Gene expression profile of NPP cluster 2.

The differentiation of epithelial cells into Non-Professional Phagocytes (NPPs) is essential for maintaining tissue homeostasis and clearing apoptotic debris. In the Drosophila ovary, epithelial follicle cells transform into NPPs following germline cell death, but the genetic mechanisms controlling this transition are not well defined. To investigate these mechanisms, we used a model in which overexpression of the active form of Notch, the Notch Intracellular Domain (NICD), induces a robust epithelial-to-NPP transition. Using single-cell RNA sequencing and trajectory analysis, we identified three transcriptional phases of NPP maturation: an early stage of metabolic activation, an intermediate stage enriched in genes related to migration and cytoskeletal remodeling, and a late stage marked by autophagy-related gene expression. These transcriptomic patterns were validated by immunostaining. SCENIC and ChiP-seq analyses identified the JNK effector Jun-related antigen (Jra) and its predicted targets, Arp2 and Arp3, which encode components of the Arp2/3 complex, as regulators of cytoskeletal remodeling. Functional assays confirmed that the JNK–Jra–Arp2/3 axis is required for cytoplasmic expansion and debris clearance during NPP differentiation.