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Fig. S1 Rescue of six1a morphant phenotypes is observed with overexpression of six1a mRNA. Immunofluorescent (IF) staining for multiple SIX family members at 24 hours post-fertilization (hpf) reveals a decrease in protein expression in the somites when six1a morpholino (MO) is injected in 1-4 cell embryos (A). Injection of sixla MO leads to downregulation of Six1 protein specifically as observed by Western blot analysis (B); with this protein expression rescued by co-injection of six1a mRNA with six1a MO. Quantification of cleaved-caspase-3 IF staining (C) at 24 hpf demonstrates that cell survival is also rescued with six1a mRNA overexpression in six1a morphants, ***p<0.0001, ANOVA with Bonferroni’s post-hoc test. Live images (D) at 24 hpf, anterior to the left, show that the U-shaped somite morphology observed in six1a morphants is rescued to normal V-shape with six1a mRNA overexpression (control: n=13/13, 13 ng six1a MO: n=8/10, 13 ng six1a MO + 100pg six1a mRNA: n=12/17). Inset images confirm stage-matching across groups.