Orphan nuclear receptors Nur77, Nurr1 and Nor1 in zebrafish (Danio rerio): Characterization, regulation, and roles in development.

摘要

The orphan nuclear receptors Nurr1, Nur77, and Nor1 (NR4A family) have been implicated in both the normal regulation and the pathophysiology of cell proliferation, differentiation, and apoptosis. In mice, NR4A receptors are required for development and maintenance of dopaminergic (DA) neurons (Nurr1), for axonal guidance and survival of hippocampal neurons (Nor1), and for development and regulation of neuroendocrine axes (Nur77). Because of the advantages of zebrafish as a model for developmental genetics, I cloned and characterized cDNAs encoding the zebrafish NR4A receptors. Sequence and phylogenetic analysis indicated a high degree of evolutionary conservation within and across metazoan taxa. Molecular homology modeling of zebrafish Nurr1 and Nur77 against crystal structures of mammalian orthologs revealed features consistent with ligand independent regulation, suggesting a conserved characteristic. Real time reverse transcriptase polymerase chain reaction (RT-PCR) analysis demonstrated that NR4A mRNAs are most abundant in the brain and eye of adult zebrafish. Levels of NR4A mRNAs in eggs, embryos and larvae revealed maternal transfer followed by gene-specific developmental programming. Whole-mount in situ hybridization analysis demonstrated gene-specific NR4A expression patterns in brain and retina as early as 2 days post-fertilization (dpf; brain regionalization stage). Consistent with a role in DA neuron development, Nurr1 mRNA was localized in established catecholaminergic neurons of the central nervous system, and in a subset of retinal amacrine cells. As demonstrated by morpholino-mediated knockdown studies, Nurr1 was necessary for development of certain Nurr1-labeled neuronal clusters and for normal expression of the neural genes tyrosine hydroxylase and neuropilin in whole embryos. NR4A mRNAs were rapidly (< 2 hour) and robustly (4--8 fold) induced by forskolin as early as 1 dpf in embryos, and similarly induced in zebrafish brain and ovary cell cultures (40--400 fold), confirming their designation as immediate early genes. In contrast, estradiol treatment (2 d) increased Nur77 (but not Nor1 or Nurr1) mRNA in brain, eye, and ovary of adult animals, but had no effect on mRNA levels in embryos. The results described in this thesis provide insight into the evolutionary origin, conserved structural features, regulation and neural functions of the NR4A family of orphan nuclear receptors, and demonstrate the utility of zebrafish as a model for further understanding their roles in neurodevelopmental signaling and human neural disease processes.