Loss-of-function approach: Analysis of SMO variants using a Zebrafish Knock Out (K/O) model rescue assay
To better understand the function of these SMO variants we performed rescue experiments involving the injection of human SMOtranscripts and measuring the biological activity of each variant on developing zebrafish embryos using a CRISPR/Cas9 genetic mutant in this study. We confirmed that the vast majority of smo-/smo-homozygous mutants (Fig.3B) manifested a shortened body trunk, curledU -type tail, heart edema, decreased eye color of the retina, and microphthalmia (Chen et al., 2001). We collected smo-/smo-homozygous mutants by mating smo+/smo - breeding pairs. We optimized this assay by empirically determining the most effective dose for phenotypic rescue using the WT human SMO . We found our ideal assay conditions to be 80pg of human WT SMO mRNA based on the observed trunk-tail shape and eye color recovery (Suppl.Table4). There were three types of phenotypic rescue: complete rescue (Fig.3B _Control and TypeI), or partial rescue, scored as primarily eye (Fig.3B _TypeII) or primarily body rescue (Fig.3B _TypeIII). The genotype was assessed by fluorescence PCR after phenotype confirmation. The abnormal phenotype of smo-/smo-homozygous is efficiently rescued by microinjection of WT SMO and all variants at 48hpf except for p.V404M (Fig.3C). Genotyped embryos showed that p.V404M was significantly deficient in its’ ability to rescue the smo-/smo- phenotype. Furthermore, the proportion of rescue types showed the percentage of partial rescue, combined percentages of eye rescue and body rescue, is significantly different only with the p.V404M variant (Fig.3D). Essentially identical results for the 7 variants were obtained at 24hpf by scoring for simple complete rescue using a much lower 10pg dose. However, the mutant phenotype began to re-appear at later time points (data not shown). Interestingly, we note that Type III rescue was not detected with p.R113Q.