Chinmo maintains adult stem cell sex identity by directly regulating Doublesex and Insulin signaling [scRNA-seq]
Ontology highlight
ABSTRACT: After sex specification during development, inappropriate sex maintenance in adults can lead to numerous defects and pathologies across many species. The molecular and cellular signals involved remain unknown. In the adult Drosophila testis, removing the JAK-STAT downstream effector Chinmo from adult somatic cyst stem cells (CySCs) causes their feminization, such that the sex-concordant interactions between male germ cells and the adjacent somatic cells are disrupted, causing gradual formation of testis germ cell tumors (GCT). Here we use single cell RNA-Seq to characterize cell-type specific transcriptional profiles for chinmo mutant testes undergoing sex transformation. This revealed both expected and novel mutant-specific cell populations and the full repertoire of transcriptomic changes accompanying sex transformation. Cell-cell communication networks were computed among all major cell types, revealing that feminized CySCs have reduced outgoing signaling to themselves and hub cells but elevated outgoing signaling including insulin signaling to germline stem cells (GSCs) /spermatogonia. Using Chinmo CUT & Tag, we identified Chinmo’s direct targets including the sex determination factor dsx and members of the insulin signaling pathway. We also found that Chinmo mainly functions as an activator in CySCs. By functional assays, we found ectopic expression of insulin pathway members in GSCs/spermatogonia phenocopies feminized chinmo mutant testes. In addition, knocking down insulin signaling in germ cells partially rescues the chinmo mutant phenotype. Altogether, we uncovered that Chinmo directly regulates canonical sex determination signals, as well as insulin signaling, to drive sex conversion of CySCs and germline tumorigenesis.
Project description:After sex specification during development, inappropriate sex maintenance in adults can lead to numerous defects and pathologies across many species. The molecular and cellular signals involved remain unknown. In the adult Drosophila testis, removing the JAK-STAT downstream effector Chinmo from adult somatic cyst stem cells (CySCs) causes their feminization, such that the sex-concordant interactions between male germ cells and the adjacent somatic cells are disrupted, causing gradual formation of testis germ cell tumors (GCT). Here we use single cell RNA-Seq to characterize cell-type specific transcriptional profiles for chinmo mutant testes undergoing sex transformation. This revealed both expected and novel mutant-specific cell populations and the full repertoire of transcriptomic changes accompanying sex transformation. Cell-cell communication networks were computed among all major cell types, revealing that feminized CySCs have reduced outgoing signaling to themselves and hub cells but elevated outgoing signaling including insulin signaling to germline stem cells (GSCs) /spermatogonia. Using Chinmo CUT & Tag, we identified Chinmo’s direct targets including the sex determination factor dsx and members of the insulin signaling pathway. We also found that Chinmo mainly functions as an activator in CySCs. By functional assays, we found ectopic expression of insulin pathway members in GSCs/spermatogonia phenocopies feminized chinmo mutant testes. In addition, knocking down insulin signaling in germ cells partially rescues the chinmo mutant phenotype. Altogether, we uncovered that Chinmo directly regulates canonical sex determination signals, as well as insulin signaling, to drive sex conversion of CySCs and germline tumorigenesis.
Project description:Spermatogonial stem cells are foundation of spermatogenesis. In our previous study, we observed a rapid degeneration of spermatogenesis by conditional knockout (cKO) of Nanos2 in adult spermatogonia, but the underlying mechanisms are largely unknown. While, conditional overexpression (cOE) of Nanos2 also inhibited germ cell development and stayed in undifferentiation status. Here we used cultured germline stem cells (GSCs) to study the mechanisms of Nanos2 in spermatogonia. We compared Nanos2 expression in cultured GSCs with in vivo isolated Nanos2 positive cells, and found the level of Nanos2 is significantly lower in GSCs as compared to Nanos2 positive cells. Thus we generated Nano2 cOE GSCs lines. By conditional inducing expression of Nanos2 we compared gene expression profile to control GSCs. These data provide the first evidence of Nanos2 regulating genes in spermatogonial stem cells.
Project description:This series compares gene expression between germ line stem cells (GSCs) purified either from bam mutant or Dpp-overexpressing ovaries, with gene expression in Kc cells Experiment Overall Design: 3 replicates of GSCs from bam mutant ovaries Experiment Overall Design: 3 replicates of GSCs from Dpp overexpressing ovaries Experiment Overall Design: 3 replicates of Kc tissue culture cells
Project description:Purpose: The goal of this sequencing is to investigate alterations in gene expression that result from impaired retinoid signaling compared with control, and how the RA signaling controls spermatogonia differentiation Methods: THY1+ spermatogonia mRNA profiles of 4-day-old control and germ cell specific impaired retinoid signaling mice were generated by High-throughput sequencing Results: Gene ontology (GO) analysis of the genes at the top of the ranked genes indicated enrichment in genes associated with roles in reproduction, transcription and spermatogenesis. In total, we identified 1633 and 742 transcripts (Reads Per Kilobase of transcript per Million mapped reads (RPKM) > 1) that were significantly (p-value < 0.05, > 1.5-fold difference) down- and up-regulated, respectively, in the germ cell mutants compared with the controls. Most importantly, we found that the majority of transcripts of replication-dependent core histone genes, histone cluster 1 (Hist1) were downregulated in germ cell mutants. THY1+ spermatogonia mRNA profiles of 4-day old germ cell specific impaired retinoid signaling and control mice were generated by deep sequencing, twice, using Illumina HiSeq 2000
Project description:The function of germ cells in somatic growth and aging is demonstrated in invertebrate models but remains unclear in vertebrates. Here, we demonstrate sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri. In females, germ-cell-removal shortened lifespan, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ-cell-removal in males improved their health state with increased vitamin D signaling. Body size increased in both sexes, but it was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different endocrine system pathways, depending on the sex, which may underlie the sexual difference in the reproductive strategies.
Project description:To futher understand the properties of CDH1+ GSCs and CDH1- GSCs, flow cytometry was used to sort the twopopulations after trypsin digestion and they were compared by total RNA sequencing (RNA-seq). The data clearlyshowed CDH1+ and CDH1- cells as having highly distinct profiles. In particular, numerous epithelial genes (e.g.Dsp, Pkp2, Krt19) were highly expressed in the CDH1+ population. In contrast, most genes known to be generalmarkers of SSCs/undifferentiated spermatogonia were downregulated (e.g. GFRA1 and ID4) or unchanged (e.g. ZBTB16and SALL4) in CDH1- GSCs. Additionally, KEGG pathway analysis revealed that the two populations exhibiteddistinctive activity in several signaling pathways including WNT and TGFb signaling. Notably, Tgfbr1, Smad2, Smad3tended to be lower in CDH1+ GSCs while Smad7, an inhibitor of TGFb signaling, was higher.The results showed thatCDH1+ GSCs were more epithelial in nature compared to CDH1- GSCs and supported the notion that CDH1+ GSCs are areable to partly overcome MET barrier because they may be in an advanced stage of MET.
Project description:Dedifferentiation is an important process to replenish lost stem cells during aging or regeneration after injury to maintain tissue homeostasis. Here we report that Enhancer of Zeste [E(z)], a component of the Polycomb Repression Complex 2 (PRC2), is required for the partially differentiated germ cell to dedifferentiate, in order to maintain a stable pool of germline stem cells (GSCs) within the niche microenvironment. During aging, germ cells with reduced E(z) activity have defects in maintaining GSCs, which is not due to increased GSC death or premature differentiation. We found evidence that the decrease of GSCs upon inactivation of E(z) in the germline is likely attributed by defective dedifferentiation. In addition, E(z) mutant germ cells fail to replenish lost GSCs during recovery from genetically manipulated GSC depletion. Together, our data suggest that E(z) acts intrinsically in germ cells for the dedifferentiation process to replenish lost GSCs during both aging and tissue regeneration.