Transcriptional control in the context of primary neurogenesis

Abstract

The Myc/Max/Mad network of bHLH-Zip transcription factors plays an essential role in a variety of cellular processes including proliferation, differentiation and apoptosis. Mxi1, a Mad family member, was previously isolated in our laboratory on the basis of its early expression pattern in the territories of primary neurogenesis. In the first part of this thesis work, a functional characterization of Xmxi during Xenopus embryogenesis was performed. Consistent with an early role in neurogenesis, Xmxi1 was found to be positively regulated by the panneural genes, and proneural genes, as well as negatively by the Notch pathway. Loss-of-function experiments demonstrated an essential role for Xmxi1 in the establishment of a mature neural state that can be activated by factors that induce neuronal differentiation, such as SoxD and X ngnr-1. Overexpression of Xmxi1 resulted in a transient inhibition of neuronal differentiation, and at early tailbud stages both endogenous and ectopic neurogenesis were observed. While Xmxi1 enhances cell proliferation and apoptosis in the early Xenopus embryo, both activities appear not to be required for the function of Xmxi1 in primary neurogenesis. During primary neurogenesis, cell to cell signaling mediated by the Notch pathway restricts the number of cells that undergo neuronal differentiation. In the second part of this thesis work, an unbiased screen to identify early target genes of the Notch effector Enhancer-of-split related 1 (ESR1) was performed. A library enriched in early ESR1 target genes was prepared by PCR subtractive amplification using Xenopus ectodermal explants and a hormone-inducible antimorphic form of ESR1 (ESR1 VP16 GR). Through microarray analysis, 2,304 clones from the library enriched in ESR1 target genes, together with an additional 25,138 cDNA clones from two unrelated libraries 9, were screened for regualtion by ESR1 VP16-GR. In total, 55 genes were identified of these, 12 are members of the Notch pathway. In whole embryos, 43 of the 55 genes were strongly induced by ESR1 VP16 GR.