Fig. S1. Movl embryos are indistinguishable from wild-type embryos at 24 hpf. (A-L) Wild-type embryos (a,c,e,g,i,k) and Movl embryos (b,d,f,h,j,l) at 24 hpf were stained for the expression of ptc1 (A,B), gli1 (c,d), nkx2.2a (e,f), tal2 (g,h), olig2 (i,j) and eng2 (k,l). All markers show similar staining patterns between wild-type and Movl embryos. Lateral views. Scale bar: 100 m.

Fig. S2. MZovl mutants have relatively normal morphology. Bright-field images of Movl control embryo (A) and MZovl mutant (B) at 30 hpf. Note that other than the body curvature phenotype, MZovl mutants have relatively normal overall morphology. Scale bar: 100 m.

Fig. S3. MZovl mutants have randomized left-right patterning. (a-e) MZovl mutants (b-e) display randomized lft2 expression compared with wild-type embryos (a). Symmetric eng2 expression (asterisks) in the midbrain-hindbrain boundary remains normal in MZovl mutants and is used as an internal landmark to compare lft2 expression between embryos. Examples of MZovl mutants with lft2 expression on the left (b), right (d), or both sides (c), or with no lft2 expression (e), are shown. Dorsal views of 22-somite stage embryos. Scale bar: 50 m. (f) Quantification of left-right defects in MZovl mutants. Of wild-type embryos, 96% (n=100) show lft2 expression on the left side, whereas MZovl mutants (n=45) show randomized lft2 expression: 24% on the left side (B), 27% on the right side (D), 40% bilaterally (C), and 9% with no expression (E).

Fig. S4. Neural and somite patterning in iguana mutants. (A-H) Control embryos (igu/+ or +/+) (a,c,e,g) and igu/igu mutants (b,d,f,h) at 24 hpf were stained for the expression of ptc1 (A,B), nkx2.2b (c,d), olig2 (e,f) and eng2 (g,h). igu/igu mutants show dampened, but expanded, ptc1 expression (B). Accordingly, nkx2.2b expression in the neural tube is lost in igu/igu mutants (D), whereas olig2 expression in the neural tube (F) and eng2 expression in the somite (H) are expanded. Lateral views. Scale bar: 100 m.

Fig. S5. iguana mutants lack primary cilia. Cilia were visualized by staining with acetylated α-tubulin antibody (red), and basal bodies were visualized by staining with γ-tubulin antibody (green). (A,B) Primary cilia in the neural tube (arrowheads in A) are absent in igu/igu mutants (B), whereas the longer floor plate cilia (arrows) are reduced in number in igu/igu mutants as compared with control embryos (igu/+ or +/+) (A). (C,D) igu/igu mutants lack short non-motile cilia (arrowhead in C) and have a reduced number of the longer tether cilia (arrows) in the otic vesicle. Note that the cluster of tether cilia at the opposite end of the otic vesicle is out of focus in the control embryo (C). Asterisks in B and D mark the staining of axons, which also label with antibody to acetylated α-tubulin. Lateral views at 24 hpf. Scale bars: 20 m.

Fig. S6. Epistasis analysis of cilia and Hh signaling in somite patterning. (A-J) Movl control embryos (a,c,e,g,i) and MZovl mutants (b,d,f,h,j) were stained for the expression of eng (green) and prox1 (red) following different manipulations. Ectopic expression of shh mRNA (C), depletion of ptc1,2 using morpholinos (E), and expression of dnPKA mRNA (I) in Movl embryos induce ectopic muscle pioneer cells (prox1⁺, strong eng⁺, arrows) and superficial slow fibers (prox1⁺) as compared with uninjected controls (a), whereas treatment with cyclopamine at 100 M (g) blocks the formation of most muscle pioneers, superficial slow fibers and medial fast fibers (prox1⁻, weak eng⁺, arrowhead in A). By contrast, MZovl embryos are insensitive to these manipulations and are indistinguishable from untreated MZovl embryos, in which medial fast fibers (arrowheads) are expanded (b,d,f,h,j). Lateral views at 24 hpf. Scale bar: 100 m.