Mapping the Shh regulatory landscape
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Sonic hedgehog (Shh) is an important signalling protein expressed extensively in development, throughout tissues of the central nervous system, gut and the posterior of the limb bud. The complicated expression pattern of Shh is regulated by a series of long-range enhancers located flanking and dispersed throughout a 1 Mb genomic desert. Disruption of SHH as a result of mutations within the gene or its enhancers has been implicated in two developmental conditions. These are Holoproencephaly (HPE3) a common developmental defect of the forebrain and frequently the mid-face in humans, and preaxial polydactyly (PPD), a congenital limb abnormality encompassing a varied phenotype affecting the digits on the anterior side of the hands and feet that has been attributed to misexpression of Shh. In order to investigate the Shh regulatory landscape and survey regulatory activity, a transposon-based chromosomal engineering strategy known as the local hopping enhancer detection (LHED) system was employed. Using this method a targeting vector containing a LacZ reporter gene as well as LoxP sites was inserted within the Shh region. The ‘hopping’ nature of the transposable element was then exploited to scatter it throughout the region. Tetraploid complementation embryos derived entirely from ES cells were generated and examined in order to gain an insight into enhancer activity. The region was found to be in an open conformation over its length and is generally susceptible to all Shh enhancers. Genes within the regulatory domain, such as the widely expressed Rnf32 gene, were found to resist Shh enhancer activities by a process of regulatory evasion by the promoter, a mechanism that may be common in large regulatory domains. Finally, at the boundaries of the region Shh activity was found to be lost incrementally at a number of genomic positions. Mouse lines were also generated to look at both enhancer activity and loss of function effects and three deletions of increasing size were generated between Shh and the furthest enhancer, the Zrs. These in turn, delete firstly a gut and pharyngeal epithelial enhancer, secondly the gut, pharyngeal enhancers as well an oral epithelial enhancer and finally all three epithelial enhancers as well as three forebrain enhancers. Reporter gene expression was found to be lost incrementally from those enhancers deleted without disrupting the rest of the region. Previously unidentified notochord enhancer(s) were found to lie within the region 100-530 kb upstream of Shh. Examination of the resultant phenotypes showed that gut and craniofacial defects were found to occur as a result of the loss of enhancers which drive expression within these tissues. Variable phenotypes were found to occur potentially as a result of temporal changes to Shh expression or as a result of threshold levels of HH being required for normal development. Other enhancers within the Shh region and outwith the deletions were not found to be disrupted by these modifications suggesting the enhancers within the region act independently of each other. The largest deletion resulted in bringing the Zrs (which drives Shh limb expression) within 170 kb of the gene, however limb development; was not, found to be affected suggesting distance is not required for Zrs function. Overall, the LHED transposon system has been utilised in order to examine the Shh region in more detail, allowing mapping of enhancer function by reporter gene expression and examination of phenotypes generated by deletion of enhancers.