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.