Selective labeling of central and peripheral sensory neurons in the developing zebrafish using P2X(3) receptor subunit transgenes.

The two paralogous P2X receptor subunit genes p2rx3.1 and p2rx3.2 are selectively expressed in overlapping, but unique, patterns of sensory neurons in the developing zebrafish. We constructed a series of transgenes derived from both genes using the recombineering technique. Transgenes utilizing either enhanced green fluorescent protein or monomeric red fluorescent protein-1 were shown to be expressed with the same spatial and temporal patterns as the native genes. The p2rx3.1-derived transgenes labeled the vast majority of the Rohon-Beard neurons in the spinal cord and neurons of the trigeminal ganglia. The p2rx3.2-derived transgene labeled fewer Rohon-Beard and trigeminal neurons than what was observed for the p2rx3.1-derived transgenes, but was also detected in neurons of the epibranchial ganglia. Three distinct populations of sensory neurons were detected: those expressing only one or the other paralog, and those expressing both paralogs. The fluorescent proteins encoded by the transgenes allowed for visualization of the neuronal somas as well as their peripheral and central projections. These reagents should prove extremely useful in providing the basis for future studies aimed at elucidating the developmental and physiological attributes of sensory neurons.

Molecular characterization of the zebrafish P2X receptor subunit gene family.

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.