Fig. 2. Mash1 is required for dI3 and dI5 neuron formation, while Ngn2 represses formation of these populations. Immunofluorescence on transverse sections of neural tubes in wild-type (A,E,I), Mash1^–/– (B,F,J), Ngn2^–/– (C,G,K) and Mash1^–/–;Ngn2^–/– (D,H,L) E10.5 mouse embryos. (A-D) Co-labeling with Brn3a and Lhx1/5 (yellow) marks dI2 neurons. There is a significant increase in dI2 neurons in the Mash1^–/– and Mash1^–/–;Ngn2^–/– embryos, but not Ngn2^–/– relative to wild type. (E-H) Isl1 identifies dI3 neurons (green). In the Mash1^–/– and Mash1^–/–;Ngn2^–/– mutant embryos, there is a dramatic decrease in dI3 neurons relative to wild type. By contrast, an increase in dI3 neurons in Ngn2^–/– is detected. (I-L) Pax2 marks dI4 and dI6 neurons (green), and Lmx1b labels dI5 neurons (red). In the Mash1^–/– and Mash1^–/–;Ngn2^–/– embryos, there is a complete loss of dI5. This is the opposite in Ngn2^–/– embryos, where there is an increase in dI5 relative to wild type. dI4/6 neurons increase in the Mash1^–/–, are unchanged in the Ngn2^–/–, and are dramatically reduced in the Mash1^–/–;Ngn2^–/– relative to wild type embryos. (M) Individual populations in each mutant were counted on at least three sections from at least three embryos and cell counts are shown in the table. Because dI4 and dI6 neuronal populations cannot be distinguished in the Mash1 null, all Pax2 cells dorsal to the boundary where the first ventral Pax2 cell was found within the ventricular zone were counted, and as such they are labeled as dI4/6. Scale bar: 50 µm.