To determine if human CLP1 is a functional orthologue of zebrafish clp1 we injected wt and mutant human CLP1 mRNA into mutant clp1 p.R44X mutant fish zygotes. The curved tail phenotype was apparent by 48 hpf so we utilized this as a readout as measured by the depth of the vertebral curve from highest to lowest point at 3 dpf. Average height was less than 50 μm in wt fish but over 300 μm in mutant fish (Figure 3E). Injection of wt human CLP1 mRNA partially rescued the average curve height to less than 200 μm, although variable from fish-to-fish. Injection of the human p.R140H mutant CLP1 mRNA did not mediate such rescue, with peak average height not less than 300 μm. In addition, we performed ISH for otx2 on wt and clp1 p.R44X mutants injected with human CLP1 mRNA. As expected, human wt CLP1, but not CLP1R140H was sufficient to prevent most loss of otx2 expression in clp1 p.R44X mutants (Figure 3F). We conclude that human CLP1 can at least partially replace the zebrafish clp1, suggesting the human and zebrafish CLP1 genes are orthologues, and that the human mutation lacks activity in vivo.
