Chinmo prevents transformer alternative splicing to maintain male sex identity.

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

Steroid signaling controls sex-specific development in an invertebrate.

In vertebrate sexual development, two important steroid hormones, testosterone and estrogen, regulate the sex-specific development of many tissues. In contrast, invertebrates utilize a single steroid hormone, ecdysone, to regulate developmental timing in both sexes. However, here we show that in Drosophila melanogaster, sex-specific ecdysone (E) signaling controls important aspects of gonad sexual dimorphism. Rather than being regulated at the level of hormone production, hormone activity is regulated cell-autonomously through sex-specific hormone reception. Ecdysone receptor (EcR) expression is restricted to the developing ovary and is repressed in the testis at a time when ecdysone initiates ovary morphogenesis. Interestingly, EcR expression is regulated downstream of the sex determination factor Doublesex (Dsx), the founding member of the Dsx/Mab3 Related Transcription Factor (DMRT) family that regulates gonad development in all animals. E signaling is required for normal ovary development1,2, and ectopic activation of E signaling in the testis antagonized stem cell niche identity and feminized somatic support cells, which were transformed into follicle-like cells. This work demonstrates that invertebrates can also use steroid hormone signaling to control sex-specific development. Further, it may help explain recent work showing that vertebrate sexual development is surprisingly cell-autonomous. For example, chickens utilize testosterone and estrogen to control sex-specific development, but when they have a mixture of cells with male and female genotypes, the male cells develop as male and the female cells develop as female despite exposure to the same circulating hormones3. Sex-specific regulation of steroid hormone signaling may well underly such cell-autonomous sexual fate choices in vertebrates as it does in Drosophila.

Integrated stress response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the integrated stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor that instructs oogenesis. We demonstrate that Atf4 regulates lipase activity to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient-sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat-body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction-directly by impacting yolk lipoprotein production and follicle maturation and systemically by regulating ovulation.

Proteomic pathway analysis reveals inflammation increases myeloid-derived suppressor cell resistance to apoptosis.

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the Integrated Stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor which instructs oogenesis. We demonstrate that Atf4 regulates the lipase Brummer to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction: directly, by impacting yolk lipoprotein production and follicle maturation, and systemically, by regulating ovulation.

The Regulation of Germline Sex Determination in Drosophila by Sex lethal.

BACKGROUND: The establishment of male or female identity (sex determination) is essential for creating the anatomical, physiological, and behavioral differences between 2 sexes of the same species (sexual dimorphism). In many organisms, including mammals and Drosophila, sex is determined by inheritance of sex chromosomes, while in other animals, sex is determined by environmental factors. Arguably the most important consequence of sex determination is the production of healthy gametes necessary for reproduction: female oocytes and male spermatids. SUMMARY: The generation of sperm and oocytes requires cooperation between 2 different cell types within the gonad: germ cells and somatic cells. Defects in sex determination in either the somatic gonad or germline lead to disorders of sexual development and infertility. In Drosophila, the gene Sex lethal (Sxl) is the key determinant of sex in both the soma and the germline. However, how Sxl controls sex determination is much more well understood in the soma than the germline. Key Mesage: This review will focus on Sxl in the germline, how it is activated specifically in female germ cells, and how it regulates germline sex determination and sexual development.

Transcriptomic analysis of feminizing somatic stem cells in the Drosophila testis reveals putative downstream effectors of the transcription factor Chinmo.

One of the best examples of sexual dimorphism is the development and function of the gonads, ovaries and testes, which produce sex-specific gametes, oocytes, and spermatids, respectively. The development of these specialized germ cells requires sex-matched somatic support cells. The sexual identity of somatic gonadal cells is specified during development and must be actively maintained during adulthood. We previously showed that the transcription factor Chinmo is required to ensure the male sexual identity of somatic support cells in the Drosophila melanogaster testis. Loss of chinmo from male somatic gonadal cells results in feminization: they transform from squamous to epithelial-like cells that resemble somatic cells in the female gonad but fail to properly ensheath the male germline, causing infertility. To identify potential target genes of Chinmo, we purified somatic cells deficient for chinmo from the adult Drosophila testis and performed next-generation sequencing to compare their transcriptome to that of control somatic cells. Bioinformatics revealed 304 and 1549 differentially upregulated and downregulated genes, respectively, upon loss of chinmo in early somatic cells. Using a combination of methods, we validated several differentially expressed genes. These data sets will be useful resources to the community.

Proliferative stem cells maintain quiescence of their niche by secreting the Activin inhibitor Follistatin.

Aging causes stem cell dysfunction as a result of extrinsic and intrinsic changes. Decreased function of the stem cell niche is an important contributor to this dysfunction. We use the Drosophila testis to investigate what factors maintain niche cells. The testis niche comprises quiescent "hub" cells and supports two mitotic stem cell pools: germline stem cells and somatic cyst stem cells (CySCs). We identify the cell-cycle-responsive Dp/E2f1 transcription factor as a crucial non-autonomous regulator required in CySCs to maintain hub cell quiescence. Dp/E2f1 inhibits local Activin ligands through production of the Activin antagonist Follistatin (Fs). Inactivation of Dp/E2f1 or Fs in CySCs or promoting Activin receptor signaling in hub cells causes transdifferentiation of hub cells into fully functional CySCs. This Activin-dependent communication between CySCs and hub regulates the physiological decay of the niche with age and demonstrates that hub cell quiescence results from signals from surrounding stem cells.

Regulation of proliferation, cell competition, and cellular growth by the Drosophila JAK-STAT pathway.

The JAK-STAT pathway is a key regulator of tissue size in Drosophila melanogaster. Here we provide an overview of its roles in processes that regulate the size of Drosophila imaginal discs, epithelia of diploid cells that proliferate and acquire specific fates in the larvae and that become functional in the adult. Drosophila has a single JAK and a single STAT gene, which has facilitated genetic dissection of this pathway. Moreover, the sophisticated genetic tools available in flies for clonal growth assays have made Drosophila an ideal organism in which to dissect the multiple roles of the JAK-STAT pathway in growth control. Studies in flies have revealed JAK-STAT pathway activity as a central node for diverse signals that control proliferation and mass accumulation. In addition, recent work has establish a new role for the pathway in cell competition, a process thought to be akin to the early stages of transformation in which more robust cells kill and take the place of less robust ones.