Recent work in mice suggests an essential function for Clp1 even before zygotic implantation; however, mice with a homozygous kinase-dead mutant allele (p.K127A) survived embryogenesis but died perinatally with spinal motor neuron degeneration (Hanada et al., 2013). To test for central nervous system requirements in a model vertebrate, we generated a germline clp1 p.R44X mutation by ENU mutagenesis in zebrafish and bred to homozygosity. Wild type fish showed strong neural clp1 expression by in situ hybridization (ISH), which was severely reduced in mutants (Figure S3A). Heterozygous and wt clutchmates were indistinguishable, but homozygous mutants did not survive past 5 days post fertilization (dpf), displayed abnormal swimming behavior, abnormal head shape and curved tail consistent with a neuromotor defect (Figure 3A-B). A second allele, representing a missense mutation near the kinase domain (p.L35R) showed a similar uniform lethality by 5 dpf (Figure S3B). We conclude that clp1 is essential in zebrafish.
