Targeted mutation of the talpid3 gene in zebrafish revealsits conserved requirement for ciliogenesis and hedgehog signalling across the vertebrates

Abstract

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chickentalpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutantalleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within amonth of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chickta3 mutants: Absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by theexpanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetricpectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary ciliaand western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largelyrescued the effects of maternal depletion, but the motile cilia of Kupffer's vesicle remained aberrant, resulting in lateralitydefects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation ofTa3 function during vertebrate evolution. © 2011.