Figure 2—figure supplement 1.

Figure 2—figure supplement 1. Characterization of CRISPR/Cas9 generated dact1−/− and dact2−/− mutants.

(A) Schematic representations of dact1 and dact2 exons, positions of guide RNA target site, introduced premature stop codon (arrow), and sequences of mutations. (B) DNA fragment analysis of dact1+/- and dact2+/- animals showing wildtype (250 and 387 bp, respectively) and mutant (228 and 380 bp, respectively). (C) Expression levels of dact1 and dact2 mRNA by RT-qPCR in 12 hpf dact1−/− mutants, dact2−/− mutants, and dact1/2−/− compound mutants. Eight embryos were pooled for mRNA isolation per sample. (D) Injection of dact1 mRNA, dact2 mRNA, or a combination of dact1 and dact2 mRNA rescues the rod-shaped ethmoid plate phenotype in dact1/2−/− compound mutants. Representative images of Alcian blue stained dact1/2−/− double mutant treated with 300 pg dact1 mRNA and 300 pg dact2 mRNA. Arrow highlights normal ethmoid plate (EP). Visceral cartilage (VC) also appeared normal. (E) Quantification of the mutant craniofacial phenotype observed in a dact1−/−,dact2+/- breeding in-cross. Without mRNA injection, the mutant phenotype was observed at approximately (35%) the expected Mendelian ratio of 25%. Injection with dact1 mRNA, dact2 mRNA, or a combination of dact1 and dact2 mRNA decreased the frequency that the mutant craniofacial phenotype was observed.