Class II Hh regulated genes in zebrafish: nkx2.9 and nkx6.2 expression and regulation by Hh. (A and B) Lateral views of 36 hpf embryos, eyes removed, dot marks optic recess. (A) nkx2.9 is expressed in the ventral hindbrain, ventral midbrain and diencephalon in a pattern nearly identical to that of nkx2.2a (see Karlstrom et al., 2003). No nkx2.9 expression was seen outside the nervous system up to 36 hpf. Inset shows expression of nkx2.9 in the CNS at the tailbud stage (10 hpf). (B) nkx6.2 is expressed in the ventral hindbrain and ventral midbrain, but is not expressed in the forebrain. In the hindbrain, nkx6.2 is expressed dorsally, presumably in cranial motor neurons (arrows). Inset shows nkx6.2 expression in the ventral CNS at tailbud stage (10 hpf). (B′) Side view of somites 4–7 in the trunk showing nkx6.2 expression in the developing pancreas at 36 hpf. (C–H) Spinal cord cross-sections of 24 hpf embryos. (C) nkx2.9 is expressed in lateral floor plate cells, but not in the medial floor plate (*), similar to nkx2.2a (F). nkx2.9 expression is dorsally expanded in shh mRNA-injected embryos. The expression of nkx2.9 is lost in smu(smo), con(disp1), yot(gli2DR), and dtr(gli1) mutants. (D) nkx6.2 is expressed in the ventral half of the CNS. In shh mRNA-injected embryos nkx6.2 expression is expanded dorsally. nkx6.2 expression is completely absent in smu(smo) mutants. In con(disp1) mutants, nkx6.2 expression is absent dorsally but remains in the medial and lateral floor plate (arrowhead). In yot(gli2DR) and dtr(gli1) mutants, nkx6.2 expression is absent from the floor plate (arrowheads), but expression remains more dorsally. (E) nkx6.1 is expressed in lateral floor plate cells, in motor neuron precursors and in most ventral interneuron progenitors. nkx6.1 expression is dorsally expanded in shh mRNA-injected embryos and is absent in cyclopamine treated embryos. The expression of nkx6.1 is not affected in yot(gli2DR), and dtr(gli1) mutant embryos. (F) nkx2.2a is expressed in the ventral-most cells of the CNS, including the lateral floor plate but not the medial floor plate (*). (G) hlxb9 is expressed in developing motor neurons just dorsal to the floor plate. hlxb9 expression is undetectable in con(disp1) (inset) and smu(smo) mutants and highly reduced in yot(gli2DR), and dtr(gli1) mutants (data not shown). (H) olig2 is expressed in the ventral spinal cord, including in MN precursor cells (pMN). The expression of olig2 is dorsally expanded in shh mRNA-injected embryos and absent in cyclopamine treated embryos. olig2 expression appears unaffected in yot(gli2DR), and dtr(gli1) mutants. di, diencephalon; fb, forebrain; hb, hindbrain; mb, midbrain; nc, notochord; s, somite; t, telencephalon. * Marks the medial floor plate cells. Double-ended arrows in each panel show dorsal and ventral extent of gene expression. Scale bars: (A and B), 50 μm, (C–H) 15 μm in large panels, 24 μm in smaller panels.

Class I gene expression in zebrafish and regulation by Hh. (A) pax3 is expressed in the dorsal-most cells of the spinal cord, including the roof plate. (B) pax3 expression is absent or restricted dorsally in shh mRNA injected embryos. (C) In smu(smo) mutants, spinal cord pax3 expression appears normal. (D–F) The expression of pax3 is unaffected in the neural tubes of con(disp1) (D), yot(gli2DR) (E), and dtr(gli1) (F) mutants. (G) pax7 is expressed in the dorsal spinal cord, but not in the roof plate. (H) pax7 expression is reduced or absent in shh mRNA-injected embryos. (I–L) The expression of pax7 is unaltered in smu(smo) (I), con(disp1) (J), yot(gli2DR) (K), and dtr(gli1) (L) mutant embryos. (M) pax6a is expressed throughout the spinal cord with the exception of the most ventral and most dorsal cells (arrow). (N) pax6a is reduced in shh mRNA-injected embryos. (O–R) pax6a expression is ventrally expanded in smu(smo) (O), con(disp1) (P), yot(gli2DR) (Q), and dtr(gli1) (R) mutants (arrows). (S) dbx1a(hlx1) is expressed in a band of cells in the middle of the D/V axis of the spinal cord. (T) dbx1a(hlx1) expression is reduced or absent after shh mRNA injection (arrow). (U–X) dbx1a(hlx1) expression is ventrally expanded in smu(smo) (U), and con(disp1) (V) mutants but is not altered in yot(gli2DR) (W), and dtr(gli1) (X) mutants. (Y) dbx2 is expressed in the mid-dorsal cells of the spinal cord. (Y) The expression of dbx2 is reduced an restricted to the dorsal most cells of the spinal cord in shh mRNA-injected embryos and dbx2 expression is expanded ventrally in cyclopamine treated embryos. dbx2 expression appears unaffected in yot(gli2DR) and dtr(gli1) mutants. (Z) irx3a is expressed in lateral and mid-dorsal cells. In shh mRNA-injected embryos, medial expression of irx3a is repressed while lateral irx3a expression remains unaffected. In cyclopamine treated embryos irx3a is expressed throughout the neural tube with the exception of the medial floor plate. The expression of irx3a appears unaffected in yot(gli2DR) and dtr(gli1) mutant embryos. All panels show spinal cord cross sections of 24 hpf embryos. Double-ended arrows show dorsal and ventral extent of gene expression. In general, labeling intensity appeared similar in wild type and mutant embryos. Scale bar: 15 μm (33 μm in Y and Z mutant panels).