CHARACTERIZATION OF ZEBRAFISH NEUROD/NEUROGENIN GENE FAMILY AND AN EARLY PROGRAM OF NEURODIFFERENTIATION

Abstract

The basic helix-loop-helix transcription factors have been shown to play important roles in cell fate determination by binding to a common DNA consensus (CANNTG) as either homo- or heterodimers. Neuron development is a highly complex process in vertebrates, and some neurogenic genes are the members of the bHLH family. As zebrafish has become an increasingly popular vertebrate model in embryonic and genetic studies and providesan excellent system to study neuron development, four neurogenic bHLH genes have been isolated from zebrafish embryonic cDNA library. All of these genes have the conserved bHLH domain and are classified into two subfamilies, three in neuroD subfamily (zfneuroD, zfNDR1 and zfNDR2) and one in neurogenin subfamily (zfNgR1), according to the amino acid sequence homology within the bHLH domain. zfneuroD shows high homology to Xenopus NeuroD (70%) and human NeuroD (75% ), and is thought as the counterpart of the human and Xenopus neuroD genes. Both zfNDR1 (61.4%) and zfNDR2 (54.4%) are highly homologous to MATH 2. zfNgR1, which belongs to the neurogenin subfamily, does not share significant homology with the rat neurogenin, X-NgR 1 or other bHLH genes and may represent a novel member in the neurogenic subfamily. Analysis of their expression patterns by whole mount in situ hybridization shows that all of these four genes are expressed in different parts or nervous system with different time tables. zfNgR1 is the first gene expressed in zebrafish embryo. Its transcripts can be detected at 9.5 hpf stage in midline of prechordal region and on both sides of the neural keel along the rostro-lateral edges of the neural plate. The zfNgR1 mRNA can also be detected in most, if not all, primary neuronal cell clusters in the spinal cord. Immediately following zfNgR1, zfneuroD starts to express at 10 hpf stage. Its transcript can be detected in trigeminal ganglia, telecephalon, diencephalon, Rohan- Beard cells and motor neurones. zfNDR1 and zfNDR2 transcripts cannot be detected until in the 28 hpf stage embryo. zfNDR1 is expressed only in telecephalon and some ganglia cells in the eye at a low level. zfNDR2 is expressed at a high level in telecephalon and at a low level in olfactory placode. These results indicate that all of these genes are involved in neuron differentiation and may play different roles during development of the nervous system.