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.

Histone proteins provide a means of packaging DNA over 10,000-fold in order to allow the accommodation of genetic material as chromatin in the nucleus of the cell. However, the chemical manipulation of histones underpins an array of additional biological functions of chromatin. The unstructured N-terminal tails of histones are covalently modified in a variety of fashions, with many of these modifications implicated in the regulation of gene expression. Lysine specific demethylase 1 (LSD1), which demethylates mono- and di-methylated histone H3 lysine 4 (H3K4) as part of a complex including CoREST and histone deacetylases (HDACs), is essential for embryonic development in the mouse beyond embryonic day (E)6.5. The aim was to determine the role of LSD1 during this early period of embryogenesis through generation and analysis of conditional knockout mouse embryonic stem (ES) cells, which are the in vitro counterpart of the epiblast. Prior analysis of post-implantation loss-of-function genetrap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in histone H3K9 and H3K56 acetylation, but only minor increases in global H3K4 methylation. Despite this biochemical perturbation, LSD1 deleted ES cells proliferate normally and retain stem cell characteristics. However, differentiation of these ES cells is associated with significant cell death. Loss of LSD1 causes the aberrant expression of 588 genes, including transcription factors with roles in anterior/posterior patterning and tissue specification. Brachyury, a key-regulator of mesodermal differentiation, is a direct target gene of LSD1 and is over-expressed in E6.5 Lsd1 genetrap embryos. Thus, LSD1 regulates the expression and appropriate timing of a key developmental regulator, as part of the LSD1/CoREST/HDAC complex, during early embryonic development. Notably, rescue experiments show that the catalytic activity of LSD1 is not required for gene repression and it is proposed that the regulatory role of LSD1/CoREST/HDAC complex is governed by the ability to target HDACs to genomic regions or to prevent promoter access of gene-activating complexes with H3K4 tri-methylation catalytic activity.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.