The Interactive Fly
Genes involved in tissue and organ development
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).
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).
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).
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, 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).
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).
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).
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).
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).
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
ftp://ncbi.nlm.nih.gov/genbank/genomes/D_melanogaster/, 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
http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker), and NAP and GAP2 (available at http://genome.cs.mtu.edu/sas.html).
BLAST programs were downloaded from the National
Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/) (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).
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).
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 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).
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).
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).
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).
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).
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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.
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.
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.
Genes Involved in spermatogenesis
Genes involved in organ development
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