Fig. 5. Essential neurogenic function of Mash1 at E11.5 and subsequent stages in the dorsal spinal cord. (A-D) Spinal cords of Mash1^–/– and control embryos were analyzed by immunohistochemistry at E12.5. (A,B) Differentiated neurons were visualized using the TuJ1 antibody (green), and YO-PRO1 (red) was used to stain nuclei. The width of the progenitor domain is indicated. (C,D) In control embryos, Lbx1+ cells (green) are rarely found in the progenitor domain, marked by the expression of Pax7 (red). In Mash1^–/– embryos, many Lbx1+ cells can be found in the progenitor domain co-expressing Lbx1 and Pax7 (yellow). (E) Numbers of Pax7+ cells in the dorsal progenitor domain were determined in Mash1^–/– (white columns) and control (black columns) embryos at the indicated time points. The number of progenitor cells is increased in Mash1^–/– at E11.5 and at subsequent stages. (F) Quantification of newborn neurons at various developmental stages; to achieve this, BrdU was injected at various time points, and the number of dorsal BrdU+/NeuN+ neurons in Mash^–/– and control embryos was determined 24 hours later. There is a decrease in the number of newborn neurons at E11.5 and at subsequent stages in Mash1^–/– embryos. (G) The proliferation index of progenitor cells (number of dorsal BrdU+ progenitor cells/number of Pax7+ progenitor cells after a 2-hour BrdU pulse) was determined in Mash1^–/– and control embryos at the indicated time points. The proliferation index of progenitor cells is not altered in Mash1^–/– embryos. (H) The differentiation index (number of BrdU+NeuN+ dorsal neurons/total number of BrdU+ dorsal cells after a 24 hours BrdU pulse) was determined in Mash1^–/– (white columns), Mash1^Ngn2/Mash1^Ngn2 (grey columns) and control (black columns) embryos at E12.5. The thickness of the optical section shown in A and B is 10 µm; the thickness of the optical sections shown in C and D is 1.2 µm. The error bars in E-H represent s.d. Scale bars: 100 µm in A,B; 50 µm in C,D.