Differentiation of Mouse Enteric Nervous System Progenitor Cells Is Controlled by Endothelin 3 and Requires Regulation of Ednrb by SOX10 and ZEB2

BACKGROUND & AIMS: Maintenance and differentiation of progenitor cells in the developing enteric nervous system are controlled by molecules such as the signaling protein endothelin 3 (EDN3), its receptor (the endothelin receptor type B [EDNRB]), and the transcription factors SRY-box 10 (SOX10) and zinc finger E-box binding homeobox 2 (ZEB2). We used enteric progenitor cell (EPC) cultures and mice to study the roles of these proteins in enteric neurogenesis and their cross regulation. METHODS: We performed studies in mice with a Zeb2 loss-of-function mutation (Zeb2D) and mice carrying a spontaneous recessive mutation that prevents conversion of EDN3 to its active form (Edn3ls). EPC cultures issued from embryos that expressed only wild-type Zeb2 (Zeb2(+/+) EPCs) or were heterozygous for the mutation (Zeb2(Delta/+) EPCs) were exposed to EDN3; we analyzed the effects on cell differentiation using immunocytochemistry. In parallel, Edn3ls mice were crossed with Zeb2(Delta/+) mice; intestinal tissues were collected from embryos for immunohistochemical analyses. We investigated regulation of the EDNRB gene in transactivation and chromatin immunoprecipitation assays; results were validated in functional rescue experiments using transgenes expression in EPCs from retroviral vectors. RESULTS: Zeb2(Delta/+) EPCs had increased neuronal differentiation compared to Zeb2(+/+) cells. When exposed to EDN3, Zeb2(+/+) EPCs continued expression of ZEB2 but did not undergo any neuronal differentiation. Incubation of Zeb2(Delta/+) EPCs with EDN3, on the other hand, resulted in only partial inhibition of neuronal differentiation. This indicated that 2 copies of Zeb2 are required for EDN3 to prevent neuronal differentiation. Mice with combined mutations in Zeb2 and Edn3 (double mutants) had more severe enteric anomalies and increased neuronal differentiation compared to mice with mutations in either gene alone. The transcription factors SOX10 and ZEB2 directly activated the EDNRB promoter. Overexpression of EDNRB in Zeb2(Delta/+) EPCs restored inhibition of neuronal differentiation, similar to incubation of Zeb2(+/+) EPCs with EDN3. CONCLUSIONS: In studies of cultured EPCs and mice, we found that control of differentiation of mouse enteric nervous system progenitor cells by EDN3 requires regulation of Ednrb expression by SOX10 and ZEB2.