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Specification and Differentiation of Muscle

As indicated by global expression patterns, yeast cells of different mating types are still rather similar. Developmental biologists do not yet know the complete set of molecules that distinguishes any one cell type (e.g., muscle) from all the other cell types in a multicellular organism. The extensive cell specification and differentiation that occur during development of animals and plants depend on both quantitative and qualitative differences in gene expression, controlled at the level of transcription, as well as on cell structures and protein activity states.

An impressive array of molecular strategies, some analogous to those found in yeast cell-type specification, have evolved to carry out the complex developmental pathways that characterize multicellular organisms. Muscle cells have been the focus of many such studies because their development can be studied in cultured cells as well as in intact animals. Early advances in understanding the formation of muscle cells (myogenesis) came from discovery of regulatory genes that could convert cultured cells into muscle cells. Then mouse mutations affecting those genes were created and studied to learn the functions of the proteins encoded by these genes, following which scientists have investigated how the muscle regulatory genes control other genes.

Twist protein is a transcription factor necessary to create muscle cells in flies and other animals. Twist turns on production of other transcription factors that in turn activate genes encoding myosin, actin, and other muscle-specific proteins. DNA microarray analysis has been applied to understand the function of twist and other regulatory genes in muscle development. For example, the expression pattern of normal Drosophila embryos recently has been compared with that of mutant embryos in which the twist gene is defective. To assess how many genes are needed to specify muscle, researchers determined the expression of about 4000 fly genes (about 30 percent of the total) in normal fly embryos and in twist mutants. Of the genes included in the microarray, about 130 (3.3 percent), including many known muscle differentiation genes, were transcribed at lower levels (or not at all) in the twist mutants. These results suggest that transcriptional changes in at least several hundred genes are associated with differentiation of a highly specialized cell type such as muscle.

Other recent microarray studies have looked for genes whose transcription differs in various subtypes of muscle in mice. These have identified 49 genes out of 3000 genes examined that are transcribed at substantially different levels in red (endurance) muscle and white (fast response) muscle. Clues to the molecular basis of the functional differences between red and white muscle are likely to come from studying those 49 genes and their products.

Here we examine the role of certain transcription factors in creating skeletal muscle in vertebrates. These muscle regulators illustrate how coordinated transcription of sets of target genes can produce differentiated cell types and how a cascade of transcriptional events and signals is necessary to coordinate cell behaviors and functions.

Embryonic Somites Give Rise to Myoblasts, the Precursors of Skeletal Muscle Cells

Vertebrate skeletal myogenesis proceeds through three stages: determination of the precursor muscle cells, called myoblasts; proliferation and in some cases migration of myoblasts; and their terminal differentiation into mature muscle (Figure 22-14). In the first stage, myoblasts arise from blocks of mesoderm cells, called somites, that are located next to the neural tube in the embryo. Specific signals from surrounding tissue play an important role in determining where myoblasts will form in the developing somite. At the molecular level, the decision of a mesoderm cell to adopt a muscle cell fate reflects the activation of genes encoding particular transcription factors.

As myoblasts proliferate and migrate, say, to a developing limb bud, they become aligned, stop dividing, and fuse to form a syncytium (a cell containing many nuclei but sharing a common cytoplasm). We refer to this multinucleate cell as a myotube. Concomitant with cell fusion is a dramatic rise in the expression of genes necessary for further muscle development and function.

The specific extracellular signals that induce determination of each group of myoblasts are expressed only transiently. These signals trigger production of intracellular

Myoblasts

Myogenin and MEFs 2| Proliferation and/or migration

Myoblasts

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Premuscle masses

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Myotube (muscle cell)

Myotube (muscle cell)

▲ FIGURE 22-14 Three stages in development of vertebrate skeletal muscle. Somites are epithelial spheres of embryonic mesoderm cells, some of which (the myotome) become determined as myoblasts after receiving signals from other tissues (1). After the myoblasts proliferate and migrate to the limb buds and elsewhere (2|), they undergo terminal differentiation into multinucleate skeletal muscle cells, called myotubes ( 3). Key transcription factors that help drive the myogenic program are highlighted in yellow. See also Figure 22-17.

factors that maintain the myogenic program after the inducing signals are gone. We discuss the identification and functions of these myogenic proteins, and their interactions, in the next several sections.

Myogenic Genes Were First Identified in Studies with Cultured Fibroblasts

Myogenic genes are a fine example of how transcription factors control the progressive differentiation that occurs in a cell lineage. In vitro studies with the fibroblast cell line designated C3H 10TV2 have played a central role in dissecting the transcription control mechanisms regulating skeletal myogenesis. When these cells are incubated in the presence of 5-azacytidine, a cytidine derivative that cannot be methylated and therefore alters transcription, they differentiate into myotubes. Upon entry into cells, 5-azacytidine is converted to 5-azadeoxycyti-dine triphosphate and then is incorporated into DNA in place of deoxycytidine. Because methylated deoxycytidine residues commonly are present in transcriptionally inactive DNA regions, replacement of cytidine residues with a derivative that cannot be methylated may permit activation of genes previously repressed by methylation.

The high frequency at which azacytidine-treated C3H 10TV2 cells are converted into myotubes suggested to early workers that reactivation of one or a small number of closely linked genes is sufficient to drive a myogenic program. To test

► EXPERIMENTAL FIGURE 22-15 Myogenic genes isolated from azacytidine-treated cells can drive myogenesis when transfected into other cells. (a) When C3H IOTV2 cells (a fibroblast cell line) are treated with azacytidine, they develop into myotubes at high frequency. To isolate the genes responsible for converting azacytidine-treated cells into myotubes, all the mRNAs from treated cells first were isolated from cell extracts on an oligo-dT column. Because of their poly(A) tails, mRNAs are selectively retained on this column. Steps 1 and 2|: The isolated mRNAs were converted to radiolabeled cDNAs. Step 3: When the cDNAs were mixed with mRNAs from untreated C3H IOTV2 cells, only cDNAs derived from mRNAs (light red) produced by both azacytidine-treated cells and untreated cells hybridized. The resulting double-stranded DNA was separated from the unhybridized cDNAs (dark blue) produced only by azacytidine-treated cells. Step 4: The cDNAs specific for azacytidine-treated cells then were used as probes to screen a cDNA library from azacytidine-treated cells (Chapter 9). At least some of the clones identified with these probes correspond to genes required for myogenesis. (b) Each of the cDNA clones identified in part (a) was incorporated into a plasmid carrying a strong promoter. Steps 1 and 2|: C3H IOTV2 cells were cotransfected with each recombinant plasmid plus a second plasmid carrying a gene conferring resistance to an antibiotic called G418; only cells that have incorporated the plasmids will grow on a medium containing G418. One of the selected clones, designated myoD, was shown to drive conversion of C3H IOTV2 cells into muscle cells, identified by their binding of labeled antibodies against myosin, a muscle-specific protein (step 3). [See R. L. Davis et al., 1987, Cell 51:987]

(a) Screen for myogenic genes

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this hypothesis, researchers isolated DNA from C3H 10TV2 cells grown in the presence of 5-azacytidine and transfected it into untreated cells. The observation that 1 in 104 cells trans-fected with this DNA was converted into a myotube is consistent with the hypothesis that one or a small set of closely linked genes is responsible for converting fibroblasts into myotubes.

Subsequent studies led to the isolation and characterization of four different but related genes that can convert C3H 10TV2 cells into muscle. Figure 22-15 outlines the experimental protocol for identifying and assaying one of these genes, called the myogenic determination (myoD) gene. C3H 10TV2 cells transfected with myoD cDNA and those treated with 5-azacytidine both formed myotubes. The myoD cDNA also was able to convert a number of other cultured cell lines into muscle. Based on these findings, the myoD gene was proposed to play a key role in muscle development. A similar approach identified three other genes—myogenin, myf5, and mrf4—that also function in muscle development.

Muscle-Regulatory Factors (MRFs)

and Myocyte-Enhancing Factors (MEFs) Act in Concert to Confer Myogenic Specificity

The four myogenic proteins—MyoD, Myf5, myogenin, and MRF4—are all members of the basic helix-loop-helix (bHLH) family of DNA-binding transcription factors (see Figure 11-22b). Near the center of these proteins is a DNA-binding basic (B) region adjacent to the HLH domain, which mediates dimer formation. Flanking this central DNA-binding/dimerization region are two activation domains. We refer to the four myogenic bHLH proteins collectively as muscle regulatory factors, or MRFs (Figure 22-16a).

(a) Structure of muscle-regulatory factors (MRFs)

DNA binding/dimerization

(a) Structure of muscle-regulatory factors (MRFs)

DNA binding/dimerization

Transactivation

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