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nautilus is the only MyoD-related gene in Drosophila. Nautilus expression begins around stage 9 at full germ-band extension in a subset of mesodermal cells organized in a stereotypic pattern in each hemisegment. The muscle founder cell marker Duf-LacZ, produced by the enhancer trap line rP298LacZ, is coexpressed in numerous Nautilus-positive cells when founders first appear. Founders entrain muscle identity through the restricted expression of transcription factors such as S59, eve, and Kr, all of which are observed in subsets of the nautilus expressing founders. We inactivated the nautilus gene using homology-directed gene targeting and Gal4/UAS regulated RNAi to determine whether loss of nautilus gene activity affected founder cell function. Both methods produced a range of defects that included embryonic muscle disruption, reduced viability and female sterility, which could be rescued by hsp70-nautilus cDNA transgenes. Our results demonstrate Nautilus expression marks early founders that give rise to diverse muscle groups in the embryo, and that nautilus gene activity is required to seed the correct founder myoblast pattern that prefigures the muscle fiber arrangement during embryonic development. more...

Schneider SL2 cells activate the myogenic program in response to the ectopic expression of daughterless alone, as indicated by exit from the cell cycle, syncytia formation, and the presence of muscle myosin fibrils. Myogenic conversion can be potentiated by the coexpression of DMEF2 and nautilus with daughterless. In RT-PCR assays Schneider cells express two mesodermal markers, nautilus and DMEF2 mRNAs, as well as very low levels of daughterless mRNA but no twist. Full-length RT-PCR products for nautilus and DMEF2 encode immunoprecipitable proteins. We used RNA-i to demonstrate that both endogenous nautilus expression and DMEF2 expression are required for the myogenic conversion of Schneider cells by daughterless. Coexpression of twist blocks conversion by daughterless but twist dsRNA has no effect. Our results indicate that Schneider cells are of mesodermal origin and that myogenic conversion with ectopic expression of daughterless occurs by raising the levels of daughterless protein sufficiently to allow the formation of nautilus/daughterless heterodimers. The effectiveness of RNA-i is dependent upon protein half-life. Genes encoding proteins with relatively short half-lives (10 h), such as nautilus or HSF, are efficiently silenced, whereas more stable proteins, such as cytoplasmic actin or beta-galactosidase, are less amenable to the application of RNA-i. These results support the conclusion that nautilus is a myogenic factor in Drosophila tissue culture cells with a functional role similar to that of vertebrate MyoD. This is discussed with regard to the in vivo functions of nautilus., (Copyright 2000 Academic Press.) more...

The formation of striated muscle in both vertebrates and invertebrates involves the activity of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors. The high degree of evolutionary conservation of MyoD-related proteins, both in the sequence of their bHLH domains and in their general developmental expression patterns, suggests that these factors are also conserved at the level of function. We have addressed this directly using MyoD and E protein factors from vertebrates, Drosophila, and Caenorhabditis elegans. Various MyoD and E factor combinations were tested for their ability to interact in vitro and to function in vivo in the myogenic conversion of 10T12 mouse fibroblasts. We found that the ability of different homo- and heterodimers to bind DNA in vitro was an accurate measure of biological activity in vivo. A second assessment of conserved function comes from the ability of these factors to rescue a C. elegans hlh-1 (CeMyoD) null mutation. We found that both Drosophila and chicken MyoD-related factors were able to rescue a C. elegans CeMyoD loss-of-function mutation. These results demonstrate a remarkable degree of functional conservation of these myogenic factors despite differences in E-protein interactions., (Copyright 1999 Academic Press.) more...

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle "pioneer" or "founder" cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. We now have utilized toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA. These results define a crucial role for nautilus in embryonic muscle formation. The application of RNA interference to a variety of known Drosophila mutations as controls gave phenotypes essentially indistinguishable from the original mutation. RNA-i provides a powerful approach for the targeted disruption of a given genetic function in Drosophila. more...

Although the ubiquitous helix-loop-helix (HLH) protein E12 does not homodimerize efficiently, the myogenic factor MyoD forms an avid DNA-binding heterodimer with E12 through the conserved HLH dimerization domain. However, the mechanism which ensures this selective dimerization is not understood at present. In our functional studies of various amino acid changes in the E12 HLH domain, we found that a single substitution in E12 helix 1 can abolish the effect of the E12 inhibitory domain and results in the efficient DNA binding of the E12 homodimer. Competition experiments revealed that the inhibitory domain, in fact, blocks the dimerization of E12 rather than DNA binding. MyoD contains two glutamic residues in helix 2 that are required for efficient dimerization with E12. More importantly, these residues were not essential for dimerization with E12 mutants in which the dimerization inhibitory domain had been relaxed, or for dimerization with E47 which does not contain the inhibitory domain owing to the use of an alternative exon. The positions of these glutamic residues are conserved among the four myogenic factors. Thus, members of the MyoD family of gene regulatory proteins can overcome the E12 dimerization inhibitory domain through a mechanism involving, in part, the negatively charged amino acid residues in helix 2. This result describes a novel mechanism facilitating the selective formation of the MyoD(MRF)-E12 heterodimer that enhances dimerization specificity and may apply to other members of the E-protein family. more...

Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor) box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos, somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2. more...

The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with E12. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with E12. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and E12 and determine the dimerization specificity of the bHLH proteins. more...

MyoD is a member of the basic helix-loop-helix (bHLH) family of muscle gene regulatory proteins that includes myogenin, myf-5, and MRF4. These proteins have been shown to heterodimerize with E2A bHLH proteins, E12/E47, and to bind to a consensus sequence known as an E-box, CANNTG, the target for transcriptional activation by these myogenic regulators. MyoD is also a phosphorylated nuclear protein that is present in muscle cells prior to the transcriptional activation of the muscle-specific genes, many of which contain E-box elements in their regulatory regions. Here we report that phosphorylated chicken MyoD, called CMD1, produced in sf9 cells using the baculovirus system, is qualitatively similar to CMD1 isolated by immunoaffinity purification from primary cultures of embryonic chick breast muscle. Functional analysis of phosphorylated and dephosphorylated CMD1 produced in sf9 cells indicates that, in the presence of magnesium, DNA binding of phosphorylated CMD1 is inhibited whereas binding in association with E12 is not affected. However, CMD1 binding alone is equally efficient when either EDTA is added in excess or dephosphorylated or bacterially expressed CMD1 is used in the assay. Our results suggest that cellular phosphorylation changes the CMD1 homodimer-heterodimer equilibrium which, in turn, modulates and/or eliminates binding site competition between CMD1 homodimers and CMD1/E-protein heterodimers in the cell. more...

We have isolated a cDNA clone, called Dmyd for Drosophila myogenic determination gene, from a 0-16 hour Drosophila embryo library that encodes a protein with structural and functional characteristics similar to the members of the vertebrate MyoD family (Paterson et al 1991). Dmyd encodes a polypeptide of 332 amino acids with 82% identity to MyoD in the 41 amino acids of the putative helix-loop-helix region and 100% identity in the 13 amino acids of the basic domain proposed to contain the essential recognition code for muscle specific gene activation. The gene is unique and maps to 95A/B on the right arm of the third chromosome. Low stringency hybridizations indicate Dmyd is not a member of a multigene family, similar to MyoD in vertebrates. Dmyd is a nuclear protein in Drosophila, consistent with its role as a nuclear gene regulatory factor, and is proposed to be a transiently expressed marker for a unique subset of muscle founder cells. We have used an 8kb promoter fragment from the gene, which contains the first 55 amino acids of the Dmyd protein, joined to lac Z to follow myogenic precursor cells into muscle fibers using antibodies to beta-galactosidase and Dmyd. Unlike the myogenic factors in vertebrate muscle cells, Dmyd appears to be expressed at a much lower level in differentiated Drosophila muscles so it cannot be followed continuously as a muscle marker. This is reflected in the loss of expression of Dmyd RNA in 12-24 hour embryos, a major period of early myogenesis, as well as in the undetectable level of the nuclear antigen in primary cultures of embryonic and adult Drosophila muscle. Functional differences between Dmyd and CMD1 are described and explained in terms of a model which may give insight to the nature of homo and heterodimer formation in the bHLH family of proteins. more...

We have isolated a cDNA clone, called Dmyd for Drosophila myogenic-determination gene, that encodes a protein with structural and functional characteristics similar to the members of the vertebrate MyoD family. Dmyd clone encodes a polypeptide of 332 amino acids with 82% identity to MyoD in the 41 amino acids of the putative helix-loop-helix region and 100% identity in the 13 amino acids of the basic domain proposed to contain the essential recognition code for muscle-specific gene activation. Low-stringency hybridizations indicate that Dmyd is not a member of a multigene family similar to MyoD in vertebrates. Dmyd is a nuclear protein in Drosophila, consistent with its role as a nuclear-gene regulatory factor, and is proposed to be a transiently expressed marker for muscle founder cells. We have used an 8-kilobase promoter fragment from the gene, which contains the first 55 amino acids of the Dmyd protein, joined to lacZ, to follow myogenic precursor cells into muscle fibers with antibodies to beta-galactosidase and to Dmyd. Unlike the myogenic factors in vertebrate muscle cells, Dmyd appears to be expressed at a much lower level in differentiated Drosophila muscles, so Dmyd cannot be followed continuously as a muscle marker. This fact is reflected in the loss of Dmyd RNA expression in 12- to 24-hr embryos, a major period of early myogenesis, as well as in the undetectable level of the nuclear antigen in primary cultures of embryonic and adult Drosophila muscle. more...

We isolated the cDNA encoding a myogenic factor expressed in embryonic chick breast muscle by virtue of its weak hybridization to the mouse MyoD1 clone. Nucleotide sequence analysis and amino acid comparison define this clone, CMD1, as encoding a protein similar to mouse MyoD1. CMD1 encodes a polypeptide smaller than MyoD1, 298 versus 318 amino acids, respectively, and is 80% concordant by amino acid sequence overall. The basic and myc domains required for myogenic conversion of mouse 10T1/2 'fibroblasts' to myoblasts with MyoD1 are completely conserved in CMD1. CMD1 is just as efficient as the mouse homolog in myogenic conversion of 10T1/2 cells and coactivates the endogenous mouse MyoD1 gene in the process. The efficiency of myoblast conversion depends on the levels of CMD1 expression and suggests that the cellular concentration of CMD1 plays a role in the onset of myogenesis. Transient expression of CMD1 in a variety of nonmuscle cells from different germ-layer origins activates both cotransfected muscle-specific promoters and, in some cases, endogenous muscle-specific genes. 5-Bromodeoxyuridine (BrdU) treatment of chicken and mouse myoblasts reduces the expression of CMD1 and MyoD1, respectively, and may explain how this thymidine analog inhibits myogenesis and the activity of transfected muscle-specific promoters in BrdU-treated myoblasts. Transient expression of CMD1 in BrdU-treated myoblasts reactivates cotransfected muscle-specific promoters. CMD1 activates muscle-specific promoters in cotransfections regardless of cell type, whereas 'housekeeping' or constitutive promoters can be activated moderately, unaffected, or repressed, depending on the promoter and cell background. The rate and degree of myogenic conversion may be more restricted by cell phenotype than by germ-layer origin. more...