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).