Transcriptional cascades that specify cell fate have been well described in invertebrates. In mammalian development, however, gene hierarchies involved in determination of cell lineage are not understood. With the recent cloning of the MyoD family of myogenic regulatory factors, a model system has become available with which to study the dynamics of muscle determination in mammalian development. Myogenin, along with other members of the MyoD gene family, possesses the apparent ability to redirect nonmuscle cells into the myogenic lineage. This ability appears to be due to the direct activation of an array of subordinate or downstream genes which are responsible for formation and function of the muscle contractile apparatus. Myogenin-directed transcription has been shown to occur through interaction with a DNA consensus sequence known as an E box (CANNTG) present in the control regions of numerous downstream genes. In addition to activating the transcription of subordinate genes, members of the MyoD family positively regulate their own expression and cross-activate one another's expression. These autoregulatory interactions have been suggested as a mechanism for induction and maintenance of the myogenic phenotype, but the molecular details of the autoregulatory circuits are undefined. Here we show that the myogenin promoter contains a binding site for the myocytespecific enhancer-binding factor, MEF-2, which can function as an intermediary of myogenin autoactivation. Since MEF-2 can be induced by myogenin, these results suggest that myogenin and MEF-2 participate in a transcriptional cascade in which MEF-2, once induced by myogenin, acts to amplify and maintain the myogenic phenotype by acting as a positive regulator of myogenin expression.The formation of skeletal muscle during vertebrate development involves the induction of mesoderm from primary ectoderm and the subsequent generation of proliferating myoblasts that ultimately terminally differentiate in response to environmental cues. The recent discovery of a family of related muscle-specific factors that can convert fibroblasts to myoblasts has contributed to rapid progress toward understanding the molecular events that underlie the establishment of the skeletal muscle phenotype (for reviews, see references 55 and 69). Members of this muscle regulatory gene family include MyoD (21), myogenin (25, 77), myf5 (9), and MRF4/herculin/myf6 (8,48,61), each of which can activate myogenesis when introduced into a wide range of nonmuscle cell types.

Expression of the myogenic helix-loop-helix (HLH) protein myogenin in muscle cell precursors within somites and limb buds is among the earliest events associated with myogenic lineage determination in vertebrates. Mutations in the myogenin promoter that abolish binding sites for myogenic HLH proteins or myocyte enhancer factor-2 (MEF-2) suppressed transcription of a linked lacZ transgene in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2 participate in separable regulatory circuits leading to myogenin transcription and provide evidence for positional regulation of myogenic regulators in the embryo.

Abstract. During vertebrate embryogenesis, the muscle-specific helix-loop-helix protein myogenin is expressed in muscle cell precursors in the developing somite myotome and limb bud before muscle fiber formation and is further upregulated during myogenesis.We show that cis-acdng DNA sequences within the 5' flanking region of the mouse myogenin gene are sufficient to direct appropriate temporal, spatial, and tissue-specific transcription of myogenin during mouse embryogenesis. Myogenin-lacZ transgenes trace the fate of embryonic cells that activate myogenin transcription and suggest that myogenic precursor cells that migrate from the somite myotome to the limb bud are committed to a myogenic fate in the absence of myogenin transcription. Activation of a myogenin-lacZ transgene can occur in limb bud explants in culture, indicating that signals required for activation of myogenin transcription are intrinsic to the limb bud and independent of other parts of the embryo. These results reve~ multiple populations of myogenic precursor cells during development and suggest the existence of regulators other than myogenic helix-loop-helix proteins that maintain cells in the early limb bud in the myogenic lineage.

Myogenin, a member of the MyoD family of helix-loop-helix proteins, can induce myogenesis in a wide range of cell types. In addition to activating muscle structural genes, members of the MyoD family can autoactivate their own and cross-activate one another's expression in transfected cells. This has led to the hypothesis that autoregulatory loops among these factors provide a mechanism for amplifying and maintaining the muscle-specific gene expression program in vivo. Here, we make use of myogenin-null mice to directly test this hypothesis. To investigate whether the myogenin protein autoregulates the myogenin gene during embryogenesis, we introduced a myogenin-acZ transgene into mice harboring a null mutation at the myogenin locus. Despite a severe deficiency of skeletal muscle in myogenin-null neonates, the myogenin-4acZ transgene was expressed normally in myogenic cells throughout embryogenesis. These results show that myogenin is not required for regulation of the myogenin gene and argue against the existence of a myogenin autoregulatory loop in the embryo.Positive autoregulation has been invoked as a mechanism through which several genes that regulate cell fate induce and maintain their own expression (1-4). Thus, the discovery that members of the MyoD family (MyoD, myogenin, Myf5, and MRF4) can autoactivate their own and cross-activate one another's expression in transfected cells led to the notion that such autoregulatory loops might provide a mechanism for amplifying the expression of these genes above the threshold required to initiate myogenesis and for stabilizing the musclespecific gene expression program (5-8). Whether these autoregulatory interactions occur during embryogenesis or are simply a tissue culture phenomenon remains to be determined. Myogenin is the only member of the MyoD family that is expressed in all skeletal muscle cells (9, 10). During embryogenesis, myogenin gene expression is detected within myogenic cells in the somitic myotomes and the limb buds and subsequently within differentiated skeletal muscle fibers throughout the body (11)(12)(13)(14)(15). In tissue culture cells, myogenin expression is rapidly upregulated when myoblasts enter into the differentiation pathway in response to withdrawal of growth factors.In contrast, MyoD and Myf5 are expressed in proliferating myoblasts prior to differentiation (16,17) and MRF4 is expressed during myofiber maturation (8,18,19).Gene targeting in transgenic mice has shown that myogenin is essential for muscle development (20,21) and that its functions are distinct from those of MyoD and Myf5 (refs. 22 and 23; reviewed in ref. 24). In the absence of myogenin, muscle-forming regions of neonatal mice are populated by cells that express MyoD but not most muscle structural genes. The expression of MyoD indicates that these cells are committed to the myogenic lineage, but the precise point in the myogenic pathway at which they are arrested is unclear.To investigate whether the myogenin protein positively autoregulates the myogenin gene...

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