Notch-hedgehog antagonism regulates stem cell niche formation through an E2-E3-E3 ubiquitin module.

Distinct microenvironments, including the stem cell niche and differentiation zone, promote stem cell self-renewal and progeny differentiation, yet how these domains are established remains unclear. In the developing Drosophila ovary, we show that Notch activation silences Hedgehog (Hh) signaling in anterior intermingled cells (ICs), specifying cap cells (CpCs) that form the germline stem cell (GSC) niche. Notch upregulates the E2 enzyme Ubc10, which partners with the RBR E3 ligase Ari-1 and a Cullin 3 (Cul3)-based RING E3 ligase to assemble an E2-E3-E3 complex, targeting Ci for degradation. By contrast, ICs posterior to CpCs retain Hh signaling and give rise to escort cells, forming the GSC differentiation zone. ARIH1, CUL3, and GLI2, the human orthologs of Ari-1, Cul3, and Ci, associate in human cells, suggesting conservation of this ubiquitin ligase module. Together, these findings identify a Notch-dependent E2-E3-E3 ubiquitin ligase module linking Notch activation to Hh silencing during niche patterning. This strategy may act broadly in cell-fate decisions and reveals a paradigm in which a developmental cue regulates an E2 enzyme to facilitate ubiquitin ligase assembly.