Dissecting the enhancer gene regulatory network in early Drosophila spermatogenesis

Cellular decision-making and tissue homeostasis are governed by transcriptional networks shaped by chromatin accessibility. Using single-nucleus multi-omics, we jointly profiled gene expression and chromatin accessibility in 10,335 cells from the Drosophila testis apical tip. This enabled inference of 138 cell type-specific enhancer regulons (eRegulons) using SCENIC+. We functionally validated key transcription factors (TFs), including ovo and klumpfuss (klu), known from other stem cell systems but not previously linked to spermatogenesis. CRISPR-mediated knockout revealed their essential roles in germline stem cell (GSC) regulation, and we provide evidence that they co-regulate shared targets through overlapping enhancer elements. We further uncovered a critical role for canonical Wnt signaling, with Pangolin/Tcf activating lineage-specific targets in the germline, soma, and niche. The Pan eRegulon links Wnt activity to cell adhesion, intercellular signaling and GSC maintenance. Together, our study defines the enhancer-driven regulatory landscape of early spermatogenesis and reveals conserved, combinatorial mechanisms of niche-dependent stem cell control. Dmel w1118 were raised at 25°C and virgins were collected. Testes from 1 day old flies were dissected and testis apical tips were cut and flashfrozen in liquid nitrogen. Finally, testis tips were pooled and nuclei were isolated according to protocol.