation, both through autonomous and non- autonomous mechanisms involving somatic support cells. Moreover, it seems likely that Tor activity may be a more general requirement for the differentiation of many stem cell types, as increased PI3K or Tor has been shown to induce differentiation in many instances. In particular, mouse long term hematopoietic stem cells are lost to differentiation when the PI3K inhibitor Pten is mutated, while Drosophila intestinal stem cells differentiate when Tor is hyperactive due to Tsc1/2 complex inactivation. Moreover, inhibition of Tor activity by Rapamycin promotes cellular reprogramming to pluripotency, while cells with increased Tor activity cannot be reprogrammed, suggesting a conserved role for Tor signaling in promoting differentiated states (Amoyel, 2016b).

    The search for Y-linked genes: Y chromosome fertility factors encode dynein heavy chain polypeptides

    The molecular identity and function of the Drosophila melanogaster Y-linked fertility factors have long eluded researchers. Although the D. melanogaster genome sequence has recently been completed, the fertility factors still are not identified, in part because of low cloning efficiency of heterochromatic Y sequences. A method for iterative BLAST searching has been used to assemble heterochromatic genes from shotgun assemblies, and kl-2 and kl-3 have been identified as 1-beta and gamma-dynein heavy chains, respectively. These conclusions are supported by formal genetics with X-Y translocation lines. Reverse transcription-PCR was successful in linking together unmapped sequence fragments from the whole-genome shotgun assembly, although some sequences were missing altogether from the shotgun effort and had to be generated de novo. A previously undescribed Y gene, polycystine-related (PRY), was also found. The closest paralogs of kl-2, kl-3, and PRY (and also of kl-5) are autosomal and not X-linked, suggesting that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes arising de novo from the autosomes (Carvalho, 2000).

    The discovery that the Y chromosome of Drosophila melanogaster contains genes essential only for male fertility dates back to the birth of Drosophila genetics and the theory of chromosomal inheritance. In 1929, Stern showed that these genes are localized in both the short (YS) and long (YL) arms of the Y chromosome, and in 1960, Brosseau used x-ray-induced mutations to identify seven complementation groups, two in YS (ks-1 and ks-2) and five in YL (kl-1 to kl-5). In 1981, Kennison obtained fertile X-Y translocation lines and used them to construct males with deletions in each of the fertility factors. With these lines, Kennison confirmed six of the seven fertility factors previously identified by Brosseau (kl-4 was not confirmed). The same lines allowed a more precise identification of the defects associated with the lack of each of the fertility factors. In particular, the lack of kl-3 or kl-5 causes the loss of the outer arm of the sperm tail axoneme, a structure known to contain the molecular motor protein dynein in other organisms. Indeed, in 1982, Goldstein showed that sperm from kl-3- and kl-5- (and also kl-2-) males lack three discrete high molecular weight proteins with mobility similar to dynein heavy chains of Chlamydomonas reinhardtii and proposed that these fertility factors are the structural genes of three different dynein heavy chain proteins. In 1993, Gepner and Hays sequenced part of kl-5 and showed that it encodes an axonemal beta-dynein heavy chain that is expressed in the testis (Carvalho, 2000 and references therein).

    Axonemal dynein heavy chains are known to be responsible for the beating of flagella and cilia, which explains why kl mutants produce immotile sperm. There are several isoforms of axonemal dynein heavy chains (alpha, beta, gamma, 1beta, 1alpha, etc.) that associate to form the inner and outer arms of the axonemes. D. melanogaster has at least seven other dynein heavy chain genes, scattered in chromosomes X, 2, and 3 (Carvalho, 2000 and references therein).

    Another important experimental breakthrough was the development of a method to discern banding patterns in Drosophila heterochromatin, which allowed the first detailed cytogenetic investigation of the Y chromosome. Gatti and Pimpinelli (1983) identified 25 heterochromatic bands on the Y and mapped the fertility factors to these bands. It became clear that some of the fertility factors, including kl-5, are unusually large [~3 megabase (Mb)]. The paradox of a conventional coding gene (e.g., kl-5), spread over a huge amount of DNA was solved by Bünemann and coworkers: in the kl-5 homolog of Drosophila hydei, some of the introns are gigantic (>1 Mb) and most likely account for the unusual size of the gene. These introns are composed of short repetitive sequences and satellite DNA. These key discoveries trace back to the extensive work on lampbrush Y chromosomes initiated by Meyer and coworkers in 1961 (Carvalho, 2000 and references therein).

    As can be seen from the above summary, the progress on the identification of Y-linked genes has been very slow. This slow progress is mainly a consequence of the technical difficulties caused by the heterochromatic state of the Y chromosome, and most of the experimental breakthroughs mentioned above actually are ingenious ways to implement standard tools used for euchromatic genes in heterochromatin. The Y chromosome does not recombine during meiosis, preventing classical genetic mapping; this problem was solved by Kennison's lines. It does not undergo polytenization, making cytogenetic studies more difficult [solved by Gatti and Pimpinelli]. P element mutagenesis was also more difficult, because the common markers are often silenced when inserted in the Y, but now there are special P constructs that make it possible to overcome this limitation (Carvalho, 2000 and references therein).

    The recent sequencing of the Drosophila genome might have yielded the final solution, but again the heterochromatic nature of the Y chromosome posed special difficulties. Most heterochromatin is composed of short repetitive sequences that are not stable in the vectors used in sequencing projects. Thus, despite comprising nearly 30% of the genome, heterochromatic sequences account for only 2% of the sequence reads. Furthermore, its repetitive nature does not allow the assembly of the individual sequence reads (~500 bp) into larger scaffolds, and these into complete chromosome arms. As a result, only 15 kb (a small portion of the kl-5 gene) have been assigned to the Y chromosome, whereas essentially all of the 120 Mb of the euchromatin have been assembled into chromosomes X, 2, 3, and 4. Besides these mapped sequences, 631 scaffolds (ranging from 1 kb to 64 kb, and totaling ~4 Mb of sequence) remain unmapped. These unmapped scaffolds most likely contain pieces of heterochromatic genes, including Y-linked ones (Carvalho, 2000 and references therein).

    Of special interest in the study has been the inventive use of iterative BLAST searching for assembly of heterochromatic sequences. The unmapped Drosophila scaffolds (called "armU" in Celera's CD-ROM release of the Drosophila genome) were downloaded from, and then an armU database was built by using the FORMATDB program of the STANDALONE BLAST. In this way BLAST searches could be restricted to the set of unmapped scaffolds. In addition to STANDALONE BLAST, extensive use was made of the programs WWWSTANDALONE BLAST (Linux version), NETBLAST, REPEATMASKER (available at, and NAP and GAP2 (available at BLAST programs were downloaded from the National Center of Biotechnology Information ( (Carvalho, 2000 and references therein).

    To see how a Y-linked gene appears in the armU sequences, the complete cDNA of kl-5 (Genbank nucleotide record: AF210453) was used as a query sequence in a BLASTN search against the armU database. In addition to the fragment already identified (CG17616 gene in the Genbank AE002688 scaffold), most of the kl-5 gene was retrieved, scattered across five scaffolds. The majority of these scaffolds contain complete exons (the exception is AE003233, which begins in the middle of an exon). Their 3' ends contain the 5' splice junctions and a variable portion of downstream intronic sequences, often ending with simple repetitive sequences. The 5' ends of scaffolds in armU have analogous structures. Some exons are missing altogether in armU. These observations fit well with the expected behavior of a gene like kl-5 in whole genome shotgun (WGS) projects: exons define unique, nonrepetitive sequences that will be cloned regularly and will be assembled into at least a small scaffold in the end of WGS. Normally sized introns will be readily cloned and assembled along with exons. Indeed, most of the kl-5 scaffolds contain several exons and the intervening short introns. However, some introns of kl-5 probably contain Mega base-sized blocks of repetitive DNA that cannot be assembled by WGS or any other available method. These fragments will rarely be cloned and sequenced and, even if sequenced, would not be assembled into a scaffold. In short, during WGS, a gene like kl-5 will be chopped into several pieces, delimited by the unclonable intronic satellite DNA. Most of the time, a gene immersed in heterochromatin will go undetected by the normal 'first pass' annotation procedures (which rely on gene prediction tools and BLASTX with high stringency), because these methods will work poorly with individual exon sequences. However, the whole gene may be retrieved if a suitable query sequence is available (the kl-5 cDNA in this case) to identify and align its pieces. Very small exons embedded in large introns will most likely be lost during the WGS, and this probably explains the lack of some kl-5 exons (Carvalho, 2000).

    To identify other fertility factors in armU, protein sequences were used as query sequences. Suitable proteins were chosen as follows. First the 631 scaffolds of armU were filtered with REPEATMASKER and a BLASTX search of each of them was performed against the nr database (all known proteins, including putative ones) with a rather high stringency (e = 10-4). There were proteins that gave hits in hundreds of scaffolds; most of them are reverse transcriptases, copia polyprotein, etc., and most likely are matching transposable elements of armU that 'escaped' REPEATMASKER. Some other proteins have hits in a few scaffolds; these are homologs of prospective Y-linked genes, chopped in pieces as kl-5. Each of these prospective proteins was used as a query sequence (as was done with the cDNA of kl-5), running TBLASTN with a lower stringency (e = 10) against the armU database. A staggered pattern results from the very large introns of Y-linked genes. Two such cases are myosin VII (AAF06035, from Dictyostelium discoideum) and gamma-dynein heavy chain, from C. reinhardtii], and were investigated further. All tested scaffolds relating to myosin VII proved to be not Y-linked (i.e., PCR produces bands when either male or female DNA is used as the template), whereas most of the dynein-related were Y-linked. Several of the Y scaffolds were identified by using gamma-dynein heavy chain (Genbank record: Q39575) as a TBLASTN query sequence with a low stringency (sometimes e = 1,000), in an attempt to retrieve missing exons. Because there is a big overlap among several of the dynein-related scaffolds, most likely two different Y-linked dynein heavy chain genes were found (Carvalho, 2000).

    Genomic DNA from Y deficient males (kl-1-, kl-2-, etc.) was used in PCR to map each of the Y-linked scaffolds identified in the previous step. It should be noted that this procedure assigns a given scaffold to a region of the Y chromosome (e.g., the kl-2 region), but it does not necessarily imply that this scaffold belongs to the actual fertility gene. This distinction is important, because a given region may contain more than one gene. For the sake of simplicity the regions of the Y are referred to by the name of the respective fertility factor they carry (Carvalho, 2000).

    Several exons of kl-2 and kl-3 genes are missing from the BLAST results. These missing exons may be absent in armU sequences (as happened with kl-5) or may have diverged enough to be no longer identified by these methods. RT-PCR was used to obtain the sequence of these missing exons and to check whether the Y sequences that were detected are expressed. RT-PCR sequences were obtained from all splice junctions between adjacent scaffolds so that they could be precisely identified. The sequencing of the gaps revealed several previously missed armU scaffolds. In kl-2, some 330 codons of the N terminus are still missing. AE003086 filled the gap between AE003157 and AE002962. There is no sequence gap between AE002962 and AE003049. AE002706 filled a small portion of the gap between AE003049 and AE003219; the remaining 2 kb were sequenced and it was found that it is entirely missing in armU. The AE003219 scaffold contains five internal, short introns and extends through the stop codon. Regarding kl-3, AE002577 and AE002776 appear to be spurious matches caused by running TBLASTN with low stringency, because no RT-PCR product could be recovered that includes these sequences. The gap between AE002917 and AE002920 was sequenced; the 948-bp sequence is missing in armU. Finally, some 230 codons in the C terminus seem to be missing. Each of the three big kl-3 scaffolds contains one internal intron (Carvalho, 2000).

    The internal introns were identified and localized with the NAP program, which aligns genomic DNA with proteins allowing for GT/AG bounded gaps (in the case presented here, armU scaffolds and the gamma-dynein Genbank sequence Q39575 were aligned). RT-PCR sequences surrounding each putative intron were obtained and aligned with the corresponding armU scaffold with the GAP2 program, which aligns genomic DNA with cDNA, again allowing for GT/AG bounded gaps. Almost all putative introns suggested by NAP were confirmed, although the inferred splice junctions frequently were not precise. Seven frame-shift sequence errors in armU sequences were pinpointed by NAP and BLASTX and were corrected by sequencing (Carvalho, 2000).

    The assembled cDNA of kl-2 and kl-3 were deposited in GenBank under the accession numbers AF313479 and AF313480. Polycystine-related (PRY) is a putative, previously unidentified Y-linked gene. During attempts to retrieve missing exons two armU scaffolds were found that map to the kl-5 region but have no similarity with the kl-5 cDNA. AE002774 seems to contain only two short pieces of transposable elements and was not further investigated. AE003011 showed a strong similarity with the product of a putative gene localized in chromosome 2 (AAF44887) and also a weaker similarity with the human polycystine protein (AAD18021). Interestingly, polycystine is similar to the sea urchin sperm receptor for egg jelly (AAB08448). Using the Drosophila hypothetical protein AAF44887 as a query sequence in TBLASTN (against armU sequences) another closely related scaffold, AE003212 was recovered. RT-PCR closed the gap between it and AE003011; thus, they most likely are part of a previously unidentified expressed Y-linked gene, which is currently being sequenced. Surprisingly, AE003212 maps to the kl-3 region. These findings imply that the breakpoint of the V24 translocation (the h4 band) cuts the PRY gene in the middle. Thus, V24 is defective for PRY -- a close examination of this line may give some clue about the function of this gene. Because the kl-3 and kl-5 regions are known to contain factors (other than the dyneins) that cause sterility when present in three copies, it is possible that PRY is responsible for this phenotype (Carvalho, 2000).

    A simple method has been described for tailoring BLAST searches in such a way that poorly assembled fragments from WGS projects may reveal genes embedded in heterochromatin. The method relies on TBLASTN searches (instead of the more usual BLASTX) to identify putative heterochromatic genes by the distinct staggered pattern they produce. It was applied and the kl-2 and kl-3 genes on the Y chromosome of D. melanogaster were successfully identified. The sequences that were found are long, transcribed open reading frames that encode dynein heavy chain polypeptides. Thus the quest for the Y dyneins initiated by Hardy is now finished, and Drosophila Y gene hunting may now move to the even more exciting task of identifying the mysterious kl-1, ks-1, and ks-2 (Carvalho, 2000).

    Heterochromatic genes are islands of unique sequence and appear in the end of WGS as isolated scaffolds that could not be assembled into chromosomes. If introns are large enough and contain heterochromatic repeat sequences, they will be sufficiently underrepresented in the WGS sequences to disrupt the assembly of flanking scaffolds. As a result, exons of the same gene are scattered in several unmapped scaffolds ('armU'), generating a staggered pattern in TBLASTN and BLASTN searches. This pattern will occur not only in the Y-linked genes but also in autosomal heterochromatin, as in the case of the Drosophila myosin VII homolog. Although heterochromatic genes pose special problems for genome sequencing, it is possible that they have an advantage over euchromatic genes: once the euchromatic sequence of a whole chromosome arm (a Mb-sized, unique sequence scaffold) is obtained, it may be very difficult to detect all of the genes it contains, whereas it is possible that a significant proportion of the unique heterochromatic sequences represents functional genes. It seems that only functional sequences resist the forces that fill heterochromatic regions with short repeats and thus remain clonable, unique sequences. During this project, 38 armU scaffolds were examined, and at least 12 of them (~1/3) seem to be part of genes. Of course, this sample is not random, but it strongly suggests that the small isolated scaffolds remaining at the end of WGS projects may be a good source of interesting genes. Indeed, it is an advantage of the WGS approach over clone-based strategies that, besides the euchromatin, it also retrieves unique sequence heterochromatin, no matter how deeply the sequence is located within the heterochromatin (Carvalho, 2000).

    A striking pattern emerges from the phylogeny of the Y dyneins: they all are closely related to other Drosophila genes, but none of these paralogous genes is X-linked. The same pattern occurs with PRY. Furthermore, the Drosophila X chromosome contains only one dynein heavy chain (Dhc 16F), in contrast with the three Y-linked ones. Thus, it seems likely that these genes were acquired from autosomes, rather than being present in the hypothetical chromosome pair that gave rise to the X and Y. This mechanism has been demonstrated for the mammalian Y, but in that case the Y chromosome also exhibits a number of X-derived genes. Another Drosophila Y-linked gene, Su(Ste), has been shown to be recently originated from an autosomal gene. Repetitive sequences also do not show any sign of X-Y homology; it has been proposed that much of the Drosophila Y is virtually a new construct, rather than a degenerated X. The data presented in this study clearly support this hypothesis. It remains to be seen whether any part of the ancestral Y was homologous to the X (as may be the case for rDNA genes, which are present in both X and Y chromosomes) or whether it is a totally new construct. This picture of the Drosophila Y may change if other, yet unidentified Y genes (kl-1, ks-1, ks-2, etc.) turn out to have X homologs. Whatever its origin, the present configuration of the Drosophila Y chromosome seems to be quite old, for at least kl-5 is present also in D. hydei and Drosophila mediopunctata, which diverged from D. melanogaster ~39 million years ago. A few Drosophila species have fertile X0 males; it will be most interesting to study the location of their axonemal dynein heavy chain genes (Carvalho, 2000).

    The absence of X homologs and the close similarity between Y and autosomal genes suggest that the former is an agglomeration of autosomal genes. This hypothesis is the most parsimonious and explains well the kl-5, kl-3, and PRY cases. However, it is also possible that Y chromosomal genes have transposed to the autosomes, and this possibility might explain the kl-2 case. The closest paralogs of kl-3 and kl-2 are the CG9492 and CG9068 genes, respectively. Dynein heavy chains have ~4,500 amino acids, whereas CG9492 and CG9068 are shorter (3,508 and 1,227, respectively) and seem to lack the C terminus. The former case results from a misannotation: BLASTX and NAP identified all of the missing ~1,000 amino acids of CG9492 (including the stop codon at position 188,944 in the AE003683 scaffold). However, CG9068 seems to be truncated, for no sign of the 'missing' C terminus could be found. Therefore, the relationship between CG9068 and kl-2 is unclear; it is possible that kl-2 originated from CG9068 and that after this the latter suffered a deletion. However, it is also possible that CG9068 results from a partial transposition (perhaps being a pseudogene) of kl-2 (Carvalho, 2000).

    The phylogeny of the dyneins strongly suggests that kl-2 encodes a 1beta-dynein, whereas kl-3 encodes a gamma-dynein. This phylogeny fits well with the known mutant phenotypes of kl genes and with the function of dynein heavy chains; kl-3- mutations (but not kl-2-) disrupt the outer arms of axonemal microtubules, and gamma-dyneins are part of these structures. 1beta-dyneins are part of the inner arms, and it remains to be explained why kl-2- mutants do not show cytological defects (Carvalho, 2000).

    It has been noted that the human Y chromosome exhibits a 'functional coherence'; besides housekeeping genes, many Y genes have male-related functions, which contrasts with the random content of the other chromosomes. It is striking that the Drosophila Y has an even stronger coherence, approaching obsession; all known fertility factors (kl-2, kl-3, and kl-5) encode proteins belonging to the same gene family (axonemal dynein heavy chain). This extreme functional coherence, coupled with the lack of X homologs (which might provide an 'historical' cause), begs for an explanation (Carvalho, 2000).

    Theoretically, the Y chromosome is expected to accumulate male-related genes; male-female antagonistic effect of genes may hamper the evolution of male-related traits, unless they are located in a male-specific region of the genome. This prediction has been demonstrated experimentally, and the findings presented here support it. Regarding the particular male fitness trait involved, the most likely advantage conferred by sperm axonemal motor proteins is sperm competitive ability. The PRY gene may also be involved in sperm competition if it has a function similar to its homolog in sea urchin. Drosophila females mate several times; thus, there is ample room for sperm competition, and clearly there is genetic variation for this trait. It is proposed that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes, most likely caused by sperm competition. This hypothesis explains the puzzling finding of a Y chromosome packed with motor proteins which are absent in the X chromosome. The large element of chance involved in the occurrence of the appropriate translocations probably explains the apparent incompleteness of the process, that is, outer arms are composed of alpha-, beta-, and gamma-dyneins, but only beta and gamma got Y counterparts (Carvalho, 2000).

    The hypothesis that natural selection has driven an accumulation on the Drosophila Y of genes related to sperm function may be tested in several ways. (1) Studies designed to quantify Y-linked variation in sperm competition are clearly needed. (2) The comparative method of looking for dynein heavy chain genes in other Diptera (including species with fertile X0 males) may reveal the intermediate steps of the birth of dynein-packed Y chromosomes. (3) the identification of the other fertility factors may yield more clues about the forces shaping Y chromosome evolution in Drosophila (Carvalho, 2000).

    Post-meiotic transcription in Drosophila testes

    Post-meiotic transcription has been thought to be essentially absent from Drosophila spermatogenesis. This study identified 24 Drosophila genes whose mRNAs are most abundant in elongating spermatids. By single-cyst quantitative RT-PCR, post-meiotic transcription of these genes was identied. It is concluded that transcription stops in Drosophila late primary spermatocytes, then is reactivated by two pathways for a few loci just before histone-to-transition protein-to-protamine chromatin remodelling in spermiogenesis. These mRNAs localise to a small region at the distal elongating end of the spermatid bundles, thus they represent a new class of sub-cellularly localised mRNAs. Mutants for a post-meiotically transcribed gene (scotti), are male sterile, and show spermatid individualisation defects, indicating a function in late spermiogenesis (Barreau, 2008).

    Many genes with unknown functions have testes-specific expression. To determine when during spermatogenesis these proteins are made, the transcript patterns of >1200 genes was examined by in situ hybridisation. The expression of spermiogenesis genes in primary spermatocytes, and the storage of transcripts for later use during spermiogenesis, means that the translation of specific mRNAs in Drosophila spermatids correlates well with their disappearance, as translation exposes stored mRNAs to the RNA degradation machinery. In summary, 529 of the 553 mRNAs detected in spermatids were transcribed in primary spermatocytes, persisted in the spermatid cytoplasm, and were degraded at various stages in elongation. Unexpectedly, 24 germ-line expressed genes were found that did not conform to this pattern. These were subdivided on the basis of subtle differences in transcript localisation patterns, and are refered to collectively as 'comets and cups' (Barreau, 2008).

    Comet and cup transcripts were detected at very low levels in primary spermatocytes by RNA in situ hybridisation, and were barely detected in early elongation spermatids. However, robust signals, with striking subcellular localisations, were evident in more elongated spermatids. Spermatid nuclei are located at one end of these elongated cells, in the basal-most region of the testis; comet and cup mRNAs were localised to the distal ends of the spermatids, in subtly different patterns. 'Comet' mRNAs localised into a ball shape at the ends of spermatid bundles, trailing away proximally to a less abundant, speckled distribution. 'Cup' transcripts localised in shallow cup-like shapes at the ends of spermatid bundle (Barreau, 2008).

    Comet and cup expression patterns are extremely unusual. The obvious explanation for the abrupt mRNA appearance during spermatid differentiation is post-meiotic transcription. Alternatively, the transcripts could be present earlier, but either (1) diffuse or (2) masked, so undetectable by in situ hybridisation (Barreau, 2008).

    To verify the post-meiotic transcription, and to determine its timing with respect to cellular differentiation events, a single-cyst quantitative reverse transcription PCR (Q-RT-PCR) protocol was developed. Testes were dissected, and individual cysts isolated, photographed, and staged according to morphology; total RNA was then isolated and first strand cDNA synthesised. Each cyst yielded cDNA for 60 Q-RT-PCR reactions. Testis-specific control genes were chosen. The CG10252 transcript conforms to the conventional pattern for a late elongation protein and CG10252 protein is detected in mature sperm. CG3927 was detected exclusively in primary spermatocytes; CG11591 was expressed in primary spermatocytes and the signal disappeared from mid-elongation spermatids. CG3927 and CG11591 controlled for cyst visual staging. For each cyst, expression levels of both staging controls and up to eight test genes were compared with the internal control CG10252. The 13 comet and cup gene transcripts assayed by isolated-cyst Q-RT-PCR showed broadly similar profiles in Q-RT-PCR assays. All transcripts were detected in primary spermatocytes and round spermatids. sunz, sowi, soti, c-cup, d-cup, wa-cup, p-cup and r-cup were low or not detected in very short elongating cysts, but were detected at high levels in a few longer spermatid cysts. hale, schuy, boly, cola and swif were detected at a basal level in almost all cysts, but were much more abundant in a few mid-elongation bundles. From these differences, two separate regulatory modules activating post-meiotic gene expression were inferred, with the hale group being transcribed in more cysts than the sunz group. Spermatid length measurements give good staging of the relative differentiation states of cysts from a single testis, but the exact length of spermatids expressing comets and cups varied between testes. The initial low-level signal in primary spermatocytes, the dip in signal intensity in early spermatids, then the dramatic appearance in later spermatids conclusively demonstrate that there is post-meiotic transcription in Drosophila testes (Barreau, 2008).

    In Drosophila, bulk histone removal initiates in the early 'canoe' stage of nuclear remodelling, and protamine deposition is complete by late canoe stage. To determine comet and cup transcription timing with respect to chromatin reorganisation, cysts were staged via combined fluorescent fusion-protein localisation and spermatid-length measurements. Cysts were isolated from flies co-expressing Mst35Ba-GFP (protamine-GFP) and H2A-mRFP1 (histone-GFP). Protamine accumulation initiates before all histones have been removed, as some nuclei fluoresced both red and green. Transcription of comet and cup genes was detected in mid-elongation cysts that were positive for histone and negative for protamine. Thus, comet and cup transcription occurs just before the deposition of protamines. Some comet and cup mRNAs were also detected in cysts positive for protamine-GFP. This could be due to ongoing transcription, or to message stability. The recently described active transcription in spermatids coincides with the comet and cup gene transcription peak (Barreau, 2008).

    These experiments were repeated using cysts isolated from flies co-expressing Tpl94D-GFP (transition protein) and H2A-mRFP1. Initiation of post-meiotic comet and cup gene expression was found in cysts lacking nuclear Tpl94D, indicating that comet and cup gene transcription initiates before the deposition of transition protein, while chromatin is presumably still nucleosomal (Barreau, 2008).

    Approximately 4 kb of genomic DNA, including the entire scotti (soti, a comet) ORF, was depleted by FLP-mediated recombination between flanking FRT-containing transposons. soti homozygous mutants were viable and female fertile, but male sterile. Phase contrast microscopy indicated no gross defects in soti testes organisation or spermatid elongation; however, empty seminal vesicles indicated spermiogenesis defects. Within each individualising spermatid cyst, 64 actin-rich investment cones move together as an individualisation complex, pushing ahead a cystic bulge of excess cytoplasm and organelles. This cytoplasm is discarded from spermatid distal ends as a waste bag. Waste bags were completely absent from mutant testes, and cystic bulges were rarely seen. FITC-phalloidin labelling revealed that investment cones formed normally in soti mutant males; however, nuclei failed to remain tightly clustered and were displaced distally along the cyst. Although investment cones progressed away from the nuclei in mutants, investment cone coupling within individualisation complexes was lost, and cones never progressed the full length of mutant spermatids. Thus, soti function is required for spermatid individualisation (Barreau, 2008).

    Post-meiotic transcription, in early spermatids, has been reported for two loci in Drosophila, hsr-omega and Hsp70; however, this study has been unable to reproduce these findings. Ninety-six percent of genes whose mRNAs were detected in spermatids are not actively transcribed in these cells (being made in spermatocytes), so what is special about the exceptional 4% - the comets and cups? These genes are found throughout the euchromatin, including the X chromosome, and their local genomic environments showed no unusual features. Their flanking genes showed no bias towards or away from testis-specific expression in adults. There are three comet and cup gene clusters, two of which clearly represent gene duplication events. The final cluster comprises hale and schuy; both encode glutamine-rich proteins, but their evolutionary history is unclear. The expression of all 10 related genes in the CG11635-CG8701 cluster was investigated. CG11635, CG18449, CG2127 and CG8701 were expressed in the conventional spermiogenesis gene pattern - transcribed in primary spermatocytes and stored until late elongation - while spaw, hubl, swif, cola, boly and whip were typical 'comets'. Q-RT-PCR confirmed that the post-meiotic transcription and RNA localisation to distal ends of spermatids were correlated (Barreau, 2008).

    In mammals, the transcription of many genes in spermatids has been described, and new reports are frequent. These mammalian genes typically, although not exclusively, encode components of the mature sperm. By contrast, Drosophila comet and cup proteins, with the exception of Boly and Pglym87, are not sperm components. Perhaps comet and cup proteins function, like Soti, during spermiogenesis, rather than in sperm. Alternatively, perhaps they are present in sperm but at a very low copy number. mRNAs of several comet and cup gene homologues were transcribed in conventional patterns, and the encoded proteins detected in sperm. orb (a comet) encodes an RNA-binding protein, potentially anchoring other comet and cup mRNAs. The other comet or cup proteins have no predicted function. PKD2 encodes a Ca2+-activated non-selective cation channel, and it is intriguing that a Drosophila PKD2 homologue (Pkd2/Amo) concentrates at the distal ends of sperm, and is important for sperm function. Sunz, Sowi and D-cup are EF-hand-containing proteins, and so could function with Pkd2 in mediating a Ca2+ signal at the spermatid tail tip. This signal could then be transduced along spermatid tails, perhaps via the mitochondrial derivative or the endoplasmic reticulum-derived axonemal sheath, which stretches the length of spermatid tails, activating the apoptotic pathway to synchronise individualisation and ensure normal investment cone progression (Barreau, 2008).

    In conclusion, there is significant transcription from several genomic loci in Drosophila spermatids, and the post-meiotically expressed transcripts localise to the growing ends of spermatids. This transcription and RNA localisation occurs before spermatid chromatin remodelling, and at least one post-meiotically-expressed gene is required for spermiogenesis (Barreau, 2008).

    The B-type lamin is required for somatic repression of testis-specific gene clusters

    Large clusters of coexpressed tissue-specific genes are abundant on chromosomes of diverse species. The genes coordinately misexpressed in diverse diseases are also found in similar clusters, suggesting that evolutionarily conserved mechanisms regulate expression of large multigenic regions both in normal development and in its pathological disruptions. Studies on individual loci suggest that silent clusters of coregulated genes are embedded in repressed chromatin domains, often localized to the nuclear periphery. To test this model at the genome-wide scale, transcriptional regulation of large testis-specific gene clusters was studied in somatic tissues of Drosophila. These gene clusters showed a drastic paucity of known expressed transgene insertions, indicating that they indeed are embedded in repressed chromatin. Bioinformatics analysis suggested the major role for the B-type lamin, LamDm(o), in repression of large testis-specific gene clusters, showing that in somatic cells as many as three-quarters of these clusters interact with LamDm(o). Ablation of LamDm(o) by using mutants and RNAi led to detachment of testis-specific clusters from nuclear envelope and to their selective transcriptional up-regulation in somatic cells, thus providing the first direct evidence for involvement of the B-type lamin in tissue-specific gene repression. Finally, it was found that transcriptional activation of the lamina-bound testis-specific gene cluster in male germ line is coupled with its translocation away from the nuclear envelope. These studies, which directly link nuclear architecture with coordinated regulation of tissue-specific genes, advance understanding of the mechanisms underlying both normal cell differentiation and developmental disorders caused by lesions in the B-type lamins and interacting proteins (Shevelyov, 2009).

    It was hypothesized that, in addition to the somatic silencing of testis-specific gene clusters, LamDm0 is also required for attachment of these clusters to the nuclear envelope. To test this hypothesis, intranuclear positions of the 60D1 and 22A1 regions was determined in the interphase nuclei of cultured S2 cells in which LamDm0 was ablated by RNAi. Fluorescence in situ hybridization (FISH) combined with immunostaining for LamDm0 confirmed RNAi-induced ablation of LamDm0, and showed approximately 2-fold decrease in frequency of the 60D1 and 22A1 loci bound to lamina. Next, association of the 60D1 gene cluster with nuclear envelope wa analyzed during male germ-line differentiation. The whole-mount testes dissected from the third instar larvae were analyzed for intranuclear localization of the 60D1 region by FISH combined with immunostaining for LamDm0. Morphologically, a group of small cells is located at the end of testis and includes spermatogonia, cyst cells and stem cells, in which the 60D1 locus is silent. In these cells, the 60D1 region is associated with nuclear lamina in 76% of nuclei, similarly to the cultured somatic S2 cells. On the contrary, in spermatocytes (identified by characteristic large nuclei), in which testis-specific genes in the 60D1 region are expressed, this region is found at the nuclear envelope in only 6% of the nuclei. Thus, transcriptional activation of the testis-specific gene cluster in male germ line is coupled to its dissociation from the nuclear envelope. Similarly, detachment from the nuclear lamina has been associated with transcriptional activation of other lamina-bound loci both in Drosophila and in mammals. These observations strongly suggest a model in which gene repression is controlled in a cell type-specific manner through regulated tethering of chromatin to the nuclear lamina. Further dissection of the mechanisms that mediate repression of lamina-bound multigenic regions and control localization of these regions at nuclear envelope will provide new insights into coordinated regulation of tissue-specific genes, thus advancing understanding of cell differentiation both in normal development and in disease (Shevelyov, 2009).

    Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

    Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? This study investigated how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. The silenced state, set up in precursor cells, was found to be relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components [specifically E(z) and Su(z)12], recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC), a tissue-specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription (Chen, 2011).

    The results suggest a stepwise series of developmentally programmed events as terminal differentiation genes convert from a transcriptionally silent state in precursor cells to full expression in differentiating spermatocytes (see Model for the developmentally programmed steps that oppose PcG repression and turn on terminal differentiation gene expression). In precursor cells, differentiation genes are repressed and associated with background levels of hypophosphorylated Pol II and H3K4me3. These genes also display elevated levels of H3K27me3 and Polycomb at the promoter region, suggesting that they are acted upon by the PcG transcriptional silencing machinery. Notably, the differentiation genes studied in precursor cells here did not show the hallmark bivalent chromatin domains enriched for both the repressive H3K27me3 mark and the active H3K4me3 mark that have been characterized for a cohort of differentiation genes in mammalian ESCs (Chen, 2011).

    The cell fate switch from proliferating spermatogonia to the spermatocyte differentiation program initiates both global and local changes in the transcriptional regulatory landscape, starting a cell type-specific gene expression cascade that eventually leads to robust transcription of the terminal differentiation genes. Globally, soon after the switch from spermatogonia to spermatocytes, core subunits of the PRC2 complex are downregulated, including E(z), the enzyme that generates the H3K27me3 mark. Locally, after male germ cells become spermatocytes, Pol II accumulates at the terminal differentiation gene promoters, although these genes still remain transcriptionally silent, with low H3K4me3 and high Polycomb protein levels near their promoters (Chen, 2011).

    The next step awaits the expression of spermatocyte-specific forms of core transcription machinery and chromatin-associated regulators, including homologs of subunits of both the general transcription factor TFIID (tTAFs) and the MIP/dREAM complex (Aly and other testis-specific components of tMAC). The tMAC complex acts either locally or globally, perhaps at the level of chromatin or directly through interaction with tTAFs, to allow recruitment of tTAFs to promoters of target terminal differentiation genes. The action of tTAFs then allows full and robust transcription of the terminal differentiation genes, partly by displacing Polycomb from their promoters (Chen, 2011).

    Strikingly, the two major PcG protein complexes appear to be regulated differently by the germ cell developmental program: whereas the PRC2 components E(z) and Su(z)12 are downregulated, the PRC1 components Polycomb, Polyhomeotic and dRing continue to be expressed in spermatocytes. The global downregulation of the epigenetic 'writer' E(z) in spermatocytes might facilitate displacement of the epigenetic 'reader', the PRC1 complex, from the differentiation genes, with the local action of tTAFs at promoters serving to select which genes are relieved of PRC1. In addition, the tTAFs act at a second level to regulate Polycomb by recruiting and accompanying Polycomb and several other PRC1 components to a particular subnucleolar domain in spermatocytes. It is not yet known whether sequestering of PRC1 to the nucleolus by tTAFs plays a role in the activation of terminal differentiation genes, perhaps by lowering the level of PRC1 that is available to exchange back on to differentiation gene promoters. Conversely, recruitment of PRC1 to the nucleolar region might have a separate function, such as in chromatin silencing in the XY body as observed in mammalian spermatocytes (Chen, 2011).

    The findings indicate that, upon the switch from spermatogonia to spermatocytes, the terminal differentiation genes go through a poised state, marked by presence of both active Pol II and repressive Polycomb, before the genes are actively transcribed. Stalled Pol II and abortive transcript initiation are emerging as a common feature in stem/progenitor cells. This mechanism may prime genes to rapidly respond to developmental cues or environmental stimuli. Stalled Pol II could represent transcription events that have initiated elongation but then pause and await further signals, as in the regulation of gene expression by the androgen receptor. Alternatively, Pol II might be trapped at a nascent preinitiation complex, without melting open the DNA, as found in some instances of transcriptional repression by Polycomb. Although ChIP analyses did not have the resolution to distinguish whether Pol II was stalled at the promoter or had already initiated a short transcript, the results with antibodies specific for unphosphorylated Pol II suggest that Pol II is trapped in a nascent preinitiation complex. The PRC1 component dRing has been shown to monoubiquitylate histone H2A on Lys119 near or just downstream of the transcription start site. It is proposed that in early spermatocytes, before expression of the tTAFs and tMAC, the local action of PRC1 in causing H2AK119ub at the terminal differentiation gene promoters might block efficient clearing of Pol II from the preinitiation complex and prevent transcription elongation (Chen, 2011).

    Removal of PRC1 from the promoter and full expression of the terminal differentiation genes in spermatocytes require the expression and action of tMAC and tTAFs. Cell type-specific homologs of TFIID subunits have been shown to act gene-selectively to control developmentally programmed gene expression. For example, incorporation of one subunit of the mammalian TAF4b variant into TFIID strongly influences transcriptional activation at selected promoters, directing a generally expressed transcriptional activator to turn on tissue-specific gene expression (Chen, 2011).

    The local action of the tTAFs to relieve repression by Polycomb at target gene promoters provides a mechanism that is both cell type specific and gene selective, allowing expression of some Polycomb-repressed genes while keeping others silent. Similar developmentally programmed mechanisms may also reverse PcG-mediated epigenetic silencing in other stem cell systems. Indeed, striking parallels between the current findings and recent results from mammalian epidermis suggest that molecular strategies are conserved from flies to mammals. In mouse epidermis, the mammalian E(z) homolog Ezh2 is expressed in stem/precursor cells at the basal layer of the skin. Strikingly, as was observed for E(z) and Su(z)12 in the Drosophila male GSC lineage, the Ezh2 level declines sharply as cells cease DNA replication and the epidermal differentiation program is turned on. Overexpression of Ezh2 in epidermal precursor cells delays the onset of terminal differentiation gene expression (Ezhkova, 2009), and removal of the Ezh2-generated H3K27me3 mark by the Jmjd3 (Kdm6b) demethylase is required for epidermal differentiation (Chen, 2011).

    In particular, the results suggest a possible explanation for the conundrum that, although PcG components are bound at many transcriptionally silent differentiation genes in mammalian ESCs, loss of function of PcG components does not cause loss of pluripotency but instead causes defects during early embryonic differentiation. In Drosophila male germ cells, events during the switch from precursor cell proliferation to differentiation are required to recruit Pol II to the promoters of differentiation genes. Without this differentiation-dependent recruitment of Pol II, loss of Polycomb is not sufficient to precociously turn on terminal differentiation genes in precursor cells. Rather, Polycomb that is pre-bound at the differentiation gene promoters might serve to delay the onset of their transcription after the mitosis-to-differentiation switch. Robust transcription must await the expression of cell type- and stage-specific components of the transcription machinery. These might in turn guide gene-selective reversal of Polycomb repression to facilitate appropriate differentiation gene expression in specific cell types (Chen, 2011).

    The poly(A) polymerase GLD2 is required for spermatogenesis in Drosophila melanogaster

    The DNA of a developing sperm is normally inaccessible for transcription for part of spermatogenesis in many animals. In Drosophila melanogaster, many transcripts needed for late spermatid differentiation are synthesized in pre-meiotic spermatocytes, but are not translated until later stages. Thus, post-transcriptional control mechanisms are required to decouple transcription and translation during spermatogenesis. In the female germline, developing germ cells accomplish similar decoupling through poly(A) tail alterations to ensure that dormant transcripts are not prematurely translated: a transcript with a short poly(A) tail will remain untranslated, whereas elongating the poly(A) tail permits protein production. In Drosophila, the ovary-expressed cytoplasmic poly(A) polymerase WISPY is responsible for stage-specific poly(A) tail extension in the female germline. This study examined the possibility that a recently derived testis-expressed WISPY paralog, GLD2, plays a similar role in the Drosophila male germline. It was shown that knockdown of Gld2 transcripts causes male sterility, as GLD2-deficient males do not produce mature sperm. Spermatogenesis up to and including meiosis appears normal in the absence of GLD2, but post-meiotic spermatid development rapidly becomes abnormal. Nuclear bundling and F-actin assembly are defective in GLD2 knockdown testes and nuclei fail to undergo chromatin reorganization in elongated spermatids. GLD2 also affects the incorporation of protamines and the stability of dynamin and transition protein transcripts. The results indicate that GLD2 is an important regulator of late spermatogenesis and is the first example of a Gld-2 family member that plays a significant role specifically in male gametogenesis (Sartain, 2011).

    Spermatogenesis is a tightly controlled developmental process that requires the stage-specific production of proteins. In animals, spermatogenesis begins when a diploid germline cell produced from the testis stem cell niche undergoes differentiation and proliferation though mitosis and meiosis to form many haploid spermatocytes. Post-meiotic development, called spermiogenesis, is a series of morphological changes that will determine the final shape and form of the mature sperm, which can vary greatly among taxa. One important phenomenon that is seen in spermatogenesis in many species is that transcription is silenced for part of the process: for example, transcription cannot occur after nuclear condensation in mice and there is some evidence for transcriptional silencing during meiosis in Drosophila. In such cases, any proteins that must be translated during the transcriptionally silent period must be synthesized from mRNAs that were transcribed earlier but remain untranslated until the appropriate stage of development. Furthermore, some transcripts needed for late spermiogenesis, such as those of don juan and Mst87F, are synthesized in spermatocytes, although they are not translated until much later (Sartain, 2011 and references therein).

    Spermatogenesis in Drosophila melanogaster is well described. Testis gonial cells originating from germline stem cell divisions undergo synchronous mitosis and meiosis with incomplete cytokinesis, resulting in a cyst of 64 round, haploid spermatids after the completion of meiosis. The spermatids undergo morphological changes, including flagellum extension and nuclear reshaping within the syncytium, until spermatid individualization occurs. The cells exit the testis as mature sperm (Sartain, 2011).

    During the final stages of spermatogenesis in Drosophila, as in many other invertebrate and vertebrate species, chromatin reorganization events cause the spermatid nuclei to become tightly compacted. Histones associated with spermatocyte chromatin are ultimately exchanged for protamines, allowing the nucleus to condense up to 200 fold. Two genes encoding protamines have been identified in Drosophila (Mst35Ba, or Protamine A; and Mst35Bb, or Protamine B). Additionally, the gene Tpl94D demonstrates functional homology to mammalian transition proteins, which bind chromatin as an intermediate step between histone-based and protamine-based chromatin organization. Therefore, nuclear compaction in Drosophila occurs as a two-step process: histones are first displaced by transition proteins, and transition proteins are later exchanged for protamines (Sartain, 2011 and references therein).

    Soon after protamine incorporation, the spermatids in a cyst become separated from one another in a process called individualization. During this process, a cone-like structure composed of cross-linked F-actin assembles around each nucleus in the cyst. The 64 cones in the cyst move as a unit down the length of the sperm tails, simultaneously pushing out excess cytoplasm and wrapping each spermatid in an individual membrane. The separated, mature sperm then roll into coils and exit the testis to be stored in the seminal vesicle (Sartain, 2011).

    In Drosophila, there are many examples of transcripts that are synthesized in spermatocytes but are not translated until after meiosis, to such an extent that transcriptional activity in the developing Drosophila sperm cell was previously thought to be predominantly limited to early spermatocytes and spermatogonia. However, recent evidence demonstrates that transcriptional activity occurs post-meiotically as well. For those transcripts that remain quiescent until post-meiotic stages, a translational control mechanism must be in place (Sartain, 2011).

    Many cell types, including oocytes and neurons, achieve translational regulation through adjusting the length of the poly(A) tail in the cytoplasm. A long poly(A) tail promotes translation of the transcript through recruitment of translation initiation factors, whereas a transcript with a short poly(A) tail remains untranslated or is degraded. Most mRNAs are extensively polyadenylated in the nucleus; however, for some transcripts that will be held in an untranslated state for a period of time, poly(A) tail modifications occur outside the nucleus. In Xenopus, transcripts destined for post-transcriptional poly(A) tail adjustment contain two consensus sequences in their 3'UTR: a cytoplasmic polyadenylation element (CPE) and the hexamer AAUAAA; these recruit a complex of proteins that alter poly(A) tail length. In Xenopus, CPE is bound by CPE-binding protein (CPEB). The cleavage and polyadenylation specificity factor (CPSF) binds to the hexamer. CPEB and CPSF recruit a cytoplasmic poly(A) polymerase (PAP) and a deadenylase, both of which work on the transcript simultaneously. However, the deadenylase is slightly more efficient than the PAP, so the net effect is a poly(A) tail that remains short. Upon a signal to activate translation, CPEB is phosphorylated, causing the deadenylase to dissociate from the complex; the PAP is then free to elongate the poly(A) tail (Sartain, 2011).

    PAPs that act in the cytoplasmic complex differ from nuclear PAPs. The Gld-2 (germline development 2) family of cytoplasmic PAPs has been described in C. elegans, Xenopus and Drosophila. Whereas nuclear PAPs contain a catalytic domain and an RNA-binding domain, Gld-2 family members have only a catalytic domain. For RNA specificity, Gld-2 associates with an RNA-binding protein, typically a Gld-3, to form a heterodimer that acts as a cytoplasmic PAP (Sartain, 2011 and references therein).

    Gld-2 family members have been shown to play roles in oogenesis in several organisms. In worms, a Gld-2 homolog is involved in the mitosis/meiosis decision to make both male and female germ cells (Kadyk, 1998). In Drosophila, the X-linked Gld-2 homolog wispy (wisp) is necessary for oogenesis and egg activation. WISP is present in ovaries but not testes and is necessary for the completion of meiosis in oocytes. WISP has been shown to polyadenylate transcripts of cortex, which is required for proper meiotic progression. WISP also polyadenylates several developmental transcripts, the protein products of which are needed for early embryogenesis, including bicoid, Toll and torso (Sartain, 2011 and references therein).

    The Drosophila genome contains an autosomal paralog of wisp called Gld2. Previous studies of Gld2 have demonstrated a role in long-term memory and show that GLD2 acts as a PAP in vitro. This study shows that Gld2 is expressed in the male, but not female, germline. It is required for the completion of spermatogenesis, specifically for the elongation and individualization stages. In GLD2 knockdown testes, the first disruption observed is post-meiotic, at the onset of spermatocyst elongation. In these testes, the nuclei in developing cysts scatter and basal bodies are not observed near nuclei. F-actin-containing individualization complexes do not assemble and nuclear compaction does not complete. Additionally, protamines are not incorporated and transcripts for both dynamin (shibire - FlyBase) and the transition protein are undetectable. These findings indicate that Gld2 arose from duplication of the wisp locus, and that this derived paralog was likely maintained in the genome owing to its essential role in spermatogenesis (Sartain, 2011).

    The genus Drosophila can be divided into subgenera, and genome sequences are available for two of these: Sophophora (which contains D. melanogaster) and Drosophila (which contains species that diverged from D. melanogaster ~63 million years ago). wisp and Gld2 orthologs have been identified in species from both subgenera. Available gene expression data show that the wisp orthologs in D. melanogaster, D. mojavensis and D. virilis have female-biased expression, whereas the Gld2 orthologs from D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. pseudoobscura and D. mojavensis have male-biased expression). This is consistent with the conservation of the oogenesis and spermatogenesis functions of WISP and GLD2, respectively, across the entire genus. All other insect species with sequenced genomes (including Anopheles gambiae and Aedes aegypti, which are the closest relatives to Drosophila with completely sequenced genomes) possess only a single ortholog of wisp/Gld2, based on best reciprocal BLAST searches. Therefore, a duplication event occurred after the most recent common ancestor (MRCA) of Drosophila and mosquitoes, but prior to the MRCA of the genus Drosophila, to give rise to either wisp or Gld2 (depending on which locus is ancestral and which is derived). A phylogenetic reconstruction of the evolutionary relationships of the insect protein coding sequences supports this hypothesis. Furthermore, the wisp/Gld2 ortholog in A. gambiae has female-biased expression, based on microarray data from whole males and females. This has led to a hypothesis that the ancestral germline function of this gene family is in ovaries, and that the testis function of GLD2 is derived (Sartain, 2011).

    The testis-expressed cytoplasmic PAP, GLD2, is required for spermatogenesis in D. melanogaster. Knockdown of GLD2 in the testes causes widespread defects in post-meiotic spermatogenesis events. In a GLD2 knockdown, the earliest defects are seen in early post-meiotic spermatids, when the basal body fails to dock at the nuclear envelope and the nuclei begin to scatter. Many late-stage events of spermatogenesis are also affected, including protamine translation and F-actin cone formation on individualization stage spermatids. Additionally, GLD2 knockdown affects the stability of dynamin transcripts and those of transition protein (Tpl94D) in the testes. Interestingly, GLD2-deficient germ cells appear to undergo normal meiosis, in contrast to mutants of other Gld-2 homologs, including the GLD2 paralog WISP (Sartain, 2011).

    There is indirect evidence that GLD2 acts as a cytoplasmic PAP in the Drosophila testes. The GLD2 protein contains a PAP/25A domain, which is shared by all known Gld-2 family proteins. Additionally, GLD2 has the ability to elongate poly(A) tails in vitro. The current study has shown that GLD2 interacts with the Gld-3 homolog BIC-C in a yeast two-hybrid assay. Furthermore, at least two transcripts are absent in GLD2 knockdown testes, which might be the result of destabilization owing to an inability to elongate their poly(A) tails. Taken together, it is concluded that Drosophila GLD2 does act as a PAP during spermatogenesis and that the defects seen in its absence are the result of failure of one or more polyadenylation events (Sartain, 2011).

    GLD2 affects many aspects of spermatogenesis in Drosophila. First, nuclear anchoring and basal body docking are defective in the absence of GLD2. Both processes occur in early post-meiotic stages of spermatogenesis in wild-type testes; however, in the absence of GLD2, spermatid nuclei scatter throughout spermatogenic cysts and basal bodies cannot assemble at the nuclear envelope. It is possible that, in the GLD2 knockdown, the failure of these events is related. For example, it might be the case that nuclear anchoring cannot occur until basal bodies have docked properly, or vice versa. Alternatively, loss of GLD2 might affect formation of the post-meiotic nuclear envelope, which might in turn have negative effects on both nuclear anchoring and basal body docking processes. Other studies of mutants that involve basal body defects have documented nuclear localization disruptions, indicating that these two processes may be linked (Sartain, 2011).

    Second, GLD2 knockdown testes show abnormalities during nuclear condensation: nuclei clearly begin to condense, but condensation stalls at the canoe stage and does not progress further. It is believed that this phenotype reflects, at least in part, the absence of Tpl94D transition protein transcripts in GLD2 knockdown testes, and thus the inability of nuclei to progress to a condensation state at which protamines would be incorporated. In addition, and perhaps contributing to the phenotype, Protamine B is not translated in the absence of GLD2, even though its RNA is present. Thus, GLD2 acts upstream of Protamine B translation. Given that no effects were detected of GLD2 knockdown on the poly(A) tail length of protamine transcripts, it seems likely that removing GLD2 causes a block in the spermatogenic developmental pathway at a stage before protamine transcripts would normally be translated. Rathke and colleagues showed that protamines are incorporated into the spermatid chromatin after the onset of transition protein incorporation, so it is possible that a lack of transition protein causes a developmental block in GLD2 knockdown testes and that the lack of protamine translation in these testes reflects this block. There is evidence that protamine transcripts are translationally repressed for a few days after their transcription and that this repression is dependent upon elements in their 5'UTR. Thus, GLD2 could be responsible for controlling the translation of a crucial element that causes the relief of repression at the protamine 5?UTR, while not affecting the poly(A) tail status of the protamine transcript itself (Sartain, 2011).

    A third defect in GLD2-deficient spermatogenesis occurs at individualization: actin cones are never detected around late-stage nuclei and the spermatids do not separate from one another. dynamin transcripts are missing in GLD2 knockdown testes. The absence of Dynamin could account for the lack of actin cones at individualization stage nuclei: previous studies have demonstrated that Dynamin is present throughout the actin cones and that disruption of Dynamin function in temperature-sensitive mutants contributes to their instability. Lack of dynamin mRNA could indicate that it is a GLD2 target: lack of dynamin polyadenylation by GLD2 could leave the transcript vulnerable to exonucleases in the cytoplasm, resulting in its degradation. Alternatively, it is possible that the absence of Dynamin in the GLD2 knockdown results from a developmental block during late spermatogenesis, at a time before dynamin RNA would be present (Sartain, 2011).

    GLD2 localization might help to identify its target transcripts. Immunofluorescence staining experiments showed that in addition to cytoplasmic localization in spermatocytes, GLD2 localizes to the distal ends of elongated spermatogenic cysts. This is where the polarized growth of the cyst occurs in accordance with axoneme extension; additionally, a group of mRNAs that are transcribed post-meiotically have been shown to localize to the distal end of the spermatogenic cyst. Interestingly, one of these late-transcribed genes is orb, which encodes the Drosophila ortholog of CPEB, the protein necessary for cytoplasmic polyadenylation in Xenopus. The presence of both the CPEB ortholog ORB and the cytoplasmic PAP GLD2 at the end of the cyst where growth is occurring might indicate an involvement of GLD2 in late spermatocyst growth. Taken together, these data suggest that the distal end of the cyst might be a major production center for cyst growth, with the necessary mRNAs regulated post-transcriptionally through cytoplasmic polyadenylation (Sartain, 2011).

    This study has shown that GLD2 plays an essential for male, but not female, gametogenesis. This is a unique finding among the literature describing other Gld-2 homologs, where Gld-2 proteins are necessary for some aspect of oogenesis and egg maturation in Drosophila, Xenopus and mice and for the proliferative stages of gametogenesis in hermaphrodite worms. Drosophila GLD2 plays a role in the male, but not female, germline, and is required in spermatid morphogenesis rather than in proliferative stages. The evidence that Gld2 was retrotransposed to the third chromosome from a duplication of the wisp locus on the X chromosome might give insight to how this unique role for a Gld-2 homolog came about. The phenomenon of meiotic sex chromosome inactivation (MSCI) might have contributed to duplication of the wisp gene and to subsequent retention of Gld2. During spermatogenesis in Drosophila and other animals, the X chromosome is transcriptionally silenced prior to autosomal silencing. Therefore, genes located on the X chromosome have a limited capacity to encode proteins involved in spermatogenesis. Interestingly, an excess of genes has been retrotransposed from the X to the autosomes, and the autosome-derived copies are hypothesized to allow for the escape from X-inactivation. The testis-biased expression and spermatogenic functions of Gld2 suggest that it was selectively retained because it performs a function unavailable to wisp because of MSCI (Sartain, 2011).

    It is interesting that Gld2 is crucial for post-meiotic spermatogenesis in Drosophila, whereas all Gld-2 family members analyzed so far in Drosophila and other species play roles specifically at meiosis. It is hypothesized that the function of GLD2 in the male germline reflects its evolutionary origin: duplication of the X-linked wisp locus allows for an autosomal copy that can be expressed in the testis during MSCI. Although this is the first example of a Gld-2 family member with its gametogenic role solely in spermatogenesis, other species might have developed similar mechanisms of translational control in the testes; for example, spermatogenesis in mice is regulated, in part, by a cytoplasmic PAP outside of the Gld-2 family called TPAP (PAPOLB - Mouse Genome Informatics). Further investigation and identification of GLD2 targets in Drosophila testes will help to elucidate how spermatogenesis can be regulated through cytoplasmic polyadenylation (Sartain, 2011).

    Replacement of histones by protamines and Mst77F during chromatin condensation in late spermatids and role of Sesame in the removal of these proteins from the male pronucleus

    Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little of this process is known for Drosophila. This study characterizes three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame (Raja, 2005).

    For mammals, the somatic set of histones are modified, as these are in part replaced by specific variants during meiotic prophase. After meiosis, histones are replaced by major transition proteins TP1 and TP2 and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements leads to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing protamines as major chromatin condensing proteins and DNA. Some mammals have only one protamine gene, while mice and humans have two genes encoding two different protamines, both of which are essential for fertility and are haploinsufficient. HILS1 (spermatid-specific linker histone H1-like protein) has been proposed to participate in chromatin remodeling in mouse and human spermiogenesis. The transition between histone removal and its replacement by protamines in mice and humans is characterized by small 6- to 10-kDa transition proteins acting as a short-term chromosomal proteins. In mice, the transition proteins TP1 and TP2 are redundant in function. In fishes and birds, transition proteins are missing and protamines directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal configuration is maintained in sperms, while protamine-like proteins have been described for mussels. These protamine-like proteins lack the typical high cysteine content necessary for disulfide bridges. Therefore, a doughnut-type chromatin structure as in mammals is unlikely to occur in mussels. It has been proposed that the protamine-like proteins in mussels belong to the histone H1 family. The sperm chromatin of mussels contain core histones and thus a nucleosomal configuration, but histone H1 is replaced by protamine-like molecules which organize the higher order structure of the chromatin (Raja, 2005).

    For Drosophila melanogaster, chromatin reorganization after meiosis has not been studied at the molecular level. At the light microscopic level, the Drosophila spermatid nucleus is initially round after meiosis and then is shaped to a thin needle-like structure with highly condensed chromatin, so that the volume of the nucleus is condensed over 200-fold. In mammals, the volume of the nucleus is reduced over 20-fold. In the mature sperms of Drosophila, core histones are not detectable by immunohistology. There is histochemical evidence for the presence of very basic proteins in sperm, but it still remains an open question whether histones are replaced by protamine-like basic proteins in Drosophila. The analysis of the Drosophila genome sequence revealed that the proteins encoded by two genes show similarity to mammalian protamines for which the male-specific transcripts Mst35Ba and Mst35Bb have been found and have been proposed to encode protamine-like proteins. Another male specifically transcribed gene, Mst77F, is a distant relative of the histone H1/H5 (linker histone) family and has been proposed to play a role either as a transition protein or as a replacement protein for compaction of the Drosophila sperm chromatin. With enhanced green fluorescent protein (eGFP) fusion for these abovementioned proteins, this study shows that Mst35Ba and Mst35Bb indeed encode protamines and Mst77F encodes a linker histone-like protein. The expression pattern of Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, during fertilization, the removal of protamines from the male pronucleus requires the function of the maternal component, Sesame, but not for the removal of Mst77F. It has been shown that sesame mutants cause impairment of the entry of histones into the male pronucleus (Raja, 2005).

    Mst35Ba and Mst35Bb are present at cytological position 35B6 and 35B6-7, respectively, on the chromosome arm 2L. These two genes are arranged in tandem, and both consist of three exons. The 5'UTR, coding region, and the 3'UTR of these genes are highly identical; they probably arose from a recent gene duplication. The encoded protamines show over 94% identity to each other (Raja, 2005).

    A remarkable feature of protamines is their ability to form intermolecular disulfide bridges, which is reflected by the conserved cysteine residues within mammalian protamines. The dProtA and dProtB are of 146 amino acids (aa) and 144 aa, respectively, and thus longer than even the human and mouse Protamine-2, which are 102 aa and 107 aa, respectively. Both Drosophila protamines contain 10 cysteines each and show significant similarity, particularly with respect to a high cysteine, lysine, and arginine content to mammalian protamines. Human and mouse Protamine-1 aligns to the N-terminal half of the Drosophila protamines (from aa positions 27 to 82), and four cysteine residues are conserved and regularly spaced. In contrast, Protamine-2 of human and mouse shows relatively high similarity to the C-terminal half of the Drosophila protamines, with four cysteines in this region that are conserved and regularly spaced, whereas one cysteine is shared with the mouse and human Protamine-1 (Raja, 2005).

    Mst77F is present at the cytological position 77F on the chromosome arm 3L and lies within the large intron of PKA-R1. Mst77F is also male specifically transcribed, and the encoded protein has been proposed to be a linker histone H1/H5 type, which could also play the role of a transition protein or a protamine. The Mst77F protein shares a significant similarity to the HILS1 protein of mouse and human HILS1, where the percentages of cysteine, lysine, and arginine are similar to that of mHILS1 and hHILS1. HILS1 protein has been recently described as a component of the mammalian sperm nucleus. Drosophila Mst77F encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of 9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F. Mst77F contains 10 cystine residues as in Drosophila protamines, and mHILS1 contains eight cystine residues, of which four residues are conserved (Raja, 2005).

    As there are considerable differences between the mammalian protamines as well as between the mammalian HILS1 proteins and the presumptive Drosophila homologue Mst77F, additional experiments are essential to clarify if these proteins are indeed involved in the condensation of sperm chromatin (Raja, 2005).

    Drosophila protamine mRNAs are transcribed at the primary spermatocyte stage, whereas in mammals protamine mRNAs are synthesized at the round spermatid stage and translationally repressed until the elongated stage, which is mediated by 3'UTR. The Drosophila ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3'UTR of the respective protamine genes. Nevertheless, the transgenic flies carrying these constructs still show repression of translation. So, in Drosophila, the region responsible for the translational repression is most likely in the 5'UTR. Deletion constructs of Mst35Bb and Mst77F 5'UTRs fused to the reporter lacZ show that the translation repression element is indeed present in the 5'UTR. This holds true also for the mRNA of the Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated concerning translational repression so far in male germ lines of Drosophila. In contrast to mammalian spermatogenesis, in Drosophila transcription ceases already with the entry into meiotic divisions. Since the protamines are made in the elongated spermatids, the transcriptional silencing in Drosophila spermatogenesis seems to be independent of protamines (Raja, 2005).

    When primary amino acid sequences of Drosophila protamines are compared to mammalian protamines, it is quite evident that Drosophila protamines are relatively large. dProtA and dProtB are over 94% identical to each other. This could explain that both the protamines may be functionally redundant. Human and mouse Protamine-1 aligns with the N terminus of both Drosophila protamines, and Protamine-2 aligns more to the C terminus. It is possible that the Drosophila protamines undergo posttranslational cleavage at the N terminus, as is known for mammals. The cytoplasmic eGFP fused at the C terminus shows clear nuclear localization, indicating that the tagged protamine is functionally intact. Drosophila protamines each contain 10 cysteine residues at identical positions, while over 4 of 10 cysteines at the N terminus and the C terminus are conserved with human and mouse Protamine-1 and Protamine-2, respectively. With nine cysteines, the content is highest in Protamine-1 of mice. Inter- or intra-disulfide bridges can be formed between the cysteine-rich protamines to condense the DNA. For mice it is shown that mutation in protamine-1 or protamine-2 is haploinsufficient and causes male sterility. A haploid situation was analyzed for the Mst35Ba and Mst35Bb genes with the deficiency Df(2L)Exel8033/+; these flies are fertile males and show normal spermatogenesis. The large amount of identity that both dProtA and dProtB exhibit can contribute to the functional redundancy (Raja, 2005).

    Chromatin reorganization is an essential feature during spermiogenesis. The functional significance of chromatin compaction during spermiogenesis is still unknown. The main explanation seems to be that compaction of the sperm nucleus is an essential factor for its mobility as well as for the penetration of sperm into the egg and genomic stability. In mammals, somatic histones are in part replaced by spermatid-specific variants during meiotic prophase, later by major transition proteins TP1 and TP2, and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements lead to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing major chromatin condensing proteins and DNA in the nucleus (Raja, 2005).

    In Drosophila, so far no proteins have been identified that are involved in the packaging of the genome in the mature sperm nucleus. One observation, that Histone3.3 variant and the somatic H3 isoform in Drosophila are vanishing at the time of chromatin condensation, supports the view of histone displacement, but it was still a question of whether it is the real absence of histones at this stage in Drosophila or whether the antibodies are not accessible to the mature sperm due to the tight packaging of the chromatin. To circumvent this problem, the GFP fusion approach was chosen, use was made of the existing His2AvDGFP, and Protamine-eGFP and Mst77F-eGFP fusion transgenic flies were generated in order to analyze the situation in Drosophila. The results clearly show that histone His2AvD is lost from the spermatid nuclei at the time of appearance of protamines and Mst77F during later stages of spermatid differentiation. The exact molecular mechanisms underlying the histone displacement, degradation, and incorporation of protamines onto the chromatin are poorly understood. For mammals, evidence has been obtained that histone H2A is ubiquitinated in mouse spermatids around the developmental time period when histones are removed from the chromatin. The mammalian HR6B ubiquitin-conjugating enzyme is the homologue of yeast RAD6, and both can ubiquitinate histones in vitro. Thus far, the mechanism of histone displacement and protamine incorporation is unknown during spermiogenesis in Drosophila. In flies as well as in mammals, many questions remain unanswered that need to be addressed about these underlying mechanisms of chromatin remodeling during spermiogenesis (Raja, 2005).

    In mammals, transition proteins act as intermediates in the histone-to-protamine transition. In mice, the onset of HILS1 and transition proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and later with Protamine-2 but HILS1 and the transition proteins are no longer present in the mature sperm. Mice lacking both TP1 and TP2 show normal transcriptional repression, histone displacement, nuclear shaping, and protamine deposition but show the loss of genomic integrity with large numbers of DNA breaks leading to male sterility. In Drosophila, histones are displaced with synchronous accumulation of protamines and Mst77F. Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has been proposed to play a role either as a transition protein or as a protamine for compaction of the Drosophila sperm chromatin. Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic amino acid content but not to mouse TP1, TP2, or H1t. Moreover, the results show that the pattern of expression of Mst77F in the nucleus is similar to that of mHILS1 in the nucleus, with the exception that Mst77F is also transiently detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In mammalian mature sperm nuclei, it is only the protamines that are the chromatin condensing proteins which persist. This again raises the question of whether Mst77F could also play the role of protamines. However, one additional copy of dProtB (dProtA and dProtB showing 94% identity may be functionally redundant) does not rescue the ms(3)nc3 phenotype, indicating that the role of Mst77F may be completely or partially different from that of protamines in the nucleus. However, a null mutation for Mst77F is required to answer this question with respect to chromatin condensation. In ms(3)nc3 mutants, the chromatin condensation with the native protamines continues to take place. When a closer look was taken at the deposition of ProtamineB-eGFP in ms(3)nc3/Df(3L)ri-79c trans-heterozygotes, it revealed that the condensed chromatin in the tid-shaped nuclei is concentrated at the two opposite ends, with a lightly stained chromatin spaced in the center. So the chromatin condensation takes place but may not be complete with the incorporation of the mutant Mst77F protein. The large amount of chromatin compaction or condensation seen in Drosophila mature sperm when compared to that of mouse and human sperm possibly could be the result of persistence of Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm nucleus contains further protamines that have not yet been properly annotated (Raja, 2005).

    ms(3)nc3 is a second-site noncomplementation (nc) mutation that was isolated in an ethylmethanesulfonate screen to identify interacting proteins involved in microtubule function in Drosophila. This study shows that ms(3)nc3 is a single missense mutation from a T>A transition, causing the substitution of threonine instead of serine at aa position 149. Mst77F shows a pattern of expression similar to protamines in the nucleus and was also seen in the flagella until the individualization stage. Since ms(3)nc3 fails to complement class I alleles at the ß2 tubulin locus, it is possible that Mst77F has a dual role to play as a chromatin condensing protein in the nucleus and for the normal nuclear shaping. Nuclear shaping is a microtubule-based event. ms(3)nc3 leads to a tid-shaped nuclear phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar defective nuclear shaping is seen with the few homozygous and heteroallelic combinations of class I alleles of ß2 tubulin. The incorporation of the defective subunit encoded by ms(3)nc3 may interfere with the function of the resulting complex. These data suggest the involvement of an Mst77F (a linker histone variant) in the microtubule dynamics during the nuclear shaping. This again complements the role of sea urchin histone H1 in the stabilization of flagellar microtubules (Raja, 2005).

    After the first steps in the fertilization process, the male gamete is still in the highly compact protamine-based chromatin structure. In a wild-type egg, the paternal pronucleus changes the shape from the needle-like to a spherical structure. Furthermore, the male pronucleus acquires a nucleosome-based structure before zygote formation and thus is transformed into a replication-competent male pronucleus. sesame is a maternal effect mutation in HIRA and had been mapped to 7C1. HIRA family of genes (named after yeast HIR genes; HIR is an acronym for 'histone regulator') includes the yeast HIR1 and HIR2 repressors of histone gene transcription in S. cerevisiae, human TUPLE-1/HIRA, chicken HIRA, and mouse HIRA. In Drosophila, HIRA is expressed in the female germ line and a high level of HIRA mRNA is deposited in the egg. Human HIRA is shown to bind to histone H2B and H4. The WD repeats present at the N-terminal part of HIRA could probably function as a part of a multiprotein complex. Xenopus HIRA proteins are also known in promoting chromatin assembly that is independent of DNA synthesis in vitro. The corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed, has been analyzed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus, which strengthens the function of HIRA in binding to the core histones. This study shows that neither Drosophila protamine is removed from the male pronucleus in sesame mutants. This leads to the proposal that the transport and incorporation of histones onto the chromatin in some manner is coupled to the removal of protamines in which HIRA could play an important role in the multiprotein complex required in this chromatin reconstitution process. Mst77F removal from the male pronucleus in contrast to protamines is independent of HIRA (Raja, 2005).

    During spermiogenesis, chromatin reorganization of the complete genome is an essential feature for male fertility. This process leads to an extremely condensed state of the haploid genome in the sperm and requires a reorganization of the paternal genome in the male pronucleus during fertilization and before zygote formation. With the characterization of the chromatin condensing proteins in Drosophila, it would be possible to gain more insight into the mechanisms of sperm chromatin reorganization during spermiogenesis and fertilization (Raja, 2005).

    Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila

    In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. This study focused on post-meiotic stages and shows that also after meiosis, histone H3 shows a high overall methylation of K9 and K27; it was hypothesised that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, histones are lost during the early canoe stage, and just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. It is believed that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, the first transition protein-like chromosomal protein to be found in Drosophila, Tpl94D, is reported. It is proposed that Tpl94D (an HMG box protein) and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, it is showm that histone modifications and removal are independent of protamine synthesis (Rathke, 2007).

    The switch between a nucleosome-based chromatin configuration and a protamine-based structure is a specialised form of chromatin remodelling in the male germline. The mammalian zinc finger protein CTCF is involved in many epigenetic processes. Furthermore, paralogous variant of CTCF which is testis-specifically expressed, called BORIS, is exclusively expressed in the mammalian male germline. The function of BORIS in this context is still not clear. Drosophila, in contrast to mammals, contains only one CTCF gene. It was therefore asked whether Drosophila CTCF is also expressed in the testes, and immunostaining and anti-histone staining was performed on testes of transgenic flies expressing protamine-eGFP. CTCF expression was observed during pre-meiotic and meiotic stages at the chromosomes as has been shown for mitotic cell division in mammalian cell culture. Shortly after meiosis, CTCF is visible in young elongating nuclei, where it co-localises with the chromatin as indicated by the histone distribution. CTCF is also present in the early and late canoe stage spermatid heads. At the early canoe stage, CTCF is very diffusely distributed in comparison to histones. CTCF does not co-localise with the chromatin which starts to condense at one side of the nucleus. This diffuse distribution is still visible at the late canoe stage when protamine-eGFP starts to be deposited to the chromatin. CTCF is no longer detectable after the canoe stage. The earlier chromatin-associated CTCF localisation might indicate a very early role in chromatin reorganisation at the switch between the nebenkern and canoe stage. Furthermore, CTCF might be associated primarily with the chromatin, which is not yet condensing during these stages. The late canoe stage is the only post-meiotic stage where distinct regions of RNA polymerase II are found with an antibody directed against a phosphorylated subunit of active polymerase, indicative of transcription. At this precise stage, only a very small set of genes is thought to be transcribed. Also CTCF expression during chromatin reorganisation in the nucleus was detected in D. hydei (Rathke, 2007).

    Sperm morphogenesis is characterised by an impressive degree of changes in cell architecture based on stored, translationally repressed mRNAs that are recruited at the appropriate time to the polysomes. Among these are mRNAs that encode Tpl94D and protamines. A dramatic switch in structure from the nucleosomal- to the protamine-based structure of chromatin takes place, and this remarkable chromatin reorganisation of the complete genome is a typical feature depending on stored mRNAs, e.g. for protamine synthesis. This process ultimately leads to an extremely condensed state of the haploid genome in the sperm, which is essential for male fertility in mammals. This study focused on the switch between a nucleosomal- and a protamine-based chromatin reorganisation. The major steps in chromatin organisation take place in the canoe stage of spermatid development. A candidate for a transition protein in Drosophila was identified. The corresponding gene tpl94D (CG31281) encodes a predicted basic high mobility group (HMG) protein of 18.8 kDa. In transgenic flies, Tpl94D-eGFP fusion proteins are expressed solely during the switch between histones and protamines, as is typical for mammalian transition proteins. Since a highly similar chain of events to those reported in mammals is observed, the Drosophila system is considered an excellent choice to study the mechanism of chromatin remodelling during male germ cell development (Rathke, 2007).

    Generally, the bulk of histones, including their diverse modifications in the N-terminal tail, appear to be removed during the canoe stage. Furthermore, the nucleus accumulates ubiquitin at the early canoe stage, when mono-ubiquitylation of histone H2A is no longer detectable. Therefore, taking into account the known presence of proteasomes in the nucleus at this stage of chromatin reorganisation and the overlap of expression shown in this study, it is hypothesised that this ubiquitylation is targeting histones for degradation. This study investigated several mutants having mutations in ubiquitin-conjugating enzymes or ubiquitin ligases, exhibiting arrested spermiogenesis during spermatid development and that are male sterile. However, in all investigated mutants, histone removal is indistinguishable from that of wild-type flies (Rathke, 2007).

    Many histone modifications were found after meiosis and were categorised into three classes (Rathke, 2007).

    1. Histone modifications that persist from pre-meiotic stages and keep the genome silent.

      The vast majority of the genome is transcriptionally silent in post-meiotic stages. This is accompanied by multiple histone modifications that persist from pre-meiotic stages and indicate silencing such as H3K9 and H3K27 methylation. These modifications do not change significantly during post-meiotic stages, which is in agreement with the hypothesis that these modifications predominantly play a role in maintaining transcriptional silencing. Previously, phosphorylation of histones have been analysed during spermatogenesis. Phosphorylated histone H4S1 and H3S10 are present during meiotic divisions. H3S10 phosphorylation is hardly detectable after meiosis, whereas phosphorylation of H4S1 persists until chromatin compaction starts.

    2. Histone modifications that persist from pre-meiotic stages and characterise transcriptionally active chromatin.

      The primary spermatocyte phase is characterised by a high level of transcriptional activity of housekeeping genes. In addition, genes are transcribed that are needed for the subsequent steps in spermatogenesis, as the majority of transcription ceases once meiotic division starts. H4 acetylation and H3K4 and H4R3 methylation of histones were investigated. These histone modifications, which are indicative of transcriptional activity, persist until histone degradation.

    3. Increasing or de novo appearance of histone modifications that decrease the affinity between histones and DNA as a prelude to histone removal.

      It might be that H4 hyper-acetylation, as postulated for mammals and/or other secondary modifications of histones are the first step towards histone removal. The fact that these modifications are conserved between mammals and flies adds support to this hypothesis. Indeed, histone H4 acetylation is very pronounced at the canoe stage and de novo mono-ubiquitylation of histone H2A is seen in round spermatids. Both types of histone modifications are proposed to be necessary for opening the chromatin and decreasing the contact between DNA and histones. The fact that histone H2A mono-ubiquitylation vanishes before the early canoe stage, thus before the hyper-acetylation of histone H4, leads to thinking about a stepwise remodelling of the chromatin. This study proposes that these histone modifications open the chromatin, so that enzymes and regulators have access to histone-based chromatin and can induce and prepare the reorganisation of the genome in the male germline.

    It remains to be clarified whether and how these histone modifications influence the topology of the chromatin as a prelude to histone removal as well as for Tpl94D, Mst77F and protamine deposition. A functional approach based on analysis of mutants of histone-modifying enzymes is difficult, as all characterised histone-modifying enzymes are already active during Drosophila development or at least in spermatogonia and spermatocytes. Therefore a tissue-specific knock-out mutant would most probably exhibit arrest of spermatogenesis before meiosis, rendering it useless for experimental purposes (Rathke, 2007).

    At the first glance, it might seem surprising that histones and all their modifications are removed. Instead of specifically reverting the differentially modified histones to their unmodified state, they are removed together with all histones. This might allow the paternal genome to form nucleosomes with unmodified histones after fertilisation and before zygote formation. Thus, the paternal genome starts embryogenesis with a nucleosomal chromatin lacking histone modifications (Rathke, 2007).

    The data show that most of the histones are removed between the early and late canoe stage; such a process requires a loosening of contact between the histones and DNA, which in turn requires an unwinding of the chromatin structure. It is proposed that this unwinding process is facilitated by DNA nicks as they were widespread at this stage of chromatin reorganisation. Finally, Tpl94D, UbcD6 and SUMO were also observed to accumulate in the chromatin during this process. DNA breaks, Tpl94D, UbcD6 and SUMO were no longer detectable when protamines were fully expressed. Thus, it is proposed that all these proteins and the DNA breaks act together in an unknown manner to allow chromatin remodelling (Rathke, 2007).

    The CTCF protein is present during pre-meiotic stages in the nucleus and stays associated with the chromosomes during meiosis. After meiosis, however, strong localisation to the nucleus is detected during the transition from round spermatid nuclei to the early canoe stage of spermiogenesis. It is speculated that CTCF might set borders in the chromatin for the histone modifications, which are characteristic of the canoe stage, such as acetylation and ubiquitylation. CTCF is visible for longer than histones and disappears together with active RNA polymerase II. CTCF might maintain chromatin accessibility to RNA polymerase II since a few genes are known to be transcribed at this time. In addition, transient occurrence of RNA polymerase II at the late canoe stage might require CTCF to insulate active genes from inactive ones. This idea needs to be tested in tissue-specific CTCF loss-of-function mutants; such mutants are, however, currently unavailable (Rathke, 2007).

    The question of whether histone removal is dependent on a signal that monitors the start of protamine and Mst77F mRNA translation was addressed. Both histone modification and degradation are indistinguishable from the wild-type in loss-of-function mutants of Mst35Ba and Mst35Bb, the genes encoding protamine A and B, respectively. Also in nc3 mutants of Mst77F, histone removal is not disturbed. It is concluded that N-terminal tail modification of histones and histone degradation, on the one hand, and protamine deposition, on the other, are controlled by different pathways in the cell (Rathke, 2007).

    In mammals, it is well known that after meiosis the nucleosomal conformation is lost. This is accompanied by the appearance of testis-specific linker histones. So far, no linker histone variants have been identified in Drosophila, but variants of H2A (H2AvD) and H3 (H3.3) are known. In mammals, histones are hyper-acetylated before being displaced from the DNA, and phosphorylation and ubiquitylation have also been proposed to occur. For Drosophila, H2A mono-ubiquitylation and a strong increase in H4 acetylation occur shortly before histone removal and degradation. In mammals, histones are replaced first by transition proteins (major types: TP1 and TP2). This study identified the high mobility group protein Tpl94D, a first probable candidate for a functional homologue of mammalian transition proteins. In mammals, transition proteins are subsequently replaced by protamines leading to chromatin with a doughnut structure. In Drosophila, it has recently been shown that the sperm nucleus also contains protamines. Protamines A and B are encoded by two closely related protamine genes, Mst35Ba and Mst35Bb. In addition, the identification of Mst77F shows that sperm nuclei contain at least one further abundant chromatin component. Moreover, in human sperm several new putative protamines have been identified by 2D gel electrophoresis and protein sequencing. In mammals, this chromatin reorganisation is essential for male fertility. Male flies carrying the deletion protDelta38.1, where both protamines as well as three additional ORFs are removed, show severely reduced fertility (Rathke, 2007).

    In summary, a step-by-step scheme is proposed for chromatin reorganisation: (1) histone modifications lead to subsequent histone removal and degradation; (2) the exposed chromatin becomes nicked, resulting in DNA breaks; (3) Tpl94D deposition constitutes an intermediate stage that triggers subsequent protamine-based chromatin organisation (Rathke, 2007).

    Since many features concerning spermiogenesis are conserved between Drosophila and mammals, it is proposed that Drosophila is an ideal system to gain further insight into the mechanism of chromatin reorganisation in spermatid nuclei, a process that is crucial for male fertility (Rathke, 2007).

    Bi-directional gap junction-mediated Soma-Germline communication is essential for spermatogenesis

    Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning, and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP, and EGF pathways are used to mediate germline-soma communication. This study demonstrates that gap junctions may also mediate direct, bi-directional signalling between the soma and germline. When gap junctions between the soma and germline are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg/Inx4 and Inx2 on the germline and the soma side, respectively. Overall, the results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germline to ensure proper spermatogenesis in Drosophila. Moreover, this study shows that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues (Smendziuk, 2015).

    The work presented in this study demonstrates that gap junctions between the soma and germline are essential for fly spermatogenesis. Previous work showing an essential role for Zpg in the fly gonads raised the possibility that signals either from the soma or from other germ cells travel through gap junctions to regulate germline survival and differentiation. Subsequent work in the fly ovaries showed that Zpg was also required for GSC maintenance. This analysis supports and extends these conclusions by finding a cell-autonomous requirement for Zpg in GSC maintenance in the fly testis and demonstrates a role for Inx2 in the soma. Furthermore, it was found that gap junction-mediated signals from the germline also play unique and essential roles in the soma during spermatogenesis, independent of general germline defects. In particular, gap junctions are required to control the proliferation of CySCs and promote the differentiation of their daughters. This work illustrates that the main type of gap junction between the soma and the germline in the fly testis is a heterotypic channel coupling Inx2 in the soma and Zpg in the germline. Importantly, disrupting gap junctions in the soma by knocking down Inx2 phenocopies the zpg mutant phenotype in both the germline and soma. Therefore gap junction-mediated soma-germline regulation in the testis is bi-directional (Smendziuk, 2015).

    Gap junctions contribute to stem cell regulation in the testis Recent work has highlighted the importance of gap junctions in stem cell regulation in a number of systems. In line with results from other stem cell models, the data illustrates a specific role for gap junctions in both GSCs and CySCs. The role of gap junctions in stem cell regulation in the testes was illustrated by the requirement for Zpg in the germline and Inx2 in the soma for GSC maintenance. Moreover, loss of Zpg or somatic knockdown of Inx2 also affected CySC proliferation. Furthermore, ultrastructural analysis revealed the presence of gap junctions between GSCs and CySCs. These results, as a whole, suggest a requirement for gap junction-mediated soma-germline communication in both stem cell populations and at the earliest stages of sperm differentiation (Smendziuk, 2015).

    Gap junctions facilitate signalling between the soma and germline Following the stem cell stage, strong expression and co-localization of Zpg and Inx2 was consistently detected starting at the 4-cell cyst stage. Expression of Zpg and Inx2 began to diminish after the early spermatocyte stages and was not detected past meiotic stages. The timing at which Inx2 and Zpg expression were most prominent corresponds to a period during which niche signals such as BMP are lost. Loss of these signals causes the germline to undergo rapid differentiation and specialization. It has been shown that as somatic cells move away from the niche and begin differentiating, the soma forms a permeability barrier around the germline, isolating the germline from the outside environment. This transition corresponds with a switch occurs whereby soma-germline communication shifts from predominantly exocrine to juxtacrine signalling. Thus, as the germline becomes increasingly isolated, it becomes more dependent on differentiation signals that arrive via gap junctions from the soma. Once the germline becomes isolated, gap junctions may also play an important nutritive role and permit the movement of essential small metabolites between the germline and soma. Similarly, the soma requires gap junction-mediated signals to allow it to accommodate the increasingly expanded, differentiated, and specialized germline (Smendziuk, 2015).

    The observations that gap junctions regulate germline differentiation and soma proliferation are in line with studies from both vertebrate and invertebrate models. In C. elegans, it was recently shown that gap junction-mediated signals are required to maintain GSCs in the niche and for germline differentiation (Starich, 2014). Similarly, work in vertebrates has shown that loss of gap junction-mediated signalling in the soma increased proliferation in post-mitotic Sertoli cells. It is therefore likely that an early role for gap junctions in coordinating soma-germline differentiation is an evolutionarily-conserved mechanism. One recurring feature of germline-soma gap junctions is the expression of different gap junction proteins, resulting in heterotypic gap junctions, exemplified by the Inx2-Zpg gap junctions observed in flies. A key problem in understanding the role of gap junctions in mediating soma-germline communication is identifying the transported signalling cargos. Some possible signals are cAMP, Ca2+, and cGMP, which have been implicated in regulating meiosis in the germline. Attempts to study cAMP and Ca2+ in the testis have proven inconclusive. However, recent work in Drosophila ovaries has suggested that somatic gap junctions may play roles in regulating pH, membrane potential, and ion transport. Overall, multiple signals are likely exchanged between the soma and germline through gap junctions and elucidating their respective functions is a complex task that should be further studied (Smendziuk, 2015).

    Based on the results presented in this study, the following model is proposed: GSCs receive multiple cues that control their behaviour, with gap junctions mostly provide a supporting role, allowing the passage of cues from the soma that facilitate long-term GSC maintenance. After stem cell division germline undergoes rapid differentiation. The germline becomes increasingly isolated from the outside environment, and a permeability barrier is formed by the soma. As outside signals from the niche are lost, the germline relies more heavily on gap junctions to allow the passage of small molecules and metabolites from the soma to promote differentiation and provide nourishment. To ensure coordinated growth and differentiation of the soma and germline, signals pass from the germline through the gap junctions into the soma. Taken together, this work defines gap junction-mediated juxtacrine signalling as an additional signalling mechanism in the fly testis. Furthermore, this study provides a clear illustration of the bi-directional regulatory action of soma-germline gap junctions. As this study demonstratea, disrupting innexins in the soma or germline leads to a specific regulatory effect in the other tissue. Therefore bi-directional gap junction-mediated signalling plays a vital role in ensuring proper coordination of the soma and germline during spermatogenesis (Smendziuk, 2015).

    Genomic and expression analysis of transition proteins in Drosophila
    This study analyzed putative protein sequences of the transition protein-like proteins in 12 Drosophila species based on the reference sequences of transition protein-like protein (Tpl94D) expressed in Drosophila melanogaster sperm nuclei. Transition proteins aid in transforming chromatin from a histone-based nucleosome structure to a protamine-based structure during spermiogenesis - the post-meiotic stage of spermatogenesis. Sequences were obtained from NCBI Ref-Seq database using NCBI ORF-Finder (PSI-BLAST). Sequence alignments and analysis of the amino acid content indicate that orthologs for Tpl94D are present in the melanogaster species subgroup (D. simulans, D. sechellia, D. erecta, and D. yakuba), D. ananassae, and D. pseudoobscura, but absent in D. persmilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi. Transcriptome next generation sequence (RNA-Seq) data for testes and ovaries was used to conduct differential gene expression analysis for Tpl94D in D. melanogaster, D. simulans, D. yakuba, D. ananassae, and D. pseudoobscura. The identified Tpl94D orthologs show high expression in the testes as compared to the ovaries. Additionally, 2 isoforms of Tpl94D were detected in D. melanogaster with isoform A being much more highly expressed than isoform B. Functional analyses of the conserved region revealed that the same high mobility group (HMG) box/DNA binding region is conserved for both Drosophila Tpl94D and Drosophila protamine-like proteins (MST35Ba and MST35Bb). Based on the rigorous bioinformatic approach and the conservation of the HMG box reported in this work, it is suggested that the Drosophila Tpl94D orthologs should be classified as their own transition protein group (Alvi, 2016).

    The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin

    In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. This study shows that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35a/Mst35b. Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals (Kimura, 2016).

    Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila

    In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. This study shows that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization (Tirmarche, 2016).

    The HMG-box-containing proteins tHMG-1 and tHMG-2 interact during the histone-to-protamine transition in Drosophila spermatogenesis

    Spermatogenesis is accompanied by a remarkable reorganization of the chromatin in post-meiotic stages, characterized by a near genome-wide displacement of histones by protamines and a transient expression of transition proteins. In Drosophila, the Transition-protein-like 94D (Tpl94D) contains an HMG-box domain and is expressed during chromatin reorganization. This study searched for additional HMG-box-containing proteins with a similar expression pattern. Two proteins specifically expressed in the testis, tHMG-1 (CG12104) and tHMG-2 (CG30356), whose expression levels were highest during the histone-to-protamine transition. Protein-protein interaction studies revealed that tHMG-1 and tHMG-2 form heterodimers in vivo. Tpl94D, tHMG-1 and tHMG-2 were shown to localize to chromatin of the male germ line, with the most abundant levels observed during post-meiotic chromatin reorganization. Analysis of a tpl94D mutant showed that the C-terminal region of Tpl94D is dispensable for fertility. These data strongly suggested either that the truncated protein, which still contains the N-terminal HMG-box domain, is functional or that other proteins act in functional redundancy with Tpl94D during spermiogenesis. A thmg-1/thmg-2 null mutant also had no detectable specific phenotype, but hmgz, which encodes the major somatic HMG-box-containing protein HMGZ, was transcriptionally up-regulated. These results showed that Drosophila spermatogenesis is characterized by continuous and overlapping expression of different HMG-box-containing proteins. It is hypothesized that the mechanism of chromatin reorganization is a process highly secured by redundancies (Gartner, 2014).

    Prtl99C acts together with protamines and safeguards male fertility in Drosophila

    The formation of motile spermatozoa involves the highly conserved formation of protamine-rich, tightly packed chromatin. However, genetic loss of protamine function in Drosophila and mice does not lead to significant decompaction of sperm chromatin. This indicates that other proteins act redundantly or together with protamines. This study identifies Prtl99C as a Drosophila sperm chromatin-associated protein that is essential for male fertility. Whereas the loss of protamines results in modest elongation of sperm nuclei, knockdown of Prtl99C has a much stronger effect on sperm nuclei. Loss of protamines and Prtl99C indicates an additive effect of these proteins on chromatin compaction, in agreement with independent loading of these factors into sperm chromatin. These data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin (Eren-Ghiani, 2015).

    Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects

    Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. This study examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. Prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH (Hentze, 2013).

    Ecdysteroids are produced as ecdysone (E) in the prothoracic gland (PG) and further metabolized to the principal molting hormone, 20-hydroxyecdysone (20E), in target tissues. In the larval stages, molting and metamorphosis are initiated by pulses of ecdysteroids, whose synthesis is stimulated by the release of prothoracicotropic hormone (PTTH) from the brain. PTTH in turn activates E synthesis in the PG through its receptor Torso. Ecdysteroids are synthesized from cholesterol (C) by a series of reactions primarily mediated by cytochrome P450 (P450) enzymes encoded by a group of genes known as the Halloween genes. The initial catalytic conversion of C to 7-dehydrocholesterol (7dC) requires a Rieske oxygenase called Neverland. Although the following possibly rate-limiting Black Box oxidation of 7dC to the 5β-ketodiol is incompletely understood, it is known to involve the action of at least two enzymes, the dehydrogenase Shroud and Spook (Spo). The final three reactions that convert the 5β-ketodiol to E are mediated by Phantom (Phm), Disembodied (Dib) and Shadow (Sad), all encoded by P450 genes. E produced and released from the PG is converted to 20E in target tissues by another P450 enzyme, Shade (Shd). These Halloween P450 enzymes have been structurally conserved in arthropods and orthologs are found in the genome of insects and even the crustacean water flea Daphnia pulex. It is believed that orthologs from different species have the same function, although functional conservation has not been demonstrated in all insects. Surprisingly, the most structurally conserved of these steroidogenic P450 enzymes, Spo, an enzyme believed to function in the rate-limiting Black Box conversion, has not been conserved as a single ortholog. Drosophila carries two paralogs of this gene, spook (spo; Cyp307a1) and spookier (spok; Cyp307a2) whereas lepidopterans seems to have a single ortholog, spo. In the honey bee, Apis mellifera, a single ortholog, spookiest (spot; CYP307B1) exists. spo and spok show about 57% primary structure similarity and encode P450 enzymes with the same function. In Drosophila, spo is expressed during embryonic development and in the adult female ovaries, but not in the larval PG. Expression in the larval PG is occupied by spok to support ecdysteroidogenesis during postembryonic development. Thus, these genes provide the same function in distinct tissues at different times during development. Although it has not been demonstrated that Spot has the same function as Spo and Spok, the fact that it is a member of the highly conserved CYP307 family strongly suggest that it is functionally conserved. In support of this, spot is the only spo-like gene found in Apis (Hentze, 2013 and references therein).

    The Halloween genes genes are required for 20E biosynthesis, and they have been conserved in insects and perhaps most arthropods. So far, the only exception is the spider mite, Tetranychus urticae, which lacks phm, and thus, the ability to perform C25 hydroxylation of ecdysteroids. Consequently, the main ecdysteroid is ponasterone A and not 20E in this species (Hentze, 2013).

    The Halloween genes are also found in Tribolium, but it has never been tested experimentally whether their function in ecdysteroidogenesis is conserved in this species. The current data show that expression of spo, phm, dib, sad and shd increases during the final larval instar which correlates with the increased ecdysteroid production necessary for molting. Reducing the expression of two of these genes, phm and spo, resulted in delayed molting or developmental arrest of Tribolium larvae, a phenotype typically observed in animals lacking ecdysteroids. Consistent with this observation, ecdysteroid levels were low in these animals demonstrating that Spo and Phm are important for ecdysteroid biosynthesis during the larval stages. This correlation suggests strongly that the Halloween genes have been functionally conserved in Tribolium (Hentze, 2013).

    Although the four Halloween enzymes mediating the final hydroxylation steps in the biosynthesis of 20E have been identified, some upstream steps in the pathway remain less well characterized e.g. the Black Box. Earlier studies have shown that the CYP307 family enzymes function in the Black Box, since supplying precursors of 20E downstream of the Black Box reaction rescues Drosophila larvae with reduced expression of spok. Moreover, these animals were not rescued by supplements of 7dC, a 20E precursor upstream of the Black Box reactions. The CYP307 paralogs are more highly conserved than the other Halloween P450 enzymes and are believed to have similar functions. In support of this notion, ectopic expression of spo, rescues Drosophila spok mutants (Michael O'Connor, personal communication to Hentze, 2013) and expression of Bombyx mori spo is sufficient to rescue Drosophila spo mutants. spo and spok belong to the CYP307A subfamily. However, the genome of some insects, including Anopheles, Apis and Tribolium, contain a CYP307B subfamily gene. Although subtle catalytic differences may exist between CYP307 enzymes, these conserved paralogs are likely to be functionally redundant products of gene duplications that occupy different spatio-temporal patterns of expression. Such a division of activity has been found in Drosophila where spo and spok exhibit different spatio-temporal expression patterns to support ecdysteroid biosynthesis in different tissues at distinct developmental stages (Ono, 2006). The data support a similar scenario in Tribolium in which case spo is expressed during development and in the adult ovaries and spot is expressed in the reproductive system of adult males. In support of the data showing lack of spot expression during development, it was confirmed that spot is not required to support ecdysteroid biosynthesis as the RNAi mediated knock down in the larval stages did not delay development (Hentze, 2013).

    This is the first data on the expression of a CYP307B family gene in any insect, suggesting that expression of these genes may be low and/or limited to a few cells in specific tissues, like the expression of spot in the TAGs of adult male Tribolium. As the genes encoding the terminal hydroxylases are conserved as 1:1 orthologs, it is puzzling why the genes of the even more conserved CYP307 family have been allowed to duplicate so that some species like Tribolium carry two paralogs. However, the available data suggest that these genes may have divided their effort to support E biosynthesis in a temporal and spatial restricted manner (Hentze, 2013).

    The highly specific expression of spot in the TAGs suggests that ecdysteroids are produced by the reproductive system of adult males. As spot seems to be the only CYP307 family member expressed at substantial levels in adult males, the TAGs may be the major site of ecdysteroidogenesis in adult males. Little is known about the function of steroid hormones in adult insects, but they are believed to be produced in the gonads, like the sex steroids of vertebrates, and it has previously been suggested that they might have a somewhat similar function. In females of some insect species the ovaries produce ecdysteroids and their synthesis is required for oogenesis and the synthesis of vitellogenin. In Drosophila mutants lacking the ability to synthesize 20E, egg development is arrested at stage 8-9. The roles of ecdysteroids and their site of synthesis in males are much less clear. Ecdysteroids have been observed in testes and the accessory gland of male grasshoppers and in the testes of adult male blowflies. Recently Schwedes (2011) has documented the activity of the ecdysone receptor/ultraspiracle (EcR/USP) complex in numerous tissues in adult male Drosophila, suggesting that ecdysteroid signaling is important in adult male insects. High activity was observed in the male accessory gland, which could be explained by synthesis of ecdysteroids in this tissue (Hentze, 2013).

    Ecdysteroid levels in adult males are generally lower compared to the high-level pulses produced by the PG that drive transitions during development and those of female insects. This is consistent with the finding that Halloween gene expression in male adults is low compared to larvae, pupae and females. Biochemical identification of ecdysteroidogenic tissue has relied on the ability of tissues to convert labeled C into E and 20E. However, the low ecdysteroidogenic capacity of males makes it difficult to detect such conversions. Molecular approaches, such as those employed in this study, have provided some insights into the identification of ecdysteroidogenic tissues of the male mosquito, Anopheles. Using semi-quantitative PCR and in situ hybridization techniques it was shown that the genes coding for the terminal hydroxylases phm, dib, sad and shd are expressed specifically in the accessory glands of male Anopheles. In Anopheles, 20E produced in the accessory glands is stored and transferred to the female during mating, but the endocrine function of this 20E in males remains conjectural. As in Tribolium, expression of the genes coding for the biosynthetic enzymes necessary for ecdysteroid biosynthesis was not observed in the testes of Anopheles. However, whereas the testes of Anopheles seems to lack significant expression of all biosynthetic enzymes, Tribolium testes lack expression of a CYP307 family gene which is indicating their inability to conduct de novo ecdysteroidogenesis. Although tissues lacking expression of a spo-like gene may be able to mediate downstream steps in the pathway, they are unlikely to have the capacity to synthesize E from C. Such capacity has been demonstrated in Locusta migratoria and Manduca sexta where biochemical analyses have shown that non-ecdysteroidogenic tissues, in addition to the PG, can convert the 5β-ketodiol, but not C, to E. These tissues presumably lack pathway activity upstream to the 5β-ketodiol, which is the ability to perform the Black Box reaction and possibly the C to 7dC conversion. Controlling the activity of spo-like genes makes sense as they participate in the possible rate-limiting black box step and expression of these genes may therefore be the determining factor for ecdysteroidogenesis. Expression of the downstream terminal hydroxylases may not require such a precise control since 'leaky' expression of these genes alone will not enable a tissue to synthesize ecdysteroid de novo. Regulation of ecdysteroid biosynthesis at the level of Spo is also supported by evidence identifying spo as the only target of PTTH signaling in the E biosynthetic pathway in Manduca. Consistent with this view, expression of phm, dib, sad and shd was detected outside the TAGs. Although it cannot be ruled out that tissues other than the TAGs in Tribolium synthesize ecdysteroids in adult males, the present data provides evidence that the TAGs are a major site (Hentze, 2013).

    Intriguingly however, the TAGs seem to lack the expression of shd, making it likely that they synthesize E rather than 20E. This is similar to the larval PG that produces E which is released and converted to 20E in target tissue. In contrast, all the genes necessary for the production of 20E, including shd, are expressed in the ovaries of Tribolium, as in Drosophila. Therefore it is likely that the ovaries primarily produce 20E, whereas the male reproductive system may synthesize E (Hentze, 2013).

    The ovaries of female Tribolium express spo and the genes for the terminal hydroxylases, including shd, required for synthesis of 20E. Male accessory glands also express a spo-like gene, spot, and the genes for the terminal hydroxylases required for synthesis of E, but not shd. However, shd expression was detected in the carcass without the reproductive system indicating that E synthesized by the accessory gland might be converted to 20E in peripheral tissues, like during the larval stages. Alternatively, E produced by the accessory gland may be involved in male-specific hormone signaling or be transferred to females during mating (Hentze, 2013).

    Although E is a precursor of 20E it induces a specific genetic response distinct from that of 20E in Drosophila larvae. In support of an E-specific role, the distribution of E and 20E varies during pupal-adult development in Manduca. During this stage a pulse of E precedes a pulse of 20E by several days, indicating that the two hormones have different functions. Another interesting observation is that E, but not 20E, induces vitellogenesis in the cockroach Blaberus craniifer. This opens the possibility that E produced by males, and transferred to females during mating, is required to stimulate vitellogenesis. Thus, E produced in the accessory gland may affect female post-mating physiology and behavior as it has been suggested in Anopheles. Alternatively, E synthesized in the accessory gland could be released into the hemolymph to affect male physiology and behavior. For example, a minor induction in the transcription of the male specific yellow protein in Schistocerca gregaria was observed in response to E, while 20E is an inhibitor, indicating that E and 20E might have sex-specific roles. Some insight into the role of ecdysteroids in adult male insects comes from recent studies of Drosophila. The reduced ecdysteroid level of DTS-3 mutants impairs memory formation in males (Ishimoto, 2009). Interestingly, ecdysteroid levels are significantly elevated in wild type males after courtship. Further, ecdysteroids affect sleep and longevity in adult Drosophila, providing evidence that they serve important physiological functions during adulthood in males as well as in females (Hentze, 2013).

    Ecdysteroids also influence male courtship behavior by regulation of the transcription factor fruitless. A recent study also shows that conditional reduction of ecdysteroid signaling in the adult stage, and not during development, causes the males to display male-male courtship, indicating that the effects of ecdysteroids is not solely explained by neuronal wiring of a male-specific circuitry during development. This phenotype is also observed in males that lack PTTH, suggesting that PTTH may regulate E production in adults. Although PTTH was first purified from adult moths and is found in adult Drosophila, its role in adults is not known. This study presents the first data showing that PTTH regulates ecdysteroid synthesis in adult insects, i.e. has functions other than eliciting and coordinating metamorphosis. Although PTTH influences the ecdysteroid levels, the data suggest that this effect does not require transcriptional regulation of the steroidogenic genes. Thus, PTTH probably mediates an acute response on steroidogenesis through post-transcriptional regulation in the adult, a scenario similar to that observed in the PG during development. The data indicating expression of the gene for the PTTH receptor Torso in the secondary cells of the accessory glands of male Drosophila provide molecular evidence that the accessory glands may be a target of PTTH. These data are supported by the recent identification of the accessory glands as the only PTTH-responsive tissue in adult Manduca, although no link was made between the PTTH-induced phosphorylation response in the accessory glands and ecdysteroidogenesis. Together with the reduced ecdysteroid levels observed in ptth and torso RNAi males, these data raise the possibility that PTTH regulates ecdysteroid production in the accessory glands (Hentze, 2013).

    Other peptide hormones, such as the insulin-like peptides, are known to be involved in the regulation of ecdysteroid signaling. In Drosophila, body size and developmental timing is influenced by the interplay between insulin and ecdysone signaling. Insulin-like peptides have been shown to stimulate ecdysteroid biosynthesis, and one specific insulin-like peptide, DILP8, may also be involved in suppressing ecdysteroid production to coordinate organ growth and maturation. In the mosquito Aedes aegypti and Drosophila, insulin has a stimulatory effect on ecdysteroidogenesi. The fact that insulin stimulates ecdysteroid production in adults is interesting considering the potential role of ecdysteroids as sex-steroids. In crayfish, that also produces ecdysteroids, silencing of an insulin-like gene confers testicular degeneration and ovarian up regulation (Hentze, 2013 and references therein).

    In several species ecdysteroids have been shown to influence spermatogenesis, by affecting the rate of mitosis and meiosis, and thus, differentiation of germ cells. Although ecdysteroids are required for female reproduction, male Drosophila dib mutants, which are unable to synthesize 20E, are fertile. Similarly, no reduced production was observed of offspring by spot-RNAi and phm-RNAi Tribolium males. It is possible that the knock down was insufficient to affect reproductive success. However, arguing against this possibility, injection of dsRNA into larvae efficiently reduced expression of phm and spot and the 20E production. One explanation is that E or 20E is not essential for male fertility. This also agrees with previous studies, finding that knock down of phm and shd does not affect fertility of male Tribolium, and that ecdysteroids are essential for female, but not male, germ cell development in Drosophila. Interestingly, a recent study found that the secondary cells of the Drosophila accessory glands produce a substance that induces a female post-mating response by influencing egg laying activity and sexual receptivity. Considering the evidence that E has been observed to be a more potent stimulator of vitellogenesis than 20E, one could speculate that E may be produced by the secondary cells in Drosophila males and passed to the female during mating (Hentze, 2013).

    As ecdysteroids are produced in the reproductive organs of adult insects, and in some species influences spermatogenesis, release of vitellogenin, memory and sex-specific physiology and behavior, they share several features with the vertebrate sex-hormones. In vertebrates, the reproductive system of males primarily synthesizes testosterone, the precursor of the female sex steroid, estradiol. The conversion of testosterone to estradiol is catalyzed by a P450 enzyme, like Shd, that mediates the conversion of E to 20E in insects. Considering that E and 20E have distinct functions and have been proposed to function as sex steroids in insects, the present study provides the first evidence that males may synthesize E for sex-specific purposes whereas females presumably produce 20E (Hentze, 2013).

    Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila accessory gland

    The paired male accessory glands of Drosophila melanogaster enhance sperm function, stimulate egg production, and reduce female receptivity to other males by releasing a complex mixture of glycoproteins from a secretory epithelium into seminal fluid. A small subpopulation of about 40 specialized secretory cells, called secondary cells, resides at the distal tip of each gland. These cells grow via mechanisms promoted by mating. If aging males mate repeatedly, a subset of these cells delaminates from and migrates along the apical surface of the glandular epithelium toward the proximal end of the gland. Remarkably, these secretory cells can transfer to females with sperm during mating. The frequency of this event increases with age, so that more than 50% of triple-mated, 18-d-old males transfer secondary cells to females. Bone morphogenetic protein signaling specifically in secondary cells is needed to drive all of these processes and is required for the accessory gland to produce its normal effects on female postmating behavior in multiply mated males. It is concluded that secondary cells are secretory cells with unusual migratory properties that can allow them to be transferred to females, and that these properties are a consequence of signaling that is required for secondary cells to maintain their normal reproductive functions as males age and mate (Leiblich, 2012).

    The secondary cells of the male fly accessory gland selectively grow during aging in adults, a process enhanced by repeated mating. These cells exhibit a range of behaviors, induced by mating, that are atypical of secretory cells in glands, including active delamination and migration. Although migrating cells were initially observed in less than 5% of repeatedly mated males, introducing a delay between two previous matings and dissecting the resulting 18-d-old males revealed migrating cells in all animals, suggesting that this process is common in aged, mated animals (Leiblich, 2012).

    The growth, delamination and migratory activities of secondary cells all require cell-autonomous BMP signaling. One or more of these BMP-regulated processes modulates long-term, postmating behavior in females, particularly when males are repeatedly mated over short periods of time, requiring rapid replenishment of luminal content in the accessory gland. Although the numbers of vacuoles in secondary cells with high levels of BMP signaling seem more variable than controls, vacuole number in Dad-expressing secondary cells appears relatively normal, suggesting that reduced BMP signaling does not simply block the general secretory machinery. However, reduced signaling presumably affects the synthesis or function of one or more secondary cell products, leading either to direct effects in mated females or to indirect effects through modulation of main cell function or products in males (Leiblich, 2012).

    Unexpectedly, some secondary cells are transferred to females after multiple matings, particularly in aged flies, raising the possibility that these delaminating cells continue to function together with sperm even outside the male. Transfer is not essential for these cells to mediate their BMP-regulated effects in females, because not all mated females receive these cells. However, it is possible that transfer could contribute to changes in accessory gland function as the glandular epithelium undergoes BMP-dependent structural alterations during aging and mating. A recent study from Minami (2012) indicates that secondary cells are required for normal male fecundity and effects on female postmating behaviors. The current work now clearly demonstrates that BMP-mediated events in secondary cells are involved in maintaining these latter functions specifically during adulthood (Leiblich, 2012).

    The data highlight some surprising parallels between the accessory gland and the prostate, in addition to those previously reported. Like the prostate, the structure of the accessory gland epithelium changes significantly with age. Furthermore, BMP signaling is implicated in normal prostate development and in the progression of prostate cancer. Importantly, prostate cells have been identified in human semen and the phenotype of these cells may be altered in prostate cancer. Although many of these cells are likely to have sloughed off from the epithelium, the current study raises the possibility that some actively delaminate into seminal fluid (Leiblich, 2012).

    The secondary cells of the accessory gland require BMP signaling to regulate the synthesis or function of one or more important components of the seminal fluid as flies age and mate. However, this signaling simultaneously drives cell loss and changes in the morphology and function of the epithelium, which appears to lack regenerative capacity in flies. The prostate gland of most human males over 50 y of age is hyperplastic, and it is tempting to speculate that this reflects a regenerative response to similar events in this organ. A more detailed analysis of secondary cell biology should help to further elucidate the processes that underlie functional changes in the accessory gland epithelium and test whether these are shared by male reproductive glands in other organisms (Leiblich, 2012).

    BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior

    Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. This paper shows that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution (Corrigan, 2014).

    Seminal fluid synthesized by male reproductive glands has a powerful influence on fertility, affecting multiple sperm activities and altering female behavior, in some cases directly conflicting with female reproductive interests. Several previous studies have revealed an important function for seminal peptides in Drosophila in these processes. However, this study presents the first in vivo evidence that exosomes also play a key role and identify a completely novel role for BMP signaling in regulating this process (Corrigan, 2014).

    Exosome biogenesis, secretion, and uptake have been previously studied in Drosophila. However, the small size of exosomes, MVBs, and fly tissues makes these processes difficult to analyze in vivo. The AG contains only nanoliter volumes of secretions, making it impractical to use standard exosome analysis techniques, such as ultracentrifugation and Nanosight Tracking Analysis. Like other studies in flies, this study used genetic and imaging approaches to test the identity of SC-specific CD63-positive puncta. In addition, Western blot analysis of transferred seminal fluid and live imaging of giant MVBs in SCs were used to test the hypothesis that SCs produce exosomes (Corrigan, 2014).

    The human CD63-GFP tetraspanin marker was used in this analysis. However, GFP-positive puncta were also observed in large secretory compartments of SCs expressing cytosolic GFP, and exosome-sized vesicles in MVBs and the AG lumen were observed in EM analysis of wild-type glands, confirming their presence in nontransgenic flies. Because exosomes can be loaded with many cellular components, the findings provide a potential explanation for the observation that AGs of several insects, including Drosophila, secrete intracellular proteins (Corrigan, 2014).

    Other evidence strongly supports the idea that CD63-positive puncta secreted from SCs are exosomes and not vesicles shed from the plasma membrane. This includes the observation that CD63-positive puncta are found inside both acidic Rab7-positive MVB-like compartments as well as nonacidic Rab11-positive vacuoles and require the ESCRT and ESCRT-associated proteins Hrs and ALiX, as well as several Rabs linked to mammalian exosome secretion, to be formed and secreted. Secreted puncta counts have been used previously in flies to study genetic control of exosome secretion. A criticism of this approach is that reduced puncta numbers may merely reflect aggregation. However, the transfer of CD63-GFP to females was drastically reduced in mutant backgrounds, arguing against a simple aggregation model. Furthermore, because genetic manipulation of ESCRT function does not alter other secretory processes in SCs, this strongly implicates the endocytic pathway in secretion of tagged CD63 (Corrigan, 2014).

    Studies of exosomes in Drosophila as well as mammals already suggest that multiple exosome subtypes exist and may be regulated differently, e.g., different roles for ALiX, Hrs, and Evi. If different exosome subtypes are made in SCs, these cells should offer an ideal system to study their differential regulation (Corrigan, 2014).

    The remarkably large size of endosomal compartments in SCs provides new opportunities to study exosome biogenesis in vivo. To date, many studies of the intracellular exosome biogenesis machinery and endolysosomal trafficking in higher eukaryotes have relied on expressing an activated form of Rab5 or addition of the ionophore monensin in cell culture to artificially enlarge the endolysosomal compartments, disrupting normal trafficking events. Hence, this new SC in vivo model should allow reinvestigation of previously reported regulators of exosome biogenesis and identify functional differences in trafficking phenotypes, as has been seen for Hrs and ALiX (Corrigan, 2014).

    This study has already revealed a surprisingly dynamic interaction between the secretory and endolysosomal systems in SCs. Communication between these compartments using vesicular transport and tubulation processes has been reported in other cell types in flies and mammals, but this study suggests that direct fusion can also be involved. Indeed, the data are also consistent with mMVBLs forming after fusion between SVs and iLEs, suggesting that fusion events may play a critical role in establishing distinct compartments within SCs. In light of this dynamic flux between compartments, it remains unclear whether CD63-GFP-labeled exosomes might be released by the classical route involving mMVBL fusion to the plasma membrane or via an intermediate secretory compartment (Corrigan, 2014).

    Although most analysis of the fly AG has highlighted roles for MC products, such as SP, in reprogramming female postmating responses, several recent studies have also suggested a central but poorly defined function for SCs. A transcriptional program regulated by the Hox gene Abd-B controls vacuole formation in SCs (Gligorov, 2013). These findings now indicate that at least one of the effects mediated by SCs, altered receptivity to remating, requires exosome secretion (Corrigan, 2014).

    It is difficult to accurately estimate the frequency of SC exosome-sperm fusion events in each female fly because they can probably only be visualized transiently, and many may involve fusion to the very long sperm tail. Sperm play an essential role as mediators of SP-dependent postmating effects in females, so it is plausible that exosome fusion to sperm may modulate specific SP functions. Another appealing hypothesis is that SC exosomes also interact with the female reproductive tract to influence female behavior. However, whatever the target tissues, the data clearly demonstrate a role for SC exosomes in female reprogramming. Furthermore, like human prostasomes, SC exosomes fuse with sperm, highlighting possible conserved roles for exosomes in male reproductive biology. In prostate cancer, prostasomes are inappropriately secreted into the bloodstream, so that other cells in the body may be subjected to these powerful reprogramming functions, potentially supporting tumor-stroma interactions and metastasis (Corrigan, 2014).

    Reducing BMP signaling in SCs inhibits exosome secretion and leads to the formation of a novel mMVBL compartment that is filled with fluorescent CD63-GFP. A simple interpretation of this result is that MVBL compartments in these cells do not mature properly, blocking exosome secretion. Consistent with this, increasing BMP signaling in these cells produces a highly enlarged acidic compartment (Corrigan, 2014).

    Previous studies have shown that blocking endosomal maturation by knockdown of the early ESCRT component Hrs increases the size of immature endosomal class E compartments lacking ILVs and also results in increased BMP signaling. The data demonstrate that elevated BMP signaling increases mMVBL size, suggesting that there is a complex bidirectional interaction between mMVBL maturation and size and the level of BMP signaling in SCs (Corrigan, 2014).

    The findings are consistent with a model in which BMP signaling also controls SC growth by driving endolysosomal trafficking and maturation events. Late endosomes and lysosomes have previously been shown to house major nutrient sensors and cell growth machinery, including the mTORC1 complex, which is activated by intraluminal amino acids. Interestingly, the growth rate of knockdown cells with reduced ESCRT function appears to correlate with mMVBL size rather than exosome secretion rate. Whether growth in these cells is mTORC1 dependent needs to be tested (Corrigan, 2014).

    Whatever the explanation for the growth defects in SCs, these data very clearly implicate BMP signaling in the regulation of endolysosomal trafficking and exosome secretion. It will now be important to test whether BMP signaling plays a similar role in mammalian glands that secrete exosomes, such as prostate and breast, and determine whether this role is affected in diseases such as cancer (Corrigan, 2014).

    Drosophila sperm surface α-l-fucosidase interacts with the egg coats through its core fucose residues

    Fucose and α-l-fucosidases have fundamental function(s) during gamete interactions. An α-l-fucosidase has been detected as transmembrane protein on the surface of spermatozoa of eleven Drosophila species. Immunofluorescence labeling showed that the protein is localized in the sperm plasma membrane over the acrosome and the tail, in Drosophila melanogaster. In the present study, efforts were made to analyze with solid phase assays the oligosaccharide recognition ability of fruit fly sperm α-l-fucosidase with defined carbohydrate chains that can functionally mimic egg glycoconjugates. The results showed that α-l-fucosidase bound to fucose residue and in particular it prefers N-glycans carrying core α1,6-linked fucose and core α1,3-linked fucose in N-glycans carrying only a terminal mannose residue. No binding was detected when α-l-fucosidase was pre-incubated with fucoidan, a polymer of α-l-fucose and the monosaccharide fucose. Furthermore, egg labeling with anti-horseradish peroxidase, that recognized only core α1,3-linked fucose, correlates with α-l-fucosidase micropylar binding. Collectively, these data support the hypothesis of the potential role of this glycosidase in sperm-egg interactions in Drosophila (Intra, 2015).

    The fatty acid elongase Bond is essential for Drosophila sex pheromone synthesis and male fertility

    Insects use a spectacular variety of chemical signals to guide their social behaviours. How such chemical diversity arises is a long-standing problem in evolutionary biology. This study describes the contribution of the fatty acid elongase Bond to both pheromone diversity and male fertility in Drosophila. Genetic manipulation and mass spectrometry analysis reveal that the loss of bond eliminates the male sex pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Unexpectedly, silencing bond expression severely suppresses male fertility and the fertility of conspecific rivals. These deficits are rescued on ectopic expression of bond in the male reproductive system. A comparative analysis across six Drosophila species shows that the gain of a novel transcription initiation site is correlated with bond expression in the ejaculatory bulb, a primary site of male pheromone production. Taken together, these results indicate that modification of cis-regulatory elements and subsequent changes in gene expression pattern is one mechanism by which pheromone diversity arises (Ng, 2015)

    Secondary cell expressed genes in the male accessory gland are needed for the female post-mating response in Drosophila melanogaster

    Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprises behavioral and physiological changes that include increased egg-laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP), and are maintained by SP's binding to, and slow-release from, sperm. The accessory gland contains two secretory cell types whose morphology and development differs. Products of these "main" and "secondary" cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, this study analyzed iab6cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, 77 genes were identified whose expression is down-regulated by a factor of >5x in iab6cocu males. By functional assays and microscopy, 20 candidate genes were tested and it was found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg-laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles' contents may be important for the machinery used to maintain the PMR (Sitnik, 2016).

    The piRNA pathway is developmentally regulated during spermatogenesis in Drosophila

    PIWI-interacting RNAs (piRNAs) are predominantly produced in animal gonads to suppress transposons during germline development. Understanding about the piRNA biogenesis and function is predominantly from studies of the Drosophila female germline. piRNA pathway function in the male germline, however, remains poorly understood. To study overall and stage-specific features of piRNAs during spermatogenesis, this study analyzed small RNAs extracted from entire wild-type testes and stage-specific arrest mutant testes enriched with spermatogonia or primary spermatocytes. It was shown that most active piRNA clusters in the female germline do not majorly contribute to piRNAs in testes, and abundance patterns of piRNAs mapping to different transposon families also differ between male and female germlines. piRNA production is regulated in a stage-specific manner during spermatogenesis. The piRNAs in spermatogonia-enriched testes are predominantly transposon-mapping piRNAs, and almost half of those exhibit a ping-pong signature. In contrast, the primary spermatocyte-enriched testes have a dramatically high amount of piRNAs targeting repeats like suppressor of stellate and AT-chX The transposon-mapping piRNAs in the primary spermatocyte stages lacking Argonaute3 expression also show a ping-pong signature, albeit to a lesser extent. Consistently, argonaute3 mutant testes also retain ping-pong signature-bearing piRNAs, suggesting that a noncanonical ping-pong cycle might act during spermatogenesis. The study found stage-specific regulation of piRNA biogenesis during spermatogenesis: An active ping-pong cycle produces abundant transposon-mapping piRNAs in spermatogonia, while in primary spermatocytes, piRNAs act to suppress the repeats and transposons (Quénerch'du, 2016).

    Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes

    The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. This study describes the identification and initial characterization of dany (CG30401), a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC (a testis-specific meiotic arrest complex (see Always early)) and tTAFs (see Cannonball), the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, it is proposed that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization (Trost, 2016).

    Meiotic sex has a deep evolutionary origin in basal eukaryotes. While meiosis has generally remained well conserved, most other aspects of sexual reproduction have diverged rapidly. In animals that differentiate a male and a female gender, male-biased genes and in particular those expressed in the germline are affected by turnover and sequence divergence that is significantly faster than in other gene classes. Analyses in Drosophila melanogaster spermatocytes have provided some of the most striking evidence for rapid evolutionary dynamics even in key elements of transcriptional networks with numerous interactions (Trost, 2016).

    The comparison of different tissues in adult D. melanogaster has clearly revealed that the number of genes with an expression apparently restricted to a single tissue is maximal in the case of testis. The large majority of these, as well as of the testis-biased genes, are transcribed in spermatocytes, i.e., in germline cells during a growth phase between the last mitotic division and the onset of the meiotic divisions and spermiogenesis. The transcription of more than 1000 of these genes depends on the function of the testis meiotic arrest complex (tMAC). tMAC is a testis-specific variant of the MIP/dREAM/SynMuvB complex, a widely conserved somatic transcriptional regulator. tMAC contains subunits encoded by testis-specific paralogs (aly, tomb) that are only present within the genus Drosophila. They were identified based on a characteristic loss-of-function phenotype in which the testes fill up with spermatocytes failing to enter meiotic divisions and postmeiotic differentiation. Several genes of comparably recent origin and with similar mutant phenotypes (topi, comr, achi, vis) encode proteins interacting with tMAC. A second protein complex of paramount importance for spermatocyte-specific transcription is formed by testis-specific TATA-binding protein (TBP)-associated factors (tTAFs) in the genus Drosophila. The tTAF genes [can, mia, nht, rye (Taf12L - FlyBase), sa] were also identified based on their mutant phenotype, which is slightly milder than that associated with tMAC loss (Trost, 2016).

    This study describes the identification of dany, yet another gene with a recent evolutionary origin and an essential role in spermatocyte-specific gene expression. dany is most similar to the Drosophila genes distal antenna (dan) and distal antenna-related (danr). Dan and Danr have recently been implicated in the control of neuroblast competence in Drosophila embryos, where they inhibit the repositioning of crucial target genes into repressive chromatin associated with the nuclear lamina (Kohwi, et al., 2013). Similarly, Dany is required not only for cell type-specific gene expression in spermatocytes but also for normal association of chromatin with the nuclear envelope (Trost, 2016).

    This identification and characterization of dany has uncovered a factor crucial for the realization of the spermatocyte-specific gene expression program in Drosophila. Loss of dany compromises the transcriptional activation of a large number of spermatocyte-specific genes and derepresses genes that are normally inactive in spermatocytes. Moreover, dany is required for normal association of spermatocyte chromatin with the nuclear periphery (Trost, 2016).

    With regard to transcriptional activation, Dany is similar to tTAFs and tMAC. Mutations in tMAC genes have a severe effect on spermatocyte-specific gene. In the case of tTAF mutants, fewer genes are affected and the reduction in transcript levels is often less severe. Loss of dany has even slightly milder effects, but there are still ~1000 genes with significantly reduced transcript levels. Many of these genes are also dependent on tMAC and tTAFs (Trost, 2016).

    The fact that Dany is just as important for silencing of non-spermatocyte genes as for activation of spermatocyte-specific genes indicates that this protein does not function like the activators tTAFs and tMAC, which have not been implicated in gene silencing. Several additional observations support this conclusion. Dany intracellular localization in early spermatocytes is distinct from that of the tTAF Sa. dany transcript and protein levels depend neither on Sa nor on the tMAC components Aly and Topi. Vice versa, mutations in dany affect neither transcript levels of tTAF and tMAC genes nor accumulation and intracellular localization of representative protein products (Sa and Can) (Trost, 2016).

    dany orthologs cannot be detected outside the genus Drosophila, whereas the primordial dan/danr genes are present throughout the insect lineage. The testis-specific tMAC and tTAF genes are comparably young in evolutionary terms. The fact that several functionally independent factors of paramount importance for the highly complex spermatocyte-specific gene expression program have a recent origin provides further testimony of the surprising evolutionary dynamics of genes that function in the male germline. After a gene duplication event, dany has evolved to control the expression of thousands of genes in the male germline. Dany is a potent regulator; ectopic expression of dany in somatic tissues is highly toxic (Trost, 2016).

    How does Dany exert its function? Dany is a nuclear protein. After ectopic expression in larval salivary glands, it binds preferentially to all euchromatic interband regions of polytene chromosomes. In maturing spermatocytes, where it is normally expressed, Dany also appears to associate preferentially with euchromatic regions within chromosome territories and not with the regions of maximal DNA staining intensity corresponding to pericentromeric heterochromatin. As the Psq motif of some other proteins, including CENPB and transposases, is involved in sequence-specific DNA binding, it is conceivable that Dany and its closest relatives Dan and Danr bind to DNA as well. The Psq domain structure of Dan as revealed by NMR is highly similar to that observed by X-ray crystallography in CENPB. However, the DNA-binding region within CENPB is not restricted to the Psq motif but includes an additional helix-turn-helix domain that is not present in Dan, Danr and Dany. Similarly, the DNA-binding region of transposases extends considerably beyond the Psq motif. By mutating three amino acid codons within the Dany Psq motif, which correspond to positions contacting bound DNA in the case of CENPB, it was possible to demonstrate the functional importance of this motif. However, DanyAAA still retains some function; it promotes spermatogenesis beyond the stage when dany null mutant spermatocytes arrest. Moreover, the intracellular localization of DanyAAA in spermatocytes appears to be normal. Additional analyses will be required to resolve whether the Psq motif of Dany indeed binds DNA. It is pointed out that the Psq motif in CENPB contacts only four base pairs. Such a limited DNA sequence specificity could not explain the observed preferential association of Dany with euchromatic regions. Additional factors will have to be identified (Trost, 2016).

    Interestingly, the first phenotypic abnormalities that this study has detected in dany mutants occur in young spermatocytes soon after the onset of dany expression. Whereas chromosome territories tend to become enwrapped in the nuclear envelope when they are formed in wild-type spermatocytes during the S3 stage, dany mutant spermatocytes are devoid of such characteristic nuclear envelope deformations at the corresponding stage. Thus, Dany appears to be required for normal chromatin association with the nuclear envelope. Since Dany is not enriched at the nuclear periphery, it obviously is very unlikely to function as a factor that directly establishes physical contact between chromatin and the nuclear envelope. But the extensive chromatin reorganization, which presumably occurs in early spermatocytes when thousands of previously repressed genes become active while precursor-specific genes are inactivated, might be abnormal in the absence of dany. Thereby, chromatin properties that normally cause localization to the nuclear periphery might also be affected (Trost, 2016).

    Dany's closest relatives Dan and Danr, which act partially redundantly in somatic tissues, are crucial for the control of intranuclear position and silencing of the hunchback (hb) genomic locus in neuroblasts during embryogenesis. hb relocalization to the nuclear lamina, which is correlated with hb silencing and termination of Hb response competence, depends on timely Dan downregulation (Trost, 2016).

    Changes in the association of genes with the nuclear periphery have previously been implicated in the control of the spermatocyte-specific gene expression program in Drosophila. In cell types other than spermatocytes, most spermatocyte-specific genes appear to be packaged into a repressive type of heterochromatin designated 'BLACK' heterochromatin, which is associated with Lam at the nuclear envelope. In case of two representative testis-specific gene clusters (at 60D1 and 22A1), loss of Lam was shown to result in their derepression in somatic larval and cultured cells, and the normal transcriptional activation was accompanied by cluster repositioning into the nuclear interior of late spermatocytes. Immuno-FISH analyses support the notion that a repressive chromatin type that is normally associated with the nuclear periphery is impaired in dany mutants. The derepression of the eye-specific inaC gene in dany mutant spermatocytes was accompanied by delocalization away from the nuclear periphery into the interior. Extensive future work will be required to clarify the molecular basis and functional significance of these initial observations (Trost, 2016).

    Even if an extensive correlation between intranuclear position and transcriptional activity were established by systematic studies, it remains to be considered whether loss of dany might affect the organization of chromatin within the nucleus indirectly. dany mutations could alter the expression program of particular chromatin regulators or proteins of the nuclear periphery. Indeed, some important chromatin regulators and nuclear envelope proteins have been shown to undergo drastic changes in expression levels during normal spermatogenesis. Su(Hw), a multi-zinc finger DNA-binding protein that can function as a transcriptional insulator and modulator of chromatin association with the nuclear lamina, and the PRC2 complex components E(z) and Su(z) are strongly downregulated in early spermatocytes, whereas Lamin C (LamC) expression is induced. However, in these particular cases, no abnormal expression program was detected in dany mutant testis by immunolabeling (Trost, 2016).

    Loss of dany also results in the derepression of genes that appear to be Pc targets. Such genes are primarily within 'BLUE' heterochromatin. Pc-mediated repression of spermatocyte-specific genes has been proposed to be counteracted in spermatocytes by the combined action of tMAC and tTAFs. Although not identical, the dany mutant phenotype shares similarities with the meiotic arrest phenotype caused by loss of tMAC and tTAF function. Future work will be required to clarify the functional interactions between Dany and these major activators of spermatocyte-specific genes (Trost, 2016).

    Extensive spatial intranuclear chromatin reorganization might guide many differentiation processes in complex eukaryotes. For example, hundreds of genes were reported to move towards or from the nuclear envelope during differentiation of mouse embryonic stem cells, concomitant with reduced or increased expression, respectively. Similar observations were made during adipogenic cell differentiation. This identification and initial characterization of dany should help support future progress towards a molecular understanding of the mechanisms that coordinate change in spatial genome organization and cell type-specific expression programs (Trost, 2016).

    Critical roles of long noncoding RNAs in Drosophila spermatogenesis

    Long noncoding RNAs (lncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although lncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. This study identified 128 testis-specific Drosophila lncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the lncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans Furthermore, gene expression profiles for five lncRNA mutants revealed that testis-specific lncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific lncRNAs evolved much faster. Moreover, lncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, these results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis (Wen, 2016).

    Localized, reactive F-actin dynamics prevents abnormal somatic cell penetration by mature spermatids

    Spermatogenesis occurs inside a somatic cell enclosure. Sperm release, the most important final step and a target for contraceptives, has been extensively studied in fixed tissue preparations. This study provides a time-lapse description of the release process in Drosophila testis ex vivo. The spermatid tails exit the somatic enclosure and enter the testicular duct first, followed by the spermatid heads. Prior to this, individual spermatid heads attempt to invade the head cyst cell, and on each occasion they are repelled by a rapid and local F-actin polymerization response from the head cyst cell. The F-actin assembly involves N-WASp, D-WIP, and Arp2/3 complex and dissipates once the spermatid head retreats back into the fold. These findings revise the existing spermiation model in Drosophila and suggest that somatic cells can actively oppose mechanical cell invasion attempts using calibrated F-actin dynamics in situ (Dubey, 2016).


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    Genes Involved in spermatogenesis

    Genes involved in organ development

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