Coutelle, Oliver; (1998) Comparative analysis of regulatory elements of the Myf-5 gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The work presented here addresses the question of how skeletal muscle formation is initiated in the mouse by dissecting the regulatory mechanisms that control the myogenic regulatory factor Myf-5. Myf-5 is expressed in the dorsal somite from E8, before the other MRFs, myogenin, MRF4 and MyoD, become activated. In the mouse Myf-5 is located 8.5kb downstream of MRF4. Previous results have shown that dispersed over the intergenic region and intragenic regions are the regulatory elements involved in directing Myf-5 expression to the different anatomical subdomains that make up its complete expression pattern. The regulatory element(s) controlling the dorsal somite expression of Myf-5 is contained in the intergenic region while ventral somite expression depends on elements in the Myf-5 gene itself. Because of the large size of this region I have isolated the MRF4 and Myf-5 genes of the teleost Fugu rubripes, which has a genome eight times smaller than that of the mouse. Although synteny is conserved in Fugu and the intergenic distance is only 3kb, noncoding sequence including the introns is poorly conserved. Focusing on the Myf-5 gene itself, sequence comparison between the mouse and human Myf-5 genes was employed sucessfully to eliminate more than 60% of the intron sequence by identifying conserved regions in the 3'half of each of the Myf-5 introns which, together with the 3'UTR can activate reporter gene expression in the ventral posterior part of the somites. EMSA analysis with embryonic protein extracts revealed several protein binding regions within the conserved intron fragments and subsequent transgenic analysis showed not only that separate genomic regions control individual anatomical domains of Myf-5 expression, but that within these regions multiple binding sites are found, adding a further level of complexity to the regulation of Myf-5. Analysis of the Fugu Myf-5 gene in transgenic mice showed remarkable similarities with the expression pattern of Myf-5 in another teleost, the zebrafish Danio rerio. Both are expressed in the presomitic mesoderm, as well as the somites, suggesting that the expression of the Fugu transgene is a reflection of its native expression pattern.

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