Fig. 5. Regulatory interactions between Phox2b and transcription factors expressed in the progenitor domain. (A-A''') Mash1 induces Islet1, but not other motoneuronal markers. Anti-Islet1/2 immunostaining (A') and in situ hybridization with Islet2 (A'') and ChAT (A''') probes at 48 h.a.e. of Mash1 on spinal cord sections. At this time point, the transfected cells have relocated to the mantle layer, as shown by in situ hybridization with a GFP probe. (A) Forced expression of Mash1 in the spinal cord induces ectopic Islet 1/2⁺ (A') cells, but neither Islet2 (A'') nor ChAT (A''). The effect of Mash1 overexpression on Islet1/2 expression has been quantitated by counting the Islet1/2⁺ cells in the transfected area and in an equivalent area of the non-transfected side, excluding the region of the Islet1/2⁺ spinal motoneurons. A total of 692 Islet1/2⁺ cells were counted on the transfected versus 296 cells on the control side, which amounts to an average difference of 9.7±0.27 cells/section (mean±s.e.m., P<0.0001, 40 sections from six embryos). (B-D'') Phox2b upregulates Nkx6.1 and Nkx6.2 and represses Pax6 and Olig2. In situ hybridization on spinal cord sections with Nkx2.2 (B'), chick Phox2b (B''), Nkx6.2 (C'), Olig2 (C''), Nkx6.1 (D') and Pax6 (D'') probes at 24 h.a.e. of mPhox2b. The extent of electroporation is shown on adjacent sections by anti-mPhox2b immunohistochemistry (B) or in situ hybridization with a GFP probe as indicated. mPhox2b misexpression induces the endogenous Phox2b gene (B'') and expands the expression domains of Nkx6.1 (D') and Nkx6.2 (C'). By contrast, Olig2 (C'') and Pax6 (D'') expression is repressed. Nkx2.2 expression is not induced by mPhox2b (B').

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