Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation

Abstract

In zebrafish, cranial sensory circuits form by 4 days post-fertilization. We used a forward genetic screen to identify genes involved in the formation of these circuits. In one mutant allele, sl23, axons arising from the epibranchial sensory ganglia do not form their stereotypical terminal fields in the hindbrain. These embryos also had small eyes and deformed jaws, suggesting a pleiotropic effect. Using positional cloning, a 20-nucleotide deletion in the carbamoyl-phosphate-synthetase2-aspartate-transcarbamylase-dihydroorotase (cad) gene was found. Injection of a CAD morpholino phenocopied the mutant and mutants were rescued by injection of cad RNA. Cad activity is required for pyrimidine biosynthesis, and thus is a prerequisite for nucleic acid production and UDP-dependent protein glycosylation. Perturbation of nucleic acid biosynthesis can result in cell death. sl23 mutants did not exhibit elevated cell death, or gross morphological changes, in their hindbrains. To determine if defective protein glycosylation was involved in the aberrant targeting of sensory axons, we treated wild type embryos with tunicamycin, which blocks N-linked protein glycosylation. Interference with glycosylation via tunicamycin treatment mimicked the sl23 phenotype. Loss of cad reveals a critical role for protein glycosylation in cranial sensory circuit formation.

A missense mutation (p.Tyr452Cys) in the CAD gene compromises reproductive success in French Normande cattle

“…Despite being alive and apparently healthy and fertile, this animal had very poor udder morphology (scored among the lowest 1% of animals with same sire; Figure 1). This is consistent with previous observations in model organisms and humans, in which deleterious mutations in CAD lead to embryonic lethality with incomplete penetrance (Norby, 1973;Falk and Nash, 1974;Willer et al, 2005;Franks et al, 2006;Cox et al, 2014;Ng et al, 2015). Of note, studies in humans and Drosophila demonstrated that uridine supplementation reduces the severity of the symptoms by the salvage pathway of pyrimidine biosynthesis (Falk and Nash, 1974;Ng et al, 2015;Koch et al, 2017).…”

The Metalloproteinase adam19b Is Required for Sensory Axon Guidance in the Hindbrain

“…Standard mapping methods using bulk segregant and meiotic recombination analysis of microsatellite markers (Green et al, 2009 ; Cox et al, 2014 ) were used to map the sl19 mutation to a critical interval on chromosome 14. Total RNA was then extracted from mutant and wild type 4 days post fertilization (dpf) larvae and cDNA synthesized with Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA).…”

“…RNA (100 pg/nl) or morpholino oligonucleotides (MO; Gene Tools, 1–2 ng/nl, Supplementary Table S2 ) were injected as previously described (Cox et al, 2014 ).…”

“…Imaging was carried out as described previously (Cox et al, 2014 ). Final brightness and/or contrast values of images were adjusted in Adobe Photoshop CC 2017.…”

“…This was performed as described in a previous article (Cox et al, 2014 ). Images were obtained by confocal microscopy as described above.…”