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.)

Transcription factors belonging to the basic helix-loop-helix (bHLH) family have been shown to control differentiation of a variety of cell types. Tissue-specific bHLH proteins dimerize preferentially with ubiquitous bHLH proteins to form heterodimers that bind the E-box consensus sequence (CANNTG) in the control regions of target genes. Using the yeast two-hybrid system to screen for tissue-specific bHLH proteins, which dimerize with the ubiquitous bHLH protein E12, we cloned a novel bHLH protein, named Dermo-1. Within its bHLH region, Dermo-1 shares extensive homology with members of the twist family of bHLH proteins, which are expressed in embryonic mesoderm. During mouse embryogenesis, Dermo-1 showed an expression pattern similar to, but distinct from, that of mouse twist. Dermo-1 was expressed at a low level in the sclerotome and dermatome of the somites, and in the limb buds at Day 10.5 post coitum (p.c.), and accumulated predominantly in the dermatome, prevertebrae, and the derivatives of the branchial arches by Day 13.5 p.c. As differentiation of prechondrial cells proceeded, Dermo-1 expression became restricted to the perichondrium. Expression of Dermo-1 increased continuously in the dermis through Day 17.5 p.c. and was also detected in the dermis of neonates, but became downregulated in adult tissues. The Dermo-1 protein bound the E-box consensus sequence in the presence of E12, but transcriptional activity was not detectable. Instead, Dermo-1 repressed transcriptional activity of myogenic bHLH proteins. The expression pattern of Dermo-1 suggests that it functions as a regulator of gene expression in a subset of mesenchymal cell lineages including developing dermis.

Mol. Cell. Biol. 9:5022-5033, 1989). We show in this study that MEF-2 is regulated by the myogenic regulatory factor myogenin and that mitogenic signals block this regulatory interaction. Induction of MEF-2 by myogenin occurs in transfected 1OT1/2 cells that have been converted to myoblasts by myogenin, as well as in CV-1 kidney cells that do not activate the myogenic program in response to myogenin. Through mutagenesis of the MEF-2 site, we further defined the binding site requirements for MEF-2 and identified potential MEF-2 sites within numerous muscle-specific regulatory regions. The MEF-2 site was also found to bind a ubiquitous nuclear factor whose binding specificity was similar to but distinct from that of MEF-2. Our results reveal that MEF-2 is controlled, either directly or indirectly, by a myogenindependent regulatory pathway and suggest that growth factor signals suppress MEF-2 expression through repression of myogenin expression or activity. The ability of myogenin to induce MEF-2 activity in CV-1 cells, which do not activate downstream genes associated with terminal differentiation, also demonstrates that myogenin retains limited function within cell types that are nonpermissive for myogenesis and suggests that MEF-2 is regulated independently of other muscle-specific genes.Differentiation of skeletal myoblasts is accompanied by transcriptional induction of a battery of unlinked musclespecific genes. The control regions of several of these genes have been characterized and shown to interact with complex sets of muscle-specific and ubiquitous nuclear factors that cooperate to direct muscle-specific transcription (for a review, see reference 50). The best-characterized musclespecific transcription factors are the myogenic helix-loophelix proteins of the MyoD family, which includes MyoD (19), myogenin (23, 68), Myf5 (4), and MRF4/herculin/Myf6 (3,43,49). When introduced into a variety of nonmuscle cell types, each of these factors has the ability to induce myogenesis to different degrees (for reviews, see references 45 and 58). Cells of mesodermal origin, and in particular 10T1/2 fibroblasts, are especially permissive for myogenic conversion by the MyoD family, whereas cells derived from other lineages show variable degrees of muscle-specific gene expression in response to these factors (54,66

Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) genes in neuronal fate determination and cell type specification. We have compared directly the expression of Mash1 and neurogenin1 (ngn1), two bHLH genes that are expressed specifically at early stages of neurogenesis. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. We also showed that in the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. Finally, we found that there is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation.