Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors. Two bHLH genes, dHAND and eHAND, have now been isolated that are expressed in the bilateral heart primordia and subsequently throughout the primitive tubular heart and its derivatives during chick and mouse embryogenesis. Incubation of stage 8 chick embryos with dHAND and eHAND antisense oligonucleotides revealed that either oligonucleotide alone had no effect on embryonic development, whereas together they arrested development at the looping heart tube stage. Thus, dHAND and eHAND may play redundant roles in the regulation of the morphogenetic events of vertebrate heart development.

SM22 alpha is a calponin-related protein that is expressed specifically in adult smooth muscle. To begin to define the mechanisms that regulate the establishment of the smooth muscle lineage, we analyzed the expression pattern of the SM22 alpha gene during mouse embryogenesis. In situ hybridization demonstrated that SM22 alpha transcripts were first expressed in vascular smooth muscle cells at about embryonic day (E) 9.5 and thereafter continued to be expressed in all smooth muscle cells into adulthood. In contrast to its smooth muscle specificity in adult tissues, SM22 alpha was expressed transiently in the heart between E8.0 and E12.5 and in skeletal muscle cells in the myotomal compartment of the somites between E9.5 and E12.5. The expression of SM22 alpha in smooth muscle cells, as well as early cardiac and skeletal muscle cells, suggests that there may be commonalities between the regulatory programs that direct muscle-specific gene expression in these three myogenic cell types.

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.

Autosomal dominant mutations in the bHLH transcription factor TWIST1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome (SCS). The molecular mechanism underlying these phenotypes is poorly understood. We show that the ectopic expression of the related bHLH factor Hand2 phenocopies Twist1 loss-of-function phenotypes in the limb, and that they display a gene dosage-dependent antagonistic interaction. Twist1 and Hand2 dimerization partner choice can be modulated by PKA and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Interestingly, multiple TWIST1 mutations associated with SCS alter PKA-mediated Twist1 phosphorylation, suggesting that misregulation of Twist1 dimerization via either stoichiometric or posttranslational mechanisms underlies SCS phenotypes.Studies of developing vertebrate limbs have yielded many insights into the process of embryonic pattern formation. Prominent among these are the identification of a growing catalog of transcription factors that orchestrate limb patterning. While the genetic and biochemical interactions of these transcription factors are clearly important for integrating patterning information, these interactions are poorly understood. Twist1 and Hand2 are basic helix-loop-helix (bHLH) transcription factors within the Twist family, and are attractive candidates for investigating such interactions. Each is required for distinct yet subtly related aspects of limb development, and biochemical studies have revealed a complex regulation of their protein-protein interactions 1-3 .Early limb bud expression of Twist1 is observed primarily in the peripheral mesenchyme, and Twist1 is required for maintenance of the overlying apical ectodermal ridge (AER) 4-7 . Twist1 haploinsufficiency in mice and humans is associated with a range of limb abnormalities. Twist1 heterozygous null mice display a partially penetrant preaxial polydactyly 8,9 . Human Correspondence should be addressed to A.B.F. tfirulli@iupui.edu (317) 278-5814 and E.L. elaufer@columbia.edu (212) Here we investigate the biochemical and genetic interactions between Twist1 and Hand2 both in vitro and during limb development. We show that PKA and B56δ-containing PP2A can regulate Twist1 and Hand2 phosphorylation at the conserved helix I residues, that hypophosphorylation and phosphorylation mimics of these residues alter bHLH dimerization affinities, and that a population of TWIST1 mutations that cause SCS in humans exhibit disregulation of this phosphoregulatory circuit. We also show that ectopic Hand2 expression phenocopies multiple SCS-like limb phenotypes, that the appropriate genetic dosage of Hand2 and Twist1 is critical for proper limb development, and that these interactions require the phosphoregulated helix I residues. These findings support a mechanism where the Twist family dimerization partner choices are modulated by both the relative levels of gene expression and the phosphorylation state of key helix I residues, thereby dictating changes i...

We cloned a portion of the mouse smooth muscle myosin heavy chain (SM-MHC) cDNA and analyzed its mRNA expression in adult tissues, several cell lines, and developing mouse embryos to determine the suitability of the SM-MHC promoter as a tool for identifying smooth musclespecific transcription factors and to define the spatial and temporal pattern of smooth muscle differentiation during mouse development. RNase protection assays showed SM-MHC mRNA in adult aorta, intestine, lung, stomach, and uterus, with little or no signal in brain, heart, kidney, liver, skeletal muscle, spleen, and testes. From an analysis of 14 different cell lines, including endothelial cells, fibroblasts, and rhabdomyosarcomas, we failed to detect any SM-MHC mRNA; all of the cell lines induced to differentiate also showed no detectable SM-MHC. In situ hybridization of staged mouse embryos first revealed SM-MHC transcripts in the early developing aorta at 10.5 days post coitum (dpc). No hybridization signal was demonstrated beyond the aorta and its arches until 12.5 to 13.5 dpc, when SM-MHC mRNA appeared in smooth muscle cells (SMCs) of the developing gut and lungs as well as peripheral blood vessels. By 17.5 dpc, SM-MHC transcripts had accumulated in esophagus, bladder, and ureters. Except for blood vessels, no SM-MHC transcripts were ever observed in developing brain, heart, or skeletal muscle. These results indicate that smooth muscle myogenesis begins by 10.5 days of embryonic development in the mouse and establish SM-MHC as a highly specific marker for the SMC lineage. The SM-MHC promoter should therefore serve as a useful model for defining the mechanisms that govern SMC transcription during development and disease. (Circ Res. 1994;75:803-812.)