Gain-of-function approach: Over-expression analysis ofSMO variants
To experimentally analyze the effect of these point mutation changes, we conducted over-expression experiments as our first approach. We microinjected wild type (WT) human SMO mRNA to determine the appropriate dose which would induce an abnormal phenotype in WT zebrafish (TAB5). However, in the pilot experiments using 100pg to 600pgSMO mRNA we did not readily induce abnormal phenotypes (Suppl. Fig.2). Based on our understanding of the hedgehog GRN described in Suppl.Fig.1 we hypothesized that the microinjected SMO was inhibited by ptch in a negative feed-back loop attenuating the biological response.