SHH/SMO co-expression approach to the variant analysis
As a first step to quantify the biological consequences of eachSMO variant, we utilized this optimal co-injection dose (Suppl.Fig.2). The percentage of abnormal phenotypes, based on their gross microscopic appearance, between each of the seven variants indicated very mild allelic differences (Fig.2B; Suppl.TableS2 and S3). Indeed, these variants were similar in bioactivity to WT SMOunder identical conditions.
To investigate any difference in biological activities between variants, we performed in situ hybridization at 18 to 24hpf using zebrafishpax2a and pax6a , as eye developmental markers, andneurod4 as a neural marker. The expression level of pax6a(Fig.2C, D) and neurod4 (Fig.2E, F) around the eye region showed that all variants have a Type II phenotype with suppression of each genes’ expression at 19hpf (pax6a ) or 24hpf (neurod4 ) (Suppl.Table3). The pax2a up-regulation (Fig.2G, H), a known antagonist of pax6a , expression pattern showed that all variants have Type II phenotype with enhancement of pax2aexpression at 24hpf (Suppl.Table3). In all markers shown here, the percent of embryos with Type II expression patterns were over 80% among all variants tested under these conditions. Although we can clearly demonstrate the expected marker changes, our ability to measure subtle differences between alleles was limited by the near saturating dose required to elicit these changes. We conclude that none of the HPE variants are complete loss-of-function under these test conditions. Furthermore, despite minor differences between variant alleles these changes in gene expression patterns are qualitatively distinct, but not at a statistically significant level.