Fig. 6. Reduced generation of dI3 neurons in Mash1^-/- embryos. (A,C) Immunohistochemical detection of Isl1 protein shows that dI3 neurons are significantly reduced in Mash1 mutants at E10.5 and E11.5. (B,D) Gsh1/Gsh2 expression is slightly reduced in E10.5 Mash1 knockouts (B, arrowhead) but is unchanged in E11.5 Mash1^-/- embryos (D). (G,H) Concomitant with the decrease in Isl1⁺ cells, there is an expansion of Foxd3⁺ (arrowhead) and Lhx1/Lhx5⁺ (asterisk) dI2 neurons into the prospective dI3 population. (E,F) At E10.5, Ngn1 expression expands ventrally into the prospective dI3 progenitors in the Mash1 mutant (arrow). (I-P) Electroporation (EP) of Mash1 in the chick neural tube; the electroporated side is shown on the right, control side on the left. (I,J) Isl1 is strongly induced after Mash1 overexpression, suggesting an induction of dI3 neurons (arrowhead). (K,L) Moreover, Mash1 misexpression leads to a reduction of Lhx1/Lhx5, indicating that Mash1 initiates the dI3 differentiation program at the expense of dI2 and dI4 neurons. (M,N) Ectopic Gsh2⁺ cells are found 24 hours after Mash1 overexpression (arrowheads in N), suggesting a positive-feedback mechanism onto Gsh2 to maintain dI3 progenitor identity. (O,P) Mash1 overexpression represses ventral and dorsal Ngn1 expression (arrow) 20 hours after electroporation.