A class of neuroD-related basic helix-loop-helix transcription factors expressed in developing central nervous system in zebrafish

Abstract

Neuronal basic helix-loop-helix (bHLH) transcription factors such as neuroD and neurogenin have been shown to play important roles in neuronal development. In the present study, several distinct bHLH DNA fragments were isolated from the zebrafish genomic DNA by a pair of degenerate polymerase chain reaction (PCR) primers deduced from the conserved bHLH domains of neuroD and neurogenins. Based on the bHLH fragments, three complete neuroD- related cDNA clones, including complete coding regions, ndr1a, ndr1b, and ndr2 (ndr for neuroD related), were isolated and assembled by 5' and 3' rapid amplification of cDNA ends (RACE). A phylogenetic analysis indicated the presence of four groups of neuroD-related genes in the neuroD subfamily in vertebrates: neuroD, ndr1a/ndr1b/MATH-2, ndr2/NDRF, and neuroM/MATH3. Expression of the newly isolated neuroD-related genes was examined by reverse transcriptase (RT)-PCR and whole-mount in situ hybridization. Unlike neuroD, which was expressed broadly in primary neurons during early zebrafish development starting from 10 h postfertilization (hpf), expression of ndr1a and ndr1b started relatively late (around 22 hpf) and was restricted to the olfactory system: olfactory bulbs in the telecephalon (ndr1a and ndr1b) and olfactory organs (ndr1b) starting around 22 hpf. Although a faint ndr2 mRNA signal was detected by RT-PCR in early embryos, no ndr2 mRNA was detected by whole-mount in situ hybridization in embryos up to 72 hpf, suggesting that it is expressed rather late. Our observations suggest that the two novel neuroD- related genes, ndr1a and ndr1b, are involved in the development of the olfactory system and perhaps contribute to its functional complexity.