MyoD is a member of the basichelix-loop-helix (bHLH) transcription factor family, which regulates muscle determination and differentiation in vertebrates. While it is now well established that the MyoD gene is regulated by Sonic hedgehog, Wnts, and other signals, it is not known how MyoD transcription is initiated and maintained in response to these signals. We have investigated the cis control of MyoD expression to identify and characterize the DNA targets that mediate MyoD transcription in embryos. By monitoring lacZ reporter gene expression in transgenic mice, we show that regulatory information contained in 24 kb of human MyoD 5 flanking sequence is sufficient to accurately control MyoD expression in embryos. Previous studies have identified two muscle-specific regulatory regions upstream of MyoD, a 4-kb region centered at ؊20 kb (designated fragment 3) that contains a highly conserved 258-bp core enhancer sequence, and a more proximal enhancer at ؊5 kb, termed the distal regulatory region (DRR), that heretofore has been identified only in mice. Here, we identify DRR-related sequences in humans and show that DRR function is conserved in humans and mice. In addition, transcriptional activity of MyoD 5 flanking sequences in somites and limb buds is largely a composite of the individual specificities of the two enhancers. Deletion of fragment 3 resulted in dramatic but temporary expression defects in the hypaxial myotome and limb buds, suggesting that this regulatory region is essential for proper temporal and spatial patterning of MyoD expression. These data indicate that regulatory sequences in fragment 3 are important targets of embryonic signaling required for the initiation of MyoD expression.

Skeletal muscle lineage determination is regulated by the myogenic regulatory genes, MyoD and Myf-5. Previously, we identified a 258 bp core enhancer element 20 kb 5′ of the MyoD gene that regulates MyoD gene activation in mouse embryos. To elucidate the cis control mechanisms that regulate MyoD transcription, we have mutagenized the entire core enhancer using linker-scanner mutagenesis, and have tested the transcriptional activity of enhancer mutants using lacZ reporter gene expression in transgenic mouse embryos. In total, 83 stable transgenic lines representing 17 linker-scanner mutations were analyzed in midgestational mouse embryos. Eight linker-scanner mutations resulted in a partial or complete loss of enhancer activity, demonstrating that MyoD is primarily under positive transcriptional control. Six of these mutations reduced or abolished transgene expression in all skeletal muscle lineages, indicating that activation of MyoD expression in trunk, limb and head musculature is regulated, in part, by shared transcriptional mechanisms. Interestingly, however, two adjacent linker-scanner mutations (LS-14 and LS-15) resulted in a dramatic reduction in transgene expression specifically in myotomes at 11.5 days. At later stages, transgene expression was absent or greatly reduced in myotomally derived muscles including epaxial muscles (deep back muscles) and hypaxial muscles of the body wall (intercostal muscles, abdominal wall musculature). In contrast, head muscles, as well as muscles of the body derived from migrating muscle progenitor cells (e.g. limb, diaphragm), were unaffected by these mutations. In Pax-3-mutant mice, LS-14 and LS-15 transgene expression was eliminated in the body, but was unaffected in the head, yielding an identical expression pattern to the endogenous MyoD gene in mice mutant for both Myf-5 and Pax-3. These data support the hypothesis that LS-14 and LS-15 define the core enhancer targets for Myf-5-dependent activation of MyoD in myotomal muscles.

No abstract