pigk Mutation underlies macho behavior and affects Rohon-Beard cell excitability.

The study of touch-evoked behavior allows investigation of both the cells and circuits that generate a response to tactile stimulation. We investigate a touch-insensitive zebrafish mutant, macho (maco), previously shown to have reduced sodium current amplitude and lack of action potential firing in sensory neurons. In the genomes of mutant but not wild-type embryos, we identify a mutation in the pigk gene. The encoded protein, PigK, functions in attachment of glycophosphatidylinositol anchors to precursor proteins. In wild-type embryos, pigk mRNA is present at times when mutant embryos display behavioral phenotypes. Consistent with the predicted loss of function induced by the mutation, knock-down of PigK phenocopies maco touch insensitivity and leads to reduced sodium current (INa) amplitudes in sensory neurons. We further test whether the genetic defect in pigk underlies the maco phenotype by overexpressing wild-type pigk in mutant embryos. We find that ubiquitous expression of wild-type pigk rescues the touch response in maco mutants. In addition, for maco mutants, expression of wild-type pigk restricted to sensory neurons rescues sodium current amplitudes and action potential firing in sensory neurons. However, expression of wild-type pigk limited to sensory cells of mutant embryos does not allow rescue of the behavioral touch response. Our results demonstrate an essential role for pigk in generation of the touch response beyond that required for maintenance of proper INa density and action potential firing in sensory neurons.

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 knockdown 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 mutations 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.