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

Neuronal basic helix-loop-helix (bHLH) transcription factors such as neuroD and neurogenin have been shown to play important roles in neuronal develop ment. In the present study, several distinct bHLH DNA fragments were isolat ed from the zebrafish genomic DNA by a pair of degenerate polymerase chain reaction (PCR) primers deduced from the conserved bHLH domains of neuroD an d neurogenins. Based on the bHLH fragments, three complete neuroD-related c DNA clones, including complete coding regions, ndr1a, ndr1b, and ndr2 (ndr for neuroD related), were isolated and assembled by 5' and 3' rapid amplifi cation of cDNA ends (RACE), A phylogenetic analysis indicated the presence of four groups of neuroD-related genes in the nerrroD subfamily in vertebra tes: neuroD ndr1a/ndr1b/MATH-2, ndr2/NDRF, and neuroM/MATH3. Expression of the newly isolated neuroD-related genes was examined by reverse transcripta se (RT)-PCR and whole-mount in situ hybridization, Unlike neuroD, which was expressed broadly in primary neurons during early zebrafish development st arting from 10 h postfertilization (hpf), expression of ndr1a and ndr1b sta rted relatively late (around 22 hpf) and was restricted to the olfactory sy stem: olfactory bulbs in the telecephalon (ndr1a and ndr1b) and olfactory o rgans (ndp1b) 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-mo unt in situ hybridization in embryos up to 72 hpf, suggesting that it is ex pressed rather late. Our observations suggest that the two novel nenroD-rel ated genes, ndr1a and ndr1b, are involved in the development of the olfacto ry system and perhaps contribute to its functional complexity.