Characterization of proneural bHLH transcription factor target genes in X. laevis

Abstract

Zc3h12c is a member of the Zc3h12 endoribonuclease family (also known as MCPIP/Regnase), which contains four members (Zc3h12a-c). This family of proteins is characterized by the presence of a single centrally located CCCH-type zinc finger domain, which mediates RNA binding, and an N-terminally located NYN/PIN domain responsible for RNase activity. Members of this family are best described for their role in modulating inflammation, through negative feedback via destruction of specific mRNAs encoding proinflammatory cytokines. In this thesis, the role of the RNase Zc3h12c as a novel regulator of early neural development in X. laevis is described. In ectodermal explants, Zc3h12c localizes as granule-like structures in the cytoplasm. Transcripts of zc3h12c are first detected by WMISH to the bilateral stripes of primary neurogenesis in open neural plate stage embryos. The temporal expression of zc3h12c is preceded by the proneural gene neurog2, but parallels that of the neural zinc finger transcription factor myt1. In tailbud stages embryos, zc3h12c transcripts are specifically expressed in the nervous system including the brain, eye and intermediate zone of the spinal cord where cells are actively undergoing differentiation. Consistent with its expression, zc3h12c is positively regulated by Neurog2, both in the embryo and in animal caps. Overexpression of Zc3h12c increases the number of primary neurons within the neurogenic stripes in an RNase dependent manner. Results from loss of function experiments in embryos and animal caps show that Zc3h12c acts downstream of Neurog2 and is essential for primary neuron differentiation. A transcriptome analysis of animal caps directed to undergo neurogenesis through Neurog2 expression in the presence or absence of Zc3h12c (MO knockdown) was performed. Zc3h12c knockdown only influenced a subset of Neurog2 downstream genes. The knockdown of Zc3h12c did not disrupt the Neurog2-induction of genes involved in the early phase of neuronal differentiation, including most neural transcription factors (e.g. ebf2, ebf3, tlx3, myt1, neurod4). However, later acting neuronal genes like tubb2b and those encoding cytoskeletal binding proteins and other aspects of neuronal maturation and function were preferentially disrupted. The knockdown of Zc3h12c resulted in the upregulation of several hes genes, indicating elevated Notch signaling. However, the loss of neuronal differentiation upon knockdown of Zc3h12c could not be rescued through the inhibition of Notch signaling suggesting additional mechanisms. Zc3h12c knockdown also upregulated genes involved in promoting the cell cycle, including several cyclins, which suggests that the failure of neural progenitors to undergo neuronal differentiation upon Zc3h12c knockdown may be due to the inability to exit the cell cycle.