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

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.

ppk23-Dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster.

Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Although degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster, the physiological functions of these proteins are still poorly understood. We found that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory bristles that are distinct from those expressing feeding-related gustatory receptors. Disrupting ppk23 or inhibiting activity of ppk23-expressing neurons did not alter gustatory responses. Instead, blocking ppk23-positive neurons or mutating the ppk23 gene delayed the initiation and reduced the intensity of male courtship. Furthermore, mutations in ppk23 altered the behavioral response of males to the female-specific aphrodisiac pheromone 7(Z), 11(Z)-Heptacosadiene. Together, these data indicate that ppk23 and the cells expressing it play an important role in the peripheral sensory system that determines sexual behavior in Drosophila.

Diverse mechanisms for assembly of branchiomeric nerves.

The formation of branchiomeric nerves (cranial nerves V, VII, IX and X) from their sensory, motor and glial components is poorly understood. The current model for cranial nerve formation is based on the Vth nerve, in which sensory afferents are formed first and must enter the hindbrain in order for the motor efferents to exit. Using transgenic zebrafish lines to discriminate between motor neurons, sensory neurons and peripheral glia, we show that this model does not apply to the remaining three branchiomeric nerves. For these nerves, the motor efferents form prior to the sensory afferents, and their pathfinding show no dependence on sensory axons, as ablation of cranial sensory neurons by ngn1 knockdown had no effect. In contrast, the sensory limbs of the IXth and Xth nerves (but not the Vth or VIIth) were misrouted in gli1 mutants, which lack hindbrain bmn, suggesting that the motor efferents are crucial for appropriate sensory axon projection in some branchiomeric nerves. For all four nerves, peripheral glia were the intermediate component added and had a critical role in nerve integrity but not in axon guidance, as foxd3 null mutants lacking peripheral glia exhibited defasciculation of gVII, gIX, and gX axons. The bmn efferents were unaffected in these mutants. These data demonstrate that multiple mechanisms underlie formation of the four branchiomeric nerves. For the Vth, sensory axons initiate nerve formation, for the VIIth the sensory and motor limbs are independent, and for the IXth/Xth the motor axons initiate formation. In all cases the glia are patterned by the initiating set of axons and are needed to maintain axon fasciculation. These results reveal that coordinated interactions between the three neural cell types in branchiomeric nerves differ according to their axial position.

Ectodermal P2X receptor function plays a pivotal role in craniofacial development of the zebrafish.

P2X receptors are non-selective cation channels operated by extracellular ATP. Currently, little is known concerning the functions of these receptors during development. Previous work from our lab has shown that zebrafish have two paralogs of the mammalian P2X3 receptor subunit. One paralog, p2rx3.1, is expressed in subpopulations of neural and ectodermal cells in the embryonic head. To investigate the role of this subunit in early cranial development, we utilized morpholino oligonucleotides to disrupt its translation. Loss of this subunit resulted in craniofacial defects that included malformation of the pharyngeal skeleton. During formation of these structures, there was a marked increase in cell death within the branchial arches. In addition, the epibranchial (facial, glossopharyngeal, and vagal) cranial sensory ganglia and their circuits were perturbed. These data suggest that p2rx3.1 function in ectodermal cells is involved in purinergic signaling essential for proper craniofacial development and sensory circuit formation in the embryonic and larval zebrafish.