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The basic helix-loop-helix muscle regulatory factor (MRF) gene family encodes four distinct muscle-specific transcription factors known as MyoD, myogenin, Myf-5, and MRF4. These proteins represent key regulatory factors that control many aspects of skeletal myogenesis. Although the MRFs often exhibit overlapping functional activities, their distinct expression patterns during embryogenesis suggest that each protein plays a unique role in controlling aspects of muscle development. As a first step in determining how MRF4 gene expression is developmentally regulated, we examined the ability of the MRF4 gene to be expressed in a muscle-specific fashion in vitro. Our studies show that the proximal MRF4 promoter contains sufficient information to direct muscle-specific expression. Located within the proximal promoter are a single MEF2 site and E box that are required for maximum MRF4 expression. Mutation of the MEF2 site or E box severely impairs the ability of this promoter to produce a muscle-specific response. In addition, the MEF2 site and E box function in concert to synergistically activate the MRF4 gene in nonmuscle cells coexpressing MEF2 and myogenin proteins. Thus, the MRF4 promoter is regulated by the MEF2 and basic helix-loop-helix MRF protein family through a cross-regulatory circuitry. Surprisingly, the MRF4 promoter itself is not transactivated by MRF4, suggesting that this MRF gene is not subject to an autoregulatory pathway as previously implied by other studies. Understanding the molecular mechanisms regulating expression of each MRF gene is central to fully understanding how these factors control developmental events.

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The distribution of myogenic and chondrogenic cells was determined for axolotl limb blastemas at the medium bud stage, before precartilage condensation occurred. Myogenic potential of midstylopod (upper) and distal-zeugopod (lower) amputation level blastemas, divided into distal, proximal-core, and proximal-peripheral regions, was assayed in vitro. Significantly more myonuclei were observed in upper level proximal-periphery cultures than in ones from any of the other regions. Since cartilage cells give rise almost exclusively to cartilage in the regenerate (Steen, 19681, a heritable cell marker, triploidy, was used to trace the location of chondrogenic cells in histological sections of upper level early and medium bud blastemas. Although there was mixing of the graft cartilage-derived cells and host soft-tissue-derived cells, the previous cartilage cells were found more frequently within the core region at early and medium bud stages. The results of these studies indicate that more chondrogenic cells are present in the core from a very early stage, and that more myogenic cells are present in the proximal periphery by the medium bud stage.