Chinmo maintains adult stem cell sex identity by directly regulating Doublesex and Insulin signaling [scRNA-seq]

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. Overall design: Single cell RNA-seq of wild-type and chinmoST testes across three timepoints (3–5, 6–8 and 9–11 days of adulthood) using 10x Genomics Chromium Single Cell 3' Reagent Kits v3