Drosophila Importin Alpha 1 (Dα1) Is Required to Maintain Germline Stem Cells in the Testis Niche.

Stem cell maintenance and differentiation can be regulated via the differential activity of transcription factors within stem cells and their progeny. For these factors to be active, they need to be transported from their site of synthesis in the cytoplasm into the nucleus. A tissue-specific requirement for factors involved in nuclear importation is a potential mechanism to regulate stem cell differentiation. We have undertaken a characterization of male sterile importin alpha 1 (Dα1) null alleles in Drosophila and found that Dα1 is required for maintaining germline stem cells (GSCs) in the testis niche. The loss of GSCs can be rescued by ectopic expression of Dα1 within the germline but the animals are still infertile, indicating a second role for Dα1 in spermatogenesis. Expression of a Dα1 dominant negative transgene in GSCs confirmed a functional requirement for Dα1 in GSC maintenance but expression of the transgene in differentiating spermatogonia did not exhibit a phenotype indicating a specific role for Dα1 within GSCs. Our data indicate that Dα1 is utilized as a regulatory protein within GSCs to facilitate nuclear importation of proteins that maintain the stem cell pool.

TOB1 and TOB2 mark distinct RNA processing granules in differentiating lens fiber cells.

Differentiation of lens fiber cells involves a complex interplay of signals from growth factors together with tightly regulated gene expression via transcriptional and post-transcriptional regulators. Various studies have demonstrated that RNA-binding proteins, functioning in ribonucleoprotein granules, have important roles in regulating post-transcriptional expression during lens development. In this study, we examined the expression and localization of two members of the BTG/TOB family of RNA-binding proteins, TOB1 and TOB2, in the developing lens and examined the phenotype of mice that lack Tob1. By RT-PCR, both Tob1 and Tob2 mRNA were detected in epithelial and fiber cells of embryonic and postnatal murine lenses. In situ hybridization showed Tob1 and Tob2 mRNA were most intensely expressed in the early differentiating fibers, with weaker expression in anterior epithelial cells, and both appeared to be downregulated in the germinative zone of E15.5 lenses. TOB1 protein was detected from E11.5 to E16.5 and was predominantly detected in large cytoplasmic puncta in early differentiating fiber cells, often co-localizing with the P-body marker, DCP2. Occasional nuclear puncta were also observed. By contrast, TOB2 was detected in a series of interconnected peri-nuclear granules, in later differentiating fiber cells of the inner cortex. TOB2 did not appear to co-localize with DCP2 but did partially co-localize with an early stress granule marker (EIF3B). These data suggest that TOB1 and TOB2 are involved with different aspects of the mRNA processing cycle in lens fiber cells. In vitro experiments using rat lens epithelial explants treated with or without a fiber differentiating dose of FGF2 showed that both TOB1 and TOB2 were up-regulated during FGF-induced differentiation. In differentiating explants, TOB1 also co-localized with DCP2 in large cytoplasmic granules. Analyses of Tob1-/- mice revealed relatively normal lens morphology but a subtle defect in cell cycle arrest of some cells at the equator and in the lens fiber mass of E13.5 embryos. Overall, these findings suggest that TOB proteins play distinct regulatory roles in RNA processing during lens fiber differentiation.

Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects.

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Deficiency of the mitochondrial ribosomal subunit, MRPL50, causes autosomal recessive syndromic premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.

MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function.

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. We have 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). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate 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, we show 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.

Alternative models for transgenerational epigenetic inheritance: Molecular psychiatry beyond mice and man.

Mental illness remains the greatest chronic health burden globally with few in-roads having been made despite significant advances in genomic knowledge in recent decades. The field of psychiatry is constantly challenged to bring new approaches and tools to address and treat the needs of vulnerable individuals and subpopulations, and that has to be supported by a continuous growth in knowledge. The majority of neuropsychiatric symptoms reflect complex gene-environment interactions, with epigenetics bridging the gap between genetic susceptibility and environmental stressors that trigger disease onset and drive the advancement of symptoms. It has more recently been demonstrated in preclinical models that epigenetics underpins the transgenerational inheritance of stress-related behavioural phenotypes in both paternal and maternal lineages, providing further supporting evidence for heritability in humans. However, unbiased prospective studies of this nature are practically impossible to conduct in humans so preclinical models remain our best option for researching the molecular pathophysiologies underlying many neuropsychiatric conditions. While rodents will remain the dominant model system for preclinical studies (especially for addressing complex behavioural phenotypes), there is scope to expand current research of the molecular and epigenetic pathologies by using invertebrate models. Here, we will discuss the utility and advantages of two alternative model organisms-Caenorhabditis elegans and Drosophila melanogaster-and summarise the compelling insights of the epigenetic regulation of transgenerational inheritance that are potentially relevant to human psychiatry.

dRTEL1 is essential for the maintenance of Drosophila male germline stem cells.

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2.

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.

'Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway'.

BACKGROUND: Snail transcription factors mediate key cellular transitions in many developmental processes, including spermatogenesis, and their production can be regulated by TGF-ß superfamily signalling. SNAI1 and SNAI2 support many cancers of epithelial origin. Their functional relevance and potential regulation by TGF-ß superfamily ligands in germ cell neoplasia are unknown. METHODS: SNAI1, SNAI2 and importin 5 (IPO5; nuclear transporter that selectively mediates BMP signalling) cellular localization was examined in fixed normal adult human and/or neoplastic testes using in situ hybridization and/or immunohistochemistry. SNAI1 and SNAI2 functions were assessed using the well-characterized human seminoma cell line, TCam-2. Cell migration, adhesion/proliferation and survival were measured by scratch assay, xCELLigence and flow cytometry following siRNA-induced reduction of SNAI1 and SNAI2 in TCam-2 cells. The potential regulation of SNAI1 and SNAI2 in TCam-2 cells by TGF-ß signalling ligands, activin A and BMP4 was evaluated following 48 hours culture, including with siRNA regulation of IPO5 to selectively restrict BMP4 signalling. RESULTS: In normal testes, SNAI1 transcript was identified in some spermatogonia and in spermatocytes, and SNAI2 protein localized to nuclei of spermatogonia, spermatocytes and round spermatids. In neoplastic testes, both SNAI1 and SNAI2 were detected in GCNIS and in seminoma cells. SNAI1 and SNAI2 reduction in TCam-2 cells by siRNAs significantly inhibited migration and survival, respectively. Exposure to BMP4, but not activin A, significantly increased SNAI2 (~18-fold). IPO5 inhibition by siRNAs decreased BMP4-induced SNAI2 upregulation (~5-fold). Additionally, SNAI2 reduction using siRNAs inhibited BMP4-induced TCam-2 cell survival. CONCLUSIONS: This is the first evidence that SNAI1 and SNAI2 are involved in human spermatogenesis, with independent functions. These outcomes demonstrate that SNAI1 and SNAI2 inhibition leads to loss of migratory and viability capacities in seminoma cells. These findings show the potential for therapeutic treatments targeting SNAIL or BMP4 signalling for patients with metastatic testicular germ cell tumours.

Esrp1 is a marker of mouse fetal germ cells and differentially expressed during spermatogenesis.

ESRP1 regulates alternative splicing, producing multiple transcripts from its target genes in epithelial tissues. It is upregulated during mesenchymal to epithelial transition associated with reprogramming of fibroblasts to iPS cells and has been linked to pluripotency. Mouse fetal germ cells are the founders of the adult gonadal lineages and we found that Esrp1 mRNA was expressed in both male and female germ cells but not in gonadal somatic cells at various stages of gonadal development (E12.5-E15.5). In the postnatal testis, Esrp1 mRNA was highly expressed in isolated cell preparations enriched for spermatogonia but expressed at lower levels in those enriched for pachytene spermatocytes and round spermatids. Co-labelling experiments with PLZF and c-KIT showed that ESRP1 was localized to nuclei of both Type A and B spermatogonia in a speckled pattern, but was not detected in SOX9+ somatic Sertoli cells. No co-localization with the nuclear speckle marker, SC35, which has been associated with post-transcriptional splicing, was observed, suggesting that ESRP1 may be associated with co-transcriptional splicing or have other functions. RNA interference mediated knockdown of Esrp1 expression in the seminoma-derived Tcam-2 cell line demonstrated that ESRP1 regulates alternative splicing of mRNAs in a non-epithelial cell germ cell tumour cell line.

RNA binding protein Musashi-2 regulates PIWIL1 and TBX1 in mouse spermatogenesis.

RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi-2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis-expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post-transcriptional regulation during male gamete development.

Rbf Regulates Drosophila Spermatogenesis via Control of Somatic Stem and Progenitor Cell Fate in the Larval Testis.

The Drosophila testis has been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. Here, we report the identification of two independent alleles for the highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third-instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. We demonstrate that Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together our observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage.

Tob1 is expressed in developing and adult gonads and is associated with the P-body marker, Dcp2.

Tob1 is a member of the BTG/TOB family of proteins with established antiproliferative function. In Danio rerio and Xenopus laevis, the Tob1 gene is expressed from the one-cell stage through to early gastrula stages, followed in later development by discrete expression in many tissues including the notochord and somites. In both mouse and human, Tob1 is expressed in many adult tissues including the testis and ovary; however, the specific cell types are unknown. We examine Tob1 gene expression in mouse in developing germ cells and in sorted male germ cells (gonocytes, spermatogonia, pachytene spermatocytes and round spermatids) by reverse transcription and droplet digital polymerase chain reaction (RT-ddPCR) and in adult ovary and testis by immunofluorescence with anti-Tob1 protein staining. By RT-ddPCR, Tob1 expression was low in developing male germ cells but was highly expressed in round spermatids. In developing female germ cells undergoing entry into meiosis, it increased 10-fold. Tob1 was also highly expressed in round spermatids and in oocytes in all stages of folliculogenesis. Notably, a marker for P-bodies, Dcp-2, was also highly expressed in round spermatids and all oocyte stages examined. The cytoplasmic presence of Tob1 protein in round spermatids and oocytes and the association of Tob1 protein with Dcp2 in both cell types suggest that Tob1 protein plays a role in post-transcriptional mechanisms.

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis.

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development.

RNA binding proteins in spermatogenesis: an in depth focus on the Musashi family.

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the complex process of mammalian spermatogenesis, male germ cells experience extended periods of the inactive transcription despite heavy translational requirements for continued growth and differentiation. Hence, spermatogenesis is highly reliant on mechanisms of posttranscriptional regulation of gene expression, facilitated by RNA binding proteins (RBPs), which remain abundantly expressed throughout this process. One such group of proteins is the Musashi family, previously identified as critical regulators of testis germ cell development and meiosis in Drosophila, and also shown to be vital to sperm development and reproductive potential in the mouse. This review describes the role and function of RBPs within the scope of male germ cell development, focusing on our recent knowledge of the Musashi proteins in spermatogenesis. The functional mechanisms utilized by RBPs within the cell are outlined in depth, and the significance of sub-cellular localization and stage-specific expression in relation to the mode and impact of posttranscriptional regulation is also highlighted. We emphasize the historical role of the Musashi family of RBPs in stem cell function and cell fate determination, as originally characterized in Drosophila and Xenopus, and conclude with our current understanding of the differential roles and functions of the mammalian Musashi proteins, Musashi-1 and Musashi-2, with a primary focus on our findings in spermatogenesis. This review highlights both the essential contribution of RBPs to posttranscriptional regulation and the importance of the Musashi family as master regulators of male gamete development.

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium.

Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.

RNA binding protein Musashi-1 directly targets Msi2 and Erh during early testis germ cell development and interacts with IPO5 upon translocation to the nucleus.

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in Drosophila and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic Msi1 testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, Msi2 and Erh, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function.

Microarray profiling to analyze the effect of Snai1 loss in mouse intestinal epithelium.

Epithelial stem cells from a variety of tissues have been shown to express genes linked to mesenchymal cell states. The Snail family of transcriptional factors has long been regarded as a marker of mesenchymal cells, however recent studies have indicated an involvement in regulation of epithelial stem cell populations. Snai1 is expressed in the stem cell population found at the base of the mouse small intestinal crypt that is responsible for generating all differentiated cell types of the intestinal epithelium. We utilized an inducible Cre recombinase approach in the intestinal epithelium combined with a conditional floxed Snai1 allele to induce knockout of gene function in the stem cell population. Loss of Snai1 resulted in loss of crypt base columnar cells and a failure to induce a proliferative response following radiation damage. We induced Snai1 loss in cultured organoids that had been derived from epithelial cells and compared gene expression to organoids with functional Snai1. Here we describe in detail the methods for generation of knockout organoids and analysis of microarray data that has been deposited in Gene Expression Omnibus (GEO):GSE65005.

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.