Summary of genes with significantly altered expression in igu mutants and cyclopamine-treated embryos. A–C: Phenotypes of wild-type (A), igu mutant (B) and cyclopamine-treated wild-type (C) embryos at 48 hpf. Arrows point to hemorrhages. Scale bar = 100 µm. D: Venn diagram of total genes significantly up- or down-regulated in treatment group as compared to their control. The purple circle represents the comparison of igu mutants and wild-type siblings and the blue circle represents the comparison of cyclopamine-treated to DMSO-treated siblings. The overlap represents 40 annotated genes that were changed in both groups. E: Heat map of 40 genes differently expressed in both igu mutant and cyclopamine-treated embryos. Green indicates down-regulation, and red indicates up-regulation. Values on scale bar indicate range of fold changes from 6.2-fold down-regulated to 2.1-fold up-regulated. igu, iguana mutant; cyc, cyclopamine-treated.

In situ hybridization of genes misregulated in igu mutants corresponds to direction of expression change on the microarray. A,A′: Nkx3.2 is absent from the notochord (arrowheads) of igu mutants. B,B′: In floor plate (arrows) and brain (arrowheads), Nkx2.2a signal is decreased in igu mutants. C,C′: Nkx2.2b signal is absent from floor plate (arrows) in igu mutants and decreased in ventral forebrain (arrowheads). D,D′: Nkx2.9 is severely reduced in the brain (arrowheads) and floor plate (arrows) of igu mutants. E,E2: FoxF2a ventral head mesoderm (arrows) signal is reduced in igu mutants compared with wild-type siblings. F,F2: FoxF2b signal in ventral head mesoderm (arrows) of wild-type sibling is greatly reduced in igu mutant. G,G′: Tbx20 transcript is decreased in the branchiomotor neurons (arrows) and heart (arrowheads) in igu mutants. H,H′: Ntn1b is decreased in the head (arrowhead) and floor plate (arrows) of igu mutants. I,I′: Ntn5 is up-regulated in the trunk (arrowheads) of igu mutants. J,J′: Fabp11b is expressed in the eye (arrowhead) and lost in igu mutants. K,K′: Urp2 is expressed in the floor plate (arrowheads) and is absent in igu mutants. L,L′: Pim1 transcript is up-regulated in the brain and eye of igu mutants. M,M2: Shha shows no change between wild-type sibling and igu mutant. N,N2: ptch2 is strongly up-regulated in igu mutants. Scale bars = 100 µm.

In situ hybridization of genes misregulated in cyclopamine-treated embryos corresponds to direction of expression change on the microarray. A,A2: Nkx3.2 is absent from the jaw joint (black arrowheads), pharyngeal arches (white arrowheads) and notochord (arrow) of cyclopamine-treated embryos. B,B2: Nkx2.2a signal is absent from the floor plate (arrows) and reduced in the brain (arrowheads) of cyclopamine-treated embryos. C,C′,D,D′: Expression of Nkx2.2b (C,C′) and Nkx2.9 (D,D′) is completely lost from brain (arrowheads) and floor plate (arrows) of cyclopamine-treated embryos. E,E′,F,F′: Ventral head expression of Foxf2a (E) and Foxf2b (F) is absent in cyclopamine-treated embryos (E2,F′). G,G2: Tbx20 transcript is decreased in branchiomotor neurons (arrows), but maintains expression in the heart (arrowheads) cyclopamine-treated embryos. H,H′: Ntn1b is decreased in the head (arrowheads) and floor plate (arrows) of cyclopamine-treated embryos. I,I′: Ntn5 is down-regulated in the trunk (arrowheads) of cyclopamine-treated embryos. J,J′: Expression of Fabp11b in the eye (arrowheads) is absent in cyclopamine-treated embryos. K,K′: Urp2 in the floor plate (arrows) is unchanged in cyclopamine-treated embryos. L,L′: Pim1 transcript expression is increased in the brain and eye of cyclopamine-treated embryos. M,M′: Shha shows no change between DMSO and cyclopamine. N,N′: ptch2 is strongly down-regulated in cyclopamine-treated embryos. Scale bars = 100 µm.