Analysis of the zebrafish perplexed mutation reveals tissue specific roles for de novo pyrimidine synthesis during development

The zebrafish perplexed mutation disrupts cell proliferation and differentiation during retinal development. In addition, growth and morphogenesis of the tectum, jaw, and pectoral fins are also affected. Positional cloning was used to identify a mutation in the carbamoyl-phosphate synthetase2-aspartate transcarbamylase-dihydroorotase (CAD) gene as possibly causative of the perplexed mutation and this was confirmed by gene knock-down and pyrimidine rescue experiments. CAD is required for de novo biosynthesis of pyrimidines that are required for DNA, RNA, and UDP-dependent protein glycosylation. Developmental studies of several vertebrate species showed high levels of cad expression in tissues where mutant phenotypes were observed. Confocal time-lapse analysis of perplexed retinal cells in vivo showed a near doubling of the cell cycle period length. We also compared the perplexed mutation with mutants that affect either DNA synthesis or UDP-dependent protein glycosylation. Cumulatively our results suggest an essential role for CAD in facilitating proliferation and differentiation events in a tissue specific manner during vertebrate development. Both de novo DNA synthesis and UDP-dependent protein glycosylation are important for the perplexed phenotypes.