Forbes-Robertson, Sarah Anne Natasha; (1996) Structure and expression of the chicken bone morphogenetic protein-2 gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of the research described in this thesis was to investigate the structure and function of the bone morphogenetic protein-2 gene in the developing chick limb. A chicken Bmp-2 genomic clone was isolated The intron-exon structure was analysed and a portion of the 5' coding region, that was missing from cDNA clones previously isolated in our laboratory, was sequenced and analysed This 5' sequence and the intron-exon structure were found to be highly conserved with other members of the TGF-β superfamily, and Bmp genes from other species. Preliminary evidence indicates that the Bmp-2 gene has multiple 5' non-coding exons, like Bmp-4 and dpp, but unlike the other TGF-β superfamily genes. In order to analyse the 5' promoter region of the Bmp-2 gene, a novel system was developed for transfecting primary chick mesoderm cells isolated from embryonic limb buds. This transfection system provides as valuable model system in which to analyse the regulation of patterning genes expressed in the limb, since it spears to mimic various aspects of the developing limb, as shown by positional and temporal expression of various marker genes tested in the system. This is particularly useful since there are at present no established chick cell lines. However, we were unable to generate any promoter constructs of the putative promoter region of the Bmp-2 gene, possibly because of a region of instability in the 5' region of the gene. The role of the Bmp-2 gene in development was therefore further investigated by attempting to inhibit Bmp-2 gene function using an antisense RNA strategy. Antisense constructs containing various different regions of the chicken Bmp-2 gene were generated in a retroviral vector, RCAS(BP), which can be targeted to specific areas of the developing chick embryo, by injection or grafting of infected cells. Stage 16-21 embryonic chick limb buds were infected with these antisense Bmp-2 constructs. In preliminary experiments, these retroviral constructs appear to be expressed at relatively high levels in the limb, and to inhibit levels of endogenous Bmp-2 transcripts, as shown by in situ hybridisation. A possible phenotypic effect of this inhibition was shown to be a fusion between the metatarsals of digits III and IV in the wing.

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