The Knock-Down (K/D) of patched: ptch MO experiments
We hypothesized that the synergy between low dose SHH andSMO reflected an attenuation of the negative inhibition exerted by endogenous zebrafish ptch . We chose p.V404M as representative of a hypo-rescued allele (see above) and evaluated three of the variants that have a mutation in each of the other SMO domains as comparative alleles. Morpholinos (MO) targeting ptch1 orptch2 were used to minimize the effect of zebrafish ptchnegative feedback inhibition towards exogenous SMO . The injection of low doses of ptch1 0.1ng and ptch2 0.1ng, or when either is injected separately, resulted in normal phenotypes (Suppl.TableS5). However, we also observed that MO doses over 0.25 ng ofptch1 or 0.2 ng of ptch2 in single injection experiments leads to observable phenotypes, especially microphthalmia and slightly curved trunk (data not shown). We found an optimal assay condition to be 100 pg of human WT SMO mRNA and the combination of 0.1 ng of eachptch1 MO and ptch2 MO based on the observed microphthalmia and trunk shape abnormality (data not shown).
Thereafter, this optimized combination injection was performed using each of the four domain-representative variant SMO mRNAs. The results show that the eye area of embryos injected with gfp are significantly larger than that of others (P <0.0001). WTSMO produced the expected micropthalmia at the test dose. The eye area of embryos injected with p.V404M is significantly smaller than that of other variants (Fig.4A, a and b ), suggesting that the p.R113Q, p.R199W, and p.P687L point mutations affect eye development less than p.V404M point mutation. However, these findings are not substantially different from the WT control.
Our measurement of the trunk angle shows that embryos injected with WT (or all four tested variants) are significantly wider than that ofgfp (P <0.0001), a known property of midline Hh signaling on somite development. In comparisons between the four variants, p.R113Q has a significantly smaller somite angle than that of p.R199W, p.P687L and gfp (Fig.4B a’ and b’ ), suggesting that the p.R113Q mutation is less bioactive under these assay conditions. Taken together, although these biometric measures were more readily quantifiable, these results do not provide unambiguous evidence for either gain or loss-of-function when directly compared to the WT control.