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Fig. S2 The differentiation of V3 interneurons depends on the knockdown of nkx2.9, nkx2.2a and nkx2.2b but not on gata2. (A,B) Control (A) and Mo-nkx2.2a/Mo-nkx2.2b-injected (B) embryos stained with sim1 probe. In contrast to the triple knockdown including nkx2.9 (see Fig. 5B,E, Fig. S1C,D), the double knockdown does not affect sim1 expression (arrow) indicating that nkx2.9 is also crucial for the development of V3 interneurons. (C,D) Control (C) and Mo-gata2-injected (D) embryos hybridized to the vglut2.1 probe. As with sim1 expression, the expression of vglut2.1 (arrow) is not affected by knockdown of gata2 indicating that V3 interneurons are independent of gata2 function. (E,F) Control (E) and Mo-nkx2.2a, Mo-nkx2.2b, Mo-nkx2.9 injected (F) embryos stained with vglut2.1 antisense probe. The vglut2.1-expressing cells (arrow) located in the lateral floor plate are missing in the knockdown embryo, whereas more dorsally located glutamatergic cells are unaffected. (G,H) Forty-eight-hour-old control (G) and Mo-gata2-injected (H) embryos stained with tal2 probe. The expression of tal2 (arrow) in the lateral floor plate is abolished completely, whereas more dorsally located tal2-expressing cells (arrowhead) are unaffected. (I,J) Forty-eight-hour-old control (I) and Mo-olig2/Mo-tal2 double-injected (J) embryos hybridized to gad67 probe. gad67-expressing cells (arrow) in the lateral floor plate and more dorsal regions (arrowhead) are missing in knockdown embryos. This shows that these morpholinos are also highly effective at 48 hpf. (K,L) Control (K) and Mo-olig2-injected (L) embryos hybridized to gata3 antisense probe. gata3-expressing KA′ cells (arrowhead) are missing in knockdown embryos. (M,N) Control (M) and Mo-gata2-injected (N) embryos stained with gata3 antisense probe. gata3 expression in the lateral floor plate (arrow) but not in the KA′ cells (arrowhead) are abolished in morphants. Embryos are 48 hpf (A-J) or 24 hpf (K-N) and oriented anterior left and dorsal up. Scale bar: 25 μm.