Transcriptional regulation of MRF4 gene expression during embryonic mouse development

Abstract

Skeletal muscle development in the vertebrate embryo critically depends on bHLH myogenic regulatory factors including MRF4 and Myf5. Both genes exhibit distinct expression patterns during embryogenesis, although they are genetically linked with multiple regulatory elements dispersed throughout the common gene locus. MRF4 has a biphasic expression profile. Initially it is expressed in somites and later in fetal skeletal muscles. Transgenic analysis performed in this study demonstrates that elements within a region between –6.6 and –5.6 kb together with the 3-kb promoter fragment direct transgene expression in the myotome of all somites during the appropriate time frame. The 2.8-kb sequence downstream of the MRF4 promoter is crucial for gene expression in the rostral cervical somites. These data provide evidence that the partly overlapping expression patterns of MRF4 and Myf5 in somites are controlled by distinct regulatory elements. MRF4 expression in fetal muscles and in myotomes is controlled by distinct mechanisms. In the present study all elements necessary for fetal expression map in the region between –11.4 kb and +15.3 kb. Deletion of the +3.9/+15.3 kb sequence results in variable transgene expression in fetal muscles among different transgenic mouse lines. This suggests that elements for stable fetal MRF4 expression involve the region between +3.9 kb and +15.3 kb downstream of the gene. It is also shown that 11.4 kb sequence upstream of MRF4 is not sufficient to prevent MRF4 activation by the strong distal Myf5 limb enhancer. The role of Myf5 in transcriptional regulation of the MRF4 gene has been studied in Myf5 deficient mice. Analysis of the Myf5 mutant embryos reveals the role of Myf5 protein for expression of myotomal markers in the central myotome. Additionally, it is found that Myf5 is required for MRF4 expression in the most rostral somites. In all other somites MRF4 activation does not require Myf5 or MyoD.