Sharpe, James Alexander; (1997) Analysis of murine hox gene regulation and creation of an insertional mutant displaying polydactyly. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The vertebrate Hox genes are essential for embryo development, and are thought to specify positional information along the anteroposterior (A-P) axis. In this study, murine transgenic technology was used to further our understanding of how Hox genes are regulated, and to test a hypothesis which might explain their clustered organisation. Two regions of the Hoxb complex were studied: the Hoxb-9 region, and the intergenic stretch betfween Hoxb-5 and Hoxb-4. In the first study, constructs were created which contained regulatory elements from near the Hoxb-9 gene coupled to the reporter gene LacZ. These allowed identification of two regulatory regions which appear to be important for normal expression of the Hoxb-9 gene. Previous work on the regulation of the Hoxb-5 gene had suggested that two regulatory elements which are probably important for Hoxb-5 may also interact with the Hoxb-4 promoter. In the second study, a double-reporter system was developed, with which it was possible to monitor the expression from two different promoters in the same construct. Constructs were created spanning the Hoxb-5-Hoxb-4 region, in which the LacZ gene was inserted into the Hoxb-5 coding region, and the human placental alkaline phosphatase (PLAP) gene was inserted into the Hoxb-4 coding region. Using this construct, and two versions in which the regulatory regions had been deleted, it was possible to show that certain elements in this stretch of DNA may be able to activate both promoters. During the course of these transgenic experiments, an insertional mutant was created which displayed preaxial polydactyly. In the last part of this study, the mutant strain was characterised with regard to its developmental and skeletal phenotype, the chromosomal localisation of the transgene, expression of the gene Sonic hedgehog in the developing limb bud, and expression of the transgenic PLAP reporter construct. Using these data the strain was compared to previously known polydactylous mutants from the hemimelia-luxate group.

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