Oogenesis and the Oocyte (Part 1 | Part2)

In many insect species, mating stimuli can lead to changes in various behavioral and physiological responses, including feeding, mating refusal, egg-laying behavior, energy demand, and organ remodeling, which are collectively known as the post-mating response. Recently, an increase in germline stem cells (GSCs) has been identified as a new post-mating response in both males and females of the fruit fly, Drosophila melanogaster. Mating-induced increase in female GSCs of D. melanogaster were extensively studied at the molecular, cellular, and systemic levels. After mating, the male seminal fluid peptide [e.g. sex peptide (SP)] is transferred to the female uterus. This is followed by binding to the sex peptide receptor (SPR), which evokes post-mating responses, including increase in number of female GSCs. Downstream of SP-SPR signaling, the following three hormones and neurotransmitters have been found to act on female GSC niche cells to regulate mating-induced increase in female GSCs: (1) neuropeptide F, a peptide hormone produced in enteroendocrine cells; (2) octopamine, a monoaminergic neurotransmitter synthesized in ovary-projecting neurons; and (3) ecdysone, a steroid hormone produced in ovarian follicular cells. These humoral factors are secreted from each organ and are received by ovarian somatic cells and regulate the strength of niche signaling in female GSCs. This review provides an overview of the latest findings on the inter-organ relationship to regulate mating-induced female GSC increase in D. melanogaster as a model (Hoshino, 2021).

Clonal dominance arises when the descendants (clones) of one or a few founder cells contribute disproportionally to the final structure during collective growth. In contexts such as bacterial growth, tumorigenesis, and stem cell reprogramming, this phenomenon is often attributed to pre-existing propensities for dominance, while in stem cell homeostasis, neutral drift dynamics are invoked. The mechanistic origin of clonal dominance during development, where it is increasingly documented, is less understood. This study investigated this phenomenon in the Drosophila melanogaster follicle epithelium, a system in which the joint growth dynamics of cell lineage trees can be reconstructed. This study demonstrated that clonal dominance can emerge spontaneously, in the absence of pre-existing biases, as a collective property of evolving excitable networks through coupling of divisions among connected cells. Similar mechanisms have been identified in forest fires and evolving opinion networks; the spatial coupling of excitable units explains a critical feature of the development of the organism, with implications for tissue organization and dynamics (Alsous, 2021).

Despite their medical and economic relevance, it remains largely unknown how suboptimal temperatures affect adult insect reproduction. This study reports an in-depth analysis of how chronic adult exposure to suboptimal temperatures affects oogenesis using the model insect Drosophila melanogaster. In adult females maintained at 18°C (cold) or 29°C (warm), relative to females at the 25°C control temperature, egg production was reduced through distinct cellular mechanisms. Chronic 18°C exposure improved germline stem cell maintenance, survival of early germline cysts and oocyte quality, but reduced follicle growth with no obvious effect on vitellogenesis. By contrast, in females at 29°C, germline stem cell numbers and follicle growth were similar to those at 25°C, while early germline cyst death and degeneration of vitellogenic follicles were markedly increased and oocyte quality plummeted over time. Finally, this study also showed that these effects are largely independent of diet, male factors or canonical temperature sensors. These findings are relevant not only to cold-blooded organisms, which have limited thermoregulation, but also potentially to warm-blooded organisms, which are susceptible to hypothermia, heatstroke and fever (Gandara, 2022).

In Drosophila ovary, niche is composed of somatic cells, including terminal filament cells (TFCs), cap cells (CCs) and escort cells (ECs), which provide extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Niche establishment begins in larval stages when terminal filaments (TFs) are formed, but the underlying mechanism for the development of TFs remains largely unknown. This study reports that transcription factor longitudinals lacking (Lola) is essential for ovary morphogenesis. Lola protein was expressed abundantly in TFCs and CCs, although also in other cells, and lola was required for the establishment of niche during larval stage. Importantly, it was found that knockdown expression of lola induced apoptosis in adult ovary, and that lola affected adult ovary morphogenesis by suppressing expression of Regulator of cullins 1b (Roc1b), an apoptosis-related gene that regulates caspase activation during spermatogenesis. These findings significantly expand understanding of the mechanisms controlling niche establishment and adult oogenesis in Drosophila (Zhao, 2022).

Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, this study conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance (Cho, 2018).

Proper specification of germline stem cells (GSCs) in Drosophila ovaries depends on niche derived non-autonomous signaling and cell autonomous components of transcriptional machinery. Stonewall (Stwl), a MADF-BESS family protein, is one of the cell intrinsic transcriptional regulators involved in the establishment and/or maintenance of GSC fate in Drosophila ovaries. This stufy reports identification and functional characterization of another member of the same protein family, CG3838/Brickwall (Brwl) with analogous functions. Loss of function alleles of brwl exhibit age dependent progressive degeneration of the developing ovarioles and loss of GSCs. Supporting the conclusion that the structural deterioration of mutant egg chambers is a result of apoptotic cell death, activated caspase levels are considerably elevated in brwl(-) ovaries. Moreover, as in the case of stwl mutants, on several instances, loss of brwl activity results in fusion of egg chambers and misspecification of the oocyte. Importantly, brwl phenotypes can be partially rescued by germline specific over-expression of stwl arguing for overlapping yet distinct functional capabilities of the two proteins. Taken together with the phylogenetic analysis, these data suggest that brwl and stwl likely share a common MADF-BESS ancestor and they are expressed in overlapping spatiotemporal domains to ensure robust development of the female germline (Shukla, 2018).

Nutrition shapes a broad range of life-history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, this study fed adult female Drosophila melanogaster diets that contained all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, this study tested whether GCN2 and TOR mediated this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, it was found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. These results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animal's true nutritional status (Alves, 2022).

The Drosophila ovary serves as a model for pioneering studies of stem cell niches, with defined cell types and signaling pathways supporting both germline and somatic stem cells. The establishment of the niche units begins during larval stages with the formation of terminal filament-cap structures; however, the genetics underlying their development remains largely unknown. This study shows that the transcription factor Lmx1a is required for ovary morphogenesis. Lmx1a is expressed in early ovarian somatic lineages and becomes progressively restricted to terminal filaments and cap cells. Lmx1a is required for the formation of terminal filaments, during the larval-pupal transition. Finally, the data demonstrate that Lmx1a functions genetically downstream of Bric-a-Brac, and is crucial for the expression of key components of several conserved pathways essential to ovarian stem cell niche development. Importantly, expression of chicken Lmx1b is sufficient to rescue the null Lmx1a phenotype, indicating functional conservation across the animal kingdom. These results significantly expand our understanding of the mechanisms controlling stem cell niche development in the fly ovary (Allbee, 2018).

Drosophila ovaries are composed of about 15-20 functional units called ovarioles that produce eggs. In each ovariole, somatic and germline stem cells are maintained by a group of specialized cells that form the niche. These niches are composed of terminal filaments (TFs) and cap cells, which provide signals required for the maintenance and function of the stem cell populations in adult ovaries, including BMPs and Hh. Although the origin of the somatic lineage in the female gonad has been established, the genetic factors underlying the morphogenetic processes required for the specification and establishment of TF-cap structures are largely unknown. Ovarian somatic cells originate from three mesoderm cell clusters on each side of the embryo. During the mid-larval third instar (ML3), TF cells start to specify, expressing the TF marker Engrailed (En). By late third larval instar (LL3), and continuing in white pre-pupae (WPP), TFs form well-organized stacks and promote the proliferation and migration of muscle precursors that will develop into the muscular sheath and delimit individual ovarioles. This process ensures that a single TF stack and a defined population of germline and somatic cells are incorporated in each unit (Allbee, 2018).

The number of ovarioles and the developmental timing of the niche have been shown to be controlled by several signaling pathways, including Insulin, Hippo, Notch, Activin and Ecdysone pathways. In addition, the two transcription factors Bab1 and Bab2, encoded by the bric-á-brac (bab) locus, are expressed in TFs and are essential for the formation of these structures. Other BTB transcription factors, Pipsqueak, Trithorax-like and Batman (Lola like), also control TFs and ovariole numbers. Finally, loss of the Engrailed transcription factor causes abnormal TF cell stacking. Although these signaling pathways and transcriptional regulators are essential for development of stem cell niches, the early genetic events coordinating TF-cap cell specification, formation and function remain largely unknown (Allbee, 2018).

This study describes the expression and function of the LIM-homeodomain transcription factor Lmx1a in developing Drosophila ovaries. LIM-homeodomain proteins have essential roles during tissue patterning and cell differentiation in metazoans, from nematodes to vertebrates. Mammalian Lmx1a and Lmx1b are pleiotropic regulators of cell differentiation and tissue development in many organs, as shown by the defects and syndromes caused by loss-of-function mutations in these genes (Doucet-Beaupré, 2015). For instance, haploinsufficiency of Lmx1b causes nail-patella syndrome (NPS), a disorder affecting dorsal limb structures, kidneys, anterior eye components and the nervous system. Studies in genetic mouse models have confirmed a regulatory function of Lmx1b in the development of these organs and the behavior of associated cell types, including serotonergic neurons, podocytes and several eye tissues. Similarly, studies of the mouse Lmx1a factor have revealed diverse developmental functions, including brain patterning and cell fate decision, dopaminergic neuron differentiation and insulin expression. Finally, previous work has implicated Lmx1a and Lmx1b in a variety of other pathologies, including non-NPS renal disease, Parkinson's disease, and various forms of cancer, including ovarian epithelial carcinoma, highlighting the complexity of Lmx1a/b biology, as well as the urgent need to understand the function of these factors better (Allbee, 2018 and references therein).

Two LIM-homeodomain proteins of the LMX subgroup are encoded in the Drosophila genome: Lmx1a/CG32105 and Lmx1b/CG4328. Lmx1a is expressed in the LL3 eye imaginal disc and its overexpression in this tissue causes eye defects (Roignant, 2010; Wang, 2016). However, the molecular, cell biological and developmental functions of this transcription factor have yet to be described. This study shows that Lmx1a is expressed in somatic lineages in the developing ovary. Its expression becomes restricted to TFs and cap cells by LL3/P0 (freshly pupariated) and is maintained in the adult stem cell niche. Analyzing a CRISPR-generated Lmx1a knockout allele as well as cell type-specific and stage-specific knockdown of Lmx1a, this study has determined that Lmx1a is required for ovary development specifically in the TF-cap cell niche at the time at which it forms. Transcriptional profiling of developing ovaries was performed, and Lmx1a was placed downstream of Bab1/2 in the specification of TF cells. Without Lmx1a, several components of signaling pathways crucial to the forming niche are not properly expressed, including Hh, the transcription factors Sox100B, Engrailed and Invected, and confirmed that these genes are required in the Lmx1a lineage. Strikingly, expression of a chicken ortholog of Lmx1a in forming TF-cap cells is sufficient to rescue the Lmx1a null phenotype. It is anticipated that these results will further elucidate the genetic and cell biological mechanisms underlying the establishment of the Drosophila ovary stem cell niche and provide insight into the role of LIM-HD factors in tissue development and patterning, homeostasis and disease (Allbee, 2018).

Despite longstanding interest in the Drosophila ovary as a model for the adult stem cell niche, the genetic mechanisms underlying the initial establishment and development of the niche remain largely unexplored. To date, only two transcription factor loci, bric-á-brac and engrailed/invected, have been identified as required for the development or proper stacking of TFs, and their function remains largely unknown. This study has characterized the function of Lmx1a, a highly conserved LIM homeobox transcription factor, in the developing Drosophila ovary. By generating an Lmx1a knockout line as well as taking advantage of a novel collection of Gal4 drivers that produce spatially restricted developing ovary expression patterns, Lmx1a was shown to be required in TF-cap structures at the time at which they form and initiate ovarian stem cell niche establishment and ovariole morphogenesis. It remains unclear whether Lmx1a plays a role in TFs, cap cells, or both. The data also suggest that Lmx1a expression is maintained in the adult niche. Further studies will be required to determine whether it is essential for the function of the adult niche, for example by controlling the expression of self-renewal factors (Allbee, 2018).

Using RNAseq, a novel list of genes enriched in LL3/P0 ovaries was generated, and from this list several pathways have been identified that are potentially affected by the loss of Lmx1a. Within this list are several transcription factors and components of signaling pathways necessary for ovarian niche development, function and maintenance, including Hh, FGF, Notch, Engrailed and Sox100B. Indeed, RNAi-mediated knockdown of several of these transcripts within the Lmx1a-Gal4 lineage results in ovary developmental defects and reduced fertility. It is therefore proposed that Lmx1a controls many aspects of the development of this tissue, including proliferation, migration or cell shape changes, by regulating a series of developmental factors and signal pathways. It is important to note that this study does not allow distinguishing between a direct transcriptional regulation of these genes by Lmx1a and an indirect effect of an impaired development of the TF-cap structure in Lmx1a mutants. The data analyzed, in young adult females, the consequence of manipulating Hh, Sox100B, Engrailed and Invected in the Lmx1a lineage. Investigating the function of these genes, specifically during the LL3-P0 transition or later during metamorphosis or adulthood, will allow testing of whether their genetic requirement and expression are compatible with a direct regulation by Lmx1a and to understand better the sequence of events required for normal ovarian morphogenesis (Allbee, 2018).

Interestingly, Engrailed has been previously suggested to be required for terminal filament (TF) stacking based on clonal analysis of TF cells homozygous mutant for both Engrailed and Invected (Bolívar, 2006). The current work suggests that the stacking defects caused by these mutations lead to a mild developmental phenotype in young adults. However, consistent with other work revealing a crucial role for Engrailed in the adult niche, this study also shows that persistent knockdown of Engrailed/Invected well into adulthood leads to an eventual loss of ovaries. It is speculated that the progressiveness of this defect is the consequence of a continuous impairment of adult niche function that worsens a mild initial developmental phenotype (Allbee, 2018).

This study investigated where Lmx1a functions in relation to the transcription factors Bab1 and Bab2. In support of earlier work, this study found that both bab1 and bab2 are required for ovary morphogenesis. In addition, a strong reduction os shown in Lmx1a mRNA expression in the ovaries of LL3/P0 bab1 homozygous mutant animals, placing Bab1 genetically and/or temporally upstream of Lmx1a. It is worth noting that, although reduced, the expression of Lmx1a is not eliminated in Bab1 mutants. It is speculated that Bab2 activity may be maintaining residual Lmx1a expression in this background.Heterozygosity for Bab1 was found to be sufficient to enhance the ovary developmental defects caused by RNAi-mediated knockdown of the Lmx1a-dependent genes tested here, consistent with a model in which the bric-ì-brac locus lies genetically upstream of Lmx1a (Allbee, 2018).

Finally, this study found that the Lmx1a null homozygous phenotype can be significantly rescued by the expression of chicken Lmx1b, in terms of TF-cap formation, sheath formation and fertility. This reveals striking conservation between the Lmx1a/b factors across the animal kingdom. Even more striking is the conserved relationship between Lmx1a/b and Engrailed. For example, Lmx1b is required for Engrailed expression during both the establishment of the isthmic organizer in the developing mouse midbrain, and the specification and differentiation of dopaminergic neurons. Additionally, simultaneous lentiviral-mediated expression of Lmx1b and Engrailed has been shown to aid in the differentiation of dopaminergic neurons in vitro. Altogether, these observations point to a likely conserved Lmx1a/b-Engrailed transcriptional module involved in tissue patterning and cell differentiation, across different developing tissues, cell types and species. It is therefore anticipated that studies on the function of Lmx1a in the Drosophila ovary will lead to a better understanding of the developmental function of Lmx1a in animals ranging from invertebrates to mammals. This study also indicates that Drosophila represents a valuable model in which to investigate the mechanisms underlying complex diseases caused by a dysfunction of Lmx1a/b, such as nail-patella syndrome, ovarian carcinoma and Parkinson's disease (Allbee, 2018).

The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). This study used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. A null allele of the forked gene was generated using the CRISPR-Cas9 system, and forked was found not to be required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, it was demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal MT-organizing centers. These results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity (Baskar, 2019).

The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. This study addresses the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. Transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. Elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. Suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. These findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development (Durdevic, 2018).

This study shows that a transient loss of vas expression during early oogenesis leads to up-regulation of transposon levels and compromised viability of progeny embryos. The observed embryonic lethality is because of DNA DSBs and nuclear damage that arise as a consequence of the elevated levels of transposon mRNAs and proteins, which are transmitted from the mother to the progeny. This study thus demonstrates that transposon silencing in the nurse cells is essential to prevent maternal transmission of transposons and DNA damage, protecting the progeny from harmful transposon-mediated mutagenic effects (Durdevic, 2018).

The finding that suppression of Chk2-mediated DNA damage signaling in loss-of-function vas mutant flies restores oogenesis, and egg production demonstrates that Chk2 is epistatic to vas. However, hatching is severely impaired, because of the DNA damage sustained by the embryos. The defects displayed by vas, mnk double mutant embryos resembled those of PIWI (piwi, aub, and ago3) single and mnk; PIWI double mutant embryos. Earlier observation that inactivation of DNA damage signaling does not rescue the development of PIWI mutant embryos led to the assumption that PIWI proteins might have an essential role in early somatic development, independent of cell cycle checkpoint signaling. By tracing transposon protein and RNA levels and localization from the mother to the early embryos, it is suggested that, independent of Chk2 signaling, de-repressed transposons are responsible for nuclear damage and embryonic lethality. This study indicates that transposon insertions occur in the maternal genome where they cause DNA DSBs that together with transposon RNAs and proteins are passed on to the progeny embryos. Transposon activity and consequent DNA damage in the early syncytial embryo cause aberrant chromosome segregation, resulting in unequal distribution of the genetic material, nuclear damage and ultimately embryonic lethality. This study shows that early Drosophila embryos are defenseless against transposons and will succumb to their mobilization if the first line of protection against selfish genetic elements in the nuage of nurse cell fails (Durdevic, 2018).

A recent study showed that in p53 mutants, transposon RNAs are up-regulated and accumulate at the posterior pole of the oocyte, without deleterious effects on oogenesis or embryogenesis. It is possible that the absence of pole plasm in vas mutants results in the release of the transposon products and their ectopic accumulation in the oocyte. Localization of transposons to the germ plasm may restrict their activity to the future germline and protect the embryo soma from transposon activity. Transposon-mediated mutagenesis in the germline would produce genetic variability, a phenomenon thought to play a role in the environmental adaptation and evolution of species. It would therefore be of interest to determine the role of pole plasm in transposon control in the future (Durdevic, 2018).

Transposon up-regulation in the Drosophila female germline triggers a DNA damage-signaling cascade. In aub mutants, before their oogenesis arrest occurs, Chk2-mediated signaling leads to phosphorylation of Vasa, leading to impaired grk mRNA translation and embryonic axis specification. Considering the genetic interaction of vas and mnk (Chk2) and the fact that Vasa is phosphorylated in Chk2-dependent manner, it is tempting to speculate that phosphorylation of Vasa might stimulate piRNA biogenesis, reinforcing transposon silencing and thus minimizing transposon-induced DNA damage. The arrest of embryonic development as a first, and arrest of oogenesis as an ultimate response to DNA damage, thus, prevents the spreading of detrimental transposon-induced mutations to the next generation (Durdevic, 2018).

Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. Previous work has shown that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, the gene was named anion exchanger 2 (ae2). This study generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, it was found that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and these studies provide new genetic tools to investigate the mechanisms by which this occurs (Benitez, 2019).

This study shows that the VRK-1 kinase BALL is required for self-renewal of germline stem cells in Drosophila, including the symmetrically amplifying PGCs of larvae and both male and female GSCs. These stem cells are actively maintained undifferentiated and they require BMP signalling for self-renewal that emanates from their cellular niche environments. In ball² mutant female GSCs, where the requirement of BMP signalling for self-renewal is most pronounced, known targets of BMP signalling are regulated as in wild type GSCs. This indicates that BALL participates neither in the transmission nor the regulation of BMP signalling, and that it is needed to maintain stem cell character in a cell autonomous manner, irrespective of the tissue-specific maintenance signals that emanate from the niches (Herzig, 2014).

The loss of self-renewing stem cells could be caused by the induction of ectopic differentiation in these cells. The differentiation pathway was blocked in ball mutant female GSCs by removing also the central differentiation factor BAM. The results suggest that ball mutant GSCs are not eliminated from the stem cell niche because they initiate germline differentiation but that the GSCs differentiate because they lost the capacity for self-renewal (Herzig, 2014).

It is unclear by which mechanism BALL mediates the ability for GSC self-renewal. In ovaries, GSCs and FSCs undergo a regular turnover and are continuously replaced in the niche either by their own daughter cells or by symmetric divisions of the neighbouring stem cells. The replacement of GSCs involves competition between stem cells. Cells lacking BAM for instance, successfully displace less competitive wild type stem cells in the niche. However, if BALL is additionally removed from bam mutant cells, they appear to loose their competitive advantage. The molecular basis of stem cell competiveness is still poorly understood. However, it has been shown that overexpression of the Drosophila dMyc transcription factor diminutive enhances the competitiveness of GSCs and causes significantly enlarged nucleoli and increased rRNA expression in epithelial cells. These observations suggest a correlation between ribosome biogenesis and GSC competitiveness. Downregulation of ribosome biogenesis appears in fact to be directly required for germ cell differentiation, since BAM activates the Mei-P26 protein which downregulates the expression of dMyc. Furthermore, when overexpressed from a transgene, dMyc abrogates the tumour growth phenotype and the size increase of nucleoli in bam mutant cells. Additional support for the proposal that increased ribosome biogenesis in stem cells is crucial for their competitiveness and for maintaining their undifferentiated state derives from studies on wicked. Wicked is an essential component of the U3 snoRNP pre-rRNA processing, which is required to maintain the self-renewal of GSC. The findings that BALL is enriched in stem cell nucleoli and required for the structural integrity of nucleoli in tumourous GSCs provide a plausible link between ribosome biogenesis and BALL-dependent competitiveness of GSCs (Herzig, 2014).

Once displaced from the niche, ball² mutant female GSCs differentiate according to their germline fate with only minor defects. This study did not address whether the differentiation of ball mutant cells is fully completed like in the respective wild type lineages in systems other than the adult female germline, but it was found, irrespective of the system examined, that BALL is not strictly required as proliferation factor. Especially the analysis of dividing follicle cells showed that BALL is not a cell cycle regulator. With two remarkable exceptions, BALL is also not essential for cellular survival. These exceptions, i.e., ball mutant PGCs at late larval stages and ball² bamΔ86 double mutant germline cells outside the ovarian niche, represent conditions in which differentiation is either not supported by the tissue or not possible due to the lack of a differentiation factor. Therefore, it is tempting to speculate that BALL becomes only essential for cellular survival, when the ball mutant stem cells are unable to 'escape' from self-renewal into differentiation. Although ball clearly has multiple functions, e.g., oocyte chromatin organization or modulation of female PGC proliferation rate, the common defect observed in all systems examined so far is a failure in maintaining pools of undifferentiated cells. Since BALL is not an essential proliferation factor, these data suggest that also the additional defects in ball mutant larvae, i.e., lacking imaginal discs and degenerate brains, could be due to premature loss of undifferentiated progenitor or stem cells. Analysis of these systems will eventually show whether BALL is broadly required to maintain the undifferentiated state of cells during development (Herzig, 2014).

Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. This study reports that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade β-catenin. Disruption of β-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and β-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration (Lin, 2020).

Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. This study demonstrates that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, it was shown that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism (Simonet, 2020).

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. This study shows that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. These results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin (DeLuca, 2020).

The work described here shows that the Drosophila female germline has multiple advantages for studying the developmental regulation of chromatin silencing both before and during differentiation. Female GSCs continuously divide to produce new undifferentiated progenitors, which expand and differentiate into nurse cells or oocytes, generating large amounts of a much simpler tissue than a developing embryo. Additionally, an inducible reporter assay compatible with the female germline was developed that sensitively responds to developmental changes in local chromatin repression in individual cells. In contrast to RNAseq, which measures steady state RNA levels, or ChIPseq, which correlates chromatin epitopes with their perceived function on gene expression, the reporters directly test how local chromatin influences the inducibility of surrounding genes, and are easily combined with tissue specific knockdowns to identify trans-acting factors contributing to reporter inducibility. Finally, a genetic engineering approach allows any construct (not just hsGFP) to be efficiently integrated into many pre-existing 'donor' sites, including those used previously with other reporters, or sites heavily silenced by repressive chromatin in differentiated cells. Although the number of different donor sites in certain types of chromatin is currently limited, new sites continue to be generated using CRISPR/Cas9 targeting and the method can ultimately be applied virtually anywhere in the genome (DeLuca, 2020).

Analysis of Polycomb repression with reporters, ChIP, and PcG-gene knockdowns provided numerous insights into how chromatin affects gene expression and female germline development in Drosophila. GSCs, the precursors of oocytes and nurse cells, contain a non-canonical, binary distribution of moderate H3K27me3 enrichment on all transcriptionally inactive loci and very low enrichment on active chromatin. A similar non-canonical H3K27me3 distribution was observed in early fly embryos, suggesting that noncanonical chromatin represents a 'ground state' for progenitors that will propagate future generations of undifferentiated germ cells or somatic cells that differentiate into specialized tissues. Such non-canonical chromatin was first identified in mouse oocytes and preimplantation embryos. If non-canonical H3K27me3 chromatin is a characteristic of undifferentiated, totipotent cells, what function might it confer to account for its conservation (DeLuca, 2020)?

Experiments confirmed previous workshowing that chromatin modified by PRC2 is essential for the Drosophila female germ cell cycle. Germline cysts lacking PRC2 are unable to stably generate oocytes, and E(z) GermLine-specific RNAi Knock Down (GLKD) nurse cells mis-express multiple genes and degenerate at about stage 5. In contrast, GSCs lacking PRC2 properly populate their niche, divide, and produce daughters that interact with female follicle cells and begin nurse cell differentiation. Removing PRC2 activity from GSCs did not generally increase the steady state abundance of genes or the inducibility of reporters in H3K27me3-enriched inactive or PcG domains. These results suggest that PRC2 and non-canonical chromatin lack vital functions in undifferentiated germline progenitors but are critical for repressing genes upon differentiation. However, a requirement for PRC2 or non-canonical chromatin under stress conditions or prolonged aging cannot be dismissed. For example, PRC2 could promote the long-term maintenance of female GSCs, similarly to how it maintains male germline progenitors in flies and mice (DeLuca, 2020).

Germline cysts and nurse cells are found in diverse animal species across the entire phylogenetic spectrum, but their function has been well studied mostly in insects such as Drosophila where they persist throughout most of oogenesis. While nurse cells have traditionally been considered germ cells rather than late-differentiating somatic cells, this study shows that that Drosophila nurse cells initiate Polycomb silencing and enrich PRC2 activity on a nearly identical collection of PcG domains as somatic cells. In more distant species, such as mice, nurse cells initially develop in a similar manner within germline cysts and contribute their cytoplasm to oocytes, but undergo programmed cell death before the vast majority of oocyte growth. Consequently, it remains an open question whether somatic differentiation plays a role in nurse cell function in mammals and many other groups (DeLuca, 2020).

The size and composition of oocyte cytoplasm are uniquely tailored to promote optimal fecundity and meet the demands of early development. In some species, including flies, nurse cells synthesize large amounts of specialized ooplasm to rapidly produce multitudes of large, pre-patterned embryos. In others, including mammals, oocytes more slowly synthesize the majority of ooplasm. Interestingly, both ooplasm synthesis strategies apparently require Polycomb silencing. However, the nurse cell-based strategy in flies primarily requires PRC2 but not PRC1 to silence hundreds of somatic genes, while the oocyte-based strategy in mice requires PRC1 but not PRC2 (DeLuca, 2020).

Different strategies of ooplasm synthesis may have evolved to be compatible with noncanonical germ cell chromatin. Staining experiments show that Drosophila oocytes maintain a widely distributed, non-canonical H3K27me3 distribution similar to pre-meiotic precursors or mouse oocytes, suggesting that non-canonical chromatin is conserved and maintained throughout the germ cell cycle. Similar to mouse oocytes, Drosophila spermatocytes also contain non-canonical chromatin and autonomously synthesize large amounts of cytoplasm by deploying PRC1 but not PRC2. Thus, three different types of germ cells are filled with large amounts of differentiated cytoplasm that requires Polycomb silencing for its synthesis, but nevertheless maintain a non-canonical, silencing-deficient PRC2 activity (DeLuca, 2020).

The conservation of undifferentiated, non-canonical chromatin despite a strong selection for Polycomb silencing during ooplasm synthesis argues that non-canonical chromatin must have a presently unappreciated fundamental purpose in germ cells. Noncanonical chromatin could regulate multigenerational processes like mutation, recombination, or transposition, that are not easily assayed in sterile individuals. Tests of these ideas will require a better understanding of how non-canonical chromatin is regulated and methods to disrupt non-canonical chromatin without disrupting other functions required for germline viability. Additionally, non-canonical chromatin could simply result from the silencing- incompetent PRC2 that was observed in progenitors (DeLuca, 2020).

Pcl was uncovered as both an inhibitor of PRC2 silencing and promoter of non-canonical chromatin in GSCs. Pcl^GLKD dramatically altered the footprint of PRC2 activity in GSCs. Pcl^GLKD favored H3K27me3 enrichment on PREs versus inactive domains, and increased the total amount of H3K27me1 by 13-fold and H3K27me2 by 1.4-fold and decreased the total amount of H3K27me3 by 1.8 fold. By binding DNA through its winged-helix domain, Pcl triples PRC2's residence time on chromatin and promotes higher states of H3K27 methylation in vitro. In GSCs, Pcl could simply change the result of each PRC2-chromatin binding event from H3K27me1 to me3. However, it is hard to imagine how an equivalent number of nucleosomes bearing a higher H3K27 methylation state could explain how Pcl inhibits silencing. Instead, it is proposed that Pcl inhibits silencing by reducing the number of PRC2-chomatin binding events per unit time by increasing the residence time of PRC2 on chromatin with each binding event. In this model, Pcl^GLKD would not only convert many H3K27me3 nucleosomes into H3K27me1 nucleosomes, it would also convert many unmethylated nucleosomes into H3K27me1 nucleosomes. Pcl^GLKD would more subtly affect H3K27me2 abundance because it simultaneously increases the number of PRC2-chromatin binding events while reducing the probability of each binding event leading to H3K27me2 versus me1 (DeLuca, 2020).

By reducing the number of PRC2 binding events, Pcl could increase the abundance of unmethylated H3K27 residues available for acetylation - a transcription promoting modification. In both flies and mammals, PRC2 transiently associates with chromatin to mono- and dimethylate H3K27 outside of traditional PcG domains, blocking H3K27 acetylation and antagonizing transcription. This study similarly found strong and widespread PRC2-dependent silencing in H3K27me1/2 enriched chromatin in nurse cells. Because inactive domain silencing was not affected by depletion of Pcl, Jarid2, or H3K27me3, it is proposed that core-PRC2, but not Pcl-PRC2 or H3K27me3, primarily silences inactive chromatin (DeLuca, 2020).

In GSCs, abundant Pcl could saturate PRC2, effectively depleting faster-sampling core-PRC2 complexes in favor of slower sampling Pcl-PRC2. In somatic embryonic cells, Pcl is present in a small fraction of PRC2 complexes. Compared to other fly tissues, Pcl mRNA is most abundant in the ovary, and within the ovary, Pcl protein is much more abundant in GSCs and nurse cell precursors than differentiated nurse cells and somatic cells. Within each differentiated germline cyst, Pcl mRNA is depleted from nurse cells and enriched in oocytes, suggesting that Pcl protein levels may be regulated by an mRNA transport mechanism induced in region 2 that also triggers the differentiation of oocytes from nurse cells (DeLuca, 2020).

Pcl, and a second PRC2-interacting protein, Scm, regulate the transition from noncanonical to canonical chromatin and initiate Polycomb repression. During nurse cell differentiation, it is proposed that Pcl depletion frees core-PRC2 to rapidly sample and silence inactive domains, while Scm (which is absent from the GSC) induction recruits high levels of PRC1 and PRC2 activity around PREs. ScmGLKD nurse cell chromatin retained a noncanonical H3K27me3 pattern characteristic of GSCs, as if differentiation at PcG domains had not occurred. In mice, Scm homologue, Scml2, similarly associates with PcG domains to recruit PRC1/2 and silence PcG targets during male germline development. However, unlike its fly orthologue in female GSCs, Scml2 is expressed in male germline precursors. This difference could explain why mammalian PGCs partially enrich PRC2 activity on CGIs while fly female GSCs do not enrich PRC2 on specific sites. While PcG domain-associated Scm is sufficient to enrich PRC2 activity above background levels found throughout inactive chromatin, a second PRC2 interacting protein, Pcl, is additionally required to promote full PRC2 and H3K27me3 enrichment on PcG domains. Because Scm oligomerizes and interacts with PRC2 in vitro, it could form an array of PRC2 binding sites anchored to PREs through Sfmbt. It is proposed that two cooperative interactions, PRC2 with PRE-tethered Scm, and Pcl with DNA, preferentially concentrate H3K27me3-generating Pcl-PRC2 versus H3K27me1/2-generating core-PRC2 on PcG domains (Figure 6D). H3K27me3 could then be further enriched by H3K27me3-induced allosteric PRC2 activation through the Esc subunit (DeLuca, 2020).

By promoting PRC1 and PRC2 concentration on PcG domains, Scm enhances silencing on PcG-localized reporters. While Scm-depleted nurse cells completed oogenesis, a subset of PcG domain-localized PcG including chinmo and the posterior Hox gene, Abd-b, escaped repression and were potentially loaded into embryos. Eggs derived from Scm^GLKD nurse cells failed to hatch, and mis-expressed Abd-b in anterior segments following germ band elongation. This defect more closely resembled maternal plus zygotic than maternal-only Scm mutants, suggesting that Scm^GLKD may deplete both maternal and zygotic Scm (DeLuca, 2020).

However, the additional possibility that mis-regulation of maternal Polycomb targets like chinmo contribute to the subtle embryonic defects observed in maternal-only Scm mutant clones cannot be excluded (DeLuca, 2020).

Further study of the Polycomb-mediated repression described in this study will help define the gene regulation program of Drosophila nurse cells and its contribution to oocyte growth. Additional characterization and perturbation of non-canonical chromatin throughout the germ cell cycle will yield further insights into its function in development. Finally, incorporating studies of other chromatin modifications, including H3K9me3-based repression, during germ cell development will contribute to a fuller understanding of how chromatin contributes to an immortal cell lineage (DeLuca, 2020).

How formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. This study shows that their functions are necessary specifically within precursor cells for TF formation during these stages. A new function was identified for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, the presence of Bab proteins in niche cells was shown to be necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary (Miscopein Saler, 2020).

Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided our understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. This study used a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, all phase portraits were enumerated of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, parameter regions were located that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches (Diegmiller, 2021).

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1-domain (LEM-D) proteins that associate with the chromatin and DNA binding protein Barrier-to-autointegration factor (BAF). This study investigated the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only D-emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where D-emerin/Otefin function is essential. Germ cell-specific BAF knockdown causes phenotypes that mirror d-emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that similar to d-emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, these findings provide evidence for a prominent NL partnership between the LEM-D protein D-emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population (Duan, 2020).

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear membrane. Comprising lamins and hundreds of associated proteins, the NL builds contacts with the genome to regulate transcription, replication and DNA repair. The NL also connects the nucleus with the cytoskeleton, facilitating transduction of regulatory information between cellular compartments. The composition of the NL is cell-type specific, providing a diverse platform for the integration of developmental regulatory signals. Changes in NL structure occur during physiological aging and disease, suggesting that maintenance of NL function is crucial for cellular health and longevity (Duan, 2020).

One prominent family of NL proteins are LEM domain (LEM-D) proteins, named after the founding human members: LAP2, emerin and MAN1. The defining feature of this conserved family is the LEM domain (LEM-D), an ~40 amino acid domain that directly interacts with the metazoan chromatin-binding protein Barrier-to-autointegration factor (BAF, sometimes referred to as BANF1). Purified human BAF directly binds double-stranded DNA, the A-type lamin and histones in vitro, suggesting that BAF also promotes chromatin-NL connections using non-LEM-D-dependent mechanisms. In dividing metazoan cells, regulated formation of complexes between LEM-D proteins, BAF and lamin controls mitotic spindle assembly and positioning, as well as the reformation of the nucleus. In non-dividing metazoan cells, LEM-D proteins and BAF cooperate to tether the genome to the nuclear periphery and form repressed chromatin. These properties highlight central connections between LEM-D proteins and BAF in NL function (Duan, 2020).

Studies in Drosophila melanogaster have begun to define the role of LEM-D proteins and BAF in development. Drosophila has three NL LEM-D proteins that bind BAF, including two emerin orthologues (Emerin/Otefin and Emerin2/Bocksbeutel) and MAN1. Each LEM-D protein is globally expressed during development. Even so, loss of individual NL LEM-D proteins causes different, non-overlapping defects in the several tissues, including the ovaries, testes, wings and the nervous system. These restricted mutant phenotypes reflect functional redundancy among the Drosophila LEM-D proteins, as loss of any two proteins is lethal. Strikingly, phenotypes of the emerin double mutants (otefin-/-; bocksbeutel-/-) phenocopy baf null mutants. Both baf and the emerin double mutants die before pupation, resulting from decreased mitosis and increased apoptosis of imaginal discs (Barton, 2014; Furukawa, 2003). In contrast, emerin/otefin; MAN1 or emerin2/bocksbeutel; MAN1 die during pupal development, without associated defects in mitosis or apoptosis. Together, genetic studies indicate that the Drosophila emerin orthologues and BAF are important partners (Duan, 2020).

This study extend investigations of the Drosophila NL LEM-D and BAF protein partnership. Using a CRISPR generated gfp-baf allele, this study confirmed that BAF is a globally expressed nuclear protein that shows strong enrichment at the NL in diploid cells. Strikingly, this NL enrichment largely depends upon one LEM-D protein, Emerin/Otefin. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where Emerin/Otefin function is essential. In germline stem cells (GSCs), loss of Emerin/Otefin causes a thickening of the NL and reorganization of heterochromatin. These structural nuclear defects are linked to activation of two kinases of the DNA damage response pathway: Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 2 (Chk2). Although oogenesis in emerin/otefin mutants is rescued by loss of these DDR kinases, canonical triggers are not responsible for pathway activation. Instead, ATR and Chk2 activation is linked to defects in NL structure itself. Given the roles of BAF in mitotic nuclear envelope formation and repair , it was reasoned that checkpoint activation in emerin/otefin mutants might result from altered BAF function. This prediction was tested using germ cell-specific RNA interference (RNAi) to knockdown BAF. This study shows that BAF depletion disrupts NL structure, blocks differentiation and promotes GSC loss, mutant phenotypes that mirror Emerin/Otefin loss. Additionally, mutation of atr or chk2 partially restores germ cell differentiation in the baf mutant background, supporting the possibility that BAF depletion activates the NL checkpoint. Taken together, these findings suggest that Emerin/Otefin plays a dominant role in the enrichment of BAF to the NL and provide evidence that BAF functions with this prominent partner in the maintenance of an adult stem cell population (Duan, 2020).

This study extended in vivo studies of the BAF and LEM-D partnership. Capitalizing on a newly generated gfp-baf allele, this study shows that NL localization of BAF largely depends upon a single LEM-D protein, Emerin/Otefin. Loss of Emerin/Otefin is sufficient to disperse BAF in cells that express the A- and B-type lamins, Emerin2/Bocksbeutel and MAN1 in the NL. These data establish the in vivo existence of a prominent NL partnership between one LEM-D protein and BAF (Duan, 2020).

The basis for the unexpected reliance on Emerin/Otefin is unknown. One possibility is that LEM-Ds have different affinities for BAF. Pairwise alignment of amino acid residues within LEM-Ds shows the highest conservation between Drosophila emerin orthologues (70% similarity). Nonetheless, all LEM-Ds are strongly conserved in BAF-binding residues (42% identical, 67% similar). A second possibility is that the interaction of LEM-D proteins with BAF depends upon how a given LEM-D protein assembles into the NL network. Self-association of emerin influences both BAF and lamin binding. Finally, post-translational modifications (PTMs) of LEM-D proteins might impact BAF partnerships. As an example, O-GlcNAcylation modification of emerin affects BAF association, representing a regulated PTM that has the potential to alter NL function in response to nutrient availability. However, such signal-dependent PTMs are likely to be tissue specific, predicting a tissue-restricted, not global, effect on the NL enrichment of BAF. Further studies are needed to resolve the basis for the strong partnership between Emerin/Otefin and BAF (Duan, 2020).

BAF is essential for viability, with dying baf null larvae exhibiting a typical mitotic mutant phenotype that is associated with high levels of apoptosis. Several observations suggest that loss of NL BAF is not equivalent to complete loss of BAF. First, emerin/otefin null animals are viable, even though there is a global loss of NL BAF. Second, emerin/otefin null animals have lower levels of apoptosis in larval tissues than baf animals, without effects on the development of adult structures. Third, emerin/otefin mutant imaginal disc cells display an unchanged nuclear shape and chromatin architecture, whereas these cells are affected in baf mutants (Furukawa, 2003). Based on these data, it is suggested that BAF function at the NL during interphase is not essential. It is predicted that the essential BAF function relates to its contributions in mitosis and depends upon both Drosophila emerin orthologues, as these double mutant animals die with a mitotic mutant phenotype (Duan, 2020).

Effects of mislocalized BAF share features resulting from BAF overexpression in other systems. In emerin/otefin mutant germ cells, BAF dispersal contributes to the aggregation of heterochromatin. Defects in HP1a distribution have also been found in human cells overexpressing BAF or expressing a BAF mutant defective in interacting with NL components. Furthermore, several diseases affecting expression and processing of lamin A alter the distribution of BAF and resemble a BAF overexpression phenotype. Together, these findings support a model in which BAF contributes to the deleterious effects resulting from lamin or LEM-D mutations (Duan, 2020).

BAF is required for maintenance of Drosophila GSCs. Germ cell-specific BAF knockdown caused GSC loss, with remaining GSCs displaying a thickened and irregular NL structure, a phenotype shared with emerin/otefin mutants. These data support a model in which Emerin/Otefin and BAF function together to build NL structure in this cell type. Such a dependence on Emerin/Otefin for NL structure is consistent with limiting levels of the second Drosophila Emerin ortholog, Emerin2/Bocksbeutel (Barton, 2014). It is predicted that, in GSCs, the Emerin/Otefin and BAF might have a shared function in nuclear reformation at the end of mitosis (Duan, 2020).

Activation of the NL checkpoint is linked to NL deformation (Barton, 2018). Strikingly, baf mutant phenotypes are partially suppressed in atr/chk2; nos>bafRNAi animals, with double mutant ovaries showing increased germ cell survival and differentiation. Yet cell death remained in the double mutant backgrounds. Based on these observations, it is predicted that BAF loss in germ cells has multiple consequences. First, NL structure is affected. Second, loss of nuclear BAF might affect transcriptional networks required for GSC maintenance, suggested from studies showing BAF is an epigenetic regulator. Notably, the maintenance of mammalian stem cells also depends on BAF. Knockdown of BAF in either mouse or human embryonic stem cells promoted premature differentiation and reduced survival, phenotypes associated with an altered cell cycle. It remains possible that loss of Drosophila BAF in GSCs perturbs mitosis, which might induce apoptosis. Additional studies are needed to elucidate cell cycle contributions of BAF in GSCs (Duan, 2020).

These studies emphasize the important role of BAF within the NL network. Evidence is presented for consequences of BAF dispersal and loss during development, showing BAF dysfunction causes cell-type specific responses. Further definition of the developmental contributions of BAF will advance understanding of laminopathies, including the Nestor-Guillermo syndrome: a rare hereditary progeroid disorder caused by a missense mutation in BAF/BANF1 (Duan, 2020).

The Drosophila ovary is recognized as a powerful model to study stem cell self-renewal and differentiation. Decapentaplegic (Dpp) is secreted from the germline stem cell (GSC) niche to activate Bone Morphogenic Protein (BMP) signaling in GSCs for their self-renewal and is restricted in the differentiation niche for daughter cell differentiation. This study reports that Switch/sucrose non-fermentable (SWI/SNF) component Osa depletion in escort cells (ECs) results in a blockage of GSC progeny differentiation. Further molecular and genetic analyses suggest that the defective germline differentiation is partially attributed to the elevated dpp transcription in ECs. Moreover, ectopic Engrailed (En) expression in osa-depleted ECs partially contributes to upregulated dpp transcription. Furthermore, it was shown that Osa regulates germline differentiation in a Brahma (Brm)-associated protein (BAP)-complex-dependent manner. Additionally, the loss of EC long cellular processes upon osa depletion may also partly contribute to the germline differentiation defect. Taken together, these data suggest that the epigenetic factor Osa plays an important role in controlling EC characteristics and germline lineage differentiation (Hu, 2021).

Stem cells divide asymmetrically to generate a stem cell and a differentiating daughter cell. Yet, it remains poorly understood how a stem cell and a differentiating daughter cell can receive distinct levels of niche signal and thus acquire different cell fates (self-renewal versus differentiation), despite being adjacent to each other and thus seemingly exposed to similar levels of niche signaling. In the Drosophila ovary, germline stem cells (GSCs) are maintained by short range bone morphogenetic protein (BMP) signaling; the BMP ligands activate a receptor that phosphorylates the downstream molecule mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) accumulates in the GSC nucleus and activates the stem cell transcription program. This study demonstrates that pMad is highly concentrated in the nucleus of the GSC, while it quickly decreases in the nucleus of the differentiating daughter cell, the precystoblast (preCB), before the completion of cytokinesis. A known Mad phosphatase, Dullard (Dd), is required for the asymmetric partitioning of pMad. Mathematical modeling recapitulates the high sensitivity of the ratio of pMad levels to the Mad phosphatase activity and explains how the asymmetry arises in a shared cytoplasm. Together, these studies reveal a mechanism for breaking the symmetry of daughter cells during asymmetric stem cell division (Sardi, 2021).

The Drosophila female germline stem cell (GSC) is an excellent model to study niche-stem cell interaction because of its well-defined anatomy and abundant cellular markers. At the tip of each ovariole, two to three GSCs adhere to a cluster of niche cells, known as cap cells (CCs). A bone morphogenetic protein (BMP) ligand, Decapentaplegic (Dpp), is secreted by CCs and is an essential factor for GSC maintenance. Dpp binds to the serine-threonine kinase receptor Thickveins (Tkv) expressed on GSCs. Activated Tkv then phosphorylates Mothers against decapentaplegic (Mad) at its two C-terminal phosphorylation sites. Phosphorylated Mad (pMad) forms a heterodimer with Medea (Med), and the complex enters the nucleus and directly binds to the promoter of the differentiation factor bag of marbles (bam) to down-regulate its transcription. The down-regulation of bam is essential for GSC self-renewal (Sardi, 2021).

It has been hypothesized that the niche signaling rapidly decreases in one of the GSC daughters, precystoblast (preCB), as it is displaced away from the CC niche. Multiple studies have defined mechanisms for fine-tuning Dpp signal strength and range. However, it is still unclear how Mad, the immediate downstream molecule of Tkv, is initially regulated during GSC division (Sardi, 2021).

This study demonstrates that pMad rapidly reaches different levels in the dividing GSCs after mitosis but before the completion of cytokinesis. Its level in the nuclei of future GSCs remains high, while its level in the nuclei of preCBs decreases. Upon activation of the niche signal receptor Tkv kinase, its substrate, Mad, is phosphorylated near the plasma membrane and then travels throughout the cytoplasm and enters the nucleus. After mitosis, GSC takes several hours to complete cytokinesis, and the abscission occurs during DNA synthesis (S) phase. Since preCB shares its cytoplasm with GSC during this time, pMad can travel freely between the cytoplasm of two daughters. How can their nuclei have different levels of pMad (Sardi, 2021)?

This study shows that the previously identified Mad phosphatase Dullard (Dd) plays an essential role in the formation of the sharply different pMad levels in GSC and preCB. It was demonstrated that Dd interacts with Mad at the nuclear pores, where it may directly or indirectly dephosphorylate pMad. Dd itself does not exhibit asymmetric localization in GSC and preCB, but, as mathematical modeling indicates, the unbiased dephosphorylation by evenly distributed Dd combined with the phosphorylation of Mad biased toward the niche (due to local activation of Tkv on the niche side) is sufficient to explain the observed pMad asymmetry (Sardi, 2021).

In summary, these results provide a mechanism by which a self-renewal program is confined to the stem cells during asymmetric division (Sardi, 2021).

During asymmetric stem cell division, two daughter cells acquire different cell fates. The Drosophila ovarian niche differentially activates an GSC and a preCB because of the distinct position of these cells, the former adjacent to the niche and the latter displaced from the niche. What is the initial cause of the difference? While many factors have been identified that can amplify and/or ensure the already existent niche signal difference, it has been unknown how Mad, the immediate downstream molecule of the niche ligand, is initially regulated during GSC division (Sardi, 2021).

Mad is phosphorylated by active receptors at the side of GSC near the niche and then travels throughout the cytoplasm before entering the nucleus. The GSC-preCB pairs continue to share the cytoplasm for at least several hours after mitosis. Consistent with previous studies, this study observed that Mad diffuses throughout the cytoplasm of the GSC and preCB. This study found, however, that during this phase, the pMad levels in the nuclei of the GSC and preCB are already asymmetric (G1/S pMad asymmetry). It was determined that local activation of the kinase at the niche-GSC contact site is not sufficient for G1/S pMad asymmetry. Moreover, neither increasing the activity of kinase nor compromising the rate of Mad degradation affected G1/S pMad asymmetry. It was discovered that the Mad phosphatase, Dd, which dephosphorylates Mad at the nuclear pores in both GSC and preCB cells, is an essential factor in the formation of early asymmetric partitioning of pMad (Sardi, 2021).

To gain insight into the mechanisms responsible for the observed G1/S asymmetry, a mathematical model was formulated that included all major factors governing the spatiotemporal dynamics of pMad, and was constrained by the experimental data obtained in this study. The model suggests that a combination of the asymmetrically localized activated kinases, which reside at the site of contact between the GSC plasma membrane and the niche, together with sufficiently active phosphatase that is distributed symmetrically between nuclear envelopes of the GSC and preCB, is sufficient to explain the experimentally observed asymmetry of pMad levels in the GSC-preCB pairs (Sardi, 2021).

Analysis of the model revealed that for pMad asymmetry to occur, it is necessary that the positive and negative regulators of pMad (the kinases and phosphatases) be separated in space, as this brings about spatial gradients of pMad. However, this condition is not sufficient, as the gradients could be shallow. The modeling showed, furthermore, that the steepness of pMad gradients is exclusively determined by the interplay of two factors: pMad dephosphorylation that steepens the gradients and pMad diffusion in the cytoplasm that levels them out. Active regulation of the pMad diffusivity, which is determined by protein size and effective viscosity of the cytoplasm, is unlikely. This makes the phosphatase activity an essential factor determining pMad asymmetry. To a lesser degree, the asymmetry also depends on the permeability of nuclear pores to pMad export from the nucleus, since the dephosphorylation of pMad occurs inside the nucleus (Sardi, 2021).

In summary, this study identifies and explains a mechanism by which a stem cell can rapidly set up an initial asymmetry with respect to an extrinsic signal thus providing a conceptual framework for understanding the dynamics of niche-stem cell signaling (Sardi, 2021).

The niche controls stem cell self-renewal and progenitor differentiation for maintaining adult tissue homeostasis in various organisms. However, it remains unclear whether the niche is compartmentalized to control stem cell self-renewal and stepwise progeny differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells form a niche for controlling germline stem cell (GSC) progeny differentiation. This study has identified four IGS subpopulations, which form linearly arranged niche compartments for controlling GSC maintenance and multi-step progeny differentiation. Single-cell analysis of the adult ovary has identified four IGS subpopulations (IGS1-IGS4), the identities and cellular locations of which have been further confirmed by fluorescent in situ hybridization. IGS1 and IGS2 physically interact with GSCs and mitotic cysts to control GSC maintenance and cyst formation, respectively, whereas IGS3 and IGS4 physically interact with 16-cell cysts to regulate meiosis, oocyte development, and cyst morphological change. Finally, one follicle cell progenitor population has also been transcriptionally defined for facilitating future studies on follicle stem cell regulation. Therefore, this study has structurally revealed that the niche is organized into multiple compartments for orchestrating stepwise adult stem cell development and has also provided useful resources and tools for further functional characterization of the niche in the future (Tu, 2020).

Stem cells maintain adult tissue homeostasis through continuous self-renewal and generation of differentiated cells. Their self-renewal is shown to be controlled by the niche in the organisms ranging from Drosophila to mammals. Recently, stem cell progeny differentiation has also been proposed to be regulated by the niche in the Drosophila ovary. The differentiation process often consists of multiple developmental steps for generating one or several functional cell types. However, it remains largely unclear how the niche controls these differentiation steps at the cellular level (Tu, 2020).

The Drosophila ovary is an effective model for studying niche functions in regulating germline stem cell (GSC) self-renewal and differentiation. At the tip of the germarium, two or three GSCs contact cap cells anteriorly and inner germarial sheath cells (IGSs) (previously known as escort cells) laterally in region 1 (see scRNA-Seq Reveals Five IGS Subpopulations in the Drosophila Germarium). Immediate GSC progeny, cystoblasts (CBs), divide four times synchronously with incomplete cytokinesis to form interconnected mitotic cysts (MCs) (2-cell, 4-cell, and 8-cell cysts) and 16-cell cysts. IGS cells wrap around CBs and MCs in region 1 as well as 16-cell cysts in region 2a. Follicle cells begin to surround 16-cell cysts in region 2b and then form stage-1 egg chambers in region 3. Cap cells and anterior IGS cells form a niche for controlling GSC self-renewal through Dpp/BMP-mediated signaling and E-cadherin-mediated cell adhesion, whereas IGS cells form a niche for promoting differentiation partly by preventing bone morphogenetic protein (BMP) signaling. IGS cells utilize Hh, Wnt, epidermal growth factor receptor (EGFR), and Jak-Stat signaling to prevent BMP signaling in GSC progeny. Long IGS cellular processes are regulated by Hh and Rho-CDC42 small GTPase signaling, and cellular-process-mediated direct interactions are important for CB differentiation and cyst formation. Ecdysone signaling prevents IGS transformation into cap cells during development and is also needed in IGS cells for cyst formation, meiosis, and egg chamber formation. Therefore, two niches coordinately control GSC development in the Drosophila ovary (Tu, 2020).

While interacting with IGS cells, newly formed 16-cell cysts in region 2a undergo three important cellular events. First, those 16-cell cysts undergo two important meiotic events: chromosomal pairing and meiotic recombination. Second, both pro-oocytes form synaptonemal complexes initially, and only one of them becomes the oocyte. Third, 16-cell cysts change their round shape into a lens-like shape to ensure exactly one cyst is packaged into an egg chamber by follicle cells. Thus, it is tantalizing to speculate that IGS cells function as a niche for regulating these three important germ cell developmental events. Consistently, ecdysone signaling functions in IGS cells to promote meiotic entry and egg chamber formation possibly by maintaining IGS identity. This study used 10x genomics single-cell RNA-sequencing (scRNA-seq) to identify IGS subpopulations, which form linearly arranged niche compartments for orchestrating GSC self-renewal, CB differentiation, meiotic recombination, timely oocyte development, and cyst shape change. Therefore, it is proposed that the niche forms distinct subcompartments to control GSC maintenance and stepwise progeny differentiation in the Drosophila ovary (Tu, 2020).

Although IGS cells and their cellular processes form a niche for controlling GSC progeny differentiation, including CB differentiation, cyst formation, and the meiotic entry, it remains unclear whether IGS cells form distinct niche compartments that control different differentiation steps. This study used scRNA-seq to identify four IGS subpopulations, IGS1-IGS4, which are organized linearly along the germarium to interact with GSCs and their early progeny. Genetic manipulations were used to show that IGS1-IGS4 subpopulations form functionally separate compartments for controlling GSC maintenance, CB differentiation, meiotic recombination, timely oocyte specification, and cyst shape change. In addition, this study has molecularly defined the FCP population, which remain poorly studied because of the lack of suitable molecular markers. Therefore, this study has identified four IGS subpopulations that form subsequential niche compartments for controlling different steps of GSC progeny differentiation and has also molecularly defined the poorly studied FCP population, which opens the door for in-depth studies of IGS and FCP populations in the future (Tu, 2020).

Two recent scRNA-seq studies on the whole Drosophila ovary have identified different somatic cell types important for oogenesis. However, those two studies failed to identify the IGS subpopulations because of high complexities of somatic cell types and similarities of IGS cells. This study used GFP-based cell sorting and scRNA-seq to successfully identify four IGS subpopulations, IGS1-IGS4 and has further used gene-specific Gal4 lines and mRNA FISH to show their linear arrangement in the anterior germarium. IGS1 and IGS2 reside in region 1 to interact with GSCs and CBs/MCs, whereas IGS3 and IGS4 are located in region 2a to contact 16-cell cysts. IGS4 is the most posterior population directly contacting FSCs and FCPs and should be the niche for FSCs on the basis of the previous studies. Unfortunately, this study has not identified unique markers for IGS1-IGS3 subpopulations except IGS4, which specifically expresses bnb and GstS1. However, the combinatory gene expression patterns can still reliably separate IGS1-IGS3 subpopulations. For example, NetA is specifically expressed in IGS1 and IGS2, whereas croc and Hf are expressed in IGS1-IGS3. In the future, the split-Gal4 strategy will be used to generate IGS-subpopulation-specific Gal4 lines for further defining their molecular signatures and functions (Tu, 2020).

Previous studies have identified three general IGS markers, c587-Gal4, 13C06-Gal4, and PZ1444. This study has identified bin, vn, Nrt, mirr, dnc, CG7194, and CG42458 as new molecular markers for IGS and FCP cells. In addition, RNAi knockdown results have demonstrated that bin and vn maintain IGS cells and promote GSC progeny differentiation. In addition to germ-cell-mediated EGFR activation in IGS cells, this study has shown that IGS-expressing neuregulin-like EGFR Vn also contributes to EGFR signaling and thus IGS maintenance. Given that Wnt, Hh, and EGFR signaling are known to maintain IGS cells, it will be important to determine whether forkhead transcription factor Bin functions downstream of these pathways to maintain IGS cells. Transmembrane adhesion molecule Dpr17 is dynamically expressed in different IGS subpopulations in different germaria, suggesting that IGS cells could change their adhesive property dynamically. This could be a potentially exciting finding given that CBs, MCs, and 16-cell cysts have to move along the germarium by disengaging one IGS subpopulation and then engaging a new IGS subpopulation. This speculation needs future experimental confirmation. Therefore, this study has provided important insight into IGS subpopulations by uncovering new markers and functions and has also improved the ability to probe new IGS functions in regulating GSC development in the future (Tu, 2020).

Although recent studies have shown that IGS cells maintain GSCs and promote CB differentiation, none of these studies have attributed the functions to any specific IGS subpopulations. This study shows that secreted signaling molecules, NetA and Hf, are expressed in IGS1 and IGS2 to maintain GSCs. However, this study could not definitively demonstrate that only IGS1-expressing NetA and Hf contribute to GSC maintenance because of the lack of IGS1-specific Gal4 lines. Although it was shown that IGS cells are needed for CB differentiation and IGS2 directly contacts CBs/MCs, it was not possible directly demonstrate that IGS2 directly controls the differentiation of CBs and MCs into 16-cell cysts because of the lack of IGS2-specific Gal4 lines (Tu, 2020).

This study also shows that IGS3 and IGS4 control meiosis, oocyte specification, and cyst shape. IGS3 and IGS4 in region 2a extend their long cellular processes to wrap around newly formed H2AvD+ pre-meiotic and meiotic 16-cell cysts. These 16-cell cysts still have two pro-oocytes, but they only retain one oocyte and also undergo the round-to-lens shape change when surrounded by follicle cells in region 2b. Knocking down IGS4-expressing wun2 and GstS1 decreases the H2AvD+ 16-cell cysts in region 2a and increases the presence of round 16-cell cysts in region 2b, which fails to become lens-shaped, suggesting that IGS4 cells regulate meiotic recombination and cyst shape change. In addition, IGS-specific knockdown of GstS1, but not wun2, causes the presence of 2 pro-oocytes in stage 1 egg chambers. Consistently, H2126-mediated bin and smo knockdown in IGS4 can also decrease the H2AvD+ 16-cell cysts and increase the frequency of stage 1 egg chambers with two pro-oocytes, further supporting that IGS4 regulates meiosis and oocyte specification. However, the possibility that IGS3 cells might also regulate meiotic recombination and timely oocyte specification could not be completely ruled because of the lack of IGS3-specific Gal4 lines given that IGS3 expresses low amounts of wun2 and GstS1 and some of them also express H2126. In addition to one previous study showing that Ecdysone signaling in IGS cells regulates meiotic entry, this study has, for the first time, shown that IGS cells regulate meiotic recombination in 16-cell cysts. Given that the oocyte was previously thought to be determined entirely by the intrinsic mechanism, the differential RNA and protein transport caused by asymmetrically localized microtubules and fusomes, this is also the first time to show that IGS cells influence timely oocyte determination in Drosophila. Therefore, this study has provided important insight into IGS subpopulations in the regulation of GSC maintenance, cyst formation, meiotic recombination, timely oocyte determination, and cyst shape change, but the underlying signaling mechanisms await future investigation through generation of new genetic tools (Tu, 2020).

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. This study shows that Nos represses somatic gene expression by inhibiting translation of maternal importin-alpha2 (impalpha2) mRNA. Mis-expression of Impalpha2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, it is proposed that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors (Asaoka, 2019).

How germ cell fate is established and maintained is a century-old question in developmental, cellular, and reproductive biology. Metazoan species have two distinct modes of germline specification. In some species, germline progenitors are characterized by inheritance of a specialized ooplasm, or the germ plasm, which contains maternal factors necessary and sufficient for germline development. In other species, germline progenitors are specified by inductive signals from surrounding tissues. Irrespective of the mode of germline specification, transcriptional repression of somatic genes is common in germline progenitors, implying that this phenomenon is critical for separation of the germline from the soma (Asaoka, 2019).

In Drosophila, the germ plasm is localized in the posterior pole of cleavage embryos (stage 1-2), and is partitioned into germline progenitors called pole cells (stage 3-4). In pole cells of blastoderm embryos (stage 4-5), the genes required for somatic differentiation are transcriptionally repressed by two maternal proteins in the germ plasm, Polar granule component (Pgc) and Nanos (Nos). Pgc is a Drosophila-specific peptide that suppresses RNA polymerase II-dependent transcription in pole cells by inhibiting the function of positive transcriptional elongation factor b (P-TEFb, a dimer of Cyclin dependent kinase 9 and Cyclin T). By contrast, Nos is an evolutionarily conserved protein that plays an essential role in germline development in various animals. For example, in Drosophila, pole cells lacking Nos (nos pole cells) can adopt a somatic, rather than a germline, fate. Furthermore, depletion of Nos is reported to show ectopic expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and the sex-determination gene Sex lethal (Sxl), in pole cells. Thus, maternal Nos is required in pole cells for repression of somatic genes and establishment of the germ/soma dichotomy. However, the mechanism by which Nos represses somatic gene expression remains unknown (Asaoka, 2019).

Nos acts as a translational repressor of mRNAs that harbor a discrete sequence motif called Nanos Response Element (NRE) in the 3' UTR. NRE contains an evolutionarily conserved Pumilio (Pum)-binding sequence, UGU trinucleotide. In abdominal patterning, Pum represses translation of maternal hunchback (hb) mRNA by binding to NREs in its 3' UTR and recruiting Nos to the RNA/protein complex. Deletion of the NREs from hb mRNA causes its ectopic translation in the posterior half of embryos, which in turn suppresses abdomen formation. Furthermore, deletion of NREs causes hb translation in pole cells, suggesting that NRE-dependent translational repression occurs in pole cells. Indeed, Nos represses translation of head involution defective (hid) mRNA in pole cells in an NRE-like-sequence-dependent manner. In addition, Nos and Pum repress Cyclin B translation in pole cells by binding to a discrete sequence containing two UGU trinucleotides (Cyclin B NRE) These findings led to a speculation that Nos, along with Pum, represses somatic gene expression in pole cells by suppressing translation of mRNAs containing NRE or UGU in their 3' UTRs (Asaoka, 2019).

This study reports that, in pole cells, Nos, along with Pum, represses translation of importin-α2 (impα2)/Pendulin/oho31/CG4799 mRNA, which contains an NRE-like sequence in its 3' UTR. The impα2 mRNA encodes a Drosophila Importin-α homologue that plays a critical role in nuclear import of karyophilic proteins. Nos inhibits expression of a somatic gene, ftz, in pole cells by repressing Impα2-dependent nuclear import of the transcriptional activator, Ftz-F1. Based on these observations, it is proposed that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing act as a 'double-lock' mechanism to repress somatic gene expression in pole cells (Asaoka, 2019).

Maternally supplied impα2 mRNA is distributed throughout cleavage embryos. When embryos develop to the blastoderm stage, impα2 mRNA is degraded in the somatic region, but not in pole cells, resulting in enrichment of impα2 mRNA in pole cells. However, this study found that expression of Impα2 protein was at background levels in pole cells. Because impα2 mRNA contains a sequence very similar to the NRE (hereafter, NRE-like sequence) in its 3' UTR, it was assumed that impα2 mRNA is a target of Nos/Pum-dependent translational repression in pole cells. To investigate this possibility, the expression of the Impα2 protein was first monitored in pole cells of embryos lacking maternal Nos or Pum (nos or pum embryos, respectively). In these pole cells, expression of Impα2 protein was higher than in those of control (nos/+) embryos. Because neither nos nor pum mutation affected the impα2 mRNA level in pole cells, these observations show that Nos and Pum repress protein expression from the impα2 mRNA in pole cells (Asaoka, 2019).

Whether this repression is mediated by the NRE-like sequence in the impα2 3' UTR was investigated. To this end, impα2 mRNA, with or without the NRE-like sequence (impα2 WT and impα2 ΔNRE, respectively), was maternally supplied to embryos, and their protein expression was examined in pole cells at the blastoderm stage. Because a triple Myc tag sequence was inserted at the C-terminal end of the coding sequence, protein expression from these mRNAs could be monitored using an anti-Myc antibody. When impα2 WT mRNA was supplied to normal (y w) embryos, the tagged protein was expressed at low levels in the soma, but was barely detectable in pole cells. By contrast, the tagged protein from impα2 ΔNRE mRNA was detected in normal pole cells. Similar protein expression was observed in pole cells lacking Nos (nos pole cells), when impα2 WT mRNA was supplied, as well as when impα2 ΔNRE mRNA was supplied. Because the frequency of tagged protein expression from impα2 ΔNRE mRNA did not increase in cells lacking Nos, these results indicate that the NRE-like sequence mediates Nos-dependent repression of Impα2 protein expression in pole cells (Asaoka, 2019).

The NRE-like sequence of impα2 mRNA contains two UGU trinucleotides. The UGU trinucleotide is a core sequence of an RNA motif (Nos-Pum SEQRS motif: 5'-HWWDUGUR) that was highly enriched in a SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes) analysis of the Nos-Pum-RNA ternary complex. Hence, it was asked whether Pum and Nos form a ternary complex with impα2 mRNA in an NRE-like sequence-dependent manner. To address this question, electrophoretic mobility shift assay (EMSA) was performed using the Pum RNA-binding domain and the Nos protein containing Zn finger motifs and C-terminal region, which are reported to form a Nos-Pum-target RNA ternary complex in vitro. Nos and Pum together, but neither alone, formed a complex with impα2 RNA containing an NRE-like sequence (WT), whereas alteration of the NRE-like sequence (mut) abolished this interaction. These results demonstrate that Nos and Pum are able to interact with the impα2 3' UTR in an NRE-like sequence-dependent manner. The observations described above led to a conclusion that Nos, along with Pum, directly represses impα2 translation in pole cells (Asaoka, 2019).

Impα2 is a Drosophila homologue of Importin-α that mediates nuclear import of karyophilic proteins with classical nuclear localization signal (NLS). It was predicted that ectopic production of Impα2 in nos pole cells would cause aberrant nuclear import of NLS-containing karyophilic proteins. To explore this possibility, this study focused on a transcriptional activator, Ftz-F1, which contains a classical NLS and is expressed throughout early embryos, including pole cells. In normal embryos, Ftz-F1 was enriched in the cytoplasm of pole cells, although it was in the nuclei of somatic cells. In the absence of maternal Nos, the percentage of embryos with Ftz-F1 signal accumulating in pole-cell nuclei was higher than in normal embryos. Furthermore, the nuclear/cytoplasmic ratio of Ftz-F1 signal intensities in nos pole cells was higher than in normal pole cells. To determine whether this aberrant concentration of Ftz-F1 was caused by mis-expression of Impα2, this study expressed Impα2 ectopically in pole cells of normal embryos. To this end, impα2 mRNA in which the 3' UTR was replaced with the nos 3' UTR, was maternally supplied under the control of the nos promoter; the mRNA was localized to the germ plasm and pole cells under the control of the nos 3' UTR. The percentage of these embryos (impα2-nos3'UTR embryos) with Ftz-F1 in pole-cell nuclei and the nuclear/cytoplasmic ratio of Ftz-F1 intensities in their pole cells were higher than those of normal pole cells. These observations suggest that mis-expression of Impα2 in pole cells caused by depletion of maternal Nos results in aberrant nuclear import of Ftz-F1 (Asaoka, 2019).

Depletion of maternal Nos results in ectopic expression of the somatic genes ftz, eve and Sxl in pole cells. Because Ftz-F1 is required for proper expression of ftz in the soma, it was asked whether mis-expression of Impα2 causes ectopic expression of ftz in pole cells. In normal embryos, ftz mRNA was expressed in seven stripes of somatic cells, but never expressed in pole cells. By contrast, in impα2-nos3'UTR embryos, ftz mRNA was rarely detectable in pole cells. It is assumed that this low frequency of ftz expression was due to Pgc-mediated silencing of global mRNA transcription. To test this idea, Impα2 was expressed in pole cells of embryos lacking maternal Pgc (pgc impα2-nos3'UTR embryos); the frequency of ftz expression was drastically increased, compared to those of impα2-nos3'UTR embryos and the embryos lacking Pgc (pgc embryos). A similar situation was observed in embryos lacking both Pgc and Nos activities (pgc nos embryos). The percentage of embryos expressing ftz in pole cells was 82.8%, an increase relative to 35.8% in pgc embryos. Furthermore, ectopic ftz expression in pgc nos pole cells was suppressed by injecting double-stranded RNA (dsRNA) against impα2. Therefore, it is concluded that ectopic expression of ftz in pole cells is cooperatively repressed by Nos-dependent suppression of Impα2 production and Pgc (Asaoka, 2019).

In addition to ftz expression, eve was expressed ectopically in pole cells of pgc impα2-nos3'UTR embryos. Ectopic eve mRNA and its protein expression were significantly higher in pgc impα2-nos3'UTR pole cells than pgc or impα2-nos3'UTR pole cells (S3 Fig). Expression of the sex-determination gene Sxl was examined in early pole cells, because Sxl is also repressed by nos in both male and female pole cells. In males, Sxl mRNA expression was rarely detectable in pole cells of nos, impα2-nos3'UTR, pgc, and pgc impα2-nos3'UTR embryos. By contrast, in females, the percentage of embryos expressing Sxl mRNA in pole cells was significantly higher in pgc impα2-nos3'UTR embryos than in impα2-nos3'UTR, and pgc embryos. These results indicate that eve and Sxl, like ftz, are cooperatively repressed in pole cells by Impα2 depletion and Pgc-dependent transcriptional silencing. Because there is no evidence for the involvement of Ftz-F1 in eve and Sxl expression, it is likely that Impα2 mediates nuclear import of other transcriptional activator(s) for eve and/or Sxl in pole cells (Asaoka, 2019).

Nos is required in pole cells for mitotic quiescence, repression of apoptosis, and proper migration to embryonic gonads. Hence, it was asked whether mis-expression of Impα2 causes defects in these processes. First, using an antibody against a phosphorylated form of histone H3 (PH3), a marker of mitosis, whether pole cells enter mitosis in stage 7-9 embryos was investigated. Premature mitosis was detected in pole cells of nos embryos, as described previously, but never in pole cells of impα2-nos3'UTR or pgc impα2-nos3'UTR embryos. Second, using an antibody against cleaved Caspase-3, a marker of apoptosis, whether pole cells enter apoptosis in stage 10-16 embryos was investigated. Pole cells never expressed the apoptotic marker in impα2-nos3'UTR embryos, whereas in pgc impα2-nos3'UTR embryos, 20.4% of pole cells expressed the apoptotic marker. The latter was statistically indistinguishable from pgc pole cells, which have been reported to enter apoptosis. These data indicate that mis-expression of Impα2 does not affect apoptosis of pole cells even in the absence of pgc function. Last, whether mis-expression of Impα2 affects pole cell migration was investigated. The ability of pole cells to migrate properly into the embryonic gonads was never impaired in impα2-nos3'UTR embryos, and the percentage of pole cells entering the gonads in pgc impα2-nos3'UTR embryos was statistically indistinguishable from that of pgc pole cells, which has been reported to exhibit migration defect. These observations indicate that mis-expression of Impα2 does not induce premature mitosis, apoptosis, or mis-migration of pole cells. This can be partly explained by the facts that Cyclin B and hid mRNAs are the targets for Nos-dependent translational repression regulating mitosis and apoptosis in pole cells, respectively (Asaoka, 2019).

During the course of the experiments described above, it was observed that impα2-nos3'UTR interacts genetically with the pgc mutation to cause dysgenic gametogenesis. Because almost all of the ovaries in females derived from pgc mothers mated with y w males were agametic, the effect of impα2-nos3'UTR in pgc/+ background was examined. The percentage of dysgenic ovaries in pgc/+ impα2-nos3'UTR females derived from pgc/+ impα2-nos3'UTR mothers mated with y w males was significantly higher than those in pgc/+ and impα2-nos3'UTR females. In the dysgenic ovaries, almost all of the egg chambers fail to complete the vitellogenic stage, and consequently only a few mature oocytes were present. Furthermore, the percentages of dysgenic and agametic testes in pgc impα2-nos3'UTR males derived from pgc impα2-nos3'UTR mothers mated with y w males were higher than those in pgc and impα2-nos3'UTR males. In these testes, the abundance of Vasa-positive germline cells was reduced (dysgenic) or absent (agametic). Because dysgenic and agametic gonads were barely detectable in females and males derived from reciprocal crosses, the data suggest that mis-expression of Impα2 from maternal transcript, concomitant with maternal pgc depletion in pole cells, causes defects in gametogenesis. However, it cannot be tested whether concomitant depletion of maternal Nos and Pgc causes a similar phenotype because nos pole cells degenerate before adulthood, even when apoptosis in these cells is genetically repressed (Asaoka, 2019).

Expression of Importin-α subtypes is spatio-temporally regulated in the soma during development in multiple animal species, including Drosophila, and they control nuclear transport of unique karyophilic proteins to activate different sets of somatic genes. Drosophila genome contains three Importin-α family genes: impα1, 2, and 3. impα1/Kap-α1/CG8548 mRNA is not detectable in pole cells during early embryogenesis, and its protein product is ubiquitously expressed at a very low level throughout embryogenesis. By contrast, maternal impα3/Kap-α3/CG9423 mRNA is detectable in germ plasm during pole cell formation, and production of Impα3 protein is upregulated during the blastoderm stage. Because Impα3 production was independent of maternal nos activity, it is likely that Nos-dependent repression of Impα2 production is solely responsible for suppression of somatic gene expression in pole cells. By contrast, pole cells become transcriptionally active during gastrulation, when Impα2 is undetectable in these pole cells. Thus, the onset of zygotic transcription in pole cells may require Impα3-dependent nuclear import of transcription factors, in addition to the disappearance of Pgc and the alteration in chromatin-based regulation. After gastrulation, maternal impα2 mRNA is rapidly degraded in pole cells, and neither impα2 mRNA nor protein is detectable in the germline before adulthood. This suggests that maternal impα2 is dispensable for germline development, and that maternal impα2 mRNA partitioned into early pole cells must be silenced by Nos and Pum in order to suppress mis-expression of somatic genes (Asaoka, 2019).

Depletion of maternal Nos activities caused mis-expression of ftz in pole cells. Although ftz expression was barely observed in pole cells lacking only maternal Nos, it was partially derepressed in pole cells in the absence of Pgc alone, probably because a trace amount of Ftz-F1 enters pole cell nuclei even in the absence of the impα2 translation. Therefore, it is proposed that a subset of somatic genes, including ftz and eve, are repressed in pole cells by two distinct mechanisms: Nos-dependent repression of nuclear import of transcriptional activators and Pgc-dependent silencing of mRNA transcription. Pgc inhibits P-TEFb-dependent phosphorylation of Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of RNA polymerase II, a modification that is critical for transcriptional elongation; thus, mRNA transcription in pole cells is globally suppressed by Pgc. By contrast, Nos inhibits transcription of particular genes by repressing Impα2-dependent nuclear import of the corresponding transcriptional activators (Asaoka, 2019).

Nos is evolutionarily conserved and expressed in the germline progenitors of various animal species. In C. elegans, nos-1 and -2 are essential for rapid turnover of maternal lin-15B mRNA, which encodes a transcription factor that would otherwise cause inappropriate transcriptional activation in primordial germ cells. In the germline progenitors of Xenopus embryos, Nos-1, along with Pum, destabilizes maternal VegT mRNA and represses its translation to inhibit somatic (endodermal) gene expression, which is activated by VegT protein. Furthermore, in the germline progenitors (small micromeres) of sea urchin embryos, Nos silences maternal mRNA encoding a deadenylase, CNOT6, to stabilize other maternal mRNAs inherited into small micromeres. This study demonstrated that Nos inhibits translation of maternal impα2 mRNA in pole cells in order to suppress nuclear import of a transcriptional activator for somatic gene expression. Based on these observations, it is proposed that Nos silences maternal transcripts that are inherited into germline progenitors but deter the proper germline development. In addition to Nos-dependent silencing of maternal transcripts, transient suppression of RNA polymerase II elongation is observed during germline development of a wide range of animals, including Drosophila, C. elegans, Xenopus, and an ascidian, Halocynthia roretzi. Therefore, it is proposed that the 'double-lock' mechanism achieved by Nos and global suppression of RNA polymerase II activity plays an evolutionarily widespread role in germline development (Asaoka, 2019).

In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. This study demonstrates that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. It is concluded that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis (Kong, 2019).

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. This study shows that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. This study has delineated a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest (Hampoelz, 2019).

Nuclear pore complexes (NPCs) bridge the nuclear envelope (NE) and mediate nucleocytoplasmic exchange. They are giant assemblies of about 110 MDa in animals with an elaborate structure and composition. About 30 different genes encode for NPC components, termed nucleoporins (Nups). Those are subclassified into scaffold Nups that assemble into a cylindrical architecture with a ~50 nm wide central channel; and intrinsically disordered phenylalanine-glycine rich FG-Nups that line this channel. Scaffold Nups assemble into the so-called Y and inner ring complexes that form the outer and inner rings, respectively (see The Nuclear Pore Complex as a Flexible and Dynamic Gate). FG-Nups (containing phenylalanine-glycine repeats) have the capacity to phase separate in vitro. In vivo, they establish a unique biophysical milieu within the central channel that is impermeable to inert molecules. FG-Nups transiently interact with nuclear transport receptors (NTRs, also called importins, exportins, or karyopherins) that form complexes with cargo and cross the permeability barrier. Transport directionality across the NE is ensured by the small GTPase Ran. RCC1, the RanGTP exchange factor (RanGEF) is chromatin associated and maintains a high RanGTP concentration in the nucleus. The Ran GTPase activating protein (RanGAP) binds to Nup358 (also called RanBP2) at the cytoplasmic face of the NPC and ensures high RanGDP levels in the cytosol. Although RanGTP displaces cargo from import complexes in the nucleoplasm, GTP hydrolysis disassembles export complexes once they arrive at the cytoplasmic face. Nup358 is absent from lower eukaryotes but essential in animals and involved in active nuclear transport, cell cycle progression, malignant transformation, and viral infection (Hampoelz, 2019).

NPC assembly is an intricate process. In multicellular organisms, two assembly pathways were described. First, the relatively rapid assembly of NPCs from pre-existing building blocks concomitantly with nuclear envelope (NE) reformation at the end of open mitosis is referred to as 'post-mitotic' assembly. This pathway is spatially directed to chromatin by the Nup Elys. Temporal control is provided by cell-cycle-dependent kinases and phosphatases. Second, interphase assembly is a relatively slow process that generates NPCs from scratch in order to double their number for the next mitosis. It proceeds from inside out through the NE and requires the active nuclear import of Nups. Here, Nup153 spatially directs the Y complex to the inner nuclear membrane (Vollmer, 2015; Hampoelz, 2019 and references therein).

Little is known about the early steps of NPC assembly that occur prior to membrane association. FG-Nups serve as a velcro for scaffold Nups. They, however, have a considerable aggregation propensity in isolation that has to be controlled during NPC biogenesis in vivo. Non-NPC-associated Nups are chaperoned by importin β. RanGTP dissociates importin β complexes and thereby releases Nups for interphase and post-mitotic NPC assembly. Likewise, RanGTP induces NPC assembly in vitro, but the in vivo relevance of this finding remains to be tested (Hampoelz, 2019).

In multicellular organisms, nuclear pores also reside in stacked membrane sheets of the endoplasmic reticulum (ER), termed annulate lamellae (AL). Those are particularly prominent in gametes and embryos of a multitude of species including Drosophila. In early fly embryos, AL insert into the NE in order to supply the rapidly growing nuclei with additional membranes and NPCs (Hampoelz, 2016). AL are therefore thought to be maternally provided NPC storage pools. How AL assemble in the absence of a nuclear compartment, which spatially coordinates the process in case of the two previously characterized pathways, remains elusive. This study has investigated AL-NPC biogenesis in vivo during Drosophila melanogaster oogenesis. AL-NPC biogenesis was shown to be vastly abundant during oogenesis. It depends on the condensation of Nups into compositionally different granules that are transported along microtubules (MTs) and regulated by Nup358 in concert with Ran and NTRs. This NPC biogenesis is mechanistically distinct from both canonical NPC assembly pathways and progresses away from chromatin. It is proposed that instead, Nup358 condensates fulfill the role of spatially directing NPC biogenesis, in the absence of a bona fide nuclear compartment (Hampoelz, 2019).

Little has been known about the biogenesis of AL and the spatial cues that allow NPC formation away from the nuclear compartment. This work addresses these questions during Drosophila oogenesis and suggests a third, non-canonical NPC assembly mechanism. Already in nurse cells, Nup358 condenses into large granules (see A Model for NPC Biogenesis beyond the Nuclear Compartment). Condensation might be fostered by local translation of nup358 transcripts that enrich at the surface of Nup358 granules in a translation dependent manner. In nurse cells, AL biogenesis is suppressed, and only limited NPC assembly is observed within Nup358 granules. This could be due to the available amount or configuration of ER membranes or because high cytoplasmic concentrations of RanGDP promote the formation of Importin-Nup complexes that prevent other Nups from condensation. Nup358 granules become assembly platforms for AL-NPCs once they travel through ring canals into the ooplasm. Scaffold and FG-Nups condense into oocyte specific granules, possibly facilitated by elevated levels of RanGTP that dissociates the respective Nups from Importin. In the oocyte, NPC precursor granule interactions are promoted by MT dependent granule dynamics. Upon interaction, granules transfer material and assemble Nups onto available ER membrane, ultimately leading to the formation of larger stacks with multiple membrane sheets. Those are inherited to the embryo where they supplement dividing nuclei with NPCs throughout early embryogenesis (Hampoelz, 2019).

The phase-separating properties of FG-Nups have been subject to extensive research in vitro. This study provides evidence for condensation of Nups in vivo. Several properties, namely the coalescence of Nup358 granules, the transfer of material between granules, the high molecular mobility within granules, and the contact shapes observed upon granule interactions, are hallmarks of biomolecular condensates. Such condensates are defined as 'non-membranous organelles'. Although AL inherently contain stacked membrane sheets, they retain at least some characteristics of a phase separated condensate such as a milieu that is distinct from the surrounding cytoplasm. These findings underline the importance of phase separation at membranes that was also observed in other biological systems (Hampoelz, 2019).

Several lines of evidence, namely 1,6-hexanediol treatment, depletion of BicD and embargoed, and the interference with the Ran nucleotide status suggest that condensation of Nups into NPC precursor granules is critical for AL biogenesis. It is further underlined by the fact that Colchicine treatment counteracts the RanQ69L phenotype by reducing the number of MT-promoted granule interactions. Condensation concentrates NPC constituents in a constrained volume within the large ooplasm and might prevent unspecific interactions of soluble Nups. MT dynamics enhances interactions of otherwise unmixed, compositionally heterogeneous NPC precursor condensates and is a prerequisite for NPC assembly from condensed granules. It also prevents unwanted fusion and relaxation of compositionally homogeneous condensates of the same type. Facilitated interactions of granules could be of particular importance in the highly viscous ooplasm, where cytoskeleton-induced streaming is critical for the efficient distribution of various components (Hampoelz, 2019).

The two canonical NPC assembly pathways rely on the stepwise and orchestrated assembly of soluble Nups or subcomplexes onto either anaphase chromatin or the NE surface during interphase, respectively. However, these spatial cues are absent in the ooplasm and alternative mechanisms to locally concentrate assembly modules must be important. It is believed that the condensation of Nups replaces the canonical cues, in line with previous work that had shown functions of natively unfolded FG-Nups to stabilize each other but also NPC scaffold components during yeast NPC assembly. Controlled interactions and material transfer between condensates might account for specific steps of assembly and even provide a certain order, although this concept remains to be further tested (Hampoelz, 2019).

The data strongly suggest Nup358 granules as assembly platforms, where NPCs are seeded onto ER membranes. Nup358 has no reported role in initiating the assembly process in both previously described pathways (Weberruss, 2016). On the contrary, during interphase Nup358 assembles rather late onto the NPC scaffold. Although such information is not available for post-mitotic assembly, its mitotic localization to kinetochores could indicate an early role for Nup358. Indeed, Nup358 is of structural importance for the pore scaffold, given that its loss destabilizes the outer Y complex at the cytoplasmic ring at NE-NPCs. It is thus conceivable that Nup358 could not only stabilize but also recruit scaffold components onto membranes. Post-mitotic and interphase NPC assembly are initiated by two distinct Nups, Elys and Nup153, respectively. Elys localizes the Y complex onto anaphase chromatin and is dispensable for interphase assembly but also for AL formation, given that its depletion induces AL. In contrast, Nup153 seeds NPCs during interphase assembly onto the inner nuclear membrane, and it has been suggested to have a similar role at the ER during AL biogenesis. This study, however, found that Nup153 is absent from AL in oocytes(Hampoelz, 2019).

Despite all molecular and conceptual differences, the common driving force for NPC biogenesis at and beyond the nucleus is Ran that coordinates the availability of Nups for assembly by dissociating them from NTRs. Nup358 binds RanGAP and directly links the NPC to Ran activity. At the NE this is eminent to ensure a sharp Ran gradient and thus efficient nucleocytoplasmic transport. This study shows that this interaction is preserved beyond nuclei, because RanGAP strongly enriches at Nup358 granules in a Nup358-dependent manner. One might speculate that within the RanGTP milieu of the ooplasm, RanGAP induces a local Ran gradient at Nup358 granules that drives NPC biogenesis; conceptually similar to the nuclear compartment for interphase or postmitotic NPC assembly (Figure 7C and 7C'). Thereby, the observed progressive dilution of Nup358 and RanGAP at Nup358 granules in the oocyte could be important to drive their progression into AL. It might be caused by ooplasmic RanGTP that favors complex formation between Crm1 and Nup358. Although this would be consistent with the observed embargoed gene silencing phenotype, the enhanced condensation of Nup358 upon global induction of RanGTP argues for an alternative interpretation: Nup358 functionally interacts with both, Importins and Crm1 under specific conditions. It is thus not clear how exactly it is being chaperoned. The phenotype observed under embargoed gene silencing conditions might be indirectly caused by disturbance of the spatial distribution of Ran and NTRs across nurse cells and oocytes, as indicated by the variety in phenotype across individuals. Yet it stresses the importance of spatially controlled Nup condensation to assemble AL-NPCs. In any scenario, NTR-mediated de-condensation of Nup358 and the consequent reduction of local RanGAP activity would regulate the progression of NPC biogenesis by determining the availability of soluble, 'assembly prone' Nups and the degree of mixing at granule interfaces (Hampoelz, 2019).

Various aspects of AL-NPC biogenesis are markedly different from both canonical NPC assembly pathways. During oogenesis, Nup condensation, local translation and MT dependent dynamics interplay with Ran activity in order to faithfully assemble AL in oocytes. They are inherited to the embryo where this pool of ready-made NPCs supplements nuclei during the rapid interphases of the blastoderm stage. Because AL are present in a plethora of species, similar mechanisms are likely to operate throughout animals (Hampoelz, 2019).

The orientation of microtubule networks is exploited by motors to deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of microtubule filaments. In cells however, microtubules are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. This study implemented variational optical flow analysis as a new approach to analyze the polarity of microtubules in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing microtubule orientation from confocal movies, it was found that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, it was found that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of microtubule plus ends is also altered. These findings lead to a proposal that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, a possible mechanism is proposed for a specialised cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds; to counteract the recruitment of Kinesin to microtubules (Drechsler, 2020).

The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. This study examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted an analysis of focal adhesion dynamics. The rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, it was found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion (Zhao, 2022).

Me31B is a protein component of Drosophila germ granules and plays an important role in germline development by interacting with other proteins and RNAs. To understand the dynamic changes that the Me31B interactome undergoes from oogenesis to early embryogenesis, this study characterized the early embryo Me31B interactome and compared it to the known ovary interactome. The two interactomes shared RNA regulation proteins, glycolytic enzymes, and cytoskeleton/motor proteins, but the core germ plasm proteins Vas, Tud, and Aub were significantly decreased in the embryo interactome. Follow-up on two RNA regulations proteins present in both interactomes, Tral and Cup, revealed that they colocalize with Me31B in nuage granules, P-bodies/sponge bodies, and possibly in germ plasm granules. It was further shown that Tral and Cup are both needed for maintaining Me31B protein level and mRNA stability, with Tral's effect being more specific. In addition, evidence is provided that Me31B likely colocalizes and interacts with germ plasm marker Vas in the ovaries and early embryo germ granules. Finally, it was shown that Me31B's localization in germ plasm is likely independent of the Osk-Vas-Tud-Aub germ plasm assembly pathway although its proper enrichment in the germ plasm may still rely on certain conserved germ plasm proteins (McCambridge, 2020).

To summarize, although Me31B's localization to the posterior of an oocyte is likely independent of Osk, Aub, and Dart5, its proper enrichment at the site may still rely on Aub. Together with a previous report that Me31B's localization pattern is not affected in vas and tud mutants, it is speculated that Me31B's localization in a developing oocyte may be independent of the Osk-Vas-Tud-Aub assembly pathway, but its proper enrichment at the posterior germ plasm may still depend on certain conserved germ plasm proteins like Aub. (McCambridge, 2020).

This speculation, together with earlier conclusions in this study, led to the proposal of a hypothetical model for Me31B localization and enrichment process in the germline cells (see Hypothetical model of Me31B localization and enrichment into germ plasm). In this model, Me31B and conserved germ plasm proteins, Osk-Vas-Tud-Aub, exist in distinct granules in the germ plasm, Osk-Vas-Tud-Aub in germ plasm granules and Me31B (possibly associated with Tral and Cup) in separate granules but in close proximity. Me31B granules use an Osk-Vas-Tud-Aub-independent mechanism to localize to the cortex and the posterior of a developing oocyte, then the posteriorly localized Me31B granules interact with the germ plasm granules, which is necessary for proper Me31B granule enrichment in the germ plasm. In the early embryos, Me31B proteins begin to degrade rapidly and become dispersed in the cytoplasm (McCambridge, 2020).

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, protein-protein associations were examined of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed identification of 26 previously unknown associations, besides recovering several well characterized interactions. Interactions were odemtofoed between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans (Bansal, 2020).

Post-transcriptional regulation of gene expression plays an essential role during oocyte maturation. This study reports that Drosophila MARF1 (Meiosis Regulator And mRNA Stability Factor 1), which consists of one RNA-recognition motif and six tandem LOTUS domains with unknown molecular function, is essential for oocyte maturation. When tethered to a reporter mRNA, MARF1 post-transcriptionally silences reporter expression by shortening reporter mRNA poly-A tail length and thereby reducing reporter protein level. This activity is mediated by the MARF1 LOTUS domain, which binds the CCR4-NOT deadenylase complex. MARF1 binds cyclin A mRNA and shortens its poly-A tail to reduce Cyclin A protein level during oocyte maturation. This study identifies MARF1 as a regulator in oocyte maturation and defines the conserved LOTUS domain as a post-transcriptional effector domain that recruits CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress their translation (Zhu, 2018).

RNA-binding proteins (RBPs) mediate post-transcriptional gene regulation by determining molecular fates of target RNAs. In addition to RNA-binding domains, RBPs often have additional auxiliary domains. These auxiliary domains may function as effector domains for post-transcriptional gene regulation directly through enzymatic activity or indirectly by mediating protein-protein interaction. Identifying these effector domains and their molecular functions is critical to understand the roles of RBPs in post-transcriptional gene regulatory mechanism (Zhu, 2018).

MARF1 is an RBP consisting of one RNA-recognition motif (RRM) followed by several tandem LOTUS domains (Limkain, Oskar, and Tudor containing proteins 5 and 7. Also called OST-HTH). Previous studies showed that mouse MARF1 is required for completion of meiosis in oogenesis by reducing protein and mRNA levels of retrotransposons and a few endogenous genes. However, the molecular mechanism by which MARF1 regulates gene expression remains unclear (Zhu, 2018).

LOTUS domains are conserved in bacteria, fungi, plants, and animals. In animals, LOTUS domain proteins are expressed almost exclusively in the germline and are implicated in RNA regulation. In Drosophila, these LOTUS domain proteins include Oskar, Tejas (human TDRD5), Tapas (human TDRD7), and MARF1 (Meiosis Regulator And mRNA Stability Factor 1 = GC17018. Human MARF1). However, the molecular function of the conserved LOTUS domain is not fully understood (Zhu, 2018).

This work studied the biological and molecular functions of Drosophila MARF1 and its LOTUS domains. MARF1 is essential for proper oocyte maturation by regulating cyclin protein levels. When tethered to a reporter mRNA, MARF1 caused shortening of reporter mRNA poly-A tail and reduced reporter protein level. This activity was mediated by MARF1 LOTUS domain. Consistent with this finding, it was found that MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain. Furthermore, it was found that MARF1 binds cyclin A mRNA, shortens its poly-A tail, and reduces Cyclin A protein level during oocyte maturation. Thus, this study uncovered the biological and molecular functions of Drosophila MARF1 and defined its conserved LOTUS domains as a post-transcriptional effector domain to recruit the CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress translation of the mRNAs (Zhu, 2018).

MARF1 is expressed in late-stage oocytes and is required for proper oocyte maturation by regulating cyclin protein levels. MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain to shorten target mRNA poly-A tails and thus reducing cyclin protein levels without changing cyclin mRNA levels. Thus, MARF1 LOTUS domain is defined as a post-transcriptional effector domain that binds the CCR4-NOT deadenylase complex (Zhu, 2018).

Recent studies by others showed that the LOTUS domains of Drosophila Oskar, Tejas, and Tapas bind germline DEAD-box RNA helicase Vasa to stimulate Vasa ATPase and helicase activities. Crystallographic studies showed that the LOTUS domain of Oskar forms a homodimer and that each of the monomer subunits binds the C-terminal domain of the Vasa DEAD-box helicase core on the side opposite to the dimerization interface. In contrast, this study shows that the MARF1 LOTUS domain binds the CCR4-NOT deadenylase complex, but does not bind Vasa, Oskar, or another molecule of MARF1 (Zhu, 2018).

The LOTUS domains found in Oskar, Tejas, and Tapas, but not MARF1, have a C-terminal extension, which is required for interaction with Vasa. Hence the LOTUS domains are divided into two subclasses: (1) extended LOTUS (eLOTUS) domain that is present in Oskar, Tejas, and Tapas, has a C-terminal extension, and binds Vasa, and (2) minimal LOTUS (mLOTUS) domain that is present in MARF1, lacks a C-terminal extension, does not bind Vasa, and instead binds the CCR4-NOT deadenylase complex. Thus, although eLOTUS and mLOTUS domains share core sequence homology except for the C-terminal extension, they mediate distinct protein-protein interactions. Interestingly, eLOTUS proteins (Oskar, Tejas/TDRD5, Tapas/TDRD7) contain a single eLOTUS domain while mLOTUS proteins (MARF1) contain multiple tandem mLOTUS domains (Zhu, 2018).

This study also showed that MARF1 binds cyclin A mRNA. In MARF1^null mutant late-stage oocytes, cyclin A mRNA poly-A tail is longer and Cyclin A protein level is increased, without change in the cyclin A mRNA level. The degradation rate of Cyclin A protein was not changed in MARF1^null oocytes compared with control oocytes in vitro. These results indicate that in control late-stage oocytes, MARF1 post-transcriptionally regulates Cyclin A protein level by binding cyclin A mRNA, shortening cyclin A mRNA poly-A tail, and reducing Cyclin A protein level. In contrast, poly-A shortening of cyclin A mRNA is lost in MARF1^null oocytes, resulting in an accumulation of Cyclin A protein. Cyclin A is the only protein found that is increased in its level in MARF1 mutant oocytes (Zhu, 2018).

Based on these findings, a model is proposed for MARF1 molecular function. The MARF1 RRM binds specific target mRNAs, such as cyclin A mRNA, and the MARF1 mLOTUS domains recruit the CCR4-NOT deadenylase complex (see Models for MARF1 function in oocytes). This results in shortening of target mRNA poly-A tail and reduction of protein level produced from target mRNAs. The multiple, tandem mLOTUS domain may recruit multiple CCR4-NOT deadenylase complexes per single MARF1 molecule and single target mRNA, enabling efficient poly-A shortening (Zhu, 2018).

Using this model, it is proposed that MARF1 reduces Cyclin A protein level in stages 12-14 oocytes. This regulated reduction of Cyclin A protein level leads to expression of Cyclin B and Cyclin B3 proteins. This cyclin proteins expresson profile leads to stabilization of CDK1 protein and phosphorylation of CDK1 Tyr15 residue. Increase CDK1 and phosphorylation of CDK1 Tyr15 residue result in phosphorylation of appropriate target proteins of CDK1 in stage 14 oocytes. Proper global protein phosphorylation profile allows germplasm localization of Vasa and Aub and normal yolk distribution in stage 14 oocytes. As stage 14 oocytes traverse through the oviduct, meiotic Metaphase I arrest is released to complete meiosis and produce normal eggs (Zhu, 2018).

Consequently, it is speculated that Cyclin A is the main and/or most upstream target of MARF1. Persisted Cyclin A protein level in MARF1 mutant late-stage oocytes arrest them in an abnormal state rather than proceeding to a normal stage 14 including decreased protein levels of Cyclin B and Cyclin B3. Dysregulation of the three cyclin proteins levels results in the decreased CDK1 protein level and the decreased Tyr15 phosphorylation of CDK1. Dysregulation of cyclins and CDK1 alters global phosphorylation pattern. The altered phosphorylation pattern results in the loss of germplasm localization of Vasa and Aub. These together cause meiotic failure and complete sterility in MARF1 mutants (Zhu, 2018).

MARF1 seems to target specific mRNAs for gene silencing in diverse species. This study identified cyclin A mRNA as a target of Drosophila MARF1. Mouse MARF1 reduces protein and mRNA levels of retrotransposons and a few endogenous genes such as PPP2CB, suggesting that they are targets of the post-transcriptional silencing by mouse MARF1. Knockdown of human MARF1 causes upregulation of IFl44L mRNA, suggesting that IFl44L mRNA a target of human MARF1. Post-transcriptional gene silencing of target mRNAs in fly, mouse, and human MARF1 suggests that mLOTUS-domain directed recruitment of the CCR4-NOT deadenylase complex may be a widely conserved mechanism (Zhu, 2018).

LOTUS domains are found not only in animals but also in bacteria, fungi, and plants. LOTUS domains found in bacteria, fungi, and plants are more similar to the animal mLOTUS domains since they lack the C-terminal extension found in the animal eLOTUS domains. This suggests that the mLOTUS domain may be more ancient than the eLOTUS domain. It will be interesting to investigate the functions of these LOTUS domains found in non-animals, particularly the function of bacterial LOTUS domains, since bacteria do not have a poly-A tail in their mRNAs or CCR4-NOT deadenylase complex (Zhu, 2018).

Kawaguchi, S., Ueki, M. and Kai, T. (2020). Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression. PLoS One 15(4): e0231114. PubMed ID: 32243476

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. This study examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. These results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. It is proposed that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation (Kawaguchi, 2020).

Nos is an evolutionary conserved protein that play important roles in early embryogenesis, formation of primordial germ cells and maintenance of germline stem cells (GSCs). However, the function of Nos during late oogenesis has not been described yet. Nos protein expression reaches the maximum around stage 10 of oogenesis and immediately reduces thereafter. This suggests that Nos expression is tightly regulated during late oogenesis. The current results indicate that dMarf1 regulates Nos expression by inhibiting its translation in late oogenesis. In the absence of dMarf1 expression during early-to-mid oogenesis, Nos might repress cycB to prevent the premature release of meiotic arrest until stage 10 of oogenesis. These results suggest that dMarf1 may coordinate oocyte maturation during late oogenesis in Drosophila; moreover, dMarf1 is dominantly expressed after stage 10, and binds to nos mRNA to repress its translation and reduce its expression. Consequently, Nos expression almost disappears at stages 12-14. Reduced expression of Nos induces the release of cycB mRNA from repression and promotes CycB translation. Subsequently, CycB binds to Cdk1 to form the active MPF complex to promote meiosis. Therefore, dMarf1 plays an important role in the release of the second meiotic arrest to drive embryogenesis (Kawaguchi, 2020).

The MARF1 gene is evolutionarily conserved in animals; most proteins of the MARF1 family contain three major domains: NYN, OST (also known as LOTUS), and RRM. Although the ribonuclease activity of NYN is essential for MARF1 function in mouse, it is not required in Drosophila dMarf1. The OST domain is present in the proteins of several species ranging from bacteria to humans. Drosophila melanogaster has has four members of OST domain-containing proteins: dMarf1, Oskar (Osk), Tejas (Tej), and Tapas (Tap). All of these proteins except dMarf1 contain a single OST domain, and are predominantly expressed in germline cells. Structural and biochemical studies of Osk, Tej, and Tap OST domains have revealed the ability of this domain to bind to Vasa, an RNA helicase expressed exclusively in germline cells. Interestingly, the OST domain(s) of MARF1 family members is smaller than those of other proteins and does not bind to Vasa. Instead, a recent study reported that dMarf1 could bind to CCR4-Not deadenylase complex via the OST domain(s); however, the importance and cooperation between multiple OST domains remains elusive. In addition to these two domains, the MARF1 family members contain one or two RRM domains. This study showed that dMarf1 translationally repressed nos mRNA. nos may not be the only target of dMarf1; other mRNAs such as tra2 and abo can also be targeted by dMarf1, although the biological significance of these interactions remains unclear. Zhu has recently reported that dMarf1 can bind to cyclin A mRNA via its RRM domain (Zhu, 2018). However, RNA-IP analysis did not detect cyclin A in the dMarf1-bound mRNA fraction. Further studies of the molecular mechanism underlying the specificity of dMarf1 RNA binding will reveal the range of mRNA regulation during oocyte maturation (Kawaguchi, 2020).

The C-terminal region of the MARF1 family members is highly conserved among higher animals, except insects, despite not forming any secondary structure. The C-terminal region of human MARF1 has been shown to directly interact with the decapping complex component Ge-1; however, this interaction was not observed in Drosophila. Moreover, the C-terminal region of the MARF1 family members is conserved among different insect species, but different from that of higher animals, indicating that it may bind to a unique partner in insects. This hypothesis was supported by results showing that transgenic dMarf1 mutant lacking 47 C-terminal residues (ΔC47) could not rescue dMarf1^KO321 mutant phenotype (Kawaguchi, 2020).

In addition to binding to the RNA decapping complex subunit, human MARF1 can localize to processing body (P-body), which is often related to translational repression and mRNA decay. Mouse MARF1 has also been shown to degrade target mRNA via its NYN domain, which is absent in Drosophila dMarf1. Transcriptome analysis of mouse MARF1 mutant oocytes revealed that 1,470 transcripts were upregulated in the steady state, whereas 103 transcripts were downregulated, indicating a global impact on RNA homeostasis in the mutant oocytes. By contrast, the expression of a few RNAs was downregulated and dp1 mRNA expression was upregulated in Drosophila dMarf1 mutant ovaries. These results suggest that mammalian MARF1 may regulate the global transcriptome predominantly by degradation, while dMarf1 represses translation of target proteins such as CycB and CycA by modulating Nos/Pumilio and the CCR4-NOT deadenylase complex, respectively (Kawaguchi, 2020).

In addition to nos mRNA, dMarf1 can bind to other mRNAs, including tra2 and abo. The mRNA expression of tra2 was not significantly affected in stage 14 dMarf1^KO312 egg chambers, suggesting that dMarf1 post-transcriptionally regulates tra2 expression. abo is a negative regulator of histone gene expression and its expression is downregulated in mature oocytes to produce more histones. The expression of abo mRNA was upregulated by approximately three-fold in stage 14 dMarf1^KO312 egg chambers. This may result in the overexpression of Abo protein in dMarf1 mutant oocytes, which in turn causes the downregulation of histone proteins that are required for embryogenesis (Kawaguchi, 2020).

The Ppp2cb gene encodes a protein phosphatase that is involved in cell cycle regulation. Ppp2cb has been previously reported as a major downstream effector of mouse MARF1. The high expression of Ppp2CB phosphatase in the MARF1 mutant ovaries of mouse can disrupt meiosis. However, the expression of mts, a Drosophila homolog of Ppp2cb, was not affected in dMarf1 mutant ovaries, suggesting that the signaling pathway for the activation of the M phase promoting factor, CycB/Cdk1, is not conserved between mouse and Drosophila. Although the direct activation of MPF in mouse MARF1 mutant oocytes by the inhibition of Ppp2cb rescued meiotic defect, embryogenesis of mutant oocytes was affected, suggesting that Ppp2cb may have additional functions in addition to MPF activation. Similarly, dMarf1^KO312 ovaries exhibited not only meiotic defects, but also translationally downregulated some proteins required for embryogenesis, such as Dhd and Gnu. In conclusion, MARF1 may trigger oocyte maturation and coordinate multiple events during late oogenesis and fertilization (Kawaguchi, 2020).

Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of tumorigenesis, but less is known about its relevance for nonpathological processes and how it affects whole-animal physiology. This study shows that metabolic reprogramming in Drosophila female germline cells affects nutrient preferences of animals. Egg production depends on the upregulation of the activity of the pentose phosphate pathway in the germline, which also specifically increases the animal's appetite for sugar, the key nutrient fuelling this metabolic pathway. Functional evidence is provided that the germline alters sugar appetite by regulating the expression of the fat-body-secreted satiety factor Fit. These findings demonstrate that the cellular metabolic program of a small set of cells is able to increase the animal's preference for specific nutrients through inter-organ communication to promote specific metabolic and cellular outcomes (Carvalho-Santos, 2020).

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases. This study reports that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size (Ahmed, 2020).

Steroidal sex hormones including oestrogen, progesterone and testosterone regulate the growth and physiology of reproductive organs during puberty, the oestrus cycle and pregnancy. Consequently, these hormones also promote tumorigenesis in the breast, uterus and prostate. Although sex-specific differences in physiology and disease predisposition extend to nearly all organs, the functions of sex-specific steroids in non-sex organs remain relatively poorly explored and controversial. Drosophila uses one major steroid hormone, 20-hydroxy-ecdysone (ecdysone, also known as 20HE) and its derivatives. Similar to vertebrate steroids, 20HE is synthesized by cytochrome P450 enzymes from cholesterol. The ecdysone receptor comprises a ligand-binding EcR subunit and a DNA-binding Usp subunit-orthologues of human farnesoid X and liver X receptors (FXR and LXR) and retinoid X receptor (RXR), respectively. In juvenile insects, 20HE regulates developmental transitions including moulting, metamorphosis and sexual maturation. In adult Drosophila, 20HE is made by the ovaries after mating, resulting in higher levels in females than in males. It acts in the adult nervous and reproductive systems and affects metabolism and lifespan, but a role in the gut has not been described (Ahmed, 2020).

Drosophila intestinal stem cells (ISCs) are more proliferative in females than in males, and females are more prone to age-dependent gut dysplasia and intestinal tumours. These sex-specific traits could be due to ISC-autonomous and/or systemic factors. Consistent with the former, stress-dependent ISC divisions, which are more frequent in females than in males, are reduced if the ISCs are masculinized by repressing the sex-determination genes sxl or tra2. Mated females support more ISC division than virgin flies, which suggests hormonal influences. Because mated females have higher titres of ecdysteroid than virgins or males, tests were performed to see whether 20HE might affect ISC proliferation. Indeed, feeding virgin females 5 mM 20HE strongly induced ISC divisions. This effect was independent of ISC sex identity, and also occurred in mated females and males. Using reporters of receptor activity, it was confirmed that exogenous 20HE promotes EcR-Usp signalling in midgut ISCs, transient progenitors known as enteroblasts (EBs) and differentiated absorptive enterocytes (ECs) (Ahmed, 2020).

Unlike stress caused by detergents, 20HE treatment induced two successive waves of ISC division. Using RNA interference (RNAi) under the control of conditional cell-type-specific Gal4 drivers, it was found that the first wave (at 6 h after 20HE feeding) required EcR only in ISCs, but that later divisions (at 16 h) also depended partially on EcR in EBs. Neither wave of division required EcR in ECs, enteroendocrine or neural cells. Isoform-specific tests revealed that EcR-A was much more important than EcR-B for the 20HE-induced division of ISCs. 20HE-induced divisions were reversible , which suggests a lack of toxicity. EcR activity was not induced by enteric infection, and EcR was dispensable for infection-induced gut regeneration, which indicates a distinct role for EcR in the gut. Loss of Usp, however, did block infection-induced ISC divisions, which suggests that Usp has EcR-independent functions (Ahmed, 2020).

It was next asked whether ISC activation by 20HE involves the Upd-Jak-Stat or Egfr-ERK signalling pathways, which are known to activate ISCs after stress. Six hours of 20HE feeding induced the Egfr ligands spi and krn and their activating protease rho, but not the upd2 or upd3 cytokines or Stat signalling. Exposure to 20HE for 16 h, however, moderately induced upd2, upd3 and Stat activity. The induction of upd2, upd3 and rho required EcR in ISCs and EBs (that is, 'progenitors'), although not in ECs. The Egfr effector ERK was also mildly activated by 16 h of 20HE exposure, mostly in progenitors but occasionally in ECs. ERK activation required upd2, which suggests a signalling relay. Notably, the induction of all of these targets (upd2, upd3, Socs36E, rho, spi and krn) by 20HE was suppressed by blocking ISC mitoses with RNAi molecules that target string (also known as stg or cdc25) or Egfr. This suggests that the observed increases in Jak-Stat and Egfr-ERK signalling are responses to epithelial stress from the early ISC divisions. In further tests, it was found that Upd2 from EBs and ECs contributed strongly to ISC divisions 16 h after 20HE feeding, but only weakly to the early divisions at 6 h. Egfr and Rho, however, were always required. It is concluded that ISC divisions are initially activated ISC-autonomously via EcR, and require Egfr and Rho, whereas later divisions depend in part on cytokines produced by EBs and ECs, perhaps in response to stress from the first mitoses. The relationship of EcR to Egfr signalling warrants further investigation (Ahmed, 2020).

Because mated females produce more ecdysone than virgins or males, whether 20HE might account for sex-specific differences in the gut was tested. Consistent with this, long-term exposure of males to 20HE phenocopied the female condition, increasing ISC mitoses, stress responsiveness, epithelial turnover and midgut size. Genetically feminizing the male ISCs did not give these effect, which suggests that 20HE acts independently of genetic sex determination. Forced expression of the ISC mitogen13 sSpi also failed to enlarge male midguts, which indicates that 20HE affects more than just the ISC mitotic rate. Long-term 20HE feeding also endowed ISCs in virgin females with proliferative characteristics similar to those seen after mating. By contrast, RNAi lines that antagonized 20HE signalling in ISCs and EBs decreased gut size in mated females and suppressed mitoses in response to detergent stress. Thus, sexually dimorphic proliferative traits of ISCs are determined in part by 20HE signalling (Ahmed, 2020).

Similar to human oestrogen and progesterone, ecdysone promotes behavioural and metabolic changes that enhance female reproduction. Dose-response assays showed that 1 mM 20HE fed to virgin females activated EcR targets and ISC mitoses to similar levels to mating. Hence, this study tested whether endogenous, mating-induced 20HE activates ISCs. Indeed, mating induced a large, transient increase in ISC division and enduring gut enlargement. This was independent of genetic sexual identity. As with exogenously fed 20HE, these effects initially required EcR only in ISCs, although EcR in EBs contributed later on. Similar to exogenous 20HE, mating also induced expression of upd2 and rho, which suggests that these are normal physiological responses (Ahmed, 2020).

To confirm the source of endogenous ecdysone, ovary-specific Gal4 drivers were used to express RNAi transgenes that target the ecdysone synthesis enzymes Dib or Spo. This suppressed mating-induced ISC divisions and midgut growth, both of which could be restored by exogenous 20HE. spo mutants also failed to resize the midgut after mating, confirming these results. To learn how the gut grows in mated females, the effects on cell size and number. Depleting EcR in ECs did not reduce EC size, but mating caused a large 20HE- and EcR-dependent increase in female ISC numbers was investigated. This expansion of the stem-cell pool could cause an increase in the total number of midgut cells. These results indicate that mating-dependent ISC division, ISC expansion and gut growth are driven by 20HE signalling from the ovaries to progenitor cells in the gut (Ahmed, 2020).

Gut growth after mating is expected to increase the absorption of nutrients by the intestine and the supply of nutrients to other organs. Because egg production is limited by nutrient availability to the ovaries, whether 20HE-dependent gut growth affected female fecundity was tested. When gut resizing was blocked by expressing EcR RNAi (EcRRNAi) in midgut ISCs, or in both ISCs and EBs, egg production was reduced by approximately 40%. This suggests that 20HE-dependent gut remodelling maximizes female reproductive fitness. However, it was also noticed that the Gal4 drivers were active in a small number of escort cells in the germarium of the ovary, which raises the possibility that these fecundity defects were due in part to a requirement for EcR in those cells (Ahmed, 2020).

A study of Drosophila juvenile hormone (JH), a sesquiterpenoid, came to conclusions similar to the current study-namely that JH promotes mating-dependent gut growth and fecundity in females. The relative roles of 20HE and JH were therefore invstigated. The JH receptors Gce and Met are essential for ISC divisions in response to not only the JH receptor agonist, methoprene, but also 20HE and infection. The mitogenic effects of methoprene were confirmed, but these were weaker than those of 20HE or mating. Furthermore, it was discovered that methoprene-stimulated divisions require 20HE, and that JH or methoprene could suppress ISC divisions driven by 20HE or other stimuli. Although these results indicate interaction between 20HE and JH, further work is required to determine their precise physiological relationship (Ahmed, 2020).

To better understand how ecdysone activates ISCs, two known EcR targets were tested: the transcription factor Broad, and the nuclear receptor Eip75B, a homologue of human PPARγ and REV-ERB17. Eip75B and broad (br) mRNA were induced in midguts by 20HE or mating, and progenitor-cell-specific depletion of either factor suppressed 20HE-induced mitoses. ISC clonal growth, however, required Eip75B but not br, highlighting that Eip75B is a more essential effector. Overexpression of Eip75B was sufficient to promote ISC division and gut epithelial turnover, whereas Eip75B loss impaired both ISC mitoses and maintenance. Progenitor-specific loss of Eip75B also blocked gut growth after mating, and compromised egg production, phenocopying the effects of EcR loss. Eip75B binds DNA to repress target genes, and also binds the nuclear receptor Hr3 to inhibit Hr3-mediated transcriptional activation. Consistent with this mechanism, overexpression of Eip75B or 20HE feeding suppressed an Hr3 activity reporter, and Hr3 overexpression suppressed ISC proliferation. Moreover, depletion of Hr3 counteracted losses in ISC proliferation caused by Eip75B depletion. Although these results indicate that Hr3 is a crucial Eip75B effector, Hr3 loss was not sufficient to activate ISC division, which indicates that Eip75B has other targets. Further tests revealed that Eip75B and Hr3 mediate 20HE-independent ISC responses to stress. Enteric infection strongly induced levels of Eip75B mRNA and suppressed Hr3 activity. Removing Eip75B or Broad from ISCs by mutation or by RNAi blocked infection-induced ISC mitoses, as did overexpression of Hr3. Eip75B was also required for ISC mitoses in response to the oxidative stress agent paraquat. Furthermore, evidence was ontained consistent with previous work that the action of Eip75B is modulated by haem (a Eip75B ligand) and nitric oxide. Functions for haem and nitric oxide in the fly gut are unknown, but potentially interesting. It is concluded that Eip75B, Broad and Hr3 integrate several inputs in addition to 20HE to control ISC proliferation (Ahmed, 2020).

As females age, they experience progressive gut dysplasia in which ISCs overproliferate and mis-differentiate, leading to high microbiota loads (dysbiosis), barrier breakdown and decreased lifespan. Age-dependent intestinal dysplasia is more pronounced in females than in males, and can be identified by increases in mitoses and mis-differentiated cells doubly positive for ISC and EC markers. Suppressing EcR, Usp or Eip75B in midgut progenitors significantly reduced both parameters of dysplasia in aged flies. Similarly, suppressing ecdysone synthesis enzymes (Dib, Spo) in the ovaries, or ubiquitously, also curtailed age-dependent gut dysplasia. This effect could be reversed by 20HE supplementation. These results indicate that age-dependent gut dysplasia is potentiated by ovary-derived ecdysone, explaining the sex bias of this condition (Ahmed, 2020).

Female Drosophila are known to be more susceptible than males to genetically induced ISC-derived tumours. This study found that ISC/EB-specific RNAi targeting Notch, a receptor required for EC differentiation, drove tumour induction in 100% of mated females but was far less tumorigenic in virgin females or males. Three results indicate that this tumour predisposition is modulated by 20HE. First, in contrast to mated females, virgins were extremely resistant to NotchRNAi-mediated tumorigenesis. Second, targeting 20HE signalling in ISCs with a dominant-negative EcR-A (EcRADN) inhibited tumour growth in mated females. Third, supplementing males or virgin females with 20HE increased tumour initiation and growth. It is surmised that the use of mating-dependent, ovary-derived 20HE to stimulate gut resizing comes at a cost: it predisposes females to gut dysplasia and tumorigenesis (Ahmed, 2020).

Gut dysplasia, tumorigenesis and egg production can all shorten lifespan, which suggests that the effects of ecdysone on the gut might adversely affect longevity. In fact, earlier reports showed that EcR mutants live longer, and proposed that reproduction can shorten lifespan by damaging the soma. The lifespan assays of this study, although subject to the same caveats as previous work, support this view: suppression of EcR in midgut progenitors extended lifespan in females but not males. In evolutionary terms, the disadvantage of a slightly shorter lifespan due to sex-specific hormonal signalling is probably insignificant relative to the reproductive fitness advantage conferred by increased egg production. This may be especially true in the wild, where gut dysplasia-dependent mortality is probably counteracted by nutrient deprivation (Ahmed, 2020).

Similarities in the reproductive biology of Drosophila and mammals suggest that these inter-organ relationships have relevance to human biology. The mitogenic effects of insect ecdysone parallel those of oestrogen and testosterone as drivers of breast, uterine and prostate growth and tumorigenesis. Yet how these steroids affect the human intestine remains poorly explored. Adaptive growth of the intestine is well documented in pregnant and lactating mammals, and might depend on oestrogen and/or progesterone. Laboratory tests with rodents and human cells, as well as some studies with human participants, have linked oestrogen, testosterone and their receptors to gastrointestinal cancers, but epidemiological studies provide conflicting evidence regarding this association. The contributions of sex steroids to intestinal physiology deserve more detailed study (Ahmed, 2020).

Gut microbiota have been shown to promote oogenesis and fecundity, but the mechanistic basis of remote influence on oogenesis remained unknown. This study reports a systemic mechanism of influence mediated by bacterial-derived supply of mitochondrial coenzymes. Removal of microbiota decreased mitochondrial activity and ATP levels in the whole-body and ovary, resulting in repressed oogenesis. Similar repression was caused by RNA-based knockdown of mitochondrial function in ovarian follicle cells. Reduced mitochondrial function in germ-free (GF) females was reversed by bacterial recolonization or supplementation of riboflavin, a precursor of FAD and FMN. Metabolomics analysis of GF females revealed a decrease in oxidative phosphorylation and FAD levels and an increase in metabolites that are degraded by FAD-dependent enzymes (e.g., amino and fatty acids). Riboflavin supplementation opposed this effect, elevating mitochondrial function, ATP, and oogenesis. These findings uncover a bacterial-mitochondrial axis of influence, linking gut bacteria with systemic regulation of host energy and reproduction (Gnainsky, 2021).

The fate and proliferative capacity of stem cells have been shown to strongly depend on their metabolic state. Mitochondria are the powerhouses of the cell being responsible for energy production via oxidative phosphorylation (OxPhos) as well as for several other metabolic pathways. Mitochondrial activity strongly depends on their structural organization, with their size and shape being regulated by mitochondrial fusion and fission, a process known as mitochondrial dynamics. However, the significance of mitochondrial dynamics in the regulation of stem cell metabolism and fate remains elusive. This study characterized the role of mitochondria morphology in female germ stem cells (GSCs) and in their more differentiated lineage. Mitochondria are particularly important in the female GSC lineage. Not only do they provide these cells with their energy requirements to generate the oocyte but they are also the only mitochondria pool to be inherited by the offspring. The undifferentiated GSCs predominantly have fissed mitochondria, whereas more differentiated germ cells have more fused mitochondria. By reducing the levels of mitochondrial dynamics regulators, it was shown that both fused and fissed mitochondria are required for the maintenance of a stable GSC pool. Surprisingly, it was found that disrupting mitochondrial dynamics in the germline also strongly affects nurse cells morphology, impairing egg chamber development and female fertility. Interestingly, reducing the levels of key enzymes in the Tricarboxylic Acid Cycle (TCA), known to cause OxPhos reduction, also affects GSC number. This defect in GSC self-renewal capacity indicates that at least basal levels of TCA/OxPhos are required in GSCs. These findings show that mitochondrial dynamics is essential for female GSC maintenance and female fertility, and that mitochondria fusion and fission events are dynamically regulated during GSC differentiation, possibly to modulate their metabolic profile (Garcez, 2020).

Oocyte composition can directly influence offspring fitness, particularly in oviparous species such as most insects, where it is the primary form of parental investment. Oocyte production is also energetically costly, dependent on female condition and responsive to external cues. This study investigated whether mating influences mature oocyte composition in Drosophila melanogaster using a quantitative proteomic approach. The analyses robustly identified 4,485 oocyte proteins and revealed that stage-14 oocytes from mated females differed significantly in protein composition relative to oocytes from unmated females. Proteins forming a highly interconnected network enriched for translational machinery and transmembrane proteins were increased in oocytes from mated females, including calcium binding and transport proteins. This mating-induced modulation of oocyte maturation was also significantly associated with proteome changes that are known to be triggered by egg activation. It is proposed that these compositional changes are likely to have fitness consequences and adaptive implications given the importance of oocyte protein composition, rather than active gene expression, to the maternal-to-zygotic transition and early embryogenesis (McDonough-Goldstein, 2021a).

The zinc finger-associated domain (ZAD) is present in over 90 C2H2 zinc finger (ZNF) proteins. Despite their abundance, only a few ZAD-ZNF genes have been characterized to date. This study systematically analyzed the function of 68 ZAD-ZNF genes in Drosophila female germ cells by performing an in vivo RNA-interference screen. Eight ZAD-ZNF genes required for oogenesis were identified, and based on further characterization of the knockdown phenotypes, defects broadly consistent with functions in germ cell specification and/or survival, early differentiation, and egg chamber maturation were uncovered. These results provide a candidate pool for future studies aimed at functionalization of this large but poorly characterized gene family (Shapiro-Kulnane, 2021).

In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. This study shows that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. The RNA-degrading Super Killer complex and No-Go Decay factor Pelota were identified as key regulators of oogenesis via targeted degradation of specific RNAs expressed in undifferentiated germ cells. These regulators target RNAs enriched for cytidine sequences that are bound by the polypyrimidine tract binding protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition that gives rise to the maternal contribution to the zygote (Blatt, 2021)

The fly genome contains a single ortholog of the evolutionarily conserved transcription factor hepatocyte nuclear factor 4 (HNF4), a broadly and constitutively expressed member of the nuclear receptor superfamily. Like its mammalian orthologs, Drosophila HNF4 (dHNF4) acts as a critical regulator of fatty acid and glucose homeostasis. Because of its role in energy storage and catabolism, the insect fat body controls non-autonomous organs including the ovaries, where lipid metabolism is essential for oogenesis. This study used dHNF4 overexpression (OE) in the fat bodies and ovaries to investigate its potential roles in lipid homeostasis and oogenesis. When the developing fat body overexpressed dHNF4, animals exhibited reduced size and failed to pupariate, but no changes in body composition were observed. Conditional OE of dHNF4 in the adult fat body produced a reduction in triacylglycerol content and reduced oogenesis. Ovary-specific dHNF4 OE increased oogenesis and egg-laying, but reduced the number of adult offspring. The phenotypic effects on oogenesis that arise upon dHNF4 OE in the fat body or ovary may be due to its function in controlling lipid utilization (Almeida-Oliveira, 2021).

Epithelia form protective permeability barriers that selectively allow the exchange of material while maintaining tissue integrity under extreme mechanical, chemical, and bacterial loads. This study in the Drosophila follicular epithelium reports a developmentally regulated and evolutionarily conserved process "patency", wherein a breach is created in the epithelium at tricellular contacts during mid-vitellogenesis. In Drosophila, patency exhibits a strict temporal range potentially delimited by the transcription factor Tramtrack69 and a spatial pattern influenced by the dorsal-anterior signals of the follicular epithelium. Crucial for growth and lipid uptake by the oocyte, patency is also exploited by endosymbionts such as Spiroplasma pulsonii. These findings reveal an evolutionarily conserved and developmentally regulated non-typical epithelial function in a classic model system (Row, 2021).

Sexual reproduction in internally fertilizing species requires complex coordination between female and male reproductive systems and among the diverse tissues of the female reproductive tract (FRT). This study reports a comprehensive, tissue-specific investigation of Drosophila melanogaster FRT gene expression before and after mating. Expression profiles were identified that distinguished each tissue, including major differences between tissues with glandular or primarily nonglandular epithelium. All tissues were enriched for distinct sets of genes possessing secretion signals that exhibited accelerated evolution, as might be expected for genes participating in molecular interactions between the sexes within the FRT extracellular environment. Despite robust transcriptional differences between tissues, postmating responses were dominated by coordinated transient changes indicative of an integrated systems-level functional response. This comprehensive characterization of gene expression throughout the FRT identifies putative female contributions to postcopulatory events critical to reproduction and potentially reproductive isolation, as well as the putative targets of sexual selection and conflict (McDonough-Goldstein, 2021b).

Intercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. This study demonstrates a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth (Stark, 2021).

Data analysis using the R package Seurat3 partitioned the cells into 8 distinct clusters (using the top 30 principal components at a resolution of 0.4). These clusters were visualized with a uniform manifold approximation and projection (UMAP) plot. A number of known cell makers were used to characterize these different cell clusters. Nearly all of the cells were negative for the germline-specific marker vasa, suggesting no significant contamination from germline cells. However, there appeared to be some contamination of cap cells, represented in the relatively small cluster 6, which were positive for the cap cell marker bab1. Two known follicle cell markers Fasciclin 3 (Fas3) and cut (ct) enabled definition of clusters 2-5 as follicle cell clusters, which were referred hereafter as a combined 'follicle cell cluster'. The remaining two large clusters 0 and 1 were likely ECs that consisted of totally 1,166 cells. As each germarium contains approximately 40-50 escort cells, the total number of profiled single ECs is more than 23 times the number of total ECs per germarium. The differentially expressed genes found in clusters 0 and 1 were subsequently assessed with Gene Ontology (GO) enrichment analysis along with that from the follicle cell cluster using Metascape (Shi, 2020).

Of note, the above analysis failed to identify a specific cluster for FSCs. This study also failed to identify any cell clusters that display increased or decreased mitotic activity using the cell-cycle scoring and regression analysis, a method that has been used to identify stem cell populations. This indicates that the mitotic activity between FSCs and ECs is not sufficiently different to be separated by this analysis. It is speculated that FSCs might be mixed within the cluster 0 cells, a possibility that will be discussed shortly (Shi, 2020).

GO analysis suggested several overlapping as well as distinct biochemical and developmental processes at work by these three different groups. As expected, the FC cluster showed specific activity in chorion-containing eggshell formation (such as Cp15, Cp19, and Fcp3C in clusters 2, 4, and 7, in particular for 4 and 7) and other processes that are known to be involved in later stages of oogenesis. Notably, the cluster 1 showed putative activity for the ecdysone biosynthetic process, although the cluster 0 showed enrichment of genes involved in vitelline membrane formation (such as Vm32e, Vm26Aa, and Vml). To further evaluate the functional relevance in germline cyst differentiation, these differentially expressed genes found in these three clusters were subjected to a small-scale RNAi screen for their requirement in ECs for egg production, and these with failed egg production were analyzed further for defects in early germline development. Genes whose depletion showed defects in the germarium were considered with high confidence of functional relevance, and the gene expression changes for these genes were summarized and presented as a heatmap. Comparative gene expression analysis among those selected genes further supports functional continuation among the three cell clusters: cluster 0 appeared to be at an intermediate state between cluster 1 and the follicle cell cluster, as about half of the selected candidate marker genes overlapped with cluster 1 and the other half overlapped with the follicle cell cluster. Of note, genes involved in ecdysone biosynthesis, including phantom (phm) and shadow (sad), were shown as cluster-1-specific genes (Shi, 2020).

To map out the spatial distribution of clusters 0 and 1, fluorescence in situ RNA hybridization (FISH) was used to determine the expression pattern of the cluster-1-specific phm and IA-2, which is expressed in cluster 0 and in a subset of follicle cells. Consistent with the scRNA-seq results, the FISH signal for phm was specifically detected in ECs at region I, but not region IIa, of the germarium, suggesting that cluster 1 represents ECs located at the anterior region roughly corresponding to region I. Also consistent with the single cell results, the FISH signal for IA-2 was specifically detected in ECs in the region IIa as well as FSCs and some early follicle progenitor cells. Taken together, these results suggest that cluster 1 and 0 represent two spatially distinct EC populations located, respectively, roughly at region I and IIa of the germarium. As mentioned earlier, the cluster 0 cells, namely ECs at region IIa, possibly contain some FSCs and their immediate progenies, as the IIa-IIb region is considered be circumvented by three lines of FSCs. The lineage-tracing experiment conducted later in this study also supports the idea that ECs at region IIa can be derived from FSCs. But for simplicity, the cluster 1 cells are termed anterior ECs (aECs) and the cluster 0 cells as posterior ECs (pECs) (Shi, 2020).

The scRNA-seq analysis therefore allowed identification of two major subpopulations of ECs that are separately distributed in the germarium, and the distinct as well as continuous gene expression characteristics among aECs, pECs (which may include FSCs), and early follicle cells indicates functional separation as well as continuation among these 3 groups of cells (Shi, 2020).

After screening a collection of GMR- and NP-GAL4 transgenic/enhancer trap strains, GMR25A11-GAL4 (aEC-GAL4 for short), which carries an enhancer element for the Wnt4 gene, was identified as a possible specific GAL4 driver line for aECs. Wnt4 has also been reported to be expressed in ECs or subsets of ECs. Additionally, wun2NP3026-GAL4 (pEC-GAL4 for short) driver, which appeared to drive UAS-GFP expression specifically in pECs and cap cells, was identified, although weak GFP expression was also found in aECs in some germaria when the image was overexposed. The EC-specific expression of these two GAL4 lines was also confirmed by co-staining with PZ1444-lacZ, a known EC marker. Further examination of the patterns of these two GAL4 lines in relationship with the stages of the germline cysts revealed that aEC-GAL4+ cells were associated with 4-cell-or-less cysts, although pEC-GAL4+ cells were associated with 8- and 16-cell cysts. qRT-PCR analysis of the sorted aEC-GAL4; UAS-GFP (aEC > GFP+) and pEC-GAL4;UAS-GFP (pEC > GFP+) cells confirmed that aEC > GFP+ cells have relatively higher expression of phm and pEC > GFP+ cells have relatively higher expression of IA-2. Note that the qRT-PCR results also showed that Wnt4 expression was higher in aECs than pECs, which is consistent with the expression pattern of its GAL4 reporter used in this study. Collectively, these results suggest that these two GAL4 lines largely mark the scRNA-seq-classified aEC and pEC populations, respectively (Shi, 2020).

Cell ablation experiments were conducted with the aEC-GAL4 and pEC-GAL4 lines to determine the specific requirements of these subsets of ECs for germline differentiation. Conditional expression of an apoptosis inducer reaper (rpr) for 7 days either in aECs or pECs effectively ablated these cells. As a positive control, total EC ablation using the c587-Gal4 driver caused a complete blockage of CB differentiation, leading to a strong CB accumulation phenotype. Ablation of aECs caused mild accumulation of CB with reduced populations of 2-, 4-, and 8-cell cysts in the germarium, suggesting that aECs function in the progression of CB differentiation into 8-cell cysts. This phenotype is considerably milder than that caused by total EC ablation, suggesting that the remaining pECs might be able to somehow partially compensate the loss of aECs. The ablation of pECs caused specific and strong accumulation of 8-cell cysts in the germarium. In addition, pEC ablation resulted in 16-cell cysts at region IIb, which were unable to be properly encapsulated by follicle cells to form egg chambers, leading to a swollen germaria (increased circumferential length at region IIb) phenotype. Collectively, these results indicate that aECs functionally impact cyst cell division during CB differentiation into 8-cell cysts, whereas pECs continue to support cyst division through to the final 16-cell cyst formation. In addition, pECs may help to facilitate the developmental transition between pEC-surrounded 16-cell cyst and follicle cell-encapsulated egg chambers (Shi, 2020).

It was also found that pECs in particular may impact the survival of the developing germline cysts: the frequency of apoptotic cysts, especially those at the posterior region, was significantly increased following pEC ablation. Given that one oocyte is specified in each newly formed 16-cell cyst, whether ablation of pECs could affect oocyte specification was examined. The oocyte marker Orb normally starts its expression in all cells in 8-cell cysts and soon confined to two pre-oocytes and then finally one oocyte in 16-cell cysts. Interestingly, in both aEC- and pEC-ablated germaria, similar to normal patterns, Orb was weakly expressed in all of the 8-cell cysts examined; one Orb+ oocyte or two Orb+ pre-oocytes were invariably present in the 16-cell cysts examined. Thus, it appears that pECs are not essential for oocyte differentiation and specification, as long as the germline cyst has developed to the 16-cell stage (Shi, 2020).

The expression of two ecdysone biosynthetic genes phm and sad in aECs indicates the ability of aECs to produce ecdysone. Ecdysone signaling has been previously implicated in regulating adult Drosophila oogenesis. In the ovary, although ecdysone is found to be mainly produced in stage 8 and later egg chambers, it is speculated that aECs may provide a local source of ecdysone to regulate early stages of germline development. phm or sad were conditionally depleted in either aECs or pECs by RNAi using the aEC- and pEC-specific GAL4 drivers, and the effect on the germline development after 7 days of treatment was examined. Conditional depletion of phm in aECs, but not pECs, caused significant increase of 8-cell cyst per germarium and concurrent decline of earlier stages of germline cysts. Similar results were found for sad RNAi. This phenotype is similar to that caused by aEC ablation in aspects of significantly shrunk I and IIa regions of the germarium; however, aEC ablation shows increased CBs and decreased 8-cell cysts, opposite to that caused by phm depletion. The difference in CBs is possibly because of different effect on GSC maintenance and proliferation in these two different conditions, as conditional depletion of phm or sad in aECs also slightly reduced the GSC number in each germarium. The increase of 8-cell cysts in aEC > phm/sad-RNAi germaria is probably due to a non-cell-autonomous effect on pECs, which will be described shortly. It is concluded that cyst cell division promoted by aECs is mediated by ecdysone signaling: aECs produce ecdysone, which then acts on the associated germline cells to promote synchronous cell division toward 8-cell cysts (Shi, 2020).

Interestingly, the single-cell data also revealed that ftz-f1 and Eip78c, two ecdysone target genes, were both expressed weakly in pECs and strongly in follicle cells; neither gene was expressed in aECs. Given the finding that the Ecdysone receptor (EcR) was expressed in all ECs, pECs can potentially receive ecdysone signals produced from aECs and other sources. Eip78c-GFP, a genomic transgenic reporter fused with GFP, was used to further examine Eip78c expression in the germarium. For the germline cells in regions I and IIa, GFP was specifically expressed in GSCs, CBs, and 16- cell cysts. For somatic cells, GFP was detectable only in cap cells, pECs, and follicle cells. An EcR activity reporter line (UAS-Stinger; hs-GAL4-usp.LBD) was used to detect EcR activity in the germarium, and this reporter line showed a largely similar expression pattern to Eip78c-GFP. Therefore, in the anterior regions of the germarium, relatively high levels of EcR activation are found in GSCs and CBs in the germline and found in pECs and follicle cells in the soma. These results are consistent with the idea that local production of ecdysone by aECs activates EcR in GSC and CB to promote cell division, and the observations also suggest that this local source of ecdysone also regulates EcR activity in pECs (Shi, 2020).

To investigate whether the observed EcR activity in pECs impacts the function of pECs, EcR was depleted specifically in pECs via RNAi and such depletion caused a specific accumulation of 8-cell cysts in EC-surrounding regions. In contrast, depletion of EcR in aECs did not cause any visible phenotypes. Depletion of ftz-f1 in pECs caused a similar 8-cell cyst accumulation phenotype, indicating that ftz-f1 is an important downstream mediator of EcR signaling in pECs; again, depletion of ftz-f1 in aECs did not yield any obvious phenotypes. EcR or ftz-f1 depletion in pECs also caused a swollen germarium phenotype, as some 16-cell cysts appeared to be stagnant at region IIb and were not timely surrounded by the follicle cells to form egg chambers. It therefore appears that the EcR activity in pECs can mediate two separate roles of pECs-promoting 16-cell cyst formation from 8-cell cysts and promoting the developmental transition between 16-cell cysts and egg chamber formation (Shi, 2020).

As some FSCs are possibly mixed into the pEC-GAL4+ cell population, genetic analysis performed with the pEC-GAL4 driver could potentially reflect the function by FSCs rather than pECs. Cell lineage tracing experiments conducted with pEC-GAL4 indeed revealed that pEC-GAL4+ cells contained FSC activity. This contamination is considered minimal, as the majority of this population of cells represents the cells escorting 8- and 16-cell cysts, and these cells have low mitotic activity. Nevertheless, to evaluate the functional contributions of FSCs in the observed phenotypes, the c306-GAL4 driver, which is known to be expressed in FSCs and early FCs, was used for genetic analysis. It was found that ftz-f1 depletion driven by the c306-GAL4 driver did not cause any obvious 8-cell cyst accumulation at the IIa/IIb boundary or any obvious defects in 16-cell cyst-to-egg chamber transition, and EcR depletion only caused a very mild accumulation of 8-cell cysts, in contrast to much stronger phenotypes caused by EcR or ftz-f1 depletion driven by the pEC-GAL4 driver. Therefore, the observed phenotypes associated with the pEC-GAL4 driver can be largely attributed to the functional perturbation of pECs, but not FSCs (Shi, 2020).

During the transition leading to egg chamber formation, the 16-cell cysts are immediately surrounded by a layer of follicle cells as they are losing contact with pECs. Although the exact process of this transition is still unclear, it is reasonable to speculate that cell-cell adhesion might influence this developmental transition. Germline-soma interaction via DE-cadherin (DE-cad)-mediated cell adhesion is known to regulate germline development, as for example in stem cell maintenance and oocyte positioning (Shi, 2020).

To determine whether EcR activity in ECs regulates cell adhesion, DE-cad expression was examined in EcR-depleted pECs. Within the I and IIa regions of wild-type germaria, the strongest DE-cad expression was observed in cap cell clusters, with prominent expression additionally evident in the ring canals of 8- and 16-cell cysts, similar to results reported previously. DE-cad expression was also found in pECs, but not aECs, at the interface between ECs and germline cysts, including the soma-germline interface at the IIa/IIb boundary region. Strikingly, depletion of EcR in pECs caused a dramatic downregulation of DE-cad expression, generally at the interface between soma and 16-cell cysts, including the soma-germline interface at the IIa/IIb boundary region. In contrast, depletion of EcR in aECs did not cause any obvious changes in DE-cad expression in the germarium. Depletion of ftz-f1 in pECs caused a similar downregulation of DE-cad at the soma-germline interface; moreover, ftz-f1 depletion in aECs did not cause any significant changes in DE-cad expression in the germarium (Shi, 2020).

It is worth noting that, although the downregulation of DE-cad at soma-germline interface was obvious, there was no significant downregulation in DE-cad expression in the ring canals of 8-cell and 16-cell cysts in the above EcR RNAi or ftz-f1 RNAi germaria, showing that the depletion of EcR or ftz-f1 in pECs specifically impacts DE-cad-mediated cell adhesion between pECs and germline cysts. These observations collectively indicate that the EcR activity that was observed in pECs regulates DE-cad-mediated soma-germline cell adhesion and additionally it is suggested that this interaction impacts the formation of follicle-cell-encapsulated cysts as they develop from EC-surrounded 16-cell cysts (Shi, 2020).

As DE-cad-mediated cell adhesion has been implicated in controlling directional cell migration, such as border cell-nurse cell adhesion during border cell migration, it is tempting to speculate that adhesive interaction between the pEC and the 16-cell cyst may help to guide the 'migration' of the cyst toward posterior, thereby facilitating the transition between 16-cell cysts and egg chamber formation. To further test this speculation, the adhesive interaction was disrupted by depleting DE-cad gene (shotgun, shg) via RNAi, by depleting α-catenin via RNAi, or by expressing a truncated form of Shg that is unable to bind β-catenin (ShgΔβ) and thus has a dominant negative effect on cell adhesion in pECs. All of these genetic manipulations caused similar cyst transition defects, as many 16-cell cysts at regions IIb and III were not timely encapsulated by follicle cells and appeared stagnant at region III of the germarium. This stagnant phenotype associated with 16-cell cysts, characterized additionally by the tilted shape and compaction, were similarly observed in germaria depleted with EcR or ftz-f1 in pECs. Interestingly, there was no apparent increase in egg chamber fusion as these 16-cell cysts developed, but egg chambers with incomplete follicle cell encapsulation could be observed occasionally. Thus, DE-cadherin-mediated cell adhesion between pECs and germline cysts is functionally important for the developmental transition between pEC-surrounded 16-cell cyst and follicle-cell-encapsulated egg chambers. Of note, unlike ecdysone signaling, the adhesive interaction appears to be not required for the final synchronous division for 16-cell cyst formation from 8-cell cyst (Shi, 2020).

Next the mechanism underlying aEC/pEC patterning was explored. As aECs and pECs are associated with germline cells at different developmental stages, it was speculated that the developmental stage of a given germline cyst may determine the spatial identity of its physically associated ECs. This using two approaches. In the first approach, GSC differentiation was induced by overexpressing a differentiation-promoting gene, bam, so that the stem cells and early germline cysts that located at the anterior region were forced to differentiate into late-stage germline cysts in situ, before eventually moving out of the germarium. During this process, the aEC/pEC cell identity remained intact. When all the germ cells were gone, the remaining ECs were physically clustered together, but their original anterior/posterior identity nevertheless remained. In the second experimental approach, aEC and pEC markers were examined in bgcn mutant germaria, wherein all of the germline cells are arrested at a CB-like stage. Interestingly, although excessive accumulation of proliferative CB-like cells caused swollen and deformed germaria, the relative positioning of aECs and pECs still remained. Therefore, the patterning of aEC and pEC domains is not dependent on the stage information of the physically associated germline cysts. In fact, the maintenance of aEC/pEC domains, at least in a short run, does not even require the existence of germline cells (Shi, 2020).

Lacking a germline influence, it follows that the patterning of aEC and pEC domains is apparently regulated via a somatic mechanism. Previous studies have revealed multiple signal molecules that are secreted from the cells either anterior or posterior to the EC resident regions, such as BMP, Hedgehog (Hh), and Wnt produced from the cap cells (anterior) and Upd produced from early follicle cells (posterior). These signals generated from one pole will defuse toward another end, and the resultant signaling activation gradient may underlie the patterning of the aEC/pEC domains. However, BMP, Hh, and Wnt can also be produced in ECs, and the expression of various previously described signaling activation reporters does not support an obvious gradient of Hh or Wnt activity within the EC populations, except in the regions immediately next to the FSC site. Interestingly, consistent with a previous observation, it was found that Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway activity, revealed by a 10XSTAT-GFP reporter, displayed a gradient pattern emanating from early follicle cells to pECs. This observation suggests that JAK/STAT signaling might participate in the aEC/pEC patterning or somehow otherwise affect pEC fate (Shi, 2020).

Pursuing this, the JAK/STAT signaling activity was genetically manipulated in either aECs or pEC by expressing the JAK kinase Hop to activate the pathway, and expressing a dominant-negative form of the receptor Dome to inhibit the pathway. Neither activation nor inhibition of the JAK/STAT pathway affected the identity, distribution, or the total number of aECs. However, inhibition of JAK/STAT signaling in pECs caused a significant reduction of pEC > GFP+ cells. In contrast, JAK/STAT signaling activation in pECs did not alter any pEC > GFP+ cell phenotypes. Considering the findings suggesting that JAK/STAT activity may potentially affect EC proliferation or survival, the total EC number was calculated in each germarium using PZ1444-lacZ, and quantitative analysis indicated that the JAK/STAT pathway does not apparently have a significant impact on the total number of ECs in the germarium, rather, JAK/STAT signaling may be required for pEC fate maintenance (Shi, 2020).

Considering that JAK/STAT and EcR activities both occur in pECs, it was asked whether there is any regulatory or functional connection between JAK/STAT and EcR in pECs. Depletion of EcR in pECs does not seem to affect JAK/STAT activity in pECs, as the 10XSTAT-GFP reporter expression remained unaltered; however, inhibiting JAK/STAT by expressing DomeDN in pECs caused significant downregulation of Eip78c-GFP expression in pECs, suggesting that JAK/STAT is required for EcR activity in pECs. Not surprisingly, inhibition of the JAK/STAT pathway also caused a dramatic decline of DE-cad expression at the interface between pECs and germline cysts. Forced activation of JAK/STAT activity by overexpressing hop in pECs did not significantly affect DE-cad expression, and this forced JAK/STAT activation also failed to restore the downregulated DE-cad level caused by EcR depletion. These results suggest that JAK/STAT functions through ecdysone signaling to regulate cell adhesion. Collectively, these results led to the proposal of a model as following: the JAK/STAT activity, which is active in pECs, allows pECs to be responsive to ecdysone signaling, whose activity then regulates cell adhesive interaction between germline and soma to promote the transition between 16-cell cyst and egg chamber formation (Shi, 2020).

Collectively, this study supports that ECs, FSCs, and early follicle cells, guided by cell-to-cell signaling communications, furnish a progressive microenvironment for the stepwise differentiation of CBs toward egg chamber formation. The aECs produce local ecdysone that acts on the germline to promote synchronous cyst division; the pECs continuously support a final cyst division. Further, owing to their JAK/STAT activity provided by early follicle cells, the pECs are responsive to ecdysone and thereby undergo increased DE-cad-mediated soma-germline interactions, which facilitate the developmental transition toward egg chamber formation. Consistent with a progressive nature for the microenvironment, the aEC, pEC (which likely includes FSCs), and follicle cell clusters depicted in the UMAP plot are continuous and overlapping. The lineage relationships among these groups of somatic cells was followed by conducting cell lineage tracing experiments using the newly characterized aEC-GAL4 and pEC-GAL4, as well as two previously reported FSC and follicle cell driver lines c306-GAL4 and 109-30-GAL4. The aEC-GAL4+ cell-derived progenies were able to slowly expand to the pEC resident region over a 2-week period; conversely, the pEC-GAL4+ cell-derived progenies were also able to expand to the aEC-resident region but happened in a much slower manner; the c306-GAL4+ and 109-30-GAL4+ cell (presumptively FSCs in both cases)-derived progenies were able to slowly migrate to pEC-resident region and occasionally aEC-resident region over 2 weeks. These observations, along with numerous published observations, collectively lead to an inclusive model for the homeostatic regulation of aECs and pECs: the homeostasis of aECs and pECs is maintained through multiple mechanisms, including self-duplication, fate exchange, and FSC activity. In addition to the recent observations that ECs and follicle cells may share a common developmental origin (Slaidina, 2020; Reilein, 2020), the intimate cell lineage relationships and fate interchangeability also support the idea of functional continuity among aECs, pECs, FSCs, and follicle cells that collectively provide a progressive niche environment for orchestrating germline differentiation (Shi, 2020).

The successful identification of two subgroups of ECs in this work is likely due to the large quantity of ECs used in the single-cell analysis. This probably explains why this is missed in some similar work done by others. However, it bears emphasis that, although the present study empirically supports the classification of ECs into two major subpopulations, each of these EC subpopulations could potentially be further divided into additional subtypes. Such further classification may be limited by the 10x Genomics single-cell approach, through which potentially informative genes expressed at low levels could be missed. For instance, Wnt4, which is known to be expressed in ECs, or dpp, which is expressed in the anterior-most ECs, was not detected in the single-cell data (Shi, 2020).

The finding of a progressive niche for germline development in Drosophila is likely conserved in mammals: a recent study revealed the presence of escort-like somatic cells and the derived granulosa cells in mouse fetal ovaries and showed that these cells interact with germ cells and developing germline cysts. Further, in the mammalian testis, the Sertoli cells, which support the entire process from spermatogonia differentiation to sperm formation, have long been proposed to have functional subdomains. It is proposed that a progressive niche environment may be a common scheme for guiding the development and differentiation of germline cells and possibly other types of adult stem cells (Shi, 2020).

Small cell clusters exhibit numerous phenomena typically associated with complex systems, such as division of labour and programmed cell death. A conserved class of such clusters occurs during oogenesis in the form of germline cysts that give rise to oocytes. Germline cysts form through cell divisions with incomplete cytokinesis, leaving cells intimately connected through intercellular bridges that facilitate cyst generation, cell fate determination and collective growth dynamics. Using the well-characterized Drosophila melanogaster female germline cyst as a foundation, this study presents mathematical models rooted in the dynamics of cell cycle proteins and their interactions to explain the generation of germline cell lineage trees (CLTs) and highlight the diversity of observed CLT sizes and topologies across species. Competing models of symmetry breaking in CLTs were analyzed to rationalize the observed dynamics and robustness of oocyte fate specification, and highlight remaining gaps in knowledge. How CLT topology affects cell cycle dynamics and synchronization were analyzed and mechanisms of intercellular coupling are highlighted that underlie the observed collective growth patterns during oogenesis. Throughout, similarities across organisms are described that warrant further investigation and comments are made on the extent to which experimental and theoretical findings made in model systems extend to other species (Diegmiller, 2022).

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B/H2A/H2Av accumulate on lipid droplets (LDs), cytoplasmic fat storage organelles. Without LD-binding, maternally provided H2B/H2A/H2Av are absent, but how LDs ensure histone storage is unclear. Using quantitative imaging, this study uncover when during oogenesis these histones accumulate, and which step of accumulation is LD-dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone-anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. These findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites (Stephenson, 2021).

Early animal embryogenesis often exhibits rapid cell cycles dominated by DNA replication, mitosis and little to no transcription. Drosophila is a dramatic case where the first 13 nuclear divisions occur every 8-20 min. This speed poses a challenge for histone biology: demand increases exponentially, yet major regulatory mechanisms that control histone expression are unavailable. To meet this demand, many embryos inherit maternally synthesized histones. This study examined the origin of H2Av, H2B and H2A stockpiles in Drosophila (Stephenson, 2021).

Expression of histone messages is increased during S10, but it was unclear when the complementary maternally deposited histone proteins accumulate. A methodological challenge is that enterocytes (ECs) have polyploid nuclei. Each of the nurse cell (NC) nuclei are estimated to contain about 500 times more DNA than diploid nuclei; the 900 follicle cells contain DNA levels about eight times higher than a diploid nucleus. Thus, the histones needed to package NC and follicle cell chromatin are at least tenfold more abundant than the histone stockpile of newly laid embryos, which are estimated to be the equivalent of 1000 diploid nuclei. Detecting accumulation of the oocyte histone stockpile in addition to other histones in the EC is therefore challenging (Stephenson, 2021).

This study followed H2Av accumulation using an imaging approach that specifically quantifies histone signal in the NC and oocyte cytoplasm. H2Av already accumulates in the cytoplasm of S9 NCs and is associated with lipid droplets (LDs). An intriguing possibility is that this LD-associated H2Av pool in the early-stage NCs might support NC endoreplication. This H2Av-LD association likely brings some H2Av into the oocytes, but quantitation indicates that transfer from NCs contributes at most one fifth of the final H2Av pool in mature oocytes. These data are consistent with the possibility that the majority of the ooplasmic H2Av is synthesized in the oocyte; reduced H2Av levels in Jabba^−/− S12 oocytes may represent early loss by degradation rather than defective transfer from NCs (Stephenson, 2021).

After dumping, total H2Av levels continue to rise from S12 through S14. It is proposed that two mechanisms contribute to this rise. First, the translational efficiency of H2Av mRNA is upregulated from S12 to S14. Second, Jabba increases from S12 to S14. As more Jabba protein becomes available, it can presumably recruit more H2Av to LDs and protect it from degradation (Stephenson, 2021).

H2A and H2B levels in embryos are Jabba dependent (Li, 2012), and the pattern of H2B accumulation during oogenesis resembles that of H2Av. Currently, good tools to determine H2A accumulation are lacking, but it is predicted to will follow the same pattern, as canonical histones are typically similarly regulated. It is proposed that increasing H2Av, H2B and H2A accumulation during late oogenesis establishes an LD-bound histone depot for the early embryo (Stephenson, 2021).

It will be interesting to determine whether H3 and H4 accumulation follows a similar pattern. As canonical histones, their transcriptional and translational regulation is likely similar to that of H2A and H2B. However, they are not LD associated nor are their embryonic levels Jabba dependent, so there must be mechanistic differences (Stephenson, 2021).

Newly laid Jabba^−/− eggs have lower H2A, H2B and H2Av levels than wild type. This divergence is established during late oogenesis; H2Av and H2B levels rise in the wild type but drop in Jabba^−/−. Experiments to inhibit proteasome activity reveal that histone degradation plays a major role in bringing about this difference. As high MG132 (proteasome inhibitor) concentrations arrest development, it is infered that under conditions that allow EC development to S14, degradation is only partially inhibited. Yet even with partial inhibition, almost half of the H2Av normally lost in Jabba^−/− is retained. It is concluded that Jabba prevents histone turnover and that a major contributor to turnover is a proteasome-dependent pathway. The remaining turnover not prevented by drug treatment may be due to a proteasome-independent mechanism (Stephenson, 2021).

In the wild type, proteasome inhibition did not increase the H2Av pool beyond levels in untreated ECs. This observation was surprising as 4x Jabba females can accumulate more H2Av than wild type, which presumably indicates that even the wild type produces excess H2Av that is degraded if not protected by Jabba. It was reasoned that the direct effects on proteasome turnover are balanced out by indirect effects that compromise histone or Jabba synthesis. Indeed, high concentrations of the inhibitor abolish any rise in H2Av. Turnover of excess histones by the proteasome is well established in yeast. It is one of numerous mechanisms that prevent accumulation of free histones and the resulting cytotoxicity. It is proposed that H2Av turnover in late oogenesis is due to this general protective machinery; Jabba allows oocytes to deploy this safety feature while also accumulating H2Av needed to provision the embryo. Accordingly, Jabba determines the H2Av pool that is protected and any excess is recognized as a potential hazard. Because the mechanism that targets excess H2Av for proteasomal degradation is not known, it cannot yet be tested what type of damage this mechanism guards against (Stephenson, 2021).

The data suggest that Jabba prevents degradation by physically protecting H2Av. First, H2Av levels scale with Jabba dose, and H2Av levels beyond normal can be achieved by simply doubling Jabba levels. Second, a version of Jabba that is mislocalized to the NC nuclei and not present in oocytes is unable to support high H2Av levels. Finally, a Jabba mutant unable to bind to H2Av resulted in H2Av levels indistinguishable from those in Jabba^−/− (Stephenson, 2021).

To further unravel how Jabba prevents degradation, it will be necessary to identify the machinery that targets excess H2Av to the proteasome. Previous work has identified E3 ligases promoting H3 and H4 turnover, but those for other histones remain uncharacterized. Jabba may physically protect H2Av by shielding a ubiquitylation site. This remains to be tested. An alternate hypothesis is that histone degradation in oocytes occurs independently of ubiquitylation. Intriguingly, during development of the mammalian male germline, histone turnover occurs via ubiquitin-independent proteasome-dependent degradation (Stephenson, 2021).

In embryos, H2Av exchanges between LDs. If similar exchange occurs in oocytes, it is unclear how transient interactions with Jabba are sufficient to prevent H2Av degradation. It is speculated that either the transit time is negligible relative to the time H2Av spends interacting with Jabba or that cytosolic H2Av is accompanied by a chaperone. LD binding promotes availability of Jabba and, consequently, of histones Our analysis provides a first answer to why some histones are stored on LDs, while others are apparently stored in the cytoplasm. Jabba, the protein necessary to stabilize the H2A, H2B and H2Av ooplasmic pool, becomes trapped in NC nuclei if it is not LD bound. Thus, it is absent from the oocyte and cannot perform its protective function. It is proposed that LD binding ensures proper intercellular transport of Jabba, safeguarding its ability to function in the ooplasm (Stephenson, 2021).

H3 and H4 ooplasmic stores are presumably bound to a partner that prevents their degradation, perhaps the histone chaperone NASP. As these histones are apparently cytoplasmic, it is unclear how they and their binding partners avoid mislocalization to NC nuclei (Stephenson, 2021).

It is also unclear why histones are stored on LDs rather than another cytoplasmic structure. A priori, any cytoplasmic organelle that is transferred to oocytes could suppress the NC nuclear import of the histone anchor and promote transport into the oocyte. The large surface area of LDs may provide a readily available and, at these developmental stages, metabolically inert platform for recruitment. Other organelles may not have enough storage capacity or might be functionally impaired by histones on their surface. Alternatively, LD association may be an evolutionary accident and many other organelles might in principle be able to store histones. Identification of a Jabba region sufficient for histone binding will make it possible to address this question in the future (Stephenson, 2021).

But why is JabbaHBR mislocalized to NC nuclei? It is suspected that the histone-binding ability of JabbaHBR leads to its mislocalization, dragged along by histone nuclear import or retained in nuclei via chromatin binding. As histone binding is important to protect against degradation, mislocalization cannot be avoided unless JabbaHBR is anchored outside the nucleus. This idea could not be tested, as JabbaHBR that is unable to interact with histones still accumulates in nuclei, in cultured cells and in NCs. It is hypothesized that a cryptic nuclear localization signal in JabbaHBR promotes nuclear transport even without histone binding (Stephenson, 2021).

This analysis may shed light on how LDs regulate other proteins. LDs can transiently accumulate proteins from other cellular compartments. This has been particularly documented for proteins involved in nuclei acid binding and/or transcriptional regulation: MLX and Perilipin 5 can either be present on LDs or move into the nucleus to regulate transcription. The bacterial transcriptional regulator MLDSR is sequestered to LDs under stress conditions. As for Jabba and H2Av, LD association of such 'refugee proteins' may prevent their premature turnover or may promote their delivery to the correct intra- or intercellular location. A role for LDs in protein delivery to distant cellular compartments might be important in neurons where recent discoveries suggest important, but largely uncharacterized, roles for LDs (Stephenson, 2021).

Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. It was recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. This study tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, it was found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. It was hypothesized that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity (Neiswender, 2021).

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. This study demonstrates that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. These results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification (Huang, 2014).

This study reveals a previously unknown transcriptional role for Enok in regulating the polarized localization of Osk during oogenesis through promoting the expression of spir and mael. Spir and Mael are required for the properly polarized MT network in oocytes from stages 8 to 10A. However, protein levels of both decreased at later stages of oogenesis, allowing reorganization of the MT network and fast ooplasmic streaming. The persistent presence of Spir extending into stage 11 led to loss of ooplasmic streaming and resulted in female infertility. These findings suggest that the temporal regulation of spir expression is crucial for oogenesis, and, interestingly, Enok protein levels were also reduced in egg chambers during stages 10-13 compared with stages 1-9. While the stability of Spir or the translation of spir mRNA may also be a target for regulation, the results suggest that Enok is involved in the dynamic modulation of spir transcript. Furthermore, the results demonstrate the importance of Enok for expression of spir and mael in both ovaries and S2 cells, suggesting that Enok may play a similar role in other Spir- or Mael-dependent processes such as heart development (Huang, 2014).

Notably, Mael is also important for the piRNA-mediated silencing of transposons in germline cells. Mutations in genes involved in the piRNA pathway, including aub and armitage (armi), result in axis specification defects in oocytes as well as persistent DNA damage and checkpoint activation in germline cells. The activation of DNA damage signaling is suggested to cause axis specification defects in oocytes, as the disruption of Osk localization in piRNA pathway mutants can be suppressed by mutations in mei-41 or mnk, which encode ATR or checkpoint kinase 2, respectively. However, mutation in mnk cannot suppress the loss of posteriorly localized Osk in the mael mutant oocyte, indicating that the oocyte polarization defect in the mael mutant is independent of DNA damage signaling. Therefore, although the possibility that the piRNA pathway is affected in enok mutants due to down-regulation of mael cannot be excluded, the Osk localization defect in the enok mutant oocyte is likely independent of mei-41 and mnk (Huang, 2014).

In addition to the osk mRNA localization defect, both spir and mael mutants affect dorsal-ventral (D/V) axis formation in oocytes. However, no defects in the D/V patterning were observed in the eggshells of enok mutant germline clone embryos. Interestingly, among the spir mutant alleles that disrupt formation of germ plasm, only strong alleles result in dorsalized eggshells and embryos, while females with weak alleles produce eggs with normal D/V patterning. Since the enok¹ and enok² ovaries still express ~25% of the wild-type levels of spir mRNA, enok mutants may behave like weak spir mutants. Similarly, the ~40% reduction in mael mRNA levels in enok mutants as compared with the wild-type control may not have significant effects on the D/V axis specification (Huang, 2014).

Redundancy in HAT functions has been reported for both Moz and Sas3, the mammalian and yeast homologs of Enok, respectively. In yeast, deletion of either GCN5 (encoding the catalytic subunit of ADA and SAGA HAT complexes) or SAS3 is viable. However, simultaneously deleting GCN5 and SAS3 is lethal due to loss of the HAT activity of the two proteins, suggesting that Gcn5 and Sas3 can compensate for each other in acetylating histone residues. Indeed, while deleting SAS3 alone had no effect on the global levels of H3K9Ac and H3K14Ac, disrupting the HAT activity of Sas3 in the gcn5Δ background greatly reduced the bulk levels of H3K9Ac and H3K14Ac in yeast. Also, mammalian Moz targets H3K9 in vivo and regulates the expression of Hox genes, but the global H3K9Ac levels are not significantly affected in the homozygous Moz mutant, indicating that other HATs have overlapping substrate specificity with Moz. In flies, a previous study had reported that the H3K23Ac levels were reduced 35% in nejire (nej) mutant embryos, which lack functional CBP/p300 . However, knocking down nej by dsRNA in S2 cells severely reduced levels of H3K27Ac but had no obvious effect on global levels of H3K23Ac. This study showed that the global H3K23Ac levels decreased 85% upon enok dsRNA treatment in S2 cells. This study also showed that the H3K23Ac levels are highly dependent on Enok in early and late embryos, larvae, adult follicle cells and nurse cells, and mature oocytes. Therefore, although Nej may also contribute to the acetylation of H3K23, the results indicate that, in contrast to its mammalian and yeast homologs, Enok uniquely functions as the major HAT for establishing the H3K23Ac mark in vivo (Huang, 2014).

The H3K23 residue has been shown to stabilize the interaction between H3K27me3 and the chromodomain of Polycomb. Therefore, acetylation of H3K23 may affect the recognition of H3K27me3 by the Polycomb complex. Another study showed that the plant homeodomain (PHD)-bromodomain of TRIM24, a coactivator for estrogen receptor α in humans, binds to unmodified H3K4 and acetylated H3K23 within the same H3 tail. Also, the levels of H3K23Ac at two ecdysone-inducible genes, Eip74EF and Eip75B, have been shown to correlate with the transcriptional activity of these two genes at the pupal stage, suggesting the involvement of H3K23Ac in ecdysone-induced transcriptional activation. This study further provided evidence for the activating role of the Enok-mediated H3K23Ac mark in transcriptional regulation (Huang, 2014).

In mammals, MOZ functions as a key regulator of hematopoiesis. Interestingly, one of the genes encoding mammalian homologs of Spir, spir-1, is expressed in the fetal liver and adult spleen, indicating the expression of spir-1 in hematopoietic cells. Thus, it will be intriguing to investigate whether the Drosophila Enok-Spir pathway is conserved in mammals and whether Spir-1 functions in hematopoiesis. Taken together, the results demonstrate that Enok functions as an H3K23 acetyltransferase and regulates Osk localization, linking polarization of the oocyte to histone modification (Huang, 2014).

Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. This study demonstrates that Hecw (CG42797), a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons (Fajner, 2021).

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. This study analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. Depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. These data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical (Soffers, 2021).

Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. The results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes (Jang, 2021).

In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. The present work made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, these results suggest an important role in reproductive success (Silva, 2021).

Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, this study characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, it was shown that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface (Pokrywka, 2021).

The Irre Cell Recognition Module (IRM) is an evolutionarily conserved group of transmembrane glycoproteins required for cell-cell recognition and adhesion in metazoan development. In Drosophila melanogaster ovaries, four members of this group - Roughest (Rst), Kin of irre (Kirre), Hibris (Hbs) and Sticks and stones (Sns) - play important roles in germ cell encapsulation and muscle sheath organization during early pupal stages, as well as in the progression to late oogenesis in the adult. Females carrying some of the mutant rst alleles are viable but sterile, and previous work has identified defects in the organization of the peritoneal and epithelial muscle sheaths of these mutants that could underlie their sterile phenotype. In this study, besides further characterizing the sterility phenotype associated with rst mutants, the role of the IRM molecules Rst, Kirre and Hbs was investigated in maintaining the functionality of the ovarian muscle sheaths. Knocking down any of the three genes in these structures, either individually or in double heterozygous combinations, not only decreases contraction frequency but also irregularly increases contraction amplitude. Furthermore, these alterations can significantly impact the morphology of eggs laid by IRM-depleted females demonstrating a hitherto unknown role of IRM molecules in egg morphogenesis (Valer, 2022).

Neuronal processing is energy demanding and relies on sugar metabolism. To nurture the Drosophila nervous system, the blood-brain barrier forming glial cells take up trehalose from the hemolymph and then distribute the metabolic products further to all neurons. This function is provided by glucose and lactate transporters of the solute carrier (SLC) 5A family. This study identified three SLC5A genes that are specifically expressed in overlapping sets of CNS glial cells, rumpel, bumpel and kumpel. This study generated mutants in all genes and all mutants are viable and fertile, lacking discernible phenotypes. Loss of rumpel causes subtle locomotor phenotypes and flies display increased daytime sleep. In addition, in bumpel kumpel double mutants, and to an even greater extent in rumpel bumpel kumpel triple mutants, oogenesis is disrupted at the onset of the vitellogenic phase. This indicates a partially redundant function between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporters, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, these results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior (Yildirim, 2022).

The survival and evolution of a species is a function of the number of offspring it can produce. In insects the number of eggs that an ovary can produce is a major determinant of reproductive capacity. Insect ovaries are made up of tubular egg-producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. This study systematically measured mRNA expression of all cells in the larval ovary at the beginning, middle and end of terminal filament formation. Somatic and germ line cells were separated during these stages and their tissue-specific gene expression was assessed during larval ovary development. The number of differentially expressed somatic genes was highest during late stages of terminal filament formation and includes many signaling pathways that govern ovary development. It was also shown that germ line tissue, in contrast, shows greater differential expression during early stages of terminal filament formation, and highly expressed germ line genes at these stages largely control cell division and DNA repair. A tissue-specific and temporal transcriptomic dataset of gene expression in the developing larval ovary is provided as a resource to study insect reproduction (Tarikere, 2021).

All females adopt an evolutionary conserved reproduction strategy; under unfavorable conditions such as scarcity of food or mates, oocytes remain quiescent. However, the signals to maintain oocyte quiescence are largely unknown. This study reports that in four different species - Caenorhabditis elegans, Caenorhabditis remanei, Drosophila melanogaster, and Danio rerio - octopamine and norepinephrine play an essential role in maintaining oocyte quiescence. In the absence of mates, the oocytes of Caenorhabditis mutants lacking octopamine signaling fail to remain quiescent, but continue to divide and become polyploid. Upon starvation, the egg chambers of D. melanogaster mutants lacking octopamine signaling fail to remain at the previtellogenic stage, but grow to full-grown egg chambers. Upon starvation, D. rerio lacking norepinephrine fails to maintain a quiescent primordial follicle and activates an excessive number of primordial follicles.This study reveals an evolutionarily conserved function of the noradrenergic signal in maintaining quiescent oocytes (Kim, 2021).

Tissue aging is a major cause of aging-related disabilities and a shortened life span. Understanding how tissue aging progresses and identifying the factors underlying tissue aging are crucial; however, the mechanism of tissue aging is not fully understood. This study showed that the biosynthesis of S-adenosyl-methionine (SAM), the major cellular donor of methyl group for methylation modifications, potently accelerates the aging-related defects during Drosophila oogenesis. An aging-related increase in the SAM-synthetase (Sam-S) levels in the germline leads to an increase in ovarian SAM levels. Sam-S-dependent biosynthesis of SAM controls aging-related defects in oogenesis through two mechanisms, decreasing the ability to maintain germline stem cells and accelerating the improper formation of egg chambers. Aging-related increases in SAM commonly occur in mouse reproductive tissue and the brain. Therefore, these results raise the possibility suggesting that SAM is the factor related to tissue aging beyond the species and tissues (Hayashi, 2022).

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Using a multidisciplinary approach, this study identified the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). Elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. These results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements (Fernandez-Espartero, 2013).

Directed cell migration plays a crucial role in many normal and pathological processes such as embryo development, immune response, wound healing and tumor metastasis. During development, cells migrate to their final position in response to extracellular stimuli in the microenvironment. To migrate towards or away from a stimulus, individual cells or groups of cells must first achieve direction of migration through the establishment of cell polarity. Guidance cues, such as growth factors, control cell polarization through the regulated recruitment and activation of receptor tyrosine kinases (RTKs) to the leading edge. A key event downstream of RTK signalling in cell migration is the localization of activated Rac at the leading edge. However, little is known about the mechanisms by which external cues regulate Rac activity during cell migration. Rac is activated by GTP exchange factors (GEFs), which facilitate the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) states. Thus, GEFs appear as excellent candidates to regulate the cellular response to extracellular cues during cell migration (Fernandez-Espartero, 2013).

Among the different Rac GEF families characterized so far, the Vav proteins are the only ones known to combine in the same molecule the canonical Dbl (DH) and pleckstrin homology (PH) domains of Rac GEFs and the structural hallmark of tyrosine phosphorylation pathways, the SH2 domain. In addition, Vav activity is regulated by tyrosine phosphorylation in response to stimulation by transmembrane receptors with intrinsic or associated tyrosine kinase activity. These features make Vav proteins ideal candidates to act as signalling transducer molecules coupling growth factor receptors to Rac GTPase activation during cell migration. In fact, a number of cell culture experiments have suggested a role for the Vav proteins in cell migration downstream of growth factor signalling. Thus, the ubiquitously expressed mammalian Vav2 is tyrosine phosphorylated in response to different growth factors, including epidermal (EGF) and platelet-derived (PDGF) growth factors, and its phosphorylation correlates with enhanced Rac activity and migration in some cell types. However, the biological relevance for many of these interactions and the cellular mechanisms by which Vav regulates in vivo cell migration remains to be determined (Fernandez-Espartero, 2013).

The Vav proteins are present in all animal metazoans but not in unicellular organisms. There is a single representative in multicellular invertebrates and urochordata species (such as C. elegans, Drosophila melanogaster and Ciona intestinalis) and usually three representatives in vertebrates. The single Drosophila vav ortholog possesses the same catalytic and regulatory properties as its mammalian counterparts. In addition, the Drosophila Vav is tyrosine phosphorylated in response to EGF stimulation in S2 cells. Furthermore, a yeast two hybrid analysis has shown that the SH2-SH3 region of Vav can bind the epidermal growth factor receptor (EGFR) and the intracellular domain of PVR, PVRi, but not a kinase-dead version of PVRi, suggesting that Vav SH2-SH3-HA::PVRi interactions depend on PVR autophosphorylation. Altogether, these results suggest that the role of mammalian Vavs as transducer proteins coupling signalling from growth factors to Rho GTPase activation has been conserved in Drosophila. Thus, this study took advantage of Drosophila to analyse vav contribution to growth factor-induced cell migration in the physiological setting of a multicellular organism (Fernandez-Espartero, 2013).

The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to study guided cell migration downstream of PVR/EGFR signalling in vivo. Each egg chamber contains one oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (FCs), known as follicular epithelium (FE). The BC cluster is determined at the anterior pole of the FE and it comprises 6-8 outer cells and two anterior polar cells in a central position. BCs delaminate from the anterior FE and migrate posteriorly between the nurse cells until they contact the anterior membrane of the oocyte. BCs use the PVR and the EGFR to read guidance cues, the PDGF-related Pvf1 and the TGFβ-related Gurken, secreted by the oocyte. The Rho GTPase Rac is required for BC migration. The current model proposes that higher levels of Rac activity present in the leading cell determine the direction of migration and that this asymmetric distribution of Rac activity requires guidance receptor input. The unconventional Rac GEF Myoblast city, Mbc, is the only identified downstream signalling effector in this context. However, although genetic analysis have led to propose that the unconventional GEF for Rac, Mbc/DOCK 180, could activate Rac downstream of PVR during BC migration, this has not been formally proven. In addition, Mbc is unlikely to be the only Rac GEF actin downstream of guidance receptors in BCs as the migration phenotype due to complete removal of mbc is not as severe as the loss of both Pvr and Egfr. Thus, other effectors are likely to contribute to the complicated task of guiding BC migration. Many candidate molecules have been tested for their requirement in BC migration, MAPK pathway, PI3K, PLC-gamma, as well as RTK adaptors, such as DOCK, Trio, and Pak, but none of these is individually required (Fernandez-Espartero, 2013).

Vav proteins were initially involved in lymphocyte ontology. Only recently, cell culture experiments have implicated these proteins in cell migration events downstream of guidance factors. Interestingly, Vav proteins can either promote or inhibit cell migration. In macrophages, Vav is required for macrophage colony-stimulating factor-induced chemotaxis. In human peripheral blood lymphocytes, Vav is involved in the migratory response to the chemokine stromal cell-derived factor-1. Conversely, in Schwann cells, Vav2 is required to inhibit cell migration downstream of the brain-derived neurotrophic factor and ephrinA5. In spite of the knowledge gained from cell culture experiments, the biological relevance for many of the above interactions has remained elusive. In recent years, Vav proteins have started to emerge as critical Rho GEFs acting downstream of RTKs in diverse biological processes. Analysis of Vav2^-/- Vav3^-/- mice revealed retinogeniculate axonal projection defects and impaired ephrin-A1-induced migration during angiogenesis, suggesting a role for Vav in axonal targeting and angiogenesis downstream of Eph receptors in vivo. This study has shown that Vav can act downstream of growth factors receptors to promote BC migration in the developing Drosophila ovary, supporting a role of this family of GEFs in transducing signals from RTKs to regulate cell migration during development (Fernandez-Espartero, 2013).

Analysis of the cellular mechanisms by which Vav regulates cell migration in vertebrates is hampered by the inaccessibility of the cells and the difficulty of visualizing them in their natural environment within the embryo. Thus, it is not yet clear how Vav proteins regulate cell migration downstream of RTKs during development. In this study, by analysing cell movement in their physiological environment, it has been possible to show that Vav is required to control the length, stabilization and life of front cellular protrusions. In addition, disruption of Vav function in vivo was found to result in a decrease in Rac activity at the leading edge. Defective signalling downstream of EGFR/PVR results in defects in the dynamics of cellular protrusion and Rac activation, which are very similar to those observed in vav^-/- BCs. In addition, this study found that ectopic activation of Vav in BCs, as it is the case for PVR/EGFR and Rac, causes non-polarized massive F-actin accumulation. Thus, it is suggested that one of the roles of Vav in directed cell migration downstream of EGF/PVF signals is to remodel the actin cytoskeleton via Rac activation, hence promoting the formation and stabilization of cellular protrusions in the direction of migration. Studies in cultured neurons, have shown that the main role for mouse Vav2 during axonal repulsion is to mediate a Rac-dependent endocytosis of ephrin-Eph. Although endocytosis has been normally shown to be involved in attenuation of RTKs signalling, in BCs it has been proposed to ensure RTKs recycling to regions of higher signalling, thus promoting directed BC movement. This is based on the fact that elimination in BCs of the ubiquitin ligase Cbl, which has been shown to regulate RTK endocytosis, leads to delocalized RTK signal and migration defects. In this context, another possible role for Vav downstream of EGFR/PVR could be to mediate RTK endocytosis, as it is the case during axonal repulsion. Further analysis will be needed to fully explore the molecular and cellular mechanisms by which Vav proteins regulate cell migration in vivo in other developmental contexts (Fernandez-Espartero, 2013).

BC migration is a complex event and activation of EGF/PDGF receptors will most likely engage different GEFs to affect the distinct cytoskeletal changes necessary to accomplish it. In fact, the migration phenotype of BCs mutant for vav is less severe than that of BCs double mutant for both EGFR and PVR. In addition, although reducing Vav function decreases the asymmetry in Rac activity between front and back present in wild-type clusters, it does not eliminate it, as it happens when the function of both guidance receptors is compromised. All these results suggest that there are other GEFs besides Vav that could act downstream of EGFR and PVR to activate Rac. Previous analysis have implicated the Rac exchange factor Mbc/DOCK180 and its cofactor ELMO on BC migration. In this context, Vav and the Mbc/ELMO complex could act synergistically as GEFs to mediate Rac activation to a precise level and/or to a precise location. This awaits the validation of the Mbc/ELMO complex as a GEF for Rac in BCs. In the future, it will be important to determine how the different GEFs contribute to Rac activation, which specific downstream effectors of Rac they activate, and ultimately what cellular aspects of the migration process they control (Fernandez-Espartero, 2013).

In summary, this work demonstrates that Vav functions downstream of RTKs to control directed cell migration during development. Furthermore, this study has unravelled the cellular and molecular mechanism by which Vav regulates cell migration in the developing Drosophila egg chamber: binding of PDGF/EGF to their receptors would induce Vav activation through tyrosine phosphorylation and its association with the activated receptors. This would lead to an increase in Rac activity at the leading edge of migrating cells, which promotes the stabilization and growth of the cellular front extensions, thus controlling directed cell migration (Fernandez-Espartero, 2013).

Regulation of Vav signalling downstream of RTKs can participate not only in development or normal physiology but also in tumorigenesis. Vav1 is mis-expressed in a high percentage of pancreatic ductular adenocarcinomas and lung cancer patients. Thus, understanding the mechanisms by which Vav controls cellular processes downstream of RTKs is likely to be relevant for both developmental and tumor biology (Fernandez-Espartero, 2013).

Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. This study reports that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits Protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. The octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but are unable to lay eggs. It has been shown previously that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells is sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. It was also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II leads to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known β adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restores ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported in this study narrow the knowledge gap and offer insight into novel strategies for insect control (Lim, 2014; PubMed).

Drosophila adipocytes, together with hepatocyte-like oenocytes, compose the fat body, a nutrient-sensing organ with endocrine roles. In the larval fat body, TOR activation downstream of amino acid sensing results in the production of unknown factors that modulate overall growth of the organism. In both the larval and adult fat body, sensing of sugars and lipids leads to the production of a leptin-like cytokine, Unpaired 2 (Upd2), which controls the secretion of brain insulin-like peptides. This study reports that partially inhibiting amino acid transport in adult adipocytes results in a specific reduction in the number of ovarian GSCs and that, surprisingly, this effect is independent of TOR signaling. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response (AAR) pathway within adipocytes, causing increased rates of GSC loss. These results indicate that amino acid sensing by adipocytes through a TOR-independent mechanism is communicated to GSCs to control their maintenance, thereby contributing to their response to diet. These findings bring to light the importance of elucidating how adipocytes contribute to the regulation of various adult stem cell types by diet, and how these mechanisms might be adversely affected in obese individuals (Armstrong, 2014).

Organs often need to coordinate the growth of distinct tissues during their development. This study analyzed the coordination between germline cysts and the surrounding follicular epithelium during Drosophila oogenesis. Genetic manipulations of the growth rate of both germline and somatic cells influence the growth of the other tissue accordingly. Growth coordination is therefore ensured by a precise, two-way, intrinsic communication. This coordination tends to maintain constant epithelial cell shape, ensuring tissue homeostasis. Moreover, this intrinsic growth coordination mechanism also provides cell differentiation synchronization. Among growth regulators, PI3-kinase and TORC1 also influence differentiation timing cell-autonomously. However, these two pathways are not regulated by the growth of the adjacent tissue, indicating that their function reflects an extrinsic and systemic influence. Altogether, these results reveal an integrated and particularly robust mechanism ensuring the spatial and temporal coordination of tissue size, cell size, and cell differentiation for the proper development of two adjacent tissues (Vachias, 2014: PubMed).

Several main conclusions can be drawn from this work. First, in each follicle, growth is intrinsically coordinated between the two tissues. Second, this growth control tends to optimize cell shape in the epithelium. This is likely to be representative of the development of many epithelia where cell shape must be maintained because it is essential for the function of the tissue. In the third place, growth control has a very important impact on differentiation timing in each tissue. Furthermore, several growth pathways can cell-autonomously influence differentiation rate but are not regulated by the adjacent tissue, indicating that they only respond to extrinsic cues. Finally, as a whole, this study reveals the robustness of the spatiotemporal pattern allowing the production of mature eggs with a normal shape and a normal size. At least two examples based on Pten somatic clones can illustrate this robustness. WT border cells migrate perfectly 'on time' in a follicle in which mutant somatic cells have induced a faster germline development. Second, a WT looking mature egg can be found in the middle of an ovariole, suggesting that all developmental steps have been faster but correctly orchestrated. This robustness probably reflects the fact that final egg size is constant, that most of the developmental steps have to occur at a specific size, and that differentiation is able to block growth when the definitive egg size is reached. These observations raise the question as to how the differentiation program regulates growth and especially growth arrest in each follicle (Vachias, 2014).

These results indicate a two-way communication between the germline and the soma to ensure their coordination. It was also observed that somatic cells can influence other somatic cells but, importantly, that such an effect depends on the relay of the germ cells. This result suggests that coordination is achieved by different signals depending on the tissue. The soma and germline could communicate via the secretion of growth factors controlling the adjacent tissue, though obvious candidates were excluded. An alternative explanation would be that, as it is proposed in mammals, the two tissues are interdependent for specific metabolites, although it would be independent of TORC1, a classical sensor of metabolic activity. Finally, an attractive hypothesis would be that growth regulation between the soma and the germline depends on a mechanical steady state. Germline growth creates a tension on the follicle cell, leading to the proposal that this tension could trigger epithelial growth. If so, it would also mean that follicle cells provide a mechanical strain limiting germline growth. The mechanical control of growth in epithelial cells is usually devoted to the Hippo pathway, which is not involved in this instance. Thus, this work does not allow favoring one or the other of these nonexclusive mechanisms (Vachias, 2014).

Altogether, these results highlight several dimensions of coordination between cell growth, cell shape, and cell identity and all this between two distinct tissues. These different functional links offer a highly robust program in space and time. The relevance for such robustness has been very recently highlighted because it probably confers the reproducibility on embryonic development. Since usual pathways controlling growth are not involved in this two-way communication, this multidimensional coordination will be a useful framework for identifying molecular actors ensuring tissue homeostasis in the recurrent context of the development of two adjacent tissues (Vachias, 2014).

Two evolutionarily conserved signaling pathways that act redundantly to regulate engulfment of dead cells: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Homology searches revealed a family of putative ced-7/ABCA1 homologs encoding ABC transporters in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1, mutants were analyzed for engulfment phenotypes in oogenesis, during which nurse cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. Genetic analyses indicate that one of the ABC transporter genes, which was named Eato (CG31731), is required for nurse cell clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the follicle cells. Additionally, Eato acts in the follicle cells to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the nurse cells for their clearance. Since ABCA class transporters, such as CED-7 and Eato, are involved in lipid trafficking, it is proposed that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. This work identifies Eato as the ced-7 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during nurse cell clearance in the ovary (Santoso, 2018).

Problems with networks of coupled oscillators arise in multiple contexts, commonly leading to the question about the dependence of network dynamics on network structure. Previous work has addressed this question in Drosophila oogenesis, where stable cytoplasmic bridges connect the future oocyte to the supporting nurse cells that supply the oocyte with molecules and organelles needed for its development. To increase their biosynthetic capacity, nurse cells enter the endoreplication program, a special form of the cell cycle formed by the iterated repetition of growth and synthesis phases without mitosis. Recent studies have revealed that the oocyte orchestrates nurse cell endoreplication cycles, based on retrograde (oocyte to nurse cells) transport of a cell cycle inhibitor produced by the nurse cells and localized to the oocyte. Furthermore, the joint dynamics of endocycles has been proposed to depend on the intercellular connectivity within the oocyte-nurse cell cluster. This study used a computational model to argue that this connectivity guides, but does not uniquely determine the collective dynamics and has identified several oscillatory regimes, depending on the time scale of intercellular transport. These results provide insights into collective dynamics of coupled cell cycles and motivate future quantitative studies of intercellular communication in the germline cell clusters (Shao, 2021).

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells, promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. This study identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionary conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. Vps13 is expressed in late nurse cells and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells (Faber, 2020).

From insects to mice, oocytes develop within cysts alongside nurse-like sister germ cells. Prior to fertilization, the nurse cells' cytoplasmic contents are transported into the oocyte, which grows as its sister cells regress and die. Although critical for fertility, the biological and physical mechanisms underlying this transport process are poorly understood. This study combined live imaging of germline cysts, genetic perturbations, and mathematical modeling to investigate the dynamics and mechanisms that enable directional and complete cytoplasmic transport in Drosophila melanogaster egg chambers. During "nurse cell (NC) dumping" most cytoplasm was discovered to be transported into the oocyte independently of changes in myosin-II contractility, with dynamics instead explained by an effective Young-Laplace law, suggesting hydraulic transport induced by baseline cell-surface tension. A minimal flow-network model inspired by the famous two-balloon experiment and motivated by genetic analysis of a myosin mutant correctly predicts the directionality, intercellular pattern, and time scale of transport. Long thought to trigger transport through "squeezing," changes in actomyosin contractility are required only once NC volume has become comparable to nuclear volume, in the form of surface contractile waves that drive NC dumping to completion. This work thus demonstrates how biological and physical mechanisms cooperate to enable a critical developmental process that, until now, was thought to be mainly biochemically regulated (Alsous, 2021).

Developing oocytes need large supplies of macromolecules and organelles. A conserved strategy for accumulating these products is to pool resources of oocyte-associated germline nurse cells. In Drosophila, these cells grow more than 100-fold to boost their biosynthetic capacity. No previously known mechanism explains how nurse cells coordinate growth collectively. This study reports a cell cycle-regulating mechanism that depends on bidirectional communication between the oocyte and nurse cells, revealing the oocyte as a critical regulator of germline cyst growth. Transcripts encoding the cyclin-dependent kinase inhibitor, Dacapo, are synthesized by the nurse cells and actively localized to the oocyte. Retrograde movement of the oocyte-synthesized Dacapo protein to the nurse cells generates a network of coupled oscillators that controls the cell cycle of the nurse cells to regulate cyst growth. It is proposed that bidirectional nurse cell-oocyte communication establishes a growth-sensing feedback mechanism that regulates the quantity of maternal resources loaded into the oocyte (Doherty, 2021).

Drosophila oocytes develop together with 15 sister germline nurse cells (NCs), which pass products to the oocyte through intercellular bridges. The NCs are completely eliminated during stages 12-14, but this study discovered that at stage 10B, two specific NCs fuse with the oocyte and extrude their nuclei through a channel that opens in the anterior face of the oocyte. These nuclei extinguish in the ooplasm, leaving 2 enucleated and 13 nucleated NCs. At stage 11, the cell boundaries of the oocyte are mostly restored. Oocytes in egg chambers that fail to eliminate NC nuclei at stage 10B develop with abnormal morphology. These findings show that stage 10B NCs are distinguished by position and identity, and that NC elimination proceeds in two stages: first at stage 10B and later at stages 12-14 (Ali-Murthy, 2021).

To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. This study reports for the first time the expression patterns during oogenesis for the Arf GTPase activating protein Drongo, the fruit fly homolog of AGFG1. Temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. This study shows a first link between the recycling endosome pathway and Drongo and finds that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11 (Catrina, 2016).

drongo mRNA presents a spatially and temporally controlled distribution during oogenesis, which in fixed egg chambers is sensitive to denaturing protocols. After transcription in the nurse cells, drongo mRNA is transported and localized mainly at the oocyte's cortex as early as stage 7. These processes appear to be dependent on the proper organization of the microtubule filaments, as drongo mRNA does not localize properly in armi mutants, where microtubule network polarization is compromised. The drongo gene was identified using enhancer trapping, technique employed when functional redundancy may exist and especially when mutant phenotypes are very mild. Attempts to isolate a drongo mutant by EMS mutagenesis failed. By comparison with AGFG1, it is likely that drongo mutants or even a drongo-null may not exhibit severe defects during oogenesis, but it is also possible that mammalian cells express redundant factors that are not encoded in the fruit fly genome. Therefore, given the current lack of a drongo RNA-null fly stock, future studies are needed to determine whether drongo mRNA localization has functional significance during oogenesis and embryogenesis, for processes such as maternal loading. However, it is proposed that drongo mRNA transport and localization to the oocyte, although highly transient, is required for the correct timing of its translation at peak (Catrina, 2016).

drongo mRNA accumulates in the oocyte early in development, but Drongo peak expression does not occur before stage 8. This is achieved by translational repression via P-bodies of the drongo transcript before mid-ogenesis and this control continues in later stages of development. Additional support for translational control of drongo gene expression comes from the observation that similar to the reported premature expression of Osk in armi mutant egg chambers, clumps of Drongo are formed in stage >6 oocytes. These clumps resemble the Drongo-EGFP aggregations were observed at the oocyte's anterior in early stages of oogenesis and they are likely due to increased amounts of endogenous Drongo. However, when Drongo-EGFP is expressed, the EGFP tag may also facilitate aggregation (Catrina, 2016).

Although its transcript lacks UTRs, Drongo-EGFP properly localizes in the oocyte. It is possible that endogenous Drongo expression is sufficient for Drongo-EGFP's correct localization. This is supported by the increase in cortical localization of Drongo-EGFP after stage 6-7, when endogenous Drongo levels naturally begin to increase at the oocyte's cortex. However, other cellular factors could also be responsible for recruiting Drongo (Catrina, 2016).

Ectopic F-actin clumps have been previously reported for oocytes of rab germline clones, where F-actin is not properly released from the early endosomes, following endocytosis. This study found that the localization of Rab5 and Rab11 in the armi mutant is similar to their localization in wild type egg chambers. Therefore, the ectopic Drongo clumps observed in armi mutant are not due to Rab5-induced or Rab11-induced defects. Taken together, these results suggest that drongo mRNA localization is not required for the Drongo protein to reach the oocyte's cortex, which may be facilitated by the slow cytoplasmic flow (Catrina, 2016).

The clathrin triskelion is composed of three Clathrin heavy chains (Chc) and three light chains (Clc). In Drosophila egg chambers, apart from its role in the formation of clathrin-coated vesicles, Chc has been reported to play a role in the polarization of the microtubule network at stage 6. Clc has been shown to direct endocytosis by providing direction for membrane internalization. After stage 7, Drongo localizes mainly at the oocyte's cortex, where it shows significant overlap with Clc-GFP, concurrent with the activation of bulk endocytosis. Close analysis and comparison of the Drongo and AGFG1 protein sequences reveals that they both contain AP2 binding/sorting motifs (DxF, YxxΦ, where Phi;=hydrophobic residue). Drongo's tight association with Clc-GFP at the oocyte's cortex, as well as its AP2 binding motifs, suggest that it is involved in endocytosis. The fact that overexpression of Drongo-EGFP only mildly affects general endocytosis during oogenesis is not surprising, as its mammalian homolog, AGFG1, was reported to mediate endocytosis of only select cargo. However, this does not exclude a role for Drongo in endocytosis and it is possible that a lack of Drongo will have a more pronounced effect (Catrina, 2016).

Drongo directly interacts with components of the Arp2/3 complex, and Drongo's cortical colocalization with Arpc3A is seen in the oocyte. Moreover, Drongo distribution near the cortex of stage 8 oocytes becomes more diffuse when Arp2/3-dependent actin branching is impaired, suggesting that F-actin is important for Drongo's tight cortical localization at mid-oogenesis. CK-666 has been reported to interfere with formation of new Arp2/3-dependent branches without affecting existing branches or the ends of microfilaments. Therefore, it is not surprising that following drug treatment, only subtle changes is seen in the cortical F-actin network at mid-oogenesis. It is possible that Drongo-EGFP localization is mildly affected by drug-induced local perturbations in F-actin organization. Even though Drongo distribution is not affected by downregulation of Arpc4 expression, it is likely that Arp2/3 and the microfilaments play an essential role in Drongo localization during oogenesis. It is possible that Arp2/3 still shows some functionality despite Arpc4 depletion (Catrina, 2016).

Although Drongo colocalizes with Arf51F, in vitro studies are needed to determine if Drongo acts as a GAP effector for Arf51F. In the current studies the decrease in FM 4-64x uptake when Drongo is overexpressed is consistent with inactivation of Arf51F by excess of GAP activity, similar to the reduction in transferrin uptake observed in HeLa cells when SMAP1 is overexpressed (GAP effector for Arf6, which is the mammalian homolog for Arf51F56). Taken together, these results indicate that Drongo may participate in endocytosis and vesicular transport, where F-actin remodeling plays an important role (Catrina, 2016).

Reports that Drongo's mammalian homolog, AGFG1, and Rab11 are both essential for Influenza A virus replication led to an analysis of Drongo and Rab11 localization. Rab11 is required early in egg chamber development for oocyte determination. During mid-oogenesis it is required for polarized endocytic recycling toward the posterior pole of the oocyte, and for the posterior localization of AP-2α. Lack of Rab11, or reduced Drongo levels in the germarium, lead to defects in oocyte specification or an additional round of cystoblast division, respectively. Therefore, it is likely that Rab11 acts upstream of Drongo during early oogenesis, and this is supported by the observation that Rab11 appears unaffected in drongoiHMJ oocytes. However, since properly localized Drongo is still observed in drongoiHMJ oocytes at mid-oogenesis, it cannot be ruled out that Rab11 localization and function at this stage may be affected in a drongo-null egg chamber (Catrina, 2016).

Rab11 is also essential for efficient osk mRNA transport, localization and anchoring, as well as osk translation. The disruption of osk mRNA trafficking and anchoring is believed to be a consequence of the failure to posteriorly concentrate the plus ends of the microtubule network at mid-oogenesis in oocytes where Rab11 function is affected. Rab11 directly interacts with Nuf (Nuclear-fallout), a unique Arf effector that contains a conserved Rab11-binding motif. Rab11 and Nuf are both required for actin recruitment during metaphase furrow formation in embryogenesis (Catrina, 2016).

It was proposed that proteins associated with recycling endosome activity (e.g., Rab11 and Nuf) are also required for delivery of actin-remodeling proteins to the plasma membrane, such as Rac1 (Rho GTPase). Moreover, Arf51F regulates endosomal recycling and cortical actin remodeling, as well as trafficking of Rac1 to the plasma membrane. In addition, when coexpressed with Rab11DN, Drongo-EGFP particles show premature streaming, which is characteristic of actin-related mutations (e.g., capu and spire) (Catrina, 2016).

Due to their size, vesicles cannot rely solely on diffusion to reach their destination; their transport is composed of an active, directed movement on the microtubules and a passive, diffusive stage. MSD analysis reports on the overall movement for all tracks, determined for particles of various sizes and for long-range transport, and it yields constrained diffusion for Drongo-EGFP and Clc-GFP oocytes. Given the predominantly passive nature of the long-range transport of Drongo-EGFP and Clc-GFP that was observed in a wild type background, it is impressive to observe the transition to a more directional movement that occurs in Rab11DN egg chambers (Catrina, 2016).

It is propose that Drongo participates in recycling endosome trafficking and this study provides evidence that Drongo is associated with intracellular trafficking where F-actin remodeling plays an important role. The results improve understanding of AGFG1's essential role in viral RNA transport. It is possible that viruses exploit the versatility of AGFG1 to achieve efficient RNA release from the perinuclear region and to facilitate their cytoplasmic transport, thereby modulating desired changes to the cytoskeleton and to trafficking pathways. Moreover, the multicellular model system this study uses is uniquely qualified to precisely identify the steps where Drongo actively participates in the cytoplasmic transport of intron-containing viral RNAs. Elucidating Drongo's cellular role(s) will help determine why its mammalian homolog is required for transport and localization of intron-containing viral RNAs. This should open new avenues for overcoming rapid viral adaptability to drug-pressure by targeting conserved interactions between viral proteins and host cofactors, which are nonessential for cellular viability (Catrina, 2016).

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. This study tested how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, these results show that an appropriate level of mechanical sensitivity is required for filamin's function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components (Huelsmann, 2016).

Egg activation is the series of events that transition a mature oocyte to an egg capable of supporting embryogenesis. Increasing evidence points toward phosphorylation as a critical regulator of these events. This study used Drosophila melanogaster to investigate the relationship between known egg activation genes and phosphorylation changes that occur upon egg activation. Using the phosphorylation states of four proteins-Giant Nuclei, Young Arrest, Spindly, and Vap-33-1-as molecular markers, this study showed that the egg activation genes sarah, CanB2, and cortex are required for the phospho-regulation of multiple proteins. This study showed that an additional egg activation gene, prage, regulates the phosphorylation state of a subset of these proteins. Finally, this study shows that Sarah and calcineurin are required for the Anaphase Promoting Complex/Cyclosome (APC/C)-dependent degradation of Cortex following egg activation. From these data, a model is presented in which Sarah, through the activation of calcineurin, positively regulates the APC/C at the time of egg activation, which leads to a change in phosphorylation state of numerous downstream proteins (Krauchunas, 2013).

Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This study used germline-specific RNAi to examine the function of 189 maternal proteins that are phosphoregulated during egg activation in Drosophila melanogaster. 53 genes were identified whose knockdown reduced or abolished egg production and caused a range of defects in ovarian morphology, as well as 51 genes whose knockdown led to significant impairment or abolishment of the egg hatchability. Different stages of developmental arrest were observed in the embryos and various defects in spindle morphology and aberrant centrosome activities in the early arrested embryos. The results revealed 15 genes with newly discovered roles in egg activation and early embryogenesis in Drosophila. It is suggested that the phosphoregulated proteins may provide a rich pool of candidates for the identification of important players in the egg-to-embryo transition (Zhang, 2018).

Collective cell migration is involved in various developmental and pathological processes, including the dissemination of various cancer cells. During Drosophila melanogaster oogenesis, a group of cells called border cells migrate collectively toward the oocyte. This study shows that members of the Arf family of small GTPases and some of their regulators are required for normal border cell migration. Notably, it was found that the ArfGAP Drongo and its GTPase-activating function are essential for the initial detachment of the border cell cluster from the basal lamina. Drongo controls the localization of the myosin phosphatase Flapwing in order to regulate myosin II activity at the back of the cluster. Moreover, toward the class III Arf, Drongo acts antagonistically to the guanine exchange factor Steppke. Overall, this work describes a mechanistic pathway that promotes the local actomyosin contractility necessary for border cell detachment (Zeledon, 2019).

Cell migration requires the precise spatiotemporal control of various determinants. In particular, motility-driving forces require the coordination of both actomyosin contractility, to generate traction forces in protrusions, and propulsive forces at the back of the cell. This spatiotemporal control is even more complex during collective cell migrations in which cells maintain contacts while migrating. Indeed, in these particular migrations, these processes need to be coordinated across the group of migrating cells. Border cell migration in the Drosophila ovary is a powerful model to investigate the mechanisms that regulate collective cell migration. Border cells (BCs) detach from the follicle epithelium surrounding the egg chambers and form a small cluster of six to ten cells that migrates invasively between the giant nurse cells that compose the center of the egg chamber, toward the oocyte. Border cells use E-cadherin to maintain cluster cohesion as well as to interact with the nurse cells. Their migration is guided by receptor tyrosine kinase (RTK) ligands that are secreted by the oocyte. During border cell migration, vesicular trafficking regulators have been involved in regulating the localization of E-cadherin between border cells, in the maintenance of active RTKs at the leading edge of the cluster, and in a cell-cell communication mechanism that restrains protrusive ability to the leader cell (Zeledon, 2019).

Vesicular transport is thus critical for the spatiotemporal control of migration determinants during border cell migration. Although previous work has focused mainly on the role of small GTPases of the Rab family, the role of Arf GTPases and their regulators during border cell migration is unknown (Zeledon, 2019).

Arf GTPases regulate the formation of vesicular transport intermediates by interacting with coatomers to bend the membrane of the donor compartment. They are grouped in three classes on the basis of amino acid similarity. Although mammals have multiple class I and class II Arfs, Drosophila possess only one Arf per class: Arf79F (class I), Arf102F (class II), and Arf51F (class III). Furthermore, another small GTPase, Sar1, is structurally related to Arfs and has also been involved in vesicular transport. In addition, there are Arl (Arf-like) proteins that are closely related to Arfs but have diverse functions. The regulation of Arfs and Arls is similar to that of other small GTPases: GDP/GTP exchange factors (GEFs) promotes their activation, while GTPase-activating proteins (GAPs) are responsible for their inactivation (Zeledon, 2019).

Class I and II Arfs and Sar1 are involved mainly in bidirectional transport within the secretory pathway. However, both class I and class II Arfs can also promote trafficking steps in the endocytic pathway. The single class III Arf (ARF6 in mammals) is present at the plasma membrane and in endosomes, where it regulates recycling to the plasma membrane (Zeledon, 2019).

Arf proteins regulate cell migration in various contexts. Notably, ARF6 regulates the recycling of integrins from dissociating focal adhesions to nascent one at the leading edge and the transport of active Rac to the plasma membrane. In mammals, a class I Arf (ARF1) regulates the formation of motile structures such as podosomes and generates actomyosin contractility by acting on different RhoGTPase. Intriguingly, these functions might be independent of the role of ARF1 in vesicular transport. Similarly, Arf regulators, in particular ArfGAPs, regulate cell migration independently of vesicular transport (Zeledon, 2019).

In Drosophila, Arf79F is required for lamellipodia formation in S2R+ cells, independently of Rac, and also in epithelial tube expansion. In the latter, the GEF Gartenzwerg (Garz) and the GAP ArfGAP1 regulate its activity. The sole member of the cytohesin family of GEFs in flies, Steppke (Step), regulates actomyosin contractility during dorsal closure. Interestingly, Step might act on both class I and III Arfs in this process (Zeledon, 2019).

To improve understanding of the vesicular machinery regulating border cell migration, an RNAi screen was performed targeting Arfs, Arls, and their potential regulators. Depletion of class I and II Arfs induced strong pleiotropic effects, while neither the expression of double-stranded RNAs (dsRNAs) against class III Arf nor against Arf-like proteins induced significant border cell migration delays. Furthermore, it was found that the depletion of several Arf regulators induced migration defects. This study focused on the ArfGAP Drongo, as it seemed to specifically affect the detachment of the border cell cluster at the onset of migration. Drongo is the ortholog of mammalian AGFG1 and was shown to be required for normal egg chamber development (Zeledon, 2019).

Drongo was found to inactivate the class III Arf at the back of the border cell cluster at the onset of border cell migration. This leads to a local decrease in the levels of myosin phosphatase and a subsequent increase in myosin II activity. In turn, this promotes contractility and allows the detachment of the border cluster from the follicle epithelium. Interestingly, it was found that Drongo acts in opposition to Step. Furthermore, it was found that this pathway seems to act independently of the kinase Par-1, which was shown to inactivate myosin phosphatase at the back of the border cell clusters. Overall, this work identifies Drongo as part of a molecular cascade promoting local actomyosin contractility by clearing the back of the cluster of the myosin phosphatase (Zeledon, 2019).

This study has shown that Arfs and several of their regulators are required for border cell migration. Although RNAi lines against Arf-like proteins and numerous regulators did not induce a phenotype, it cannot be concluded that they are not involved in border cell migration, as this study has not tested the efficiency of depletion in border cell of each potential false negatives. Downregulation of either class I or class II Arfs or of the GEF Garz in border cells induces pleiotropic effects, making it difficult to ascertain their specific role in border cell migration. However, it was found that Drongo has a specific function at the initiation of border cell migration, which requires its ArfGAP activity (Zeledon, 2019).

The results indicate that Drongo regulates contractility at the back of the cluster by controlling the localization of the myosin phosphatase. In the absence of Drongo, myosin phosphatase levels increase at the back of the cluster and consequently reduce the activity of myosin II, which is required for the detachment of the cluster. Furthermore, Drongo localizes at the trailing edge at the time of detachment, suggesting a direct role in the removal of myosin phosphatase from the back of the cluster (Zeledon, 2019).

In addition to its role in regulating myosin phosphatase at the back of the cluster during detachment, Drongo might be involved in the migration process per se. Indeed, it was found in the rescue experiments that when the Drosophila melanogaster form of Drongo that is still targeted by the interfering RNA was expressed the activity of myosin II is restored at the back of the cluster, but the migration of border cell is still incomplete. Interestingly, Drongo colocalizes partially with active myosin II (p-Sqh) both at detachment and during the migration of border cells (Zeledon, 2019).

The mechanisms by which Drongo acts on myosin phosphatase are not clear. Previous work showed that the detachment of the border cell cluster requires Notch signaling and that the polarity protein Par-1 regulates myosin phosphatase activity through the direct phosphorylation of Mbs by Par-1 (Zeledon, 2019).

Drongo depletion has no effects on Par-1 and Par-3 and it does not regulate Notch activity. As Par-1 acts directly on Mbs, it is concluded that Drongo acts in parallel to Par-1 and Par-3 by regulating the localization of myosin phosphatase. These results also indicate that Drongo is not acting upstream of Notch. It could be interesting to determine if Notch regulates drongo expression. Indeed, border cells express higher levels of drongo compared with the rest of the follicle cells (Borghese, 2006), and its human ortholog AGFG1 was found to be a direct transcriptional target of Notch1 in T cell acute lymphoblastic leukemia. Hence, drongo might be part of a subset of genes regulated by Notch to ensure the detachment of the border cell cluster (Zeledon, 2019).

Several ArfGAPs have been described as regulators of the actin cytoskeleton. Some act through direct binding of actin regulators and effectors, independently of their GAP activity. For example, ASAP1 directly interact with non-muscle myosin IIA to promote cell migration. The current results indicate that the ArfGAP activity of Drongo toward Arf51F is required for border cell migration. Furthermore, it was found that Drongo functions in opposition to the ArfGEF Step. Thus, Drongo might promote contractility by maintaining Arf51F in an inactive state to keep the rear of the cluster free of myosin phosphatase. Interestingly, Step was shown to inhibit actomyosin contractility during developmental cellularization and dorsal closure. It would be interesting to determine if Drongo could also counterbalance the activity of Step to regulate contractility during these two processes (Zeledon, 2019).

The way in which the balance between Drongo and Step regulates the localization of myosin phosphatase remains unknown. As Arf51F is involved, it is appealing to hypothesize that a specific vesicular transport event might regulate the localization of myosin phosphatase. However, no evidence was obtained that myosin phosphatase is transported to or cleared from the back of the cluster through vesicular trafficking. In particular, Mbs localized in vesicular structure was not observed, neither in control conditions nor after depletion of Drongo. Still, this does not rule out that trafficking might regulate myosin phosphatase. Indeed, this study might have overlooked the trafficking of the myosin phosphatase subunits because of technical limitations. Alternatively, it is possible that a regulator of myosin phosphatase activity is trafficked or that Arf51F might directly recruit the myosin phosphatase or a regulator of myosin phosphatase. In both cases, such a regulator is probably different than the polarity proteins Par-1 and Par-3, as their distribution was found to be unaltered after Drongo depletion. Finally, Drongo and Arf51F might remodel the protein or lipid content of the plasma membrane to allow the recruitment of Mbs. For example, ARF6, the mammalian ortholog of Arf51F, has the ability to modify the lipid composition of membranes. Further work will be necessary to discriminate among these possibilities. For example, it would be possible to try to determine if perturbing various vesicular trafficking steps by independent means affects detachment and contractility. Alternatively, it would be interesting to analyze the interactome of Arf51F in its active and inactive forms to determine if active Arf51F may directly recruit the myosin phosphatase or one of its regulators (Zeledon, 2019).

Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. This study combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. A reduction was observed in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, these results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation (Hu, 2019).

In almost all animals, mature oocytes are arrested in meiosis at the end of oogenesis and require an external trigger to be activated and transition to start embryogenesis. This 'egg activation' involves multiple events that transition eggs to embryogenesis, including meiosis resumption and completion, maternal protein modification and/or degradation, maternal mRNA degradation or translation, and egg envelope changes (Hu, 2019).

Triggers of egg activation vary across species. In vertebrate and some invertebrate species, fertilization triggers egg activation. However, changes in pH, ionic environment, or mechanical pressure can also trigger egg activation in other invertebrate species. A conserved response to these triggers is a rise of intracellular free Ca2+ levels in the oocyte. This calcium rise is due to influx of external calcium and/or release from internal storage, depending on the organism. The elevated Ca2+ concentration is thought to activate Ca2+- dependent kinases and/or phosphatases, which in turn change the phospho-proteome of the activated egg, initiating egg activation events (Hu, 2019).

Drosophila eggs activate independent of fertilization and the trigger is mechanical pressure. When mature oocytes exit the ovary and enter the lateral oviduct, they experience mechanical pressure from reproductive tract tract. As the oocytes swell due to the influx of oviductal fluid, their envelopes become taut. Drosophila oocytes can be activated in vitro by incubation in a hypotonic buffer, although some egg activation events do not proceed completely normally in vitro. Intracellular calcium levels rise in oocytes occur egg activation, as observed with the calcium sensor GCaMP. This calcium rise takes the form of a wave that starts at the oocyte pole(s) and traverses the entire oocyte. Initiation of this calcium wave requires influx of external Ca2+, as chelating external Ca2+ in in vitro egg activation assays blocks the calcium wave and egg activation. Propagation of the calcium wave relies on the release of internal Ca2+ stores, likely through an Inositol 1,4,5-trisphosphate (IP3) mediated pathway, as knocking down the endoplasmic reticulum (ER) calcium channel IP3 receptor (IP3R) prevents propagation of the calcium wave (Hu, 2019).

How mechanical forces trigger calcium entry during Drosophila egg activation was unknown. However, the lack of initiation of a calcium wave in the presence of Gd3+, an inhibitor of mechanosensitive ion channels, and N-(p-Amylcinnamoyl) anthranilic acid (ACA), an inhibitor of TRP-family ion channels, suggested that TRP family ion channels are likely involved. Further supporting this idea is the recent discovery that a TRP family channel, TRPM7, is needed for calcium influx that leads to calcium oscillations in activating mouse eggs. The Drosophila genome encodes 13 TRP family channels, but according to RNAseq data, only 3 (Painless, Trpm, and Trpml) are expressed in the ovary. This study used specific inhibitors, existing mutants, germline- specific RNAi knockdown, and new knockouts that were created with CRISPR/Cas9 to screen these 3 candidates for their roles in the initiation of the calcium wave. Trpm, the single Drosophila ortholog of mouse TRPM7, was shown to mediate the calcium wave initiation, whereas the other two TRP channels are not necessary to initiate the calcium wave (Hu, 2019).

Calcium wave phenotypes are normal in oocytes from pain or trpml null mutants. However, the frequency of the calcium wave is diminished in wildtype oocytes in the presence of Trpm inhibitors and in oocytes from trpm germline knockdown or knockout mutants. These results consistently indicated that Trpm mediates the calcium influx that initiates the calcium wave during Drosophila egg activation. trpm germline knockout females also displayed significantly decreased egg hatchability, due to defects after cell cycle resumption. The reduced hatchability suggested that maternal trpm function or the calcium wave is required for further embryogenesis after egg activation (Hu, 2019).

TRP family ion channels are non-selective and respond to a wide array of environmental stimuli. Drosophila Trpm has been reported to play multiple roles throughout larval development, including maintaining Mg2+ and Zn2+ homeostasis (Georgiev, 2010; Hofmann, 2010), and sensing noxious cold in larval Class III md neurons (Turner, 2016). However, the role of Trpm in reproduction had not been investigated because of the pupal lethality of trpm null mutants. In this study germline specific RNAi knockdown and CRISPR/Cas9 mediated knockout revealed three novel functions of Drosophila Trpm: supporting early oogenesis, mediating influx of environmental calcium to initiate the calcium wave during egg activation, and maternally supporting embryonic development after egg activation (Hu, 2019).

A previous study suggested that calcium influx during Drosophila egg activation is mediated through mechanosensitive ion channels (Homer, 2008). Both Drosophila Trpm and its mouse ortholog TRPM7 are reported to be constitutively active and permeable to a wide range of divalent cations (Georgiev, 2010). Mouse TRPM7 is known to respond to mechanical pressure, but further study will be needed to determine whether Drosophila Trpm is similarly responsive to mechanical triggers, such as those that occur during ovulation (Hu, 2019).

Germline knockout of trpm significantly reduced the frequency of observing calcium waves in in vitro egg activation assays and egg hatchability. However, this reduced egg hatchability was not due to failure of cell cycle resumption during egg activation. There are two possible explanations for the reduced egg hatchability of trpm germline knockout females (Hu, 2019).

First, it is possible that trpm plays a maternal role, independent of its role in initiating the calcium wave, such that lack of maternally deposited Trpm proteins leads to defects during embryogenesis. In mouse, TRPM7 is also required for normal early embryonic development, apart from its role in calcium oscillations. Inhibition of TRPM7 function impairs pre-implantation embryo development and slows progression to the blastocyst stage. Drosophila trpm mutant lethality had been reported to occur during the pupal stage. However, those homozygous mutants were offspring of heterozygous mothers, and thus did not lack maternal Trpm function. Germline specific depletion of trpm reveals a maternal role for Trpm in embryogenesis (Hu, 2019).

Alternatively, or in addition, it is possible that oocytes lacking Trpm do not take up sufficient Ca2+ from the environment to form a calcium wave, but that at least some events of egg activation can occur despite this. In mouse, an initial calcium rise is induced by sperm-delivered PLCζ via the IP3 pathway. Yet although sperm from PLCζ null males fails to trigger normal calcium oscillations, some eggs fertilized by those sperm develop. Multiple oscillations following fertilization require influx of external calcium, mediated by TRPM7 and CaV3.2 (Bernhardt, 2018). Even though these oscillations were reported to be needed for multiple post-fertilization events, some TRPM7 and CaV3.2 double-knockout embryos still develop, albeit not completely normally. Together, these data suggest that egg activation can still occur in mouse with diminished intracellular calcium rises, analogous to what is seen in Drosophila in the absence of maternal Trpm function (Hu, 2019).

Insufficient influx of calcium in the absence of Trpm function could disrupt later (but maternally- dependent) embryogenesis. The oocyte-to-embryo transition involves multiple events. In mouse egg activation, these events take place sequentially as calcium oscillations progress, with developmental progression associated with more oscillations and more total calcium signal. Some of the events start after a certain number of oscillations but require additional oscillations to complete. It is possible that mechanisms critical for Drosophila embryo development also depend on reaching a precise level of calcium. A low-level calcium rise might be sufficient to trigger some egg activation mechanisms such as vitelline membrane crosslinking and cell cycle resumption, but high-levels of calcium may be required for further progression (Hu, 2019).

Given the importance of calcium in egg activation, it was surprising that although trpm knockout eggs lacked a calcium wave in vitro, in vivo such eggs could progress in cell cycles and even, sometimes, hatch. There may be insufficient calcium influx in the absence of Trpm for full and efficient development, but some egg activation events may still occur. Alternatively, it is possible that redundant mechanisms permit a sufficient calcium-level increase without producing a detectable wave form. Despite being able to trigger a series of egg activation events including meiosis resumption and protein translation, osmotic pressure during in vitro activation may have different properties from mechanical pressure exerted on mature oocytes during ovulation. The latter might allow opening of other calcium channels to initiate a normal calcium rise and complete egg activation. Two channels, TRPM7 and Cav3.2, are needed for the calcium oscillations following mouse fertilization, but the Drosophila ortholog of mouse Cav3.2, Ca-α1T, is not detectably expressed in fly ovaries. Other unknown channels might play this redundant role in vivo. In this light it is noted that levels of basal GCaMP fluorescence varied among oocytes incubated in IB that were inhibited from forming a wave, suggesting the possibility of a calcium increase by a redundant mechanism (Hu, 2019).

It was intriguing that Drosophila Trpm is essential for the calcium rise at egg activation, and that its mouse ortholog, TRPM7, was recently reported to be required (along with CaV3.2) for the calcium influx needed for post-fertilization calcium oscillations that are in turn required for egg activation events. This apparent conservation in mechanisms in egg activation involving orthologous Trpm channels in a protostome (Drosophila) and a deuterostome (mouse) prompts asking whether Trpm-mediated calcium influx is a very ancient and basal aspect of egg activation, with other more variable aspects such as sperm-triggered calcium rises being more derived, if better known, features. It is interesting in this light that a sperm-delivered TRP channel (TRP-3) has also been reported to mediate calcium influx and a calcium rise in another protostome, C. elegans (Hu, 2019).

This study has shown that before egg activation, the cortical actin marker Moesin is less concentrated at the posterior end of the oocyte where calcium waves generally initiate and decrease in concentration at the cortex. It is possible that lack of actin rigidity is linked to calcium channel opening: The initial sparse site of actin allows calcium channels to open and leads to calcium influx, which in turn regulates the dynamic changes in actin to form a wavefront following calcium wave. The interlinked nature of these two pathways suggests a positive feedback loop that facilitates the progression and completion of the waves. This could be achieved as follows: (a) actin disperses and calcium enters the oocyte; (b) this calcium rise causes further actin dispersion through regulation of actin-binding proteins; (c) more calcium then enters the activating egg which results in the complete calcium wave and actin reorganization in a wavelike manner. Overall, it is proposed that in Drosophila egg activation, actin is able to modulate the intracellular calcium rise, and calcium waves in turn regulate the reorganization of actin (York-Andersen, 2019).

Early work on de novo gene discovery in Drosophila was consistent with the idea that many such genes have male-biased patterns of expression, including a large number expressed in the testis. However, there has been little formal analysis of variation in the abundance and properties of de novo genes expressed in different tissues. This study investigated the population biology of recently evolved de novo genes expressed in the D. melanogaster accessory gland, a somatic male tissue that plays an important role in male and female fertility and the post mating response of females, using the same collection of inbred lines used previously to identify testis-expressed de novo genes, thus allowing for direct cross tissue comparisons of these genes in two tissues of male reproduction. Using RNA-seq data this study identified candidate de novo genes located in annotated intergenic and intronic sequence and determine the properties of these genes including chromosomal location, expression, abundance, and coding capacity. Generally, major differences were found between the tissues in terms of gene abundance and expression, though other properties such as transcript length and chromosomal distribution are more similar. Differences between regulatory mechanisms of de novo genes in the two tissues were also explored and how such differences may interact with selection to produce differences in D. melanogaster de novo genes expressed in the two tissues (Cridland, 2021).

Vitellogenesis (yolk accumulation) begins upon eclosion and continues through the process of sexual maturation. Upon reaching sexual maturity, vitellogenesis is placed on hold until it is induced again by mating. However, the mechanisms that gate vitellogenesis in response to developmental and reproductive signals remain unclear. This study has identified the neuropeptide allatostatin-C (AstC)-producing neurons that gate both the initiation of vitellogenesis that occurs post-eclosion and its re-initiation post-mating. During sexual maturation, the AstC neurons receive excitatory inputs from Sex Peptide Abdominal Ganglion (SAG) neurons. In mature virgin females, high sustained activity of SAG neurons shuts off vitellogenesis via continuous activation of the AstC neurons. Upon mating, however, Sex Peptide inhibits SAG neurons, leading to deactivation of the AstC neurons. As a result, this permits both JH biosynthesis and the progression of vitellogenesis in mated females. This work has uncovered a central neural circuit that gates the progression of oogenesis (Zhang, 2022).

Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. This study demonstrates that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc (Hu, 2020a).

The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho-proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. This study shows that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. Neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels (Hu, 2020b).

Fertility depends on the progression of complex and coordinated postmating processes within the extracellular environment of the female reproductive tract (FRT). Molecular interactions between ejaculate and FRT proteins regulate many of these processes, including sperm motility, migration, storage, and modification, along with concurrent changes in the female. Although extensive progress has been made in the proteomic characterization of male-derived components of sperm and seminal fluid, investigations into the FRT have remained more limited. To achieve a comparable level of knowledge regarding female-derived proteins that comprise the reproductive environment, this study utilized semiquantitative mass spectrometry-based proteomics to study the composition of the FRT tissue and, separately, the luminal fluid, before and after mating in Drosophila melanogaster. This approach leveraged whole-fly isotopic labelling to delineate female proteins from those transferred male ejaculate proteins. the results revealed several characteristics that distinguish the FRT fluid proteome from the FRT tissue proteome: the fluid proteome is encoded by genes with higher overall levels of FRT gene expression and tissue specificity, including many genes with enriched expression in the fat body, fluid-biased proteins are enriched for metabolic functions and the fluid exhibits pronounced postmating compositional changes. The dynamic mating-induced proteomic changes in the FRT fluid informs understanding of secretory mechanisms of the FRT, serves as a foundation for establishing female contributions to the ejaculate-female interactions that regulate fertility and highlights the importance of applying proteomic approaches to characterize the composition and dynamics of the FRT environment (McDonough-Goldstein, 2021).

This study shows that the precocious onset of mitochondrial respiratory quiescence causes a reprogramming of progeny metabolic state. The premature onset of mitochondrial respiratory quiescence drives the lowering of Drosophila oocyte NAD⁺ levels. NAD⁺ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine in embryos and lower levels of histone H3 lysine 27 trimethylation, resulting in enhanced intestinal lipid metabolism in progeny. In addition, this study shows that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells (Hocaoglu, 2021).

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. This study used physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. T he swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na(+) channels. Furthermore, through pharmacological and genetic disruption, this study reveals a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. These data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants (York-Andersen, 2021).

Discs large 5 (Dlg5) is a member of the MAGUK family of proteins that typically serve as molecular scaffolds and mediate signaling complex formation and localization. In vertebrates, Dlg5 has been shown to be responsible for polarization of neural progenitors and to associate with Rab11-positive vesicles in epithelial cells. In Drosophila, however, the function of Dlg5 is not well-documented. This study identified dlg5 as an essential gene that shows embryonic lethality. dlg5 embryos display partial loss of primordial germ cells (PGCs) during gonad coalescence between stages 12 and 15 of embryogenesis. Loss of Dlg5 in germline and somatic stem cells in the ovary results in the depletion of both cell lineages. Reduced expression of Dlg5 in the follicle cells of the ovary leads to a number of distinct phenotypes, including defects in egg chamber budding, stalk cell overgrowth, and ectopic polar cell induction. Interestingly, loss of Dlg5 in follicle cells results in abnormal distribution of a critical component of cell adhesion, E-cadherin, shown to be essential for proper organization of egg chambers (Reilly, 2015).

dlg5 was shown to be essential for normal division or maintenance of FSCs and GSCs, and is required in later stage follicle cells. Reduction of Dlg5 levels disrupts egg chamber formation, indicating that dlg5 is involved in processes fundamental to egg chamber organization (Reilly, 2015).

When Dlg5 was depleted in follicle cells for 4 d, epithelial follicle cells showed relatively normal cell shape and polarity, but the polar and stalk cells showed strong abnormalities in number, localization, and overall organization. The induction of ectopic polar cells and stalk cell overgrowth observed in these ovaries is significant because it has been suggested that the differentiation of these two cell types are controlled by similar signaling events, which distinguishes them from the other follicle cells. Loss of Dlg5 may interfere with the pathways that specify stalk and polar cell differentiation and maturation and, consequently, egg chamber organization (Reilly, 2015).

The significantly enhanced phenotype observed upon depletion of Dlg5 in follicle cells for 10 d agrees with the dlg5^D48 clonal phenotype and is consistent with the proposition that the function of dlg5 is completely lost in dlg5^D48. Further, complete loss of dlg5 function results in embryonic lethality, possibly also as a result of abnormal organization and integration of specific embryonic cells (Reilly, 2015).

Analysis of primitive embryonic gonad formation in dlg5^D48 embryos suggested that it likely functions during germ cell migration and gonad coalescence. These data are reminiscent of requirements of Dlg5 in the maintenance of the cohesion of migrating border cells in the ovary (Aranjuez, 2012). In this regard, it is interesting to note that proper gonad coalescence has been shown to depend on cell adhesion molecule E-cadherin. E-cadherin is consistently upregulated during late stages of gonad formation. Because loss of dlg5 results in altered distribution of E-cadherin in follicle cells, it is conceivable that aberrant E-cadherin levels and/or localization could also contribute to the embryonic gonad-specific phenotypes. In this context, it will be interesting to analyze further the precise requirement and cell-type specificity of Dlg5 function in controlling cell adhesion and migration (Reilly, 2015).

Many of the dlg5 phenotypes could result from aberrant E-cadherin distribution. For instance, oocyte mislocalization is often seen as a result of loss or aberrant homophilic adhesion mediated by E-cadherin between posterior follicle cells and the oocyte. Formation of interfollicular stalks is also dependent on dynamic E-cadherin accumulation: E-cadherin accumulates first at the apical boundary of prefollicular cells, followed by the establishment of lateral cell contacts to initiate and complete intercalation to form a single wide stalk. Additionally, E-cadherin has been demonstrated to be required for recruiting and anchoring stem cells to their niche prior to adulthood in the Drosophila ovary. By clonal analysis, this study showed that both germline and follicle dlg5 ovary stem cells are unable to give rise to normal daughter cells, indicating that the gene is essential in these cells. This may suggest several possibilities. There may be a cell-autonomous requirement for dlg5 in follicle cells; however, the loss of stem cells may also be an indication of the requirement for E-cadherin-mediated adhesion to the stem cell niche. Finally, the lack of cohesion in migrating germ cells in dlg5^D48 embryos is consistent with a perturbation in cell-cell adhesion, as described above. The observed phenotypes, therefore, and the role of Dlg5 in E-cadherin distribution may be related; however, more work is needed to determine the nature of the participation of Dlg5 in E-cadherin distribution before any further conclusions may be drawn (Reilly, 2015).

Dlg5 is involved in vesicle trafficking in vertebrates and has been reported to colocalize with several Rab GTPases. The punctate distribution of Dlg5 in the Drosophila ovary is consistent with a similar association of the protein with endosomes. Therefore, it seems possible that Dlg5 is involved in endosomal trafficking. The abnormal distribution of E-cadherin, which is recycled through the endosome, observed in Dlg5 KD ovaries supports this hypothesis. Further, reduction of Dlg5 in the mammalian epithelial cell line LLc-PK1 resulted in lower levels of E-cadherin, but it is not clear how Dlg5 controls E-cadherin levels (Reilly, 2015).

Thus, Dlg5, like its human homolog and Dlg1, may be involved in endosomal recycling of E-cad. But based on this characterization of dlg5 and its functional requirement, there are fundamental differences between these two genes. Although dlg5 shows embryonic lethality and is essential in ovarian stem cells, dlg1 larvae can survive for >10 d and show overgrowth phenotypes in larvae and follicle cells. Persistent dlg1 germ line clones develop into eggs, whereas follicle cells lacking dlg1 sometimes show tumor-like invasion into the interior of the egg chamber, a phenotype this study not observe in Dlg5 KD. The task of figuring out what each of these Dlg proteins contributes to apical protein trafficking and cell survival should prove informative and stimulating (Reilly, 2015).

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In this study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, s36 chorionic protein regulates the number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress (Velentzas, 2016).

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. This study reports that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). It was shown that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. These findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms (Ulmschneider, 2016).

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, a genetic screen through a collection of 126 mutants in essential genes on the X chromosome was performed to identify candidates that increase or decrease competition for the FSC niche. Approximately 55% and 6% of the mutations screened were obtained as putative FSC hypo- or hypercompetitors, respectively. A large majority of mutations were found in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypocompetition alleles, and the hypocompetition phenotype was confirmed for four of these alleles. Sec16 and another COP II vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, it was demonstrated that although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells (CySCs) of the testis, there are important tissue-specific differences. These results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and a number of putative candidates for further exploration were identified (Cook, 2017).

The follicle stem cells in the Drosophila ovary are a highly tractable model of stem cell niche competition. The Drosophila ovary is comprised of long strands of developing follicles, called ovarioles, and a pair FSCs resides at the anterior tip of each ovariole in a structure called the germarium. These FSCs divide during adulthood to provide the follicle cells that surround germ cell cysts during follicle formation. FSCs are regularly lost and replaced during adulthood, and several studies have identified genes that increase the rate of FSC loss. In most cases, the mutations investigated in these studies disrupt the ability of the mutant FSC to adhere to the niche or transduce niche signals and thus are presumed to cause the mutant stem cell to be lost in a cell-autonomous manner. However, the suggestion that stem cells may compete with the daughters of neighboring stem cells for niche occupancy raises the possibility that a mutation in a competing mutant lineage could act in a noncell-autonomous manner to influence the likelihood that a neighboring wild-type lineage will be lost and replaced (Cook, 2017).

This study has generated a new resource for investigating the genetic basis of stem cell niche competition. The collection of confirmed and candidate competition mutations demonstrates the breadth of cell functions that influence niche competition, and will allow for further study into many different facets of this process. One hyper-competition mutation and seven additional candidate hyper-competition mutations were generated in genes involved in a variety of functions, including mitochondrial function (sicily, tumor suppression (Rbf), and protein ubiquitination (bendless). The diversity of this list of candidates suggests that effects on many different cellular processes can lead to a hyper-competition phenotype. Yet the vast majority of mutations that were studied do not cause hyper-competition, suggesting that the ultimate cause(s) of hyper-competition are more constrained, and that the mechanism of hyper-competition may be similar in these diverse mutants (Cook, 2017).

A previously unstudied aspect of niche competition revealed by this screen is the involvement of vesicle trafficking genes. The Sec16^A, shi^A, wus^A, and Crag^A mutations that that were examined in this study represent a broad range of vesicle trafficking functions. Specifically, Sec16 is a central regulator of exocytosis, whereas wurst and shibire function primarily during endocytosis, and Crag is required in follicle cells for trafficking of basement membrane components to the basal surface. The shi^A (K10X) and wus^A (Q307X) alleles, which produced the most severe effects on DE-cad localization, contain nonsense mutations and are most likely to be amorphic alleles; while Crag^A (C1372S) and Sec16^A (P1926S) contain missense mutations and are more likely to be hypomorphic alleles. Comparison of the relatively mild phenotypes caused by homozygosity for Sec16^A to the more severe phenotypes caused by RNAi knockdown of Sec16^A further support the conclusion that Sec16^A is a hypomorphic allele. Interestingly, despite these differences, all four mutations caused strong hypo-competition phenotypes in the FSC lineage. This suggests that the process of niche competition is very sensitive to the function of these genes and is able to efficiently eliminate stem cell lineages with even only mild defects in vesicle trafficking (Cook, 2017).

The finding of this study suggest at least two possible reasons why impaired vesicle trafficking causes hypo-competition. First, DE-cad is known to be required for FSC maintenance, so the disruption of DE-cad localization to the membrane in vesicle trafficking mutants could at least partially account for the hypo-competition phenotype. Second, vesicle trafficking is required for functional EGFR signaling in MDCK cells, and this study found decreased pERK levels in the vesicle trafficking mutant clones. Since EGFR signaling is also essential for FSC self-renewal, the reduction in EGFR signaling may be another factor that contributes to the hypo-competition phenotype. However, EGFR signaling is normally downregulated as cells exit the FSC niche region, so it is unclear whether the decreased pERK levels in these mutant clones are a cause of the hyper-competition phenotype or a consequence of reduced association with the niche (for example, because DE-cad levels on the membrane are low). In addition, it is also unclear why the loss of detectable pERK in these mutant clones is not associated with a cell polarity defect, as has been observed in EGFR null clones. It may be that the vesicle trafficking alleles investigated in this study reduce the levels of EGFR signaling to a level that is below the limit of detection but sufficient to maintain cell polarity; or that the vesicle trafficking mutants impair the branch of EGFR signaling leading to ERK phosphorylation, but not the branch going through LKB1 and AMPK that is important for the maintenance of cell polarity in the FSC lineage. Alternately, it could be that the decrease in EGFR signaling is more gradual in the vesicle trafficking mutant clones than it is in EGFR mutant clones, so the vesicle trafficking mutant clones are able to grow normally for some time before EGFR signaling decreases to the point at which it is both undetectable by pERK staining and unable to promote FSC self-renewal or follicle cell polarity. Additional studies of these and other FSC niche competition mutants identified here will be important to clarify these issues (Cook, 2017).

In the testis, knockdown of Sec16 also affected CySC niche competition rather than self-renewal or differentiation, whereas knockdown of shi likely impaired CySC self-renewal or survival. Interestingly, knockdown of Crag had no effect on CySC retention in the niche but caused an unusual differentiation defect in which mutant cells failed to move out of the niche region. These differences indicate that there are tissue-specific aspects to the process of self-renewal and niche competition, and provide additional evidence that the hypo-competition phenotypes observed in the FSC niche are not due to a generic defect that would affect all cells equally. Overall, these studies demonstrate that mutations in multiple types of vesicle trafficking genes cause hypo-competition in both the ovary and testis. Vesicle trafficking is essential for a diverse array of cellular functions, and thus may function as a node, integrating outputs from these different cellular functions into a readout that influences the overall fitness of the cell for occupying the niche. Further investigation will make it possible to organize these and other mutations that cause niche competition phenotypes into common pathways and, ultimately, to understand whether and how stem cell niche competition promotes the maintenance of a healthy population of stem cells in each tissue throughout adulthood (Cook, 2017).

In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. This study found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. These results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division (Mathieu, 2022).

Monensin-sensitive 1 (Mon1) is an endocytic regulator that participates in the conversion of Rab5-positive early endosomes to Rab7-positive late endosomes. In Drosophila, loss of mon1 leads to sterility as the mon1 mutant females have extremely small ovaries with complete absence of late stage egg chambers - a phenotype reminiscent of mutations in the insulin pathway genes. This study shows that expression of many Drosophila insulin-like peptides (ILPs) is reduced in mon1 mutants and feeding mon1 adults an insulin-rich diet can rescue the ovarian defects. Surprisingly, however, mon1 functions in the tyramine/octopaminergic neurons (OPNs) and not in the ovaries or the insulin-producing cells (IPCs). Consistently, knockdown of mon1 in only the OPNs is sufficient to mimic the ovarian phenotype, while expression of the gene in the OPNs alone can 'rescue' the mutant defect. Last, ilp3 and ilp5 were assessessed as critical targets of mon1. This study thus identifies mon1 as a novel molecular player in the brain-gonad axis and underscores the significance of inter-organ systemic communication during development (Dhiman, 2019).

Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. This study analysed stage-specific gradients of pHi and Vmem. Distinct alterations were found of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, inhibitors were used of Na⁺/H⁺-exchangers and Na⁺-channels, V-ATPases, ATP-sensitive K⁺-channels, voltage-dependent L-type Ca²⁺-channels, Cl(-)-channels and Na⁺/K⁺/2Cl(-)-cotransporters. Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na⁺/H⁺-exchangers and amiloride-sensitive Na⁺-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na⁺/K⁺/2Cl(-)-cotransporters or ATP-sensitive K⁺-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca²⁺-channels or ATP-sensitive K⁺-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, strong influences were found on the A-P and D-V pHi- and/or Vmem-gradients. These data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation (Weiss, 2019).

During Drosophila oogenesis, the follicular epithelium differentiates into several morphologically distinct follicle-cell populations. Characteristic bioelectrical properties make this tissue a suitable model system for studying connections between electrochemical signals and the organisation of the cytoskeleton. This study analysed the patterns of basal microfilaments (bMF) and microtubules (MT) in relation to electrochemical signals. The bMF- and MT-patterns in developmental stages 8 to 12 were visualised using labelled phalloidin and an antibody against acetylated alpha-tubulin as well as follicle-cell specific expression of GFP-actin and GFP-alpha-tubulin. In order to test whether cytoskeletal modifications depend directly on bioelectrical changes, inhibitors of ion-transport mechanisms were used that have previously been shown to modify pHi and Vmem as well as the respective gradients. While relative alkalisation and/or hyperpolarisation stabilised the parallel transversal alignment of bMF, acidification led to increasing disorder and to condensations of bMF. On the other hand, relative acidification as well as hyperpolarisation stabilised the longitudinal orientation of MT, whereas alkalisation led to loss of this arrangement and to partial disintegration of MT. It is conclude that the pHi- and Vmem-changes induced by inhibitors of ion-transport mechanisms simulate bioelectrical changes occurring naturally and leading to the cytoskeletal changes observed during differentiation of the follicle-cell epithelium. Therefore, gradual modifications of electrochemical signals can serve as physiological means to regulate cell and tissue architecture by modifying cytoskeletal patterns (Weiss, 2019).

The follicle stem cells (FSCs) in the Drosophila ovary are an important experimental model for the study of epithelial stem cell biology. Although decades of research support the conclusion that there are two FSCs per ovariole, a recent study used a novel clonal marking system to conclude that there are 15-16 FSCs per ovariole. This study performed clonal analysis using both this novel clonal marking system and standard clonal marking systems, and several problems were identified that may have contributed to the overestimate of FSC number. In addition, new methods were developed for accurately measuring clone size, and FSC clones were found to produce, on average, half of the follicle cells in each ovariole. These findings provide strong independent support for the conclusion that there are typically two active FSCs per ovariole, though they are consistent with up to four FSCs per germarium (Fadiga, 2019).

How extracellular matrix participates to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. This study found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell autonomous control of F-actin fibers orientation relies on the previous BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a critical organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation (Campos, 2020).

Deciphering the mechanisms underlying tissue morphogenesis is crucial for fundamental understanding of development and also for regenerative medicine. Building organs generally requires the precise modeling of a basement membrane extracellular matrix (ECM), which in turn can influence tissue shape. However, the mechanisms driving the assembly of a specific basement membrane (BM) and how this BM then feeds forward on morphogenesis are still poorly understood. Drosophila oogenesis offers one of the best tractable examples in which such a morphogenetic process can be studied. Each ovarian follicle, which is composed of a germline cyst surrounded by the somatic follicular epithelium, undergoes a dramatic growth, associated with tissue elongation, starting from a little sphere and ending with an egg in which the anteroposterior (AP) axis is 3-fold longer than the mediolateral (ML) axis. This elongation is roughly linear from the early to the late stages, but can be separated in at least two mechanistically distinct phases. The first phase (from stage 3 to stage 8; hereby 'early stages') requires a double gradient of JAK-STAT pathway activity that emanates from each pole and that controls myosin II-dependent apical pulsations. In the second phase, from stage 7-8, elongation depends on the atypical cadherin Fat2 that is part of a planar cell polarity (PCP) pathway orienting the basal domain of epithelial follicle cells. Earlier during oogenesis, Fat2 gives a chirality to the basal domain cytoskeleton in the germarium, the structure from which new follicles bud. This chirality is required to set up a process of oriented collective cell migration perpendicularly to the elongation axis that induces follicle revolutions from stage 1 to stage 8. From each migrating cell, Fat2 also induces, in the rear adjacent cell, the formation of planar-polarized protrusions that are required for rotation. These rotations allow the polarized deposition of BM fibrils, which involves a Rab10-dependent secretion route targeted to the lateral domain of the cells. These BM fibrils are detectable from stage 4 onwards and persist until late developmental stages. Follicle rotation also participates in the planar cell polarization of integrin-dependent basal stress fibers that are oriented perpendicularly to the AP axis. Moreover, at stage 7-8, a gradient of matrix stiffness controlled by the JAK-STAT pathway and Fat2 contributes to elongation. Then, from stage 9, the epithelial cell basal domain undergoes anisotropic oscillations, as a result of periodic contraction of the oriented stress fibers, which also promotes follicle elongation . To explain the impact of fat2 mutations on tissue elongation, it is generally accepted that oriented stress fibers and BM fibrils act as a molecular corset that constrains follicle growth in the ML axis and promotes its elongation along the AP axis. However, the exact contribution of F-actin versus BM to this corset is still unclear, as is whether the orientations of stress fibers and of BM fibrils are causally linked (Campos, 2020).

This analyzed the function of Dystrophin (Dys) and Dystroglycan (Dg) during follicle elongation. Dys and Dg are the two main components of the Dystrophin-associated protein complex (DAPC), an evolutionarily conserved transmembrane complex that links the ECM (via Dg) to the F-actin cytoskeleton (via Dys). This complex is expressed in a large variety of tissues and is implicated in many congenital degenerative disorders. Loss-of-function studies in model organisms have revealed an important morphogenetic role for Dg during development, usually linked to defects in ECM secretion, assembly or remodeling . A developmental role for Dys is less clear, possibly because of the existence of several paralogs in vertebrates. As Drosophila has only one Dg and one Dys gene, it is a promising model for their functional study during development and morphogenesis (Campos, 2020).

Dg and Dys were found to be required for follicle elongation and proper BM fibril formation early in fly oogenesis. During these early stages, DAPC loss and hypomorphic fat2 conditions similarly delay stress fiber orientation. However, DAPC promotes this alignment more locally than Fat2. Moreover, DAPC genetically interacts with fat2 in different tissues, suggesting that they belong to a common morphogenetic network. Later in oogenesis, Dg and Dys are required for stress fiber orientation in a cell-autonomous manner. This is the period when the main elongation defect is seen in these mutants, arguing for a more determinant role for stress fibers compared with BM fibrils in the elongation process. Nonetheless, this latter function depends on the earlier DAPC function in BM fibril deposition. It is proposed that BM fibrils serve as a PCP memory for the late stages that are used as a template by the DAPC for F-actin stress fiber alignment (Campos, 2020).

Genetic data has already demonstrated that follicle elongation relies on at least two different and successive mechanisms. The first is controlled by JAK-STAT and involves the follicle cell apical domain, whereas the second is controlled by Fat2 and involves the basal domain and the BM. Between these phases, around stage 7-8, JAK-STAT and Fat2 seem to be integrated in a third mechanism based on a BM stiffness gradient. Interestingly, a very recent report suggests that this gradient may not directly influence tissue shape but rather do so by modifying the properties of the follicle cells underneath. This study shows that the DAPC influences elongation mainly at very late stages, suggesting the existence of a fourth mechanistic elongation phase. Of note, elongation at these late stages is also defective in fat2 mutants. This is consistent with the fact that rotation is required for polarized BM fibril deposition, and that this deposition depends on and is required for DAPC function. The existence of multiple and interconnected mechanisms to induce elongation, a process that initially appeared to be very simple, highlights the true complexity of morphogenesis, and the necessity to explore it in simple models (Campos, 2020).

Fat2 is clearly part of the upstream signal governing the basal planar polarization. However, how this polarization leads to tissue elongation is still debated. It has been proposed that elongation relies on a molecular corset that could be formed, non-exclusively, by BM fibrils or F-actin stress fibers. The initial observation that rotation is required for both elongation and BM fibrils favored a direct mechanical role for these structures. Recent data showing that BM fibrils are stiffer than the surrounding ECM supports this view. Moreover, increasing the BM fibril number and size can lead to hyper-elongation. Finally, addition of collagenase induces follicle rounding, at least at some stages, and genetic manipulation of the ECM protein levels also influences elongation. However, these experiments did not discriminate between the function of the fibril fraction and a general BM effect. Moreover, they do not demonstrate whether their impact on elongation is direct and mechanic or, indirect by a specific response of the epithelial cells. Fat2 and rotation are also required for the proper orientation of the stress fibers. The F-actin molecular corset is dynamic with follicle cells undergoing basal pulsations, and perturbation of both these oscillations and of the stress fiber structure affect elongation. In the DAPC mutants, a faint but significant elongation defect was observed during mid-oogenesis and a stronger one after stage 12. These defects are clearly correlated with the stress fiber orientation defects observed in the same mutants, both temporally and in terms of intensity. Moreover, although overexpression of Rab10 in a Dys loss-of-function mutant restores BM fibrils, it does not rescue elongation, indicating that stress fiber orientation is instrumental (Campos, 2020).

Thus, if the role of the BM fibrils as a direct mechanical corset appears limited, what is their function? One possibility could have been that they promote rotation, acting by positive feedback and explaining the speed increase over time. However, the rotation reaches the same speed in WT and DAPC mutants, excluding this possibility. Similarly, increasing the fibril fraction also has no effect on rotation speed (Campos, 2020).

The results strongly argue that BM fibrils act as a cue for the orientation of stress fibers, which then generate the mechanical strain for elongation. This appears clear in late stages when the function of the DAPC for stress fiber orientation is dependent on the previous BM fibril deposition. Although it is unknown why the cells lose their orientation from stages 10 to 12, the BM fibrils provide the long-term memory of the initial PCP of the tissue, allowing stress fiber reorientation. Such a mechanism appears to be a very efficient way to memorize positional cues, and could represent a general BM function in many developmental processes (Campos, 2020).

DAPC was found to impact the two key actors at the basal domain of the follicle cells: the BM and the stress fibers linked to the BM. All the defects of Dg null mutants were also observed in Dys mutants, demonstrating a developmental and morphogenetic role for this gene. In vertebrates, at least in some tissues, Dg presence appears to be essential for BM assembly. However, BM formation on the follicular epithelium does not require Dg, suggesting the existence of alternative platforms for its general assembly. The genetic data suggest that Rab10 is epistatic to Dg for BM fibril deposition. The usual interpretation of such a result would be that Dg is involved in the targeting of ECM secretion upstream of Rab10 rather than in ECM assembly in the extracellular space. In Caenorhabditis elegans, Dg acts as a diffusion barrier to define a precise subcellular domain for ECM remodeling. One could imagine that the DAPC has a similar function in follicle cells, by defining the position where the Rab10 secretory route is targeted. However, in DAPC loss of function, some ECM is still secreted between cells, suggesting that the lateral Rab10 route is not affected. Moreover, ECM proteins do not abnormally accumulate between cells in such mutants, suggesting that they are able to leave this localization but without forming BM fibrils. Therefore, the functional interplay between Rab10 and the DAPC is still unclear (Campos, 2020).

As mentioned before, Dg has often been proposed to act as a scaffold to promote BM assembly in mice. Deletion of the Dg intracellular domain is only sub-lethal in mice, whereas complete loss of this protein is lethal very early during development, indicating that the abolishment of Dg's interaction with Dys affects its function only partially. In these mice, laminin assembly can still be observed, for instance in the brain and retina. Similar results were also obtained in cultured mammary epithelial cells. Thus, despite the existence of Dys paralogs that could mask some effects on ECM and the fact that the same ECM alteration was observed in Dg or Dys mutant fly follicles, not all the Dg functions related to ECM assembly or secretion involve Dys. It is possible that Dys is required when Dg needs a very specific subcellular targeting for its function, whereas a more general role in ECM assembly would be independent of Dys. The results suggest that some specific effects of Dys on ECM could have been underestimated and this could help to explain the impact of its loss of function on tissue integrity maintenance. For instance, as it has been reported that Dg influences ECM organization in fly embryonic muscles, it would be interesting to determine whether this also involves Dys (Campos, 2020).

The DAPC is involved in planar polarization of the basal stress fibers and its ability to read ECM structure to orchestrate integrin-dependent adhesion could play a role in many developmental and physiological contexts. The link between the ECM and F-actin provided by this complex is likely required for this function, although this remains to be formally demonstrated. BM fibrils could provide local and oriented higher density of binding sites for Dg, and the alignment could then be transmitted to the actin cytoskeleton. Alternatively, DAPC function could rely on sensing the mechanical ECM properties. The hypothesis that the DAPC could act as a mechanosensor is a long-standing proposal, partly due to the presence of spectrin repeats in Dys. The basal domain of the follicle cells may offer an amenable model to combine genetics and cell biology approaches to decipher such function (Campos, 2020).

Altogether, this work provides important insights on the role of the BM during morphogenesis, by acting as a static PCP cue retaining spatial information while cells are highly dynamic. It also reveals important functions of the DAPC, including Dys, that may be broadly involved during animal development and physiology (Campos, 2020).

Drosophila melanogaster females undergo a variety of post-mating changes that influence their activity, feeding behavior, metabolism, egg production and gene expression. These changes are induced either by mating itself or by sperm or seminal fluid proteins. In addition, studies have shown that axenic females-those lacking a microbiome-have altered fecundity compared to females with a microbiome, and that the microbiome of the female's mate can influence reproductive success. This study investigated fecundity and the post-mating transcript abundance profile of axenic or control females after mating with either axenic or control males. Interactions between the female's microbiome and her mating status were observed: transcripts of genes involved in reproduction and genes with neuronal functions were differentially abundant depending on the females' microbiome status, but only in mated females. In addition, immunity genes showed varied responses to either the microbiome, mating, or a combination of those two factors. It was further observed that the male's microbiome status influences the fecundity of both control and axenic females, while only influencing the transcriptional profile of axenic females. These results indicate that the microbiome plays a vital role in the post-mating switch of the female's transcriptome (Delbare, 2020).

It is unknown how growth in one tissue impacts morphogenesis in a neighboring tissue. To address this, the Drosophila ovarian follicle, in which a cluster of 15 nurse cells and a posteriorly located oocyte are surrounded by a layer of epithelial cells, was used. It is known that as the nurse cells grow, the overlying epithelial cells flatten in a wave that begins in the anterior. This study demonstrates that an anterior to posterior gradient of decreasing cytoplasmic pressure is present across the nurse cells and that this gradient acts through TGFβ to control both the triggering and the progression of the wave of epithelial cell flattening. These data indicate that intrinsic nurse cell growth is important to control proper nurse cell pressure. Finally, it was revealed that nurse cell pressure and subsequent TGFβ activity in the stretched cells combine to increase follicle elongation in the anterior, which is crucial for allowing nurse cell growth and pressure control. More generally, the results reveal that during development, inner cytoplasmic pressure in individual cells has an important role in shaping their neighbors (Lamire, 2020).

Programmed cell death and cell corpse clearance are an essential part of organismal health and development. Cell corpses are often cleared away by professional phagocytes such as macrophages. However, in certain tissues, neighboring cells known as nonprofessional phagocytes can also carry out clearance functions. This study used the Drosophila melanogaster ovary to identify novel genes required for clearance by nonprofessional phagocytes. In the Drosophila ovary, germline cells can die at multiple time points. As death proceeds, the epithelial follicle cells act as phagocytes to facilitate the clearance of these cells. An unbiased kinase screen was performe to identify novel proteins and pathways involved in cell clearance during two death events. Of 224 genes examined, 18 demonstrated severe phenotypes during developmental death and clearance while 12 demonstrated severe phenotypes during starvation-induced cell death and clearance, representing a number of pathways not previously implicated in phagocytosis. Interestingly, it was found that several genes not only affected the clearance process in the phagocytes, but also non-autonomously affected the process by which germline cells died. This kinase screen has revealed new avenues for further exploration and investigation (Lebo, 2021).

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. It has been reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. The NRT-wg flies were used to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but it was found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. It is concluded that Wg protein spreads from its source cells in the germarium to promote FSC proliferation (Wang, X., 2021).

Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6-aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. This study used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion (Fic, 2021).

The form and function of epithelial cells depends on their polarization into distinct apical, lateral, and basal domains by conserved polarity factors. This polarity is then maintained by mutual antagonism between apical polarity factors such as atypical PKC (aPKC) and lateral factors such as Lethal (2) giant larvae (Lgl) and Par-1. While many aspects of the polarity machinery are now well understood, it is still unclear how the apical domain is initiated and what role cell division control protein 42 (Cdc42) plays in this process. Cdc42 was identified for its role in establishing polarity in budding yeast, where it targets cell growth to the bud tip by polarizing the actin cytoskeleton and exocytosis toward a single site. It has subsequently been found to function in the establishment of cell polarity in multiple contexts. For example, Cdc42 recruits and activates the anterior PAR complex to polarize the anterior-posterior axis in the Caenorhabditis elegans zygote and the apical-basal axis during the asymmetric divisions of Drosophila neural stem cells≈Cdc42 also plays an essential role in the apical-basal polarization of epithelial cells, where it is required for apical domain formation. Cdc42 is active when bound to GTP, which changes its conformation to allow it to bind downstream effector proteins that control the cytoskeleton and membrane trafficking. An important Cdc42 effector in epithelial cells is the Par-6-aPKC complex. Par-6 binds directly to the switch 1 region of Cdc42 GTP through its semi-CRIB domain (Cdc42 and Rac interactive binding). This induces a change in the conformation of Par-6 that allows it to bind to the C-terminus of another key apical polarity factor, the transmembrane protein Crumbs, which triggers the activation of aPKC's kinase activity. As a result, active aPKC is anchored to the apical membrane, where it phosphorylates and excludes lateral factors, such as Lgl, Par-1, and Bazooka (Baz). In addition to this direct role in apical-basal polarity, Cdc42 also regulates the organization and activity of the apical cytoskeleton through effectors such as neuronal Wiskott-Aldrich syndrome protein (N-WASP), which promotes actin polymerization, and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK; Genghis khan [Gek] in Drosophila), which phosphorylates the myosin regulatory light chain to activate contractility (Fic, 2021).

This crucial role of active Cdc42 in specifying the apical domain raises the question of how Cdc42-GTP itself is localized apically. In principle, this could involve activation by Cdc42 guanine nucleotide exchange factors (Cdc42GEFs) that are themselves apical or lateral inactivation by Cdc42GAPs. The Cdc42GEFs Tuba, intersectin 2, and Dbl3 have been implicated in activating Cdc42 in mammalian epithelia. Only Dbl3 localizes apical to tight junctions, however, as Tuba is cytoplasmic and enriched at tricellular junctions and intersectin 2 localizes to centrosomes. Thus, GEF activity may not be exclusively apical, suggesting that it is more important to inhibit Cdc42 laterally. Although nothing is known about the role of GAPs in restricting Cdc42 activity to the apical domain of epithelial cells, this mechanism plays an instructive role in establishing radial polarity in the blastomeres of the early C. elegans embryo. In this system, the Cdc42GAP PAC-1 is recruited by the cadherin adhesion complex to sites of cell-cell contact, thereby restricting active Cdc42 and its effector the Par-6-aPKC complex to the contact-free surface (Fic, 2021).

This study has analyzed the roles of Cdc42GAPs in epithelial polarity using the follicle cells that surround developing Drosophila egg chambers as a model system. By generating mutants in a number of candidate Cdc42GAPs, this study identified the Pac-1 orthologue, RhoGAP19D, as the GAP that restricts active Cdc42 to the apical domain. In the absence of RhoGAP19D, lateral Cdc42 activity leads to an expansion of the apical domain and a high frequency of epithelial invasion into the germline tissue, a phenotype that mimics the early steps of carcinoma formation (Fic, 2021).

In the absence of RhoGAP19D, both N-WASP and Gek are recruited to the lateral membrane, indicating that Cdc42 is ectopically activated there. This implies that RhoGAP19D is the major Cdc42GAP that represses Cdc42 laterally, because no other GAPs can compensate for its loss. This also suggests that the GEFs that activate Cdc42 are not restricted to the apical domain and can turn it on laterally once this repression is removed. This is consistent with the identification of multiple vertebrate GEFs with different localizations that contribute to apical Cdc42 activation. The current results therefore identify RhoGAP19D as a new lateral polarity factor. This leads to a revised network of polarity protein interactions in which RhoGAP19D functions as the third lateral factor that antagonizes the activity of apical factors, alongside Lgl, which inhibits aPKC, and Par-1, which excludes Baz/Par-3 (Fic, 2021).

The function of RhoGAP19D is very similar to that of its orthologue PAC-1, which inhibits Cdc42 at sites of cell contact in early C. elegans blastomeres to generate distinct apical and basolateral domains. Both RhoGAP19D and PAC-1 are recruited to the lateral domain by E-cadherin complexes, although the exact mechanism is slightly different. RhoGAP19D recruitment is strictly dependent on α-catenin, which links it through β-catenin to the E-cadherin cytoplasmic tail, whereas α-catenin (HMP-1) and p120-catenin (JAC-1) play partially redundant roles in recruiting PAC-1 to E-cadherin (HMR-1) in the worm. Nevertheless, in both cases, the recruitment of the Cdc42GAP translates the spatial cue provided by the localization of cadherin to sites of cell-cell contact into a polarity signal that distinguishes the lateral from the apical domain. Classic work on the establishment of polarity MDCK cells grown in suspension has revealed that the recruitment of cadherin (uvomorulin) to sites of cell-cell contact is the primary cue that drives the segregation of apical proteins from basolateral proteins. Furthermore, the expression of E-cadherin in unpolarized mesenchymal cells is sufficient to induce this segregation, although the mechanisms behind this process are only partially understood. The observation that RhoGAP19D directly links cadherin adhesion to the polarity system in epithelial cells extends the results of Klompstra (2015) in early blastomeres, strongly suggesting that PAC-1/RhoGAP19D plays an important role in the first steps in epithelial polarization (Fic, 2021).

Although PAC-1 and RhoGAP19D perform equivalent functions in early blastomeres and epithelial cells, there is one important difference between their mutant phenotypes. In pac-1 mutants, Par-6 and aPKC are mislocalized to the contacting surfaces of C. elegans blastomeres where Cdc42 is ectopically active. By contrast, Par-6 and aPKC are not mislocalized laterally in rhogap19d mutant Drosophila epithelial cells, even though lateral Cdc42-GTP does recruit two other Cdc42 effectors, N-WASP and Gek. Thus, lateral Cdc42 activity is sufficient to recruit Par-6/aPKC to the lateral domain in early blastomeres, but not in epithelial cells. Instead, it was observed that lateral Cdc42 activity in rhogap19d mutant follicle cells acts at a distance to expand the size of the apical domain. A likely explanation for this difference is the presence of Crumbs in epithelial cells. The interaction between Cdc42-GTP and Par-6 alters the conformation of Par-6 so that it can bind to Crumbs, which anchors the Par-6-aPKC complex to the apical membrane and activates aPKC's kinase activity. Although Par-6 presumably binds to Cdc42 laterally in rhogap19D mutants and undergoes the conformational change, it cannot be anchored laterally in the absence of Crumbs. This activated Par-6-aPKC complex can then diffuse until it is captured by Crumbs in the apical domain, thereby increasing apical aPKC activity, providing an explanation for why the apical domain expands in rhogap19d mutant cells. C. elegans has three Crumbs orthologues, but removal of all three simultaneously has no effect on viability or polarity. Thus, in contrast to Drosophila epithelial cells, C. elegans Crumbs proteins are not required for Par-6/aPKC localization and activation, suggesting that some other mechanism, such as Cdc42 binding, is sufficient to activate aPKC (Fic, 2021).

If the failure of active Cdc42 to recruit aPKC laterally in rhogap19d mutant cells is due to the absence of Crumbs in this region, there must be a mechanism to exclude Crumbs from the lateral domain. One proposed mechanism depends on Yurt (Moe and EPB41L5 in vertebrates), which is restricted to the lateral domain by aPKC and binds to Crumbs to antagonize its activity. However, no lateral recruitment of aPKC was observed in rhogap19d;yurt double-mutant cells. Thus, there must be some parallel mechanism that excludes Crumbs, Par-6, and/or aPKC from the lateral domain (Fic, 2021).

Although loss of RhoGAP19D only leads to a partial disruption of polarity, it causes the follicular epithelium to invade the adjacent germline tissue with 40% penetrance. This invasive behavior is not driven by an epithelial-to-mesenchymal transition, because the cells retain their apical adherens junctions and epithelial organization. Instead, the deformation of the epithelium seems to be driven by the combination of an increase in lateral contractility and an expansion of the apical domain, because reducing the dosage of Gek, which activates myosin II to drive the contractility, significantly reduces the frequency of this phenotype, as does halving the dosage of any of the apical polarity factors. The expansion of the apical domain makes the domain too long for the cells to adopt the lowest-energy conformation, giving them a tendency to become wedge shaped, which could drive the evagination. It is also possible that buckling of the epithelium contributes to invasion. Recent work has shown that epithelial monolayers under compressive stress and constrained by a rigid external scaffold have a tendency to buckle inward. The follicular cell layer is surrounded by an ECM that constrains the shape of the egg chamber and that should therefore resist expansion. In addition, the pulses of lateral contractility are likely to generate compressive stress because transiently reducing cell height while maintaining a constant volume will increase the cells' cross-sectional area, thereby exerting a pushing force on the neighboring cells. This compression coupled to the tendency to become wedge shaped due to apical expansion could therefore trigger the rare buckling events that initiate invasion. In support of this view, lateral contractility has been shown to drive the folding of the imaginal wing disc between the prospective hinge region and the pouch. This phenotype provides an example of how a partial disruption of polarity can induce cell shape changes that lead to major alterations in tissue morphogenesis (Fic, 2021).

The rhogap19d phenotype resembles the defects earliest observed in the development of ductal carcinoma in situ. In flat epithelial atypia (FEA), the ductal cells are still organized into an epithelial layer, but they display apical protrusions that are strongly labeled by the apical polarity factor Par-6. This suggests that the apical domain has expanded and bulges out of the cell, just as was observed in the rhogap19d mutant follicular cells. In the next stage, atypical ductal hyperplasia (ADH), the ductal cells start to invade the lumen of the duct while retaining aspects of normal apical-basal polarity. This again resembles the invasive phenotype of rhogap19d mutants, although overproliferation of the ductal cells probably also contributes to invasion in this case. Thus, these abnormalities, which can sometimes progress to ductal carcinoma in situ and breast cancer, mirror the effects of lateral Cdc42 activation. The RhoGAP19D human orthologues, ARHGAP21 and ARHGAP23, have been shown to bind directly to α-catenin and localize to cell-cell junctions. Furthermore, low expression of ARHGAP21 or ARHGAP23 correlates with reduced survival rates in several cancers of epithelial origin. It would therefore be interesting to determine whether these orthologues perform the same functions in epithelial polarity as RhoGAP19D and if their loss contributes to tumor development (Fic, 2021).

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, this study developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system the study demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing key genes were identified involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, this study experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed a better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression (Jevitt, 2021).

During oogenesis, a group of specialized follicle cells, known as stretched cells (StCs), flatten drastically from cuboidal to squamous shape. While morphogenesis of epithelia is critical for organogenesis, genes and signaling pathways involved in this process remain to be revealed. In addition to formation of gap junctions for intercellular exchange of small molecules, gap junction proteins form channels or act as adaptor proteins to regulate various cellular behaviors. In invertebrates, gap junction proteins are Innexins. Knockdown of Innexin 2 but not other Innexins expressed in follicle cells attenuates StC morphogenesis. Interestingly, blocking of gap junctions with an inhibitor carbenoxolone does not affect StC morphogenesis, suggesting that Innexin 2 might control StCs flattening in a gap-junction-independent manner. An excessive level of βPS-Integrin encoded by myospheroid is detected in Innexin 2 mutant cells specifically during StC morphogenesis. Simultaneous knockdown of Innexin 2 and myospheroid partially rescues the morphogenetic defect resulted from Innexin 2 knockdown. Furthermore, reduction of βPS-Integrin is sufficient to induce early StCs flattening. Taken together, this data suggests that βPS-Integrin acts downstream of Innexin 2 in modulating StCs morphogenesis (Huang, 2021).

Gap junction (GJ) proteins, the primary constituents of GJ channels, are conserved determinants of patterning. Canonically, a GJ channel, made up of two hemi-channels contributed by the neighboring cells, facilitates transport of metabolites/ions. This study demonstrates the involvement of GJ proteins during cuboidal to squamous epithelial transition displayed by the anterior follicle cells (AFCs) from Drosophila ovaries. Somatically derived AFCs stretch and flatten when the adjacent germline cells start increasing in size. GJ proteins, Innexin2 (Inx2) and Innexin4 (Inx4), functioning in the AFCs and germline respectively, promote the shape transformation by modulating calcium levels in the AFCs. These observations suggest that alterations in calcium flux potentiate STAT activity to influence actomyosin-based cytoskeleton, possibly resulting in disassembly of adherens junctions. These data have uncovered sequential molecular events underlying the cuboidal to squamous shape transition and offer unique insight into how GJ proteins expressed in the neighboring cells contribute to morphogenetic processes (Sahu, 2021).

Morphogenesis involves complex cellular interactions, regulated via short- or long-range influences, which operate both at the local as well as global level. Interactions between different cell types have acquired extra-ordinary significance during proper execution of complex morphogenetic processes including organogenesis. Interacting cellular partners of heterogenous origins participate in generation of a number of coordinated cues. Such cues either biochemical or mechanical in nature, operate both in localized and widespread manner to ultimately orchestrate the formation of simple as well as complex multicellular assemblies. Variety of signals that control the process, and underlying mechanisms that synthesize as well as propagate such signals are being investigated in different developmental contexts (Sahu, 2021).

This study demonstrates that Inx2, a gap junction protein, controls cuboidal to squamous cell shape transition in the follicular epithelium. Effect of Inx2 is mediated by its ability to regulate STAT activity, which in turn can influence the cytoskeleton. Supporting the conclusion, changes in STAT or alterations in the cytoskeletal machinery components, can either enhance or mitigate the phenotypic consequences induced by the loss of Inx2. Importantly, Inx2 engineers this shape transition likely by acting as a component of a calcium channel. It seems to achieve this by partnering with Zpg, another gap junction protein, belonging to the same family (Sahu, 2021).

Gap junction proteins have been shown to be required in a number of different biological processes including establishment of epithelial integrity and morphogenesis. For example, Inx2 has been shown to control formation of proventriculus, during foregut formation in the flies. Previous reports have also suggested that Inx2 can form homomeric as well as heteromeric gap junction channels. However, the molecular mechanism of Inx2 mediated epithelial morphogenesis has remained elusive (Sahu, 2021).

This study advances mechanistic understanding of how the gap junction channels contribute to morphological changes in the epithelial cells. Though calcium has been implicated in regulation of cell shape transition in various developmental contexts, the precise molecular mechanism underlying calcium dependent cell shape transition has not been documented. These findings demonstrate that calcium dependent JAK-STAT signaling regulates Myosin distribution to engineer cell shape transition. Interestingly previous work has shown that the same molecular cassette is used for proper specification of BCs. Together these data suggest that Inx2-zpg mediates the calcium flux in the follicle cells, which probably regulates JAK-STAT signaling in a context specific manner to achieve diverse outcomes (Sahu, 2021).

A heterotypic combination of Inx2-Zpg gap junction channel has been reported to regulate germline-soma interaction during early Drosophila spermatogenesis. In addition, heterotypic combination between Inx2 with other somatic gap junction protein, Inx3 has been reported in developing Drosophila embryo during the regulation of epithelial morphogenesis. This study has assayed the ability of a putative Inx2-Zpg heterotypic GJ channel responsible for cell shape transition underlying egg chamber morphogenesis. The results suggest that via Zpg, nurse cells induce the follicle cells to elevate free calcium levels as germline specific depletion of Zpg affects the absolute intensity and duration of calcium flux in the follicle cells. It is thus believed that optimum amount of calcium is critical for mediating proper shape transition of AFCs from cuboidal to squamous fate. Although, the nurse cells exhibit high level of GCaMP6 fluorescence, signal transmission to the adjacent follicle cells is relatively infrequent. This observation suggests that the transmission of the signal via Inx2-Zpg is spatially and temporally calibrated during oogenesis. Nurse cells are relatively of a larger size and, as such a direct physical contact may be established between a single nurse cell with a number of follicle cells via formation of a heterotypic GJ channel(s). Contact dependent assembly of Inx2-Zpg channel likely confers selectivity on the release and strength of the calcium flux. Taken together these data are consistent with the calcium flux being a crucial player during this process while the precise identity of the signal, remains to be determined. Whether calcium itself is the elusive 'signal' needs to be investigated further. It is possible that the 'signal' is more complex and consists of several components with calcium being one of the pieces of the puzzle. In such a scenario, future experiments ought to focus on uncovering the identity of the other components of the signal. It will also be important to determine as to how the non-autonomous 'signal' secreted by the nurse cells, is conveyed to the somatic neighbors to modulate the calcium flux. Follicle cells express many gap junction proteins including Inx2 on their cell surface and are competent to communicate with one another. Thus, possible contribution of neighboring follicle cells, not in direct contact with the nurse cells, may also be an important determinant (Sahu, 2021).

Recently product of tao gene has been shown to regulate follicle cell flattening by modulating the removal of lateral membrane component Fas2 by endocytosis. On similar lines, the current data shows that Inx2 permits the disassembly, and perhaps also the disappearance of adherens junction component DE-cadherin from cuboidal cells to facilitate the follicle cell stretching. Since calcium is known to regulate the rate of endocytosis, it is proposed that calcium may regulate the levels of DE-Cadherin via endocytosis to mediate the shape change in the AFCs. Partial rescue of inx2 phenotype in the heterozygous shg mutant background suggests that the accumulation of DE-Cadherin is indeed functionally connected to the phenotype. Further experiments will help reveal mechanistic connection between calcium flux and adherens junction assembly and its influence on cell shape transitions (Sahu, 2021).

Epidermal stratification during development of mammalian skin depends on cell division, collective migration and controlled differentiation. Together these three processes are responsible for the basal to squamous fate transformation. It is reported that this fate change is also accompanied by increase in the levels of GJ proteins and inter- as well as intracellular calcium. The results have implicated combined involvement of GJ proteins and calcium flux in mediating cell shape changes. It will be worthwhile to examine if similar mechanisms are employed by the differentiating basal cells. Intriguingly, several skin disorders have been linked to mutations in connexins (vertebrate counterparts of GJ proteins). These results thus provide a readily accessible genetic platform to compare and contrast the molecular mechanisms underlying epithelial cell shape transitions in diverse cellular contexts (Sahu, 2021).

Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. This study shows that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events (Reilein, 2021).

Adult stem cells generally act as a community, coordinating the activities of multiple individual cells to replenish tissues throughout life. Their behavior is invariably regulated by external signals from other cell types, known as niche cells. It is therefore essential to establish adult stem cells in suitable numbers and niche environments during development to ensure appropriately regulated community activity in adults. Understanding the underlying developmental processes would facilitate identification of genetic developmental defects that disrupt adult tissue repair or confer cancer susceptibility, and creation of expandable organoids maintained by stem cells for therapeutic use, drug studies, or mechanistic investigations (Reilein, 2021).

The development of terminally differentiated adult cell types commonly results from a series of successive, definitive, and largely irreversible decisions that are stabilized by potentially long-lasting shifts in gene expression and chromatin modification. However, the development of adult stem cells has not been studied extensively and presents a number of special considerations. First, stem cells typically continue to proliferate in adulthood and often exhibit malleable relationships with their products, with the potential for marked changes in inter-conversion rates in either direction. Does the general paradigm of definitive cell-type specification at a fixed time in development still apply, despite this marked flexibility of the final product? Second, do adult tissues and the stem cells that maintain the tissue derive from separate or common precursors? Third, is there coordination between the development of adult stem cells and their niche cells, potentially serving to arrange appropriate proportions and juxtaposition of the two cell types (Reilein, 2021)?

Drosophila ovarian follicle stem cells (FSCs) present a particularly interesting and experimentally favorable paradigm to investigate because the organization, behavior, and regulation of these adult stem cells have been studied thoroughly, revealing many similarities to the important mammalian paradigm of gut stem cells. FSCs directly produce two different types of cell in adult ovaries, follicle cells (FCs) and escort cells (ECs). Both of these cell types also act as niche cells, producing key JAK-STAT and Wnt signals, respectively. Similarly, Paneth cells in the mammalian small intestine are both stem cell products and niche cells. FSCs also provide a window into sustaining a tissue supported by two different types of stem cells. The mechanisms of coordination among FSCs, germline stem cells (GSCs), and their products in the adult are not well understood but must rely on an organization that is established during development (Reilein, 2021).

GSCs and FSCs are housed in the germarium, which is the most anterior region of each of the 30-40 ovarioles comprising the adult ovary. Somatic terminal filament (TF) and cap cells at the anterior of the germarium are stable sources of niche signals for GSCs and FSCs, including bone morphogenetic proteins (BMPs), Hedgehog, and Wnt proteins, with GSCs directly contacting cap cells. Cystoblast daughters of GSCs divide four times with incomplete cytokinesis to yield a progression of 2-, 4-, 8-, and finally, 16-cell germline cysts. Their maturation is accompanied by posterior movement and depends on interactions with neighboring quiescent, somatic ECs. ECs also act as niche cells for adjacent or nearby GSCs and FSCs (Reilein, 2021).

A rounded stage 2a 16-cell cyst loses EC contacts as it forms a lens-shaped stage 2b cyst spanning the width of the germarium and subsequently recruits FCs. The earliest FCs can be recognized by expression of high levels of the surface adhesion protein, Fasciclin 3 (Fas3). The stage 2b cyst rounds to a stage 3 cyst as surrounding FCs proliferate to form an epithelium and segregate specialized polar and stalk cells, which allow budding of the FC-enveloped cyst as an egg chamber from the posterior of the germarium. Polar cells produce the JAK-STAT ligand, Unpaired, which patterns the development of FCs along the anterior-posterior (AP) axis of each developing egg chamber and creates a gradient of signaling activity across the FSC domain, which is critical for FSC divisions and for FSC conversion to FCs. Egg chambers bud roughly every 12 hr in each ovariole of well-fed flies, progressively enlarge and mature towards egg formation and deposition, as they move posteriorly (Reilein, 2021).

There are about 16 FSCs that reside in three rings or layers around the inner germarial surface between ECs and FCs on the AP axis. The most posterior FSCs directly become FCs, while anterior FSCs directly become ECs, but FSCs also exchange AP locations, allowing a single FSC lineage to include both ECs and FCs. Individual FSCs are frequently lost and frequently amplify, with no evident temporal or functional link between division and differentiation, so that the representation and output (of ECs and FCs) of any one FSC lineage is highly variable. All of these behaviors are largely regulated by extracellular signals that are graded along the AP axis, with especially prominent roles for JAK-STAT ligand derived from polar cells and Wnt ligands derived from both cap cells and ECs. The number of FSCs, together with the spatially regulated signals that guide division rate, is matched to steady-state production of 5-6 FCs every 12 hr, while the number and location of ECs is important not only to deliver some niche signals directly but may also be important to keep FSCs at a suitable distance from the cap cell source of Wnt and Hh signals (Reilein, 2021).

Previous studies have outlined how germline and somatic ovary precursors are first selected during embryogenesis, then migrate and coalesce to form a nascent gonad, followed by specification of anterior TF cells and separation into individual developing ovarioles during larval development. Understanding of pupal development was limited, until recently, by technical difficulties associated with dissecting and imaging pupal ovaries. This study used morphological examination of developing pupal ovaries through fixed and live images, together with cell lineage studies, to examine how adult FSCs, ECs, and FCs are produced and organized from precursors present at the end of larval stages (Reilein, 2021).

Marked lineages were clustered along the AP axis, with larger clones towards the posterior, suggesting limited AP dispersion and an AP gradient of proliferation. FSC lineages almost always included marked FCs and mostly included ECs, especially if initiated early, indicating that specific stem cell precursors are not determined early; instead, FSCs likely simply acquire their characteristics according to AP location at the end of pupal development. Combined lineage and morphological studies showed that ECs and FSCs, as well as some FCs, derive from intermingled cell (IC) precursors that are interspersed with germline cells throughout pupal development, while an expanding population of cells posterior to ICs become the FCs of the first-formed egg chamber. Live imaging showed that the first pupal egg chamber is formed by migration of a germline cyst out of the developing germarium into an accumulation of future FCs, contrasting with the emergence of an FC-encased germline from the germarium that characterizes adult egg chamber budding. Thus, in this paradigm, stem cells are not specified at a fixed time in development. They arise from precursors that also build the niche and the adult tissue they support. The use of delayed specification and shared precursors, together with an evolving spatial gradient of proliferation, may be an effective general developmental strategy to juxtapose and produce appropriate numbers of stem and niche cells in the adult tissue (Reilein, 2021).

An important general objective, exemplified in this study by FSCs, is to ascertain the origin of adult stem cells and surrounding niche cells in order to understand how their number and organization are instructed during development. Investigations of changing morphology, cell movements, and cell lineage in the Drosophila ovary reveal a gradual and flexible adoption of these cell types according to AP location throughout pupal development, rather than early definitive cell fate decisions. This contrasts with the development of many other types of differentiated adult cells (Reilein, 2021).

Specifically, adult EC niche cells and FSCs develop from a common group of dividing precursors (ICs) that are interspersed with developing germline cysts. ICs express TJ and are located posterior to cap cells, which serve as adult niche cells for both GSCs and FSCs, and are already specified by the start of pupation. The most anterior ICs become regional subsets of ECs (r1 and r2a), which likely have distinctive roles in the adult. These precursors markedly attenuate division, starting from the most anterior locations, during the second half of pupation. Thus, the characteristic quiescence of adult ECs is acquired gradually and in a spatially graded manner throughout most of pupation. ICs further from the anterior produce both ECs (mainly r2a) and FSCs (as well as FCs). More posterior ICs produce FSCs and FCs, while the most posterior ICs produce only FCs. There is a progressive restriction of fates over time. That likely reflects simply the reduced width of AP territory occupied by derivatives of a single precursor cell as development proceeds, rather than any discrete and irreversible cell specification event. This pattern of germarium development ensures the juxtaposition of ECs and FSCs and may contribute to producing those cell types in appropriate proportions, with EC numbers limited by a progressive decline in proliferation that spreads from the anterior (Reilein, 2021).

Germarium development is likely driven initially by the early establishment of TF and cap cells as a source of anterior organizing signals. The behavior of adult FSCs, including conversion to FCs, additionally relies on a signaling source posterior to the germarium, namely the anterior polar cells of the most recently budded egg chamber. Polar cells were observed on the first budded egg chamber by 60 hr APF. However, at the start of pupation there are no budded egg chambers, so formation of the first FCs and the first egg chamber must proceed by a different route. We found that those first FCs derive from precursors that have accumulated posterior to ICs by 36 hr APF and differ from ICs by expressing Fas3, by lack of association with germline cells and by being restricted to produce only FCs, and not ECs or FSCs. Thus, precursors of the first FCs, some of which will become polar cells that act as posterior niche cells, have segregated from FSC precursors by 36 hr APF. After both anterior and posterior organizing centers for adult FSCs have been established and two egg chambers have budded, the architecture and Fas3 expression patterns of the germarium resemble those of adults, suggesting that the developmental process for FSCs and niche cells is complete (Reilein, 2021).

Over the course of pupal development, the entire somatic cellular complement of the adult tissue that is subsequently maintained by stem cell activity derives from an initially small set of precursors that also produce adult FSCs, with outcomes dictated simply by progressive AP cell positioning. Preliminary understanding of other adult stem cells suggests that derivation of tissues and their stem cells from a common source may be frequent but not universal. There is evidence that both adult mouse gut stem cells and adult neural stem cells of the dentate gyrus derive from a precursor pool that also gives rise to the adult epithelial tissue maintained by the stem cell. By contrast, subventricular zone neural stem cells appear to be specified during embryogenesis and remain quiescent, while the adult tissue it can replenish is built from other precursors (Reilein, 2021).

This study examined Drosophila ovary development comprehensively during pupal stages through lineage analyses initiated at different times, and by systematic analysis of fixed specimens and live imaging. Each experimental approach included some challenges and leaves some issues unresolved, but several key findings appear conclusive and supported by all three approaches (Reilein, 2021).

The challenge of labeling only a single precursor among many is common in lineage studies. Here it was met by using very mild heat-shocks to initiate clones through hs-flp induction and a multicolor labeling technique that reduces the frequency of a specific color combination substantially relative to single-color clones. The frequency of single-cell lineages was also estimated from both the overall frequency of ovariole labeling and the frequency of clone patterns (exceptional, discontinuous clones), allowing derivation of mathematical estimations of strictly single-cell lineage frequencies and yields. The results led to robust conclusions that many precursors at the start of pupation yield only ECs, a smaller proportion yield both ECs and FSCs (as well as FCs), and very few produce FSCs with no ECs. Common precursors for ECs and FSCs persist for at least the first 72 hr of pupation (later times were not tested), although they are exceeded in number at 72 hr APF by precursors that produce FSCs but not ECs. Almost all FSC lineages also included marked FCs in newly eclosed adults (Reilein, 2021).

Deducing the locations of precursors required integrating lineage and morphological studies. Each lineage was restricted in its AP extent, but the whole collection of lineages included overlapping domains that spanned the whole length of the ovariole of a newly eclosed adult, from the most anterior r1 EC to FCs and the basal stalk of the terminal egg chamber, suggesting that precursors are arranged in a continuum along the AP axis throughout pupal development. EdU labeling was directly observed in the most anterior ICs in early pupae, so it can be deduced that the most anterior of the inferred continuum of dividing precursors subject to lineage labeling lie immediately adjacent to cap cells and give rise to adult r1 ECs. The posterior extent of ovariole precursors was deduced by direct observation of the production of the first FCs, as summarized below. Those spatial insights also allowed the deduction that FSC precursors are ICs, interspersed with germline cells in the germarium throughout pupal development (Reilein, 2021).

The precursors that give rise to FCs surrounding the most posterior egg chamber of newly eclosed adults were identified by a combination of fixed and live images. These cells all express Fas3 strongly by 24 hr APF and comprise two distinguishable groups. One group was named extra-germarial crown (EGC) cells in recognition of their location, appearance and TJ expression. Cells of the second, more posterior group do not express TJ and are largely intercalated into a stalk of 1-2 cell's width at 48 hr APF prior to egg chamber budding. During budding, the most mature germline cyst leaves the germarium devoid of accompanying ICs, which do not yet express Fas3. The cyst then enters EGC territory and moves a considerable distance from the germarium as it is enveloped by both EGC and basal stalk cells (Reilein, 2021).

Towards the end of the budding process, cells posterior to the new egg chamber are stacked in single file, forming a basal stalk, which is stably retained into adulthood. Several lineages were observed that included labeled cells in the adult basal stalk and in an FC patch on the terminal egg chamber, reflecting contributions of pupal basal stalk cells. Some EGC derivatives also remain on the first budded egg chamber, while others migrate anteriorly beyond the budded cyst to form a secondary EGC between the germarium and the budded egg chamber. Secondary EGCs provide cells to partially coat the second cyst to emerge from the germarium. The second cyst appears from live imaging to move out of the germarium together with some associated ICs. Lineage studies also suggest that FCs of the second budded egg chamber derive from both EGC cells (producing FCs in egg chambers 4 and 3) and ICs (producing FCs in egg chamber 3 and more anterior locations) (Reilein, 2021).

There is no longer a recognizable EGC after two egg chambers have budded, and the two most mature germline cysts are surrounded by strongly Fas3-positive cells within the germarium, as in adults. Budding of the third, and subsequent, egg chamber is therefore likely similar to the process in adults, with cysts emerging from the germarium completely encased by FCs. Consistent with this inference, the two most posterior germarial cysts are surrounded by Fas3-positive IC cells by the time the second egg chamber starts to bud. Precursors of those FC-forming ICs were almost certainly resident in the germarium throughout pupation because we never observed cells in the expanding EGC population entering the germarium. FSC precursors, which are anterior to FC precursors, must therefore also be ICs, residing within the germarium throughout pupation. This important deduction is supported also by consideration of cell numbers. Over 60 TJ-positive somatic ICs were seen in the germarium as the first germline cyst leaves the germarium at about 48 hr APF. This exceeds the total number of ECs (about 40 on average) plus FSCs (about 16) in an adult germarium, even though many of these precursors are still dividing and amplifying towards producing a full complement of adult ECs and FSCs. Thus, the ICs in the germarium at about 48 hr APF must include all EC and FSC precursors, as well as a significant number of FC precursors (Reilein, 2021).

A recent study used single-cell RNA sequencing to explore the diversity of precursors in late larval ovaries. It has been suggested that a distinct group of cells posterior to ICs and characterized by bond expression give rise to FSCs and FCs but not ECs. This study repeated the supporting studies of lineages initiated from cells expressing bond-GAL4. By scoring FSCs directly, counting the number of labeled ECs and FSCs in each sample, and comparing the results to those for heat-shock induced single-cell lineages, it was possible to deduce that ovarioles almost always harbored more than one lineage initiated by bond-GAL4. This allowed estimation of the composition of single-cell lineages, and it was found that they were very similar to those induced by marking any dividing cell at pupariation. This inference is compatible with the observation that bond RNA and bond-GAL4 are expressed at low levels throughout the IC region in mid-third-instar larvae and with RNA in situs showing bond RNA in posterior IC regions of late third-instar larvae. In those lineages, which appear to be initiated throughout the IC region, rather than specifically in cells posterior to ICs, it was found that labeled FSCs almost always included labeled ECs, compatible with a common precursor. It was previously assumed that bond-GAL4 lineages were derived selectively from cells posterior to ICs in late third-instar larvae and that the presence of both labeled ECs and FSCs in the same ovariole resulted from the simultaneous presence of a lineage dedicated to production of each cell type. This study found evidence that bond-GAL4 also targets cells that become FCs of the first-formed egg chamber very late in pupal development. It remains uncertain whether bond might be a selective marker of EGC or basal stalk FC precursors during the first half of pupation (Reilein, 2021).

Ovary development not only organizes suitable numbers of FSCs, GSCs, and their niche cells into an adult germarium, ready to fuel lifelong adult oogenesis, but also initiates oogenesis early, so that the first eggs can be laid shortly after eclosion. This requires organized progression of germline development. Ecdysone acts positively in somatic cells of late third-instar larvae to initiate GSC differentiation, manifest by induction of the differentiation factor, Bag-of-marbles (Bam) and then cystocyte formation, revealed by the transition of the rounded spectrosome to a branched fusome. Prior studies did not reveal whether Bam-GFP expression or PGC division initiates simultaneously in all PGCs distant from cap cells or in an anterior-posterior gradient, or how germline differentiation proceeds thereafter. This study found that there is a clear anterior to posterior polarity of increasingly developed cysts that produced a mature steady-state pattern by 48 hr APF. This was deduced by counting the number of germline cells linked by fusomes, with 16 cell cysts first appearing 18 hr APF, and by observing the exclusion of EdU DNA replication signals from the most posterior regions of the germarium from 36 hr onwards, indicating occupancy by only 16 cell cysts. Moreover, a single posterior 16-cell cyst emerged for the first time between 36 hr and 48 hr APF (Reilein, 2021).

These observations raise the question of what underlies the spatially organized initiation or progression of differentiation of PGCs that are in different locations at the larval/pupal transition. The BMP signals that maintain anterior pre-GSCs are likely highly spatially restricted, as in adults, while hormonal ecdysone signals may be spatially uniform, suggesting that there must be other important differentiation signals. Potential sources of relevant signals are the adjacent somatic cell precursors, which will later become ECs. There is some evidence that the ordered posterior movement of germline cysts in adult germaria depends on EC interactions. Perhaps pupal EC precursors, even while still dividing during early pupation, have spatially graded adhesive properties or send graded differentiation signals according to their AP position (Reilein, 2021).

It is common to think of developmental processes and cell types in terms of establishment of transcriptional networks and key markers that bear witness to permanent decisions. Investigations of adult FSCs, and other adult stem cell paradigms such as the mammalian gut, as well as the observations described here regarding FSC development, reveal a more flexible process, in which environmental signals reflecting current cell locations may be more significant than stable expression of master regulators of transcription. The transcription factor TJ is selectively expressed in many developing and mature ovarian somatic cells, and it has been shown to be important for the specification of cap cells and many IC derivatives. It is therefore suspected that EGC cells, posterior to ICs, might be FC precursors because they also expressed TJ. Although that suspicion was supported by live imaging studies, the same studies showed that TJ-negative basal stalk cells also became FCs. Thus, TJ neither defines ICs nor EC/FSC/FC precursors. It is thought likely that studying the spatial distribution of external signals and their interpretation by somatic cells in the developing ovariole will be essential to understand why some cells initially mix with germline cells to become ICs, while others segregate away, and how these cells become ECs, FSCs, and FCs just in time to serve the appropriate functional role (Reilein, 2021).

Epithelial tissues are lined with a sheet-like basement membrane (BM) extracellular matrix at their basal surfaces that plays essential roles in adhesion and signaling. BMs also provide mechanical support to guide morphogenesis. Despite their importance, little is known about how epithelial cells secrete and assemble BMs during development. BM proteins are sorted into a basolateral secretory pathway distinct from other basolateral proteins. Because BM proteins self-assemble into networks, and the BM lines only a small portion of the basolateral domain, it was hypothesized that the site of BM protein secretion might be tightly controlled. Using the Drosophila follicular epithelium, this study shows that kinesin-3 and kinesin-1 motors work together to define this secretion site. Similar to all epithelia, the follicle cells have polarized microtubules (MTs) along their apical-basal axes. These cells collectively migrate, and they also have polarized MTs along the migratory axis at their basal surfaces. This study found follicle cell MTs form one interconnected network, which allows kinesins to transport Rab10+ BM secretory vesicles both basally and to the trailing edge of each cell. This positions them near the basal surface and the basal-most region of the lateral domain for exocytosis. When kinesin transport is disrupted, the site of BM protein secretion is expanded, and ectopic BM networks form between cells that impede migration and disrupt tissue architecture. These results show how epithelial cells can define a subdomain on their basolateral surface through MT-based transport and highlight the importance of controlling the exocytic site of network-forming proteins (Zajac, 2022).

The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. This study has identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). FSC maintenance and escort cell function were shown to specifically require the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. It was further demonstrated that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, this study shows that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation (Siddall, 2022).

The posterior end of the follicular epithelium is patterned by midline (MID) and its paralog H15, the Drosophila homologs of the mammalian Tbx20 transcription factor. Two perviously identified cis-regulatory modules (CRMs) recapitulate the endogenous pattern of mid in the follicular epithelium. Using CRISPR/Cas9 genome editing, this study demonstrated redundant activity of these mid CRMs. Although the deletion of either CRM alone generated marginal change in mid expression, the deletion of both CRMs reduced expression by 60%. Unexpectedly, the deletion of the 5' proximal CRM of mid eliminated H15 expression. Interestingly, expression of these paralogs in other tissues remained unaffected in the CRM deletion backgrounds. These results suggest that the paralogs are regulated by a shared CRM that coordinates gene expression during posterior fate determination. The consistent overlapping expression of mid and H15 in various tissues may indicate that the paralogs could also be under shared regulation by other CRMs in these tissues (Stevens, 2022).

Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs), are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in twelve hours during midoogenesis. Previously, it was reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. This study reports that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for the SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study single-cell RNA sequencing data was used to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened (Jia, 2022).

Apical-basal polarity is an essential epithelial trait controlled by the evolutionarily conserved PAR-aPKC polarity network. Dysregulation of polarity proteins disrupts tissue organization during development and in disease, but the underlying mechanisms are unclear due to the broad implications of polarity loss. This study uncovered how Drosophila aPKC maintains epithelial architecture by directly observing tissue disorganization after fast optogenetic inactivation in living adult flies and ovaries cultured ex vivo. Fast aPKC perturbation in the proliferative follicular epithelium produces large epithelial gaps that result from increased apical constriction, rather than loss of apical-basal polarity. Accordingly, it is possible to modulate the incidence of epithelial gaps by increasing and decreasing actomyosin-driven contractility. The origin of these large epithelial gaps were traced to tissue rupture next to dividing cells. Live imaging shows that aPKC perturbation induces apical constriction in non-mitotic cells within minutes, producing pulling forces that ultimately detach dividing and neighboring cells. It was further demonstrated that epithelial rupture requires a global increase of apical constriction, as it is prevented by the presence of non-constricting cells. Conversely, a global induction of apical tension through light-induced recruitment of RhoGEF2 to the apical side is sufficient to produce tissue rupture. Hence, this work reveals that the roles of aPKC in polarity and actomyosin regulation are separable and provides the first in vivo evidence that excessive tissue stress can break the epithelial barrier during proliferation (Osswald, 2022).

In Drosophila melanogaster, the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the oocyte posterior end, while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. Past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, neither the molecular identity nor the nature of the signal is known. This study provides evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. Furthermore, evidence is provided that PFC contact maintains Par-1 and oskar mRNA localization and microtubule cytoskeleton polarity in the oocyte. These observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex (Milas, 2023).

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development (Shah, 2022).

A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. This study found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and it, as well as other JNK pathway genes, were found to be essential for differentiation of FSC daughter cells. The data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, it was demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, these findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage (Tatapudy, 2021).

The segregation of FSC and daughter cell fates requires the orchestration of multiple developmental signaling pathways to ensure that cell fate specification and proliferation occurs at the correct time and place. This study has identified a new layer of regulation in which a single gene, ben, has multiple distinct roles in promoting differentiation and proliferation in the early FSC lineage (Tatapudy, 2021).

First, the findings demonstrate that ben functions along with the rest of the JNK signaling pathway to promote pFC (posterior follicle cell) differentiation. The phenotypes observed in JNK pathway mutants strongly suggest that the primary defect is a failure to differentiate into mature stalk cells. The range of tube-like and expanded stalk morphological phenotypes that were observed could both be explained by an inability of cells in the stalk regions between follicles to facilitate the pinching off of follicles from the germarium or to intercalate into a single row after the pinching off process has been initiated. The possibility cannot be ruled out that differentiation toward the polar cell fate or main body cell fate are also impaired upon loss of JNK signaling, but no evidence was seen for this with the markers used. Notably, although RNAi knockdown of ben or hep impaired the activity of a reporter for Notch signaling, NRE-GFP, in Region 2b pFCs and mature polar cells, this did not appear to interfere with the ability of mutant pFCs to differentiate into polar cells, so the significance of this effect on cell fate specification is unclear. Additional reporters of Notch signaling may help to determine the extent to which Notch signaling is disrupted in these mutant conditions. It was also found that pERK is retained in pFCs of ovarioles with impaired JNK signaling, and that constitutive activation of pERK phenocopies the differentiation defects observed in JNK pathway mutants. These observations suggest that JNK signaling promotes pFC differentiation into stalk cells by inhibiting the activation of ERK in pFCs (Tat in a JNK-independent manner to help shape the gradient of Hh signaling in FSCs and pFCs. Indeed, it was found that levels of Ptc-pelican-GFP, indicative of Hh signaling, are higher in ben mutant FSCs compared with wild type, and the Ptc-pelican-GFP reporter remains detectable throughout Regions 2b and 3 of tapudy, 2021).

Second, ben functions in the germarium. Likewise, it was found that RNAi knockdown of ben caused the main body follicle cells to retain zfh1 expression, a downstream target of Hh signaling, throughout the germarium and even into Stage 2 in some cases, where it is completely absent in wild-type tissue. However, it was also found that ben mutant main body follicle cells were able to mature into a Cas+, Eya- state, indicating proper differentiation with respect to these two markers. Thus, although loss of ben does not fully impair main body follicle cell differentiation, it may influence the process through its role as an upstream regulator of zfh1 expression. Further study will be required to understand the functional significance of decreased cas and zfh1 expression during main body follicle cell differentiation (Tatapudy, 2021).

JNK signaling has been found to be involved in a wide variety of biological processes in a cell type- and context-dependent manner. Some of the most well-known functions of JNK signaling involve responses to non-homeostatic conditions, such as stress, cellular damage, infection and tumor growth. However, JNK signaling has also been found to be important for processes that occur during normal development or adult homeostasis. Indeed, hep mutants were originally described for their role in dorsal closure of the epidermis in the embryo, and subsequent studies have clearly established the importance of the JNK signaling pathway in this process. Likewise, JNK signaling is also important during adult homeostasis in mammalian stem cell lineages, for example to promote self-renewal of human hematopoietic stem cells and differentiation in the mouse intestinal stem cell and human neural stem cell lineages. In line with this type of role for JNK signaling, the current findings reveal new pathway interactions for JNK signaling in the regulation of cell fate decisions in an epithelial stem cell lineage (Tatapudy, 2021).

In addition to regulating cell fate decisions, a JNK-independent role was identified for Ben in the regulation of proliferation rate and FSC niche competition. Previous studies have established that increased proliferation causes hypercompetition, and that the proliferative response to Hh signaling is the key mediator of the FSC niche competition phenotypes in Hh pathway mutants. Therefore, the hypercompetition phenotype of ben mutant clones is likely caused, in large part, by the increased Hh signaling levels and proliferation rates in these mutants. The findings that overexpression of dap or RNAi knockdown of smo was sufficient to suppress the hypercompetition phenotype of benA mutant clones are consistent with this. It is possible that delayed differentiation in newly-produced ben mutant pFCs also contributes to the hypercompetition phenotype. However, the observation that hep^G0107 mutant clones are not hypercompetitive indicates that the hypercompetition phenotype of ben mutant clones is not caused by a block in the JNK-dependent functions of Ben in pFC differentiation (Tatapudy, 2021).

Taken together, these findings indicate that Ben has JNK-dependent and -independent roles in regulating pFC cell fate decisions as they differentiate into polar, stalk and main body follicle cells. Specifically, the findings support a model in which JNK signaling promotes the differentiation of pFCs during normal homeostasis by helping to downregulate EGFR signaling in early pFCs. Previous studies found that pERK inhibits Groucho and that Groucho is required for the upregulation of Notch signaling in pFCs. Thus, the retention of pERK in pFCs may explain why Notch signaling is not upregulated in JNK pathway mutants. Separately, Ben inhibits Hh signaling in a JNK-independent manner, thereby controlling pFC proliferation, regulating FSC niche competition, and possibly contributing to main body follicle cell differentiation. Thus, this study has revealed a new layer of regulation within the signaling network that controls cell behavior in the early FSC lineage. The recently published single-cell atlases of the adult Drosophila ovary revealed a continuum of changes in gene expression that occur during cellular differentiation in the early FSC lineage, and provide the tools for characterizing this signaling network in a cell-type specific manner. In future studies, it will be interesting to apply these new single-cell approaches to mutants such as those described in this study to further elucidate the molecular basis of cell fate decisions in the FSC lineage (Tatapudy, 2021).

Intracellular RNA localization is a widespread and dynamic phenomenon that compartmentalizes gene expression and contributes to the functional polarization of cells. Thus far, mechanisms of RNA localization identified in Drosophila have been based on a few RNAs in different tissues, and a comprehensive mechanistic analysis of RNA localization in a single tissue is lacking. By subcellular spatial transcriptomics this study has identified RNAs localized in the apical and basal domains of the columnar follicular epithelium (FE) and the mechanisms mediating their localization were analyzed. Whereas the dynein/BicD/Egl machinery controls apical RNA localization, basally-targeted RNAs require kinesin-1 to overcome a default dynein-mediated transport. Moreover, a non-canonical, translation- and dynein-dependent mechanism mediates apical localization of a subgroup of dynein-activating adaptor-encoding RNAs (BicD, Bsg25D, hook). Altogether, this study identifies at least three mechanisms underlying RNA localization in the FE, and suggests a possible link between RNA localization and dynein/dynactin/adaptor complex formation in vivo (Cassella, 2022).

Only few examples of localizing RNAs in the FE have been described to date, with little mechanistic insight. To explore the extent of RNA localization in a somatic tissue in vivo and gain insight into the mechanisms underlying the phenomenon, laser-capture microdissection of apical and basal subcellular fragments of columnar follicle cells was used coupled with RNA-seq to identify localizing RNAs in this tissue. This allowed investigation in detail the landscape of mechanisms that mediate both apical and basal RNA localization in the FE. This study found that basal RNA localization is mechanistically analogous to posterior RNA localization in the oocyte (represented by osk), reflecting MT plus end enrichment. Khc, aTm1 (atypical Tropomyosin-1 isoform I/C), and the Exon Junction Complex (EJC) appear to be core components of a general basal RNA localization machinery. These results are in line with previous findings on osk RNA indicating that Khc/aTm1 bind to the 3'UTR20 and the EJC activates kinesin-1 transport through association with the coding sequence (Cassella, 2022).

According to this analysis, deposition of the EJC is necessary but not sufficient to determine RNA localization, as it was found that the EJC is deposited on both apically- and basally-directed RNAs. Interestingly, another study found that the EJC specifically localizes to the basal body of the primary cilium in mono-ciliated cells, where it controls the centrosomal localization of NIN RNA towards MT minus ends. In contrast, the current data suggest that in columnar follicle cells, which are characterized by non-centrosomal MTOCs, the EJC may play a role in MT plus end-directed (basal) RNA transport by acting synergistically with kinesin-1 and aTm1. Strikingly, the localization of osk RNA to the posterior pole of the oocyte also relies on the presence of MTs generated from non-centrosomal MTOCs65.Therefore, the mammalian EJC might have acquired a specific role in the localization of NIN RNA at basal bodies of mono-ciliated cells, while the Drosophila EJC appears to contribute to the MT plus end-directed localization of several RNAs through a centrosome-independent mechanism both in the somatic follicular epithelium (basal RNAs) and in the germline (osk RNA) (Cassella, 2022).

Interestingly, when either component of the kinesin-1 transport complex was lacking, basal RNAs were mislocalized to the apical domain in a dynein-dependent process. Therefore, dynein-mediated apical localization represents a default mechanism that must be overcome by kinesin-1 to drive basal RNA localization. Two possible scenarios could explain dynein-mediated apical mislocalization upon kinesin inhibition. Dynein and kinesin-1 could be engaged in a tug-of-war, pulling the RNAs in opposing directions, a phenomenon observed in the transport of vesicles and lipid droplets67. Alternatively, the dynein complex could be kept in an inhibited state and activated upon disruption of kinesin-1 and its regulators. If the tug-of-war scenario were correct, a change would be expected in zip RNA localization in all RNAi conditions including egl RNAi alone, namely a shift to a more basal localization due to the enhanced Khc-dependent motility. However, since no a significant change was seen in zip localization when only Egl was knocked down, the tug-of-war hypothesis appears to be less likely than the inhibition hypothesis. In addition, this phenomenon recalls osk RNA mislocalization to the oocyte anterior upon disruption of kinesin-1, aTm1 or EJC components which was hypothesized to occur due to a failure to inactivate dynein-mediated RNA transport (Cassella, 2022).

Apical RNA localization, on the other hand, can be divided into two mechanistically distinct categories, both based on dynein-mediated transport. The first category includes those RNAs that are transported apically by the dynein/BicD/Egl machinery, a well characterized RNA transport complex that directs RNAs towards MT minus ends in a variety of tissues. The data suggest that the majority of apically localizing RNAs may belong to this class, as the localization of most of the randomly chosen apical RNAs was affected in both Dhc RNAi and egl RNAi conditions. This hypothesis is consistent with previous studies that identified several apical RNAs as BicD/Egl cargoes, in a variety of Drosophila tissues. The BicD/Egl machinery has been hypothesized to be part of a larger RNP complex that ensures a tight translational control of the transported RNA. The finding that basal RNAs are on average translated more than apical RNAs suggests that RNAs transported apically in the FE by the dynein/BicD/Egl transport complex might indeed be kept in a translationally silent state until they have reached their final destination (Cassella, 2022).

The second category of dynein-dependent apical RNAs does not involve Egalitarian activity for their localization. This includes a subgroup of dynein-activating adaptors, namely BicD, hook, and Bsg25D (BICD2, HOOK1-3, and NIN/NINL in mammals). Common features of their apical RNA localization include sensitivity to puromycin and partial co-localization with cortical dynein foci containing also Dhc RNA. Moreover, both Bsg25D74 and BicD RNA constructs containing the CDS alone are sufficient for the accumulation of their encoded protein at MT minus ends. Puromycin causes the disassembly of the translational machinery and the release of the N-terminal peptides emerging from ribosomes. As the N-terminal portion of these adaptors binds dynein or dynactin subunits, it is proposed that the apical localization of BicD, hook, and Bsg25D depends on the co-translational association between dynein components and nascent adaptors at cortical dynein foci. This process might also be conserved in mammals, since the localization of both BICD2 and NIN RNA was shown to be puromycin-sensitive. Previous studies have shown that the presence of either BICD2, HOOK3 or NIN/NINL promotes the formation of highly processive dynein/dynactin complexes. Therefore, it is possible that co-translational assembly of components of the dynein-adaptor complexes is necessary to overcome dynein auto-inhibition. BicD, hook, and Bsg25D may co-translationally associate with dynein soon after nuclear export of the RNA, promoting its apical transport in a manner similar to what has been proposed for PCNT RNA targeting at centrosomes. Alternatively, since dynein can also function as a MT-tethered static anchor in mid-oogenesis oocytes and follicle cells, the interaction between dynein and nascent adaptor proteins could occur after the RNA has reached the cell cortex by dynein-mediated transport. Indeed, puromycin treatment did not completely abolish the apical enrichment of adaptor-encoding RNAs, despite causing a marked decrease in their signal close to the apical cortex, where they decorate dynein cortical foci (Cassella, 2022).

In vitro studies have shown that full-length BicD/BICD2 adopts an autoinhibitory conformation resulting from CC1/2 folding onto the CTD-containing CC3. Although the leading hypothesis in the field is that cargo binding to the CTD is responsible for the alleviation of auto-inhibition by freeing up the N-terminal dynein-binding domain, it is possible that in vivo both nascent BicD interaction with dynein and cargo binding to the CTD might cooperate in preventing BicD intramolecular inhibition in the cellular environment. Strikingly, whereas the mechanism underlying oocyte localization of BicD RNA during mid-oogenesis resembles that observed in follicle cells, the nurse cell-to-oocyte transport of BicD RNA appears to be governed by a different, translation-independent mechanism that may not involve interaction with Dhc/Dhc RNA particles, consistent with a previous study indicating that BicD RNA is translationally inhibited by Me31B in the nurse cells. In contrast to early egg chambers in which the MT network emanates from a posteriorly-positioned microtubule organizing center in the oocyte, mid-stage oocytes and columnar follicle cells are both characterized by non-centrosomal MTs (ncMTs) tethered to the cell cortex. Therefore, the establishment of ncMTs could be at the basis of the mechanistic switch from translation-independent to co-translational BicD RNA localization in these compartments. A recent report has shown that NIN RNA (the mammalian ortholog of Bsg25D) localizes at ncMTs and its expression is essential for apico-basal MT formation and columnar epithelial shape. Therefore, it is possible that the co-translational transport of adaptor-encoding RNAs may be important for correct ncMT nucleation at the apical cortex of the follicular epithelium (Cassella, 2022).

During endocytosis, molecules are internalized by the cell through the invagination of the plasma membrane. Endocytosis is required for proper cell function and for normal development in Drosophila. One component of the endocytic pathway is the retromer complex, which recycles transmembrane proteins to other parts of the cell such as the plasma membrane and the trans-Golgi network. Previous studies have shown that mutations to the retromer complex result in developmental defects in Drosophila. In humans, retromer dysfunction has been implicated in Alzheimer's and Parkinson's disease, but little is known about the role of the retromer complex in Drosophila oogenesis. This project examined the role of the retromer protein Vps26 in oogenesis by characterizing the phenotype of vps26 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins and vesicular trafficking markers in mutant egg chambers. vps26 germline clones exhibit a signaling defect between the germline cells and follicle cells indicated by an increase in LysoTracker staining of the border cells in the mutants. This signaling defect in vps26 mutants may be the result of impaired Notch signaling based on the misexpression of multiple proteins in the Notch signaling pathway in vps26 mutants (Starble, 2017).

Theoretical studies suggest that many of the emergent properties associated with multicellular systems arise already in small networks. However, the number of experimental models that can be used to explore collective dynamics in well-defined cell networks is still very limited. This study focused on collective cell behavior in the female germline cyst in Drosophila melanogaster, a stereotypically wired network of 16 cells that grows by approximately 4 orders of magnitude with unequal distribution of volume among its constituents. Multicellular growth was quantified with single-cell resolution, and it was shown that proximity to the oocyte, as defined on the network, is the principal factor that determines cell size; consequently, cells grow in groups. To rationalize this emergent pattern of cell sizes, a tractable mathematical model is proposed that depends on intercellular transport on a cell lineage tree. In addition to correctly predicting the divergent pattern of cell sizes, this model reveals allometric growth of cells within the network, an emergent property of this system and a feature commonly associated with differential growth on an organismal scale (Imran, 2017).

Hemoglobins (Hbs) are evolutionarily conserved heme-containing metallo-proteins of the "Globin" protein family which harbour the characteristic "globin fold". Hemoglobins have been functionally diversified during evolution and its usual property of oxygen transport is rather a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2 and glob3). and earlier work has reported that adequate expression of glob1 is required for the various aspects of development and also to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of Drosophila globin1 in progression of oogenesis. A dynamic expression pattern is reported of glob1 in somatic and germ cell derivatives of developing egg chambers during various stages of oogenesis which largely confines around the F-actin rich cellular components. Reduced expression of glob1 leads to various types of abnormalities during oogenesis which were primarily mediated by the inappropriately formed F-actin based cytoskeleton. Subsequent analysis in the somatic and germ line clones shows cell autonomous role of glob1 in the maintenance of the integrity of F-actin based cytoskeleton components in the somatic and germ cell derivatives. This study establishes a novel role of glob1 in maintenance of F-actin based cytoskeleton during progression of oogenesis in Drosophila (Yadav, 2016).

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. This study identified one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand understanding of the regulation of LINC complexes and NE architecture (Ding, 2017).

The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, an atomic force microscopy-based method was developed to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. These data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. This study proves that BM softens around the squamous cells and that this softening depends on the TGFbeta pathway (the ligands Gbb and Dpp signalling to follicle cells). It was also demonstrated that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes (Chlasta, 2017).

Organs are sculpted by extracellular as well as cell-intrinsic forces, but how collective cell dynamics are orchestrated in response to environmental cues is poorly understood. This study applied advanced image analysis to reveal extracellular matrix-responsive cell behaviors that drive elongation of the Drosophila follicle, a model system in which basement membrane stiffness instructs three-dimensional tissue morphogenesis. Through in toto morphometric analyses of wild type and round egg mutants, this study found that neither changes in average cell shape nor oriented cell division are required for appropriate organ shape. Instead, a major element is the reorientation of elongated cells at the follicle anterior. Polarized reorientation is regulated by mechanical cues from the basement membrane, which are transduced by the Src tyrosine kinase to alter junctional E-cadherin trafficking. This mechanosensitive cellular behavior represents a conserved mechanism that can elongate edgeless tubular epithelia in a process distinct from those that elongate bounded, planar epithelia (Chen, 2019).

Follicles have an architecture that is typical of a number of animal organs, with several components that associate to form a 3D acinar epithelium surrounding a lumen. At the same time, the simplicity and highly regular development of the follicle lend themselves to comprehensive analyses. The follicle exhibits straightforward and symmetric geometry for much of its development, while its cells originate from only two stem cell populations and show limited differential fates. Follicles can be genetically manipulated using the powerful Drosophila toolkit, and are well-suited for imaging either in fixed preparations or when cultured live ex vivo (Chen, 2019).

Development of the follicle involves several conserved morphogenetic behaviors including initial primordial assembly, epithelial diversification, and collective cell migration. A major focus for mechanistic studies has been follicle elongation, during which the initially spherical organ transforms into a more tube-like ellipsoid shape. ~2-fold elongation is seen in ~40 h between follicle budding at stage 3 to the end of stage 8; eventually there is ~2.5-fold overall elongation when the egg is laid ~25 h later. This degree of elongation is similar to that in paradigmatic morphogenetic systems such as the amphibian neural plate and mesoderm, or the Drosophila germband. In the latter tissues, the main cellular behavior that drives elongation is convergent extension, as cells intercalate mediolaterally toward a specific landmark that is defined anatomically and/or molecularly. However, these tissues have defined borders, which create boundary conditions to instruct and orient cell behaviors. No such boundary is evident along the edgeless epithelium of the Drosophila follicle, and the cellular changes that drive elongation of this acinar organ are not known (Chen, 2019).

Recent work has shown that mechanical heterogeneity patterned not within the cells of the follicle, but instead within its underlying basement membrane (BM), instructs organ shape (Crest, 2017). Specifically, a gradient of matrix stiffness that is low at the poles and peaks in the organ center provides differential resistance to luminal expansion, leading to tissue elongation. Construction of this pattern relies in part on a collective migration of cells around the follicle equatorial axis, leading to global tissue rotation. But how the cells of the epithelium respond to stiffness cues and engage in the dynamics that actually elongate the organ along the anterior-posterior (A-P) axis remains unexplored (Chen, 2019).

This study has identified an unexpected cell behavior that drives follicle elongation and demonstrates its control by a regulatory axis that responds to BM stiffness cues, thus connecting extracellular mechanical properties to intracellular signaling that drives intercellular morphogenesis in vivo (Chen, 2019).

All three morphogenetic behaviors that are engines for tissue elongation in other systems (cell shape changes, cell division, and cell rearrangement) are seen during follicle elongation. Average cellular elongation changes little during follicle elongation, and an initial cell shape anisotropy-lengthening around the organ circumferential axis-is not perturbed in a round egg mutant. Additionally, tissue rotation alone is insufficient to direct elongation-driving cell dynamics. The manipulations carried out in this study show that oriented cell division is not essential for elongation, and its absence can be compensated by other polarized cell behaviors, as also seen in e.g., the pupal thorax. Moreover, addition of cells to the elongating axis of the follicle continues at stage 7, after all mitoses have ceased. Instead, the data suggest that the major tissue-elongating behavior involves changes in cell orientation. In particular, cells in the follicle anterior shift the direction of their long axis towards the A-P axis. Without changing average cellular elongation, this reorientation of anisotropically-shaped cells changes neighbor relationships; it is suggested that this results in net cellular intercalation along the A-P axis (Chen, 2019).

The number of frank intercalations that occur during follicle elongation appears relatively limited. For instance, of the ~850 cells in a post-mitotic stage 7 follicle, only ~3 are added to the meridional arc during ~8 h prior to stage 8, inducing a ~10% change in aspect ratio. This relative paucity of intercalation events contrasts not only with the rapidly developing Drosophila germband, but also with other elongating tissues such as the Drosophila pupal wing and thorax and the vertebrate neural tube and mesoderm. Three major differences bear consideration here. First, significant growth (~9-fold) of both epithelial cells and the follicle overall occur during the 24-34 h that span stages 4 to 8, while the other systems largely rearrange a constant tissue volume. Second, the other systems have defined boundaries to impose vectorial orientation of cell behaviors, while the topologically continuous follicle epithelium lacks a boundary in the equatorial axis. Third, follicle elongation does not require conventional PCP morphogenetic signaling, nor is there evidence of PCP Myosin localization that remodels cell junctions. Instead, the instructive force seems to be patterned anisotropic resistance to growth, wherein softer BM at the poles triggers regional changes in cell behavior. Indeed, the largest changes in follicle cell orientation are seen at the anterior pole, and genetic manipulation in this region alone can prevent elongation. It is important to note that the data do not identify BM stiffness differences between the anterior and the posterior prior to cell orientation changes in the former, nor differences in pSrc levels, but do identify differences in Ecad dynamics in the latter. It is speculated that A-P patterned fates in the follicle epithelium prevent elongation behaviors in the posterior, consistent with the observation that follicles lacking posterior fate specification elongate at both poles. Overall, these differences with bounded epithelia undergoing elongation via convergent extension emphasize the new perspectives required for analysis of elongating tubular organs (Chen, 2019).

How do cells sense BM stiffness to change their orientations? The elongation-defective phenotype of Rack1-depleted follicles points to one mechanism involving Src. Rack1 is a Src-inhibiting protein, and its loss, with associated increases in cellular Src activity, perturbs follicle elongation. In Rack1-depleted follicles, initial cell orientation is not greatly altered, but anterior follicle cells remain static and are unable to shift their orientation during stages 6-8. This suggests that proper regulation of Src is required for the dynamic reapportionment of cell shape that is reflected in this switch. One familiar regulatory target of Src is integrins and their associated proteins, but no defects in integrin-dependent cell migration are seen when Rack1 is depleted from follicle cells. However, Src has also been implicated in directly regulating AJ remodeling through effects on Ecad trafficking, and FRAP analysis reveals compromised Ecad dynamics when follicles cells lack Rack1. Src is considered a mechanotransducer, and pSrc levels anticorrelate with BM stiffness in wild type, mutant and manipulated follicles. It is therefore proposed that Src mediates the instructive cue provided by BM stiffness, inducing AJ remodeling to drive morphogenesis. Whether the AJ-regulating apical Src seen in follicles is directly phosphorylated by basal integrin activation, or results from an indirect intracellular signaling cascade, remains to be investigated (Chen, 2019).

The defective tissue topology and reduced Ecad mobility seen in Rack1-depleted follicles suggests that Src is required for both elongation-driving and tissue disorder-minimizing cell rearrangements. This role of Src in follicle elongation raises interesting parallels with a second edgeless epithelium: the Drosophila trachea. In this established tubulogenesis model, gain as well as loss of Src42A activity results in shortened but broader tubules, which result from inappropriately oriented cells. Interestingly, depletion of Src42A from the follicle, like its activation, also induced elongation defects, while Src controls tracheal Ecad dynamics, perhaps in response to an ECM, albeit apically localized. Thus, in both organs Src42A could mediate stiffness-cued AJ dynamics that change the orientation of cell eccentricity. Since mammalian kidney tubule development also shows Src-dependence, these results suggest that ECM-mediated control of cell junctions via Src may be a general mechanism for morphogenesis of edgeless epithelia (Chen, 2019).

The data described in this work stem from a comprehensive analysis of follicle morphogenesis, which was enabled by ImSAnE software. ImSAnE allowed identification of critical cell dynamics near the follicle poles, which are most subject to distortion from conventional analyses, and distinguished the cellular basis underlying overtly similar elongation phenotypes. For instance, it revealed that under fat2 depletion, cells throughout the follicle are defective in planar polarized cell orientation from early stages, whereas in Rack1 follicles defective orientation is limited to the anterior and occurs only later, when elongation behaviors initiate. Furthermore, some of the ImSAnE-based morphometric findings are concordant with those recently reported based on conventional imaging. This work focuses on stages 4 through 8, and does not address cell dynamics that elongate the follicle prior to and following these stages. However, it does provide a framework for true in toto analysis of this simple organ, at multiple developmental stages and genotypes. As genetic screens and biophysical studies in the follicle are extended, the quantitative imaging platform reported here will provide a bridge towards mathematical and mechanical modeling of this flourishing system for in toto organ morphogenesis (Chen, 2019).

Protein components of the invertebrate occluding junction-known as the septate junction (SJ) - are required for morphogenetic developmental events during embryogenesis in Drosophila melanogaster. In order to determine whether SJ proteins are similarly required for morphogenesis during other developmental stages, this study investigated the localization and requirement of four representative SJ proteins during oogenesis: Contactin, Macroglobulin complement-related, Neurexin IV, and Coracle. A number of morphogenetic processes occur during oogenesis, including egg elongation, formation of dorsal appendages, and border cell migration. All four SJ proteins are expressed in egg chambers throughout oogenesis, with the highest and most sustained levels in the follicular epithelium (FE). In the FE, SJ proteins localize along the lateral membrane during early and mid-oogenesis, but become enriched in an apical-lateral domain (the presumptive SJ) by stage 10B. SJ protein relocalization requires the expression of other SJ proteins, as well as Rab5 and Rab11 in a manner similar to SJ biogenesis in the embryo. Knocking down the expression of these SJ proteins in follicle cells throughout oogenesis results in egg elongation defects and abnormal dorsal appendages. Similarly, reducing the expression of SJ genes in the border cell cluster results in border cell migration defects. Together, these results demonstrate an essential requirement for SJ genes in morphogenesis during oogenesis, and suggests that SJ proteins may have conserved functions in epithelial morphogenesis across developmental stages (Alhadyian, 2021).

Ovarian reactive oxygen species (ROS) are believed to regulate ovulation in mammals, but the details of ROS production in follicles and the role of ROS in ovulation in other species remain underexplored. In Drosophila ovulation, matrix metalloproteinase 2 (MMP2) is required for follicle rupture by degradation of posterior follicle cells surrounding a mature oocyte. MMP2 activation and follicle rupture are regulated by the neuronal hormone octopamine (OA) and the octopamine receptor in mushroom body (OAMB). This study investigated the role of the superoxide-generating enzyme NADPH oxidase (NOX) in Drosophila ovulation. Nox is highly enriched in mature follicle cells, and Nox knockdown in these cells leads to a reduction in superoxide and to defective ovulation. Similar to MMP2 activation, NOX enzymatic activity is also controlled by the OA/OAMB-Ca(2+) signaling pathway. In addition, this study reports that extracellular superoxide dismutase 3 (SOD3) is required to convert superoxide to hydrogen peroxide, which acts as the key signaling molecule for follicle rupture, independent of MMP2 activation. Given that Nox homologs are expressed in mammalian follicles, the NOX-dependent hydrogen peroxide signaling pathway that is described in this study could play a conserved role in regulating ovulation in other species (Li, 2018).

Ovulation is a key step in animal reproduction and involves multiple endocrine, paracrine, and autocrine signaling molecules, such as progesterone, epidermal growth factors, and prostaglandins. These molecules ultimately activate proteinases that break down the ovarian follicle wall, releasing a fertilizable oocyte. Several lines of evidence indicate that reactive oxygen species (ROS) also play indispensable roles in mammalian ovulation. However, there is no genetic evidence to support an in vivo role of ROS in ovulation, and the enzymes responsible for ROS production during ovulation are still unknown (Li, 2018).

ROS are oxygen-derived, chemically reactive small molecules and include superoxide anion (O2*-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH*). The physiological generation of ROS can occur as a byproduct of aerobic metabolism or as the primary function of the family of NADPH oxidases (NOXs). NOX enzymes transfer an electron across the cell membrane from NADPH in the cytosol to oxygen (O2) in the luminal or extracellular space. This movement of an electron generates O2*-, which can be rapidly converted into H2O2 by superoxide dismutases (SODs) (Li, 2018).

The mammalian NOX family comprises seven members (NOX1-5 and DUOX1-2), which have marked differences in tissue distribution and play a variety of physiological roles. Members of this family are also expressed in mammalian ovaries. Nox4 and Nox5, for example, are expressed in human granulosa cells. NOX4 and its accessory proteins in human granulosa cells show age-dependent reductions in protein expression, which correlates with low fertility. Importantly, pharmacological inhibition of NOX enzymes blocks follicle-stimulating hormone-induced oocyte maturation in mouse cumulus–oocyte complex in vitro. Despite these observations, a role for NOX in mammalian ovulation has not been demonstrated (Li, 2018).

The NOX family of enzymes is evolutionarily conserved across species. The Drosophila genome contains one Nox gene encoding NOX and one Duox gene encoding DUOX. DUOX has an additional peroxidase domain and has been well studied in gut-microbe interaction, wing formation, and wound healing. Much less is known about Nox. Earlier work reported that Nox regulates ovarian muscle contraction, which somehow influences ovulation. However, the mechanism of NOX regulation of ovulation and the cellular localization of NOX in Drosophila remain unclear (Li, 2018).

Recent work challenges the concept that ovulation is controlled by ovarian muscle contraction in Drosophila. Instead, Drosophila ovulation involves active proteolytic degradation of the follicle wall and follicle rupture and shares much in common with mammalian ovulation. Like in mammals, each oocyte in Drosophila is encapsulated in a layer of somatic follicle cells to form an egg chamber, which develops through 14 distinct stages to become a mature follicle (stage-14 egg chamber) in ovarioles. In mature follicles, the zinc finger transcription factor Hindsight (HNT) induces the expression of matrix metalloproteinase 2 (MMP2) in posterior follicle cells and octopamine receptor in mushroom body (OAMB) in all follicle cells. During ovulation, octopamine (OA) is released from neuron terminals in the ovary and binds to its receptor OAMB in stage-14 follicle cells. OAMB receptor activation causes an increase in intracellular calcium that activates MMP2 enzymatic activity, which breaks down posterior follicle cells and induces follicle rupture. Strikingly, the entire process of follicle rupture can be recapitulated ex vivo by culturing isolated mature follicles with OA in the absence of ovarian muscles and oviducts. This work casts doubt on the proposed involvement of ovarian muscles in follicle rupture/ovulation (Li, 2018).

This study investigated the role of Nox in Drosophila ovulation. Surprisingly, it was found that ovarian muscle Nox does not play a major role in ovulation but rather that Nox is enriched in mature follicle cells and is essential for follicle rupture/ovulation. OA/OAMB-Ca2+ signaling activates NOX enzymatic activity to produce extracellular O2*-, which is converted into H2O2 by an extracellular SOD3. These results suggest that NOX-produced ROS in mature follicles play a conserved role in regulating follicle rupture/ovulation across species (Li, 2018).

Ovarian ROS are indispensable for ovulation in mice. However, the site of production of ROS is unknown and it is unclear whether ROS play a conserved role in ovulation across species. This study provides genetic evidence that follicular ROS are required for ovulation in Drosophila. NOX, whose activity is regulated by follicular adrenergic signaling, regulates follicle rupture and ovulation by producing O2*- in the extracellular space of mature follicle cells. In addition, the data suggest that an extracellular SOD3 converts this O2*- into H2O2, which is the key signaling molecule responsible for regulating follicle rupture. H2O2 can partially mimic LH in regulating cumulus expansion and gene expression in mammalian follicles. It is thus plausible that H2O2 plays a conserved role in regulating follicle rupture/ovulation from insects to mammals (Li, 2018).

Members of the NOX family are also expressed in mouse and human granulosa cells and are functional in producing ROS. Norepinephrine, the mammalian counterpart of OA, is highly enriched in human follicular fluid and causes ROS generation in human granulosa cells. It will be interesting to determine whether norepinephrine plays a similar role as OA in generating ROS through regulating NOX activity during follicle rupture/ovulation in mammals (Li, 2018).

Why would Drosophila mature follicles use NOX to generate ROS during follicle rupture? ROS can be generated through the mitochondrial respiratory chain and membrane-bound NOX family enzymes, as well as by a host of intracellular enzymes, such as xanthine oxidase, cyclooxygenases, cytochrome p450 enzymes, and lipoxygenases that produce ROS as part of their normal enzymatic function. As high-level cytoplasmic ROS are detrimental to cell function and viability, limiting O2*-/H2O2 production in the extracellular environment may be essential for cell viability and function. This is consistent with the finding that overexpression of Sod1, which presumably produces extra-cytoplasmic H2O2, led to a disruption in follicle rupture and egg laying. Interestingly, Nox-knockdown follicles overexpressing Sod1 had normal follicle rupture, likely due to compensation of NOX-generated H2O2 by intracellularly produced H2O2, whereas bathing Nox-knockdown follicles in H2O2 did not rescue the defect in OA-induced follicle rupture. These findings suggest that local ROS production is essential for cellular physiology, while global ROS may be detrimental (Li, 2018).

Interestingly, Sod3 knockdown alone was sufficient to cause follicle rupture defects in Drosophila, yet mice lacking SOD3 are healthy and fertile. It is possible that SOD1 can compensate for the loss of SOD3 in mouse follicles, as mice lacking SOD1 or both SOD1 and SOD3 are subfertile or infertile, respectively (Li, 2018).

This study solved a conundrum in Drosophila ovulation. Previous work demonstrated that follicle rupture requires OA/OAMB induction of MMP2 activity in posterior follicle cells. However, OA/OAMB induces a rise in intracellular Ca2+ in all mature follicle cells. What is the role of OA/OAMB-Ca2+ in nonposterior follicle cells? This work demonstrated that OA/OAMB-Ca2+ signaling activates NOX in all follicle cells to produce O2.*- and H2O2, which are important for follicle rupture. NOX-generated ROS had a minimal effect on MMP2 activity, implying that these ROS regulate an independent pathway that is required for follicle rupture. Further studies should test whether region-specific Nox knockdown, such as only in nonposterior follicle cells, causes a follicle rupture defect (Li, 2018).

The targets of H2O2 in regulating follicle rupture are still unknown. Biological redox reactions catalyzed by H2O2 typically affect protein function by promoting the oxidation of cysteine residues. The best-characterized examples of H2O2-mediated signal transduction include several protein tyrosine phosphatases in growth factor signaling pathways, such as platelet-derived growth factor, epidermal growth factor (EGF), insulin, and B cell receptor signaling. Oxidation of the cysteine residue in the active-site motif of these phosphatases reversibly inactivates phosphatase activity and promotes growth factor signaling. The timing of H2O2 production and follicle rupture makes it unlikely that H2O2 promotes follicle rupture in Drosophila follicle cells by regulating growth factor signaling. The peak production of O2*- (and presumably of H2O2) is ~30-40 min after OA stimulation, which coincides with the beginning of follicle rupture. There is not enough time to allow growth factor signaling-mediated transcription and translation to occur before rupture happens. Alternatively, H2O2 is also involved in the activation of the ADAM (a disintegrin and metalloprotease) family of metalloproteases, possibly through direct oxidation of a cysteine residue that prevents the inhibition of catalytic domain by the prodomain of the enzyme. The idea is favored that NOX-generated H2O2 activates ADAM or other proteinases to regulate follicle rupture in addition to MMP2 activation. Microarray and RNA-sequencing analysis identified multiple proteinases that are up-regulated in Drosophila follicle cells during ovulation, and at least six different proteinases have been suggested to be involved in mammalian ovulation. Recent bioinformatics and large-scale proteomic analyses have predicted >500 proteins containing redox-active cysteine residues, some of which could serve as the downstream effectors of H2O2 for follicle rupture (Li, 2018).

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. This study describes a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction (Luo, 2021).

It is proposed that during cellular reorganization and repolarization, as in the oocyte, the establishment of a dynamic, subtly biased MT network is a widespread phenomenon and provides a general mechanism by which strong cell polarity can be initiated and maintained while efficiently handling the transport requirements of cargoes distributed throughout the cytoplasm. The tools developed in this study to quantitate global or local bias in a complex field of MTs can now be applied widely to other oocytes and other cell types to test the generality of the proposed biased random model for MT organization (Parton, 2011).

Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. Previous work has shown that cytoplasmic dynein is required for Drosophila oocyte growth and has assumed that it simply transports cargoes along microtubule tracks from nurse cells to the oocyte. This study reports that instead of transporting individual cargoes along stationary microtubules into the oocyte, cortical dynein actively moves microtubules within nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. This robust microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein performs bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. It is proposed that the dynein-driven microtubule flow could serve as a novel mode of fast cytoplasmic transport (Lu, 2022).

The risk of meiotic segregation errors increases dramatically during a woman's thirties, a phenomenon known as the maternal age effect. In addition, several lines of evidence indicate that meiotic cohesion deteriorates as oocytes age. One mechanism that may contribute to age-induced loss of cohesion is oxidative damage. In support of this model, it has been reported that the knockdown of the reactive oxygen species (ROS)-scavenging enzyme, superoxide dismutase (SOD), during meiotic prophase causes premature loss of arm cohesion and segregation errors in Drosophila oocytes. If age-dependent oxidative damage causes meiotic segregation errors, then the expression of extra SOD1 (cytosolic/nuclear) or SOD2 (mitochondrial) in oocytes may attenuate this effect. To test this hypothesis, flies were generatee that contain a UAS-controlled EMPTY, SOD1, or SOD2 cassette, and expression was induced using a Gal4 driver that turns on during meiotic prophase. The fidelity of chromosome segregation was compared in aged and non-aged Drosophila oocytes for all three genotypes. As expected, p{EMPTY} oocytes subjected to aging exhibited a significant increase in nondisjunction (NDJ) compared with non-aged oocytes. In contrast, the magnitude of age-dependent NDJ was significantly reduced when expression of extra SOD1 or SOD2 was induced during prophase. These findings support the hypothesis that a major factor underlying the maternal age effect in humans is age-induced oxidative damage that results in premature loss of meiotic cohesion. Moreover, this work raises the exciting possibility that antioxidant supplementation may provide a preventative strategy to reduce the risk of meiotic segregation errors in older women (Perkins, 2019).