A Temporal Signature of Epidermal Growth Factor Signaling Regulates the Differentiation of Germline Cells in Testes of Drosophila melanogaster

Tissue replenishment from stem cells follows a precise cascade of events, during which stem cell daughters first proliferate by mitotic transit amplifying divisions and then enter terminal differentiation. Here we address how stem cell daughters are guided through the early steps of development. In Drosophila testes, somatic cyst cells enclose the proliferating and differentiating germline cells and the units of germline and surrounding cyst cells are commonly referred to as cysts. By characterizing flies with reduced or increased Epidermal Growth Factor (EGF) signaling we show that EGF triggers different responses in the cysts dependent on its dose. In addition to the previously reported requirement for EGF signaling in cyst formation, a low dose of EGF signaling is required for the progression of the germline cells through transit amplifying divisions, and a high dose of EGF signaling promotes terminal differentiation. Terminal differentiation was promoted in testes expressing a constitutively active EGF Receptor (EGFR) and in testes expressing both a secreted EGF and the EGFR in the cyst cells, but not in testes expressing either only EGF or only EGFR. We propose that as the cysts develop, a temporal signature of EGF signaling is created by the coordinated increase of both the production of active ligands by the germline cells and the amount of available receptor molecules on the cyst cells.     

gone early,a Novel Germline Factor,Ensures the Proper Size of the Stem Cell Precursor Pool in the Drosophila Ovary

In order to sustain lifelong production of gametes, many animals have evolved a stem cell–based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell–cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.     

Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila

In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

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

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

Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.     

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4‐encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline‐specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis. genesis 50:914–920, 2012. © 2012 Wiley Periodicals, Inc.     

Evidence for Chromatin-Remodeling Complex PBAP-Controlled Maintenance of the Drosophila Ovarian Germline Stem Cells

In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.     

Dmp53 is sequestered to nuclear bodies in spermatogonia of Drosophila melanogaster

p53 family members have been implicated in regulation of genomic integrity and apoptosis in a variety of tissues. The Drosophila family member, Dmp53, primarily functions to regulate apoptosis in developing and regenerating tissues but loss of function mutants are viable and fertile. Dmp53 exhibits a striking expression pattern in the male germline with high levels found in nuclear bodies in pre-meiotic germ cells. The localisation of Dmp53 to nuclear bodies is dependent upon Dmp53 complexes being able to bind DNA, and although dmp53 mutants do not affect germline stem cell (GSC) maintenance or differentiation, GSCs are sensitive to overexpression of Dmp53 but maturing spermatogonia are not. Dmp53 thus has differential effects depending upon the stage of male germline maturation.     

Autonomous differentiation of Drosophila spermatogonia in vitro

Spermatogenesis is a complex process that produces functional sperm by establishing male germline stem cells (mGSCs) in adult testes. To study Drosophila spermatogenesis in vitro , we examined various culture conditions of spermatogonia. Spermatogonia from larval testes began to differentiate soon after culture, whereas mGSCs did not undergo self-renewal division. Strikingly, 16-cell spermatogonia from early and late larval testes differentiated into motile spermatids autonomously. Furthermore, individual spermatogonia developed into motile spermatids even after mechanical dissociation from encapsulating cyst cells. This is the first study to report that spermatogonia in larval testes retain the ability to differentiate into spermatids in the absence of gonadal tissue. Our in vitro system should provide an excellent opportunity to study spermatogenesis in detail and apply genetic manipulation.     

The Drosophila DOCK family protein sponge is involved in differentiation of R7 photoreceptor cells

The Drosophila sponge (spg)/CG31048 gene belongs to the dedicator of cytokinesis (DOCK) family genes that are conserved in a wide variety of species. DOCK family members are known as DOCK1–DOCK11 in mammals. Although DOCK1 and DOCK2 involve neurite elongation and immunocyte differentiation, respectively, the functions of other DOCK family members are not fully understood. Spg is a Drosophila homolog of mammalian DOCK3 and DOCK4. Specific knockdown of spg by the GMR-GAL4 driver in eye imaginal discs induced abnormal eye morphology in adults. To mark the photoreceptor cells in eye imaginal discs, we used a set of enhancer trap strains that express lacZ in various sets of photoreceptor cells. Immunostaining with anti-Spg antibodies and anti-lacZ antibodies revealed that Spg is localized mainly in R7 photoreceptor cells. Knockdown of spg by the GMR-GAL4 driver reduced signals of R7 photoreceptor cells, suggesting involvement of Spg in R7 cell differentiation. Furthermore, immunostaining with anti-dpERK antibodies showed the level of activated ERK signal was reduced extensively by knockdown of spg in eye discs, and both the defects in eye morphology and dpERK signals were rescued by over-expression of the Drosophila raf gene, a component of the ERK signaling pathway. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in and around the plasma membrane of the eye disc cells. Together, these results indicate Spg positively regulates the ERK pathway that is required for R7 photoreceptor cell differentiation and the regulation is mediated by interaction with Rap1 during development of the compound eye.     

Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation

Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. Here we identify that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. We found that Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, we found that Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances our understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.