Fig. 2. Pax6 is both necessary and sufficient to regulate endogenous Ngn2 expression and activate the E1 enhancer. (A-D) Double immunocytochemistry with an -ß-galactosidase antibody (green) and an -Nkx6.2 antibody (red, A,B), or an -Nkx6.1 antibody (red, C,D), on transverse sections of spinal cord from E10.5 embryos, heterozygous for the Ngn2^KIlacZ allele, and either wild-type (A,C) or homozygous Sey mutants (B,D) at the Pax6 locus. ß-gal expression is down-regulated in Nkx6.2-expressing cells (p1 domain, arrowhead in B) and dorsal Nkx6.1-expressing cells (p2 domain, arrowhead in D). (E-J) Dorsal views of whole-mount chick neural tubes labelled for Ngn2 (E), Pax6 (F,J), ß-gal (G,I) and GFP (H). Embryos were harvested 6 hours after being electroporated with a CMVPax6 vector (E,F,I,J), an E1ßglobinlacZ vector (G-J) or a CMVGFP vector (H). The electroporated side of neural tubes is at the bottom of the panels. Only a few Ngn2-positive and ß-gal-positive cells are detectable at this stage (arrowhead in the unelectroporated side of the neural tube in E, and electroporated side in G, respectively), where endogenous levels of Pax6 are low (top in F). In the presence of high exogenous levels of Pax6 protein (bottom in F and J), the number of cells expressing endogenous Ngn2 (E) and activating the E1 element (I) is strongly increased. The inset in J shows two cells co-expressing ß-gal and high levels of Pax6. Dashed lines in left panels outline the neural tube.

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