In mouse, multiple phenotypes observed in Clp1 mutants are rescued by allelic removal of p53, suggesting that the cellular apoptosis observed in clp1 mutants is p53-dependent (Hanada et al., 2013). In order to test this in zebrafish, we performed p53 morpholino knockdown using a published reagent (Langheinrich et al., 2002) in clp1 wt and mutants, then performed otx2 ISH as a marker for cell loss. We observed rescued otx2 expression in p53-knockdown clp1 mutant zebrafish, suggesting the neural apoptosis is p53-dependent (Figure S3D). We also performed zygotic knockdown of the single hspc117 zebrafish orthologue, the gene proposed to mediate the redundant splicing pathway, with either ATG- or splice-blocking morpholinos, achieving near complete loss of spliced transcript with the latter. However, we found no phenotype in any of the morphants (not shown). We additionally performed hspc117 knockdown in clp1 p.R44X mutants, but found no exacerbation of the clp1 mutant phenotype (not shown), suggesting that, at least in zebrafish, hspc117 does not play an essential role, or genetically interact with clp1.
