Optimization of the biological response to SMOover-expression
To minimize the inhibition of exogenous SMO via endogenous zebrafish ptch , we co-injected a small amount of WT humanSHH mRNA with SMO mRNA. We identified an optimal combination to induce abnormal phenotypes is the co-injection of 2pg ofSHH mRNA and 200pg SMO mRNA (see the dose-response pilot study, Suppl.Fig.2). We could independently demonstrate that 2pg ofSHH mRNA (alone) or 200pg of SMO mRNA (alone) has minimal phenotypic consequences that were similar to our gfp control mRNA-injected embryos (Suppl.TableS1). We could demonstrate that co-injection induced a striking synergistic effect for embryos that was consistent with our understanding of the Hh GRN. The majority of co-injected embryos resulted in decreased pigmentation of the retina at 24 to 48 hours post fertilization (hpf). Cyclopia as a severe phenotype was observed in a few embryos at 24hpf (Fig.2A, B).