Hill, K.V.; (2007) Regulation of Nkx2.2 gene expression in the vertebrate neural tube; a target of graded Sonic hedgehog signalling. Doctoral thesis , University of London.

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Abstract

Sonic hedgehog (Shh) is a morphogen implicated in the developmental patterning of many vertebrate tissues. One such tissue is the neural tube (NT). In ventral regions of the NT distinct neuronal subtypes emerge in precise spatial order from progenitor cells arrayed along its dorsal-ventral axis. Shh regulates this process by controlling the expression patterns of transcription factors in progenitor cells. In addition, cross- repressive interactions between pairs of transcription factors, expressed in adjacent regions, ensures the generation of defined domains. The regulation of Nkx2.2 and Nkx2.9 expression represents an example of this mechanism. The expression of these genes is restricted to a ventral (p3) domain, comprising neural progenitors dorsal to the floor plate. Induction of Nkx2.2 and Nkx2.9 requires high levels of Shh signalling. In part this appears to be because the homeodomain protein Pax6 must be repressed to allow Nkx2.2/2.9 induction. We have analysed the regulatory regions of the Nkx2 genes in order to understand the molecular mechanisms underpinning their expression pattern. The 5' flanking region of Nkx2.2 and Nkx2.9 contains a 250bp block of highly conserved DNA (CNCR) that is found in human, mouse, Fugu and zebrafish. This region includes a binding site for the transcriptional regulators of the Shh pathway: Gli (GBS). Using a BAC homologous recombination system and assays in zebrafish, we provide evidence that the CNCR is required to direct Nkx2.2a-like gene expression. Mouse in vivo reporter assays using fragments containing the CNCR of zNkx2.2a, indicate the CNCR is sufficient to direct reporter gene expression in the p3 domain of the NT. Mutational analysis indicates that the GBS is necessary but not sufficient to account for this expression profile. In vivo assays further suggest correct Nkx2.2 expression requires input from additional transcriptional activators as well as a floor plate repressor. All these factors appear to act through regulatory elements within the CNCR.

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