Dissecting the enhancer gene regulatory network in early Drosophila spermatogenesis
- Patrick van Nierop y Sanchez
- Pallavi Santhi Sekhar
- Kerem Yildirim
- Tim Lange
- Laura Zoe Kreplin
- Vigneshwarr Muruga Boopathy
- Stephanie Rosswag de Souza
- Kim Dammer
- David Ibberson
- Qian Wang
- Katrin Domsch
- Anniek Stokkermans
- Shubhanshu Pandey
- Petra Kaspar
- Rafael Martinez-Gallegos
- Xuefan Gao
- Aakriti Singh
- Natalja Engel
- Fillip Port
- Michael Boutros
- Josephine Bageritz
- Ingrid Lohmann
2025-07-22
Cellular decision-making and tissue homeostasis are governed by transcriptional networks shaped by chromatin accessibility. Using single-nucleus multi-omics, we jointly profile gene expression and chromatin accessibility in 10,335 cells from the Drosophila testis apical tip. This enables inference of 147 cell type-specific enhancer-gene regulons using SCENIC + . We functionally validate key transcription factors, including ovo and klumpfuss , known from other stem cell systems but not previously linked to spermatogenesis. CRISPR-mediated knockout reveals their essential roles in germline stem cell regulation, and we provide evidence that they co-regulate shared targets through overlapping enhancer elements. We further uncover 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 germline stem cell maintenance. Together, our study defines the enhancer-driven regulatory landscape of early spermatogenesis and reveals conserved, combinatorial mechanisms of niche-dependent stem cell control.