Cooh

Myogenin-interacting domain

▲ FIGURE 22-16 General structures of two classes of transcription factors that participate in myogenesis. MRFs (muscle regulatory factors) are bHLH (basic helix-loop-helix) proteins produced only in developing muscle. MEFs (myocyte-enhancing factors), which are produced in several tissues in addition to developing muscle, belong to the MADS family. The myogenic activity of MRFs is enhanced by their interaction with MEFs.

bHLH proteins form homo- and heterodimers that bind to a 6-bp DNA site with the consensus sequence CANNTG (N = any nucleotide). Referred to as the E box, this sequence is present in many different locations within the genome (on a purely random basis the E box will be found every 256 nucleotides). Thus some mechanism(s) must ensure that MRFs specifically regulate muscle-specific genes and not other genes containing E boxes in their transcription control regions. One clue to how this myogenic specificity is achieved was the finding that the DNA-binding affinity of MyoD is tenfold greater when it binds as a heterodimer complexed with E2A, another bHLH protein, than when it binds as a homodimer. Moreover, in azacytidine-treated C3H 10TV2 cells, MyoD is found as a heterodimer complexed with E2A, and both proteins are required for myogenesis in these cells. The DNA-binding domains of E2A and MyoD have similar but not identical amino acid sequences, and both proteins recognize E box sequences. The other MRFs also form heterodimers with E2A that have properties similar to MyoD-E2A complexes. This het-erodimerization restricts activity of the myogenic transcription factors to genes with closely linked E boxes.

Since E2A is expressed in many tissues, the requirement for E2A is not sufficient to confer myogenic specificity. Subsequent studies suggested that specific amino acids in the bHLH domain of all the MRFs confer myogenic specificity by allowing MRF-E2A complexes to bind specifically to another family of DNA-binding proteins called myocyte enhancing factors, or MEFs. MEFs were considered excellent candidates for interaction with MRFs for two reasons. First, many muscle-specific genes contain recognition sites for both MEFs and MRFs. Second, although MEFs cannot induce myogenic conversion of azacytidine-treated C3H 10TV2 cells by themselves, they enhance the ability of MRFs to do so. This enhancement requires physical interaction between a MEF and MRF-E2A heterodimer. MEFs belong to the MADS family of transcription factors and to contain a MEF domain, adjacent to the MADS domain, that mediates interaction with myogenin (Figure 22-16b). The synergistic action of the MEF homodimer and MRF-E2A heterodimer is thought to drive high-level expression of muscle-specific genes.

Knockout mice and Drosophila mutants have been used to explore the roles of MRF and MEF proteins in conferring myogenic specificity in intact animals, extending the work in cell culture. These experiments demonstrated the importance of three of the MRF proteins and of MEF proteins for distinct steps in muscle development (see Figure 22-14). The function of the fourth myogenic protein, Mrf4, is not entirely clear; it may be expressed later and help maintain differentiated muscle cells and by combinatorial control to ensure that only muscle-specific genes are activated.

Terminal Differentiation of Myoblasts Is Under Positive and Negative Control

Powerful developmental regulators like the MRFs cannot be allowed to run rampant. In fact, their actions are circum scribed at several levels. First, production of the muscle regulators is activated only in mesoderm cells, at the right place and time in the embryo, in response to spatial regulators of the sorts that are described in Chapter 15. Other proteins mediate additional mechanisms for assuring tight control over myogenesis: chromatin-remodeling proteins are needed to make target genes accessible to MRFs; inhibitory proteins can restrict when MRFs act; and antagonistic relations between cell-cycle regulators and differentiation factors like MRFs ensure that differentiating cells will not divide, and vice versa. All these factors control when and where muscles form.

Activating Chromatin-Remodeling Proteins MRF proteins control batteries of muscle-specific genes, but can do so only if chromatin factors allow access. Remodeling of chro-matin, which usually is necessary for gene activation, is carried out by large protein complexes (e.g., Swi/Snf complex) that have ATPase and perhaps helicase activity. These complexes are thought to recruit histone acetylases that modify chromatin to make genes accessible to transcription factors (Chapter 11). The hypothesis that remodeling complexes help myogenic factors was tested using dominant-negative versions of the ATPase proteins that form the cores of these complexes. (Recall from Chapter 9 that a dominant-negative mutation produces a mutant phenotype even when a normal allele of the gene also is present.) When genes carrying these dominant-negative mutations were transfected into C3H 10TV2 cells, the subsequent introduction of myogenic genes no longer converted the cells into myotubes. In addition, a muscle-specific gene that is normally activated did not exhibit its usual pattern of chromatin changes in the doubly transfected C3H 10TV2 cells. These results indicate that transcription activation by myogenic proteins depends on a suitable chromatin structure in the regions of muscle-specific genes.

Inhibitory Proteins Screens for genes related to myoD led to identification of a related protein that retains the HLH dimerization region but lacks the basic DNA-binding region and hence is unable to bind to E box sequences in DNA. By binding to MyoD or E2A, this protein inhibits formation of MyoD-E2A heterodimers and hence their high-affinity binding to DNA. Accordingly, this protein is referred to as Id, for inhibitor of DNA binding. Id prevents cells that produce MyoD and E2A from activating transcription of the muscle-specific gene encoding creatine kinase. As a result, the cells remain in a proliferative growth state. When these cells are induced to differentiate into muscle (for instance, by the removal of serum, which contains the growth factors required for proliferative growth), the Id concentration falls. MyoD-E2A dimers now can form and bind to the regulatory regions of target genes, driving differentiation of C3H 10TV2 cells into myoblast-like cells.

Recent work shows that histone acetylases and deacety-lases are also crucial for regulating muscle-specific genes. As explained in Chapter 11, acetylation of histones in chromatin is necessary to activate many genes; in contrast, histone deacetylases cause transcriptional repression (see Figure 11-32). MEF2 recruits histone acetylases such as p300/CBP, through another protein that serves as a mediator, thus activating transcription of target genes. Chromatin immunopre-cipitation experiments with antibodies against acetylated histone H4 show that the acetylated histone level associated with MEF2-regulated genes is higher in differentiated my-otubes than in myoblasts (see Figure 11-31). The role of histone deacetylases in muscle development was revealed in experiments in which scientists first introduced extra myoD genes into cultured C3H 10TV2 cells to raise the level of MyoD. This resulted in increased activation of target genes and more rapid differentiation of the cells into myotubes. However, when genes encoding histone deacetylases also were introduced into the C3H 10TV2 cells, the muscle-inducing effect of MyoD was blocked and the cells did not differentiate into myotubes.

The explanation for how histone deacetylases inhibit MyoD-induced muscle differentiation came from the surprising finding that MEF2 can bind, through its MADS domain, to a histone deacetylase. This interaction, which can prevent MEF2 function and muscle differentiation, is normally blocked during differentiation because the histone deacetylase is phosphorylated by a calcium/calmodulin-dependent protein kinase; the phosphorylated deacetylase then is moved from the nucleus to the cytoplasm. Taken together, these results indicate that activation of muscle genes by MyoD and MEF2 is in competition with inactivation of muscle genes by repressive chromatin structures and that nuclear versus cytoplasmic localization of chromatin factors is a key regulatory step.

Cell-Cycle Proteins The onset of terminal differentiation in many cell types is associated with arrest of the cell cycle, most commonly in G1, suggesting that the transition from the determined to differentiated state may be influenced by cell-cycle proteins including cyclins and cyclin-dependent kinases (Chapter 21). For instance, certain inhibitors of cyclin-dependent kinases can induce muscle differentiation in cell culture, and the amounts of these inhibitors are markedly higher in differentiating muscle cells than in non-differentiating ones in vivo. Conversely, differentiation of cultured myoblasts can be inhibited by transfecting the cells with DNA encoding cyclin D1 under the control of a con-stitutively active promoter. Expression of cyclin D1, which normally occurs only during G1, is induced by mitogenic factors in many cell types and drives the cell cycle (see Figure 21-28). The ability of cyclin D1 to prevent myoblast differentiation in vitro may mimic aspects of the in vivo signals that antagonize the differentiation pathway. The antagonism between negative and positive regulators of G1 progression is likely to play an important role in controlling myogenesis in vivo.

Cell-Cell Signals Are Crucial for Muscle Cell-Fate Determination and Myoblast Migration

As noted already, after myoblasts arise from somites, they must not only proliferate but also move to their proper locations and form the correct attachments as they differentiate into muscle cells (Figure 22-17). Myogenic gene expression often follows elaborate events that tell certain somite cells to delaminate from the somite epithelium and which way to move. A transcription factor, Pax3, is produced in the subset of somite cells that will form muscle. Pax3 appears to be at the top of the regulatory hierarchy controlling muscle formation in the body wall and limbs. Myoblasts that will migrate, but not cells that remain behind, also produce a transcription factor called Lbx1. If Pax3 is not functional, Lbx1 transcripts are not seen and myoblasts do not migrate. Both Pax3 and Lbx1 can affect expression of myoD.

The departure of myoblasts from somites depends upon reception of a secreted protein signal appropriately called scatter factor, or hepatocyte growth factor (SF/HGF). This signal is produced by embryonic connective tissue cells (mesenchyme) in the limb buds to which myoblasts migrate. Production of SF/HGF is previously induced by other secreted signals such as fibroblast growth factor and Sonic hedgehog, which are critical to limb development (Chapter 15). The cell-surface receptor for SF/HGF, which is expressed by myoblasts, belongs to the receptor tyrosine kinase (RTK) class of receptors (Chapter 14). Cells migrate from the somites at the regions along the head-to-tail body axis where limbs will form, and not

Dermamyotome Neural tube

Dermamyotome Neural tube

Sclerotome

(gives rise to skeletal structures such as vertebrae)

Sclerotome

(gives rise to skeletal structures such as vertebrae)

▲ FIGURE 22-17 Embryonic determination and migration of myoblasts in mammals. After formation of the neural tube, each somite forms sclerotome, which develops into skeletal structures, and dermomyotome. The dermomyotome gives rise to the dermis of the skin and to the muscles. Lateral myoblasts migrate to the limb bud; medial myoblasts develop into the trunk muscles. The remainder of a dermomyotome gives rise to the connective tissue of the skin. [Adapted from M. Buckingham, 1992, Trends Genet. 8:144.]

elsewhere, due to the presence of SF/HGF at limb locations and not elsewhere. If the SF/HGF signal or its receptor is not functional, somite cells will produce Lbx1 but not go on to migrate; thus no muscles will form in the limbs. Expression of the myogenin gene, which is necessary for myotube formation, and of mrf4, which is necessary for muscle fiber differentiation, does not begin until migrating myoblasts approach their limb-bud destination; these steps in terminal muscle differentiation are presumably not compatible with migration.

We have touched on just a few of the many external signals and transcription factors that participate in development of a properly patterned muscle. The function of all these regulatory molecules must be coordinated both in space and in time during myogenesis.

bHLH Regulatory Proteins Function in Creation of Other Tissues

Four bHLH transcription factors that are remarkably similar to the myogenic bHLH proteins control neurogenesis in Drosophila. Similar proteins appear to function in neurogen-esis in vertebrates and perhaps in the determination and differentiation of hematopoietic cells.

The neurogenic Drosophila bHLH proteins are encoded by an «100-kb stretch of genomic DNA, termed the achaete-scute complex (AS-C), containing four genes designated achaete (ac), scute (sc), lethal of scute (l'sc), and asense (a). Analysis of the effects of loss-of-function mutations indicate that the Achaete (Ac) and Scute (Sc) proteins participate in determination of neuronal stem cells, called neuroblasts, while the Asense (As) protein is required for differentiation of the progeny of these cells into neurons. These functions are analogous to the roles of MyoD and Myf5 in muscle determination and of myogenin in differentiation. Two other Drosophila pro teins, designated Da and Emc, are analogous in structure and function to vertebrate E2A and Id, respectively. For example, heterodimeric complexes of Da with Ac or Sc bind to DNA better than the homodimeric forms of Ac and Sc. Emc, like Id, lacks a DNA-binding basic domain; it binds to Ac and Sc proteins, thus inhibiting their association with Da and binding to DNA. The similar functions of these myogenic and neurogenic proteins are depicted in Figure 22-18.

A family of bHLH proteins related to the Drosophila Achaete and Scute proteins has been identified in vertebrates. One of these, called neurogenin, which has been identified in the rat, mouse, and frog, controls the formation of neuroblasts. In situ hybridization experiments showed that neurogenin is produced at an early stage in the developing nervous system and induces production of NeuroD, another bHLH protein that acts later (Figure 22-19a). Injection of large amounts of neurogenin mRNA into Xenopus embryos further demonstrated the ability of neurogenin to induce neurogenesis (Figure 22-19b). These studies suggest that the function of neurogenin is analogous to that of the Achaete and Scute in Drosophila; likewise, NeuroD and Asense may have analogous functions in vertebrates and Drosophila, respectively.

In addition to neurons, the nervous system contains a large number of glial cells, which also arise from the neuro-ectoderm. Glial cells support and insulate neurons; they also provide guidance and contact surfaces for migrating neurons during development and send a signal to neurons that promotes formation of synapses. Neurogenins control the fates of precursor cells that are capable of making either neurons or glial cells by promoting neural development and repressing glial development. Neurogenins are therefore switches that control the decision between two alternative cell fates, just as the yeast mating-type regulators select among three cell types.

► FIGURE 22-18 Comparison of genes that regulate Drosophila neurogenesis and mammalian myogenesis. bHLH transcription factors have analogous functions in determination of precursor cells (i.e., neuroblasts and myoblasts) and their subsequent differentiation into mature neurons and muscle cells. In both cases, the proteins encoded by the earliest-acting genes (left) are under both positive and negative control by other related proteins (blue type). [Adapted from Y N. Jan and L. Y Jan, 1993, Cell 75:827.]

Fly neurogenesis ac, scmRNA

achaete scute

Proneural genes

Vertebrate myogenesis myf5, myoD mRNA

myf5 myoD (others)

Promuscular genes

E2A (general)

Id as mRNA

asense (others)

Neurons

Neural precursor genes myogenin mRNA

myogenin mRNA

Muscle precursor genes

Muscle precursor genes

Precursor determination

Differentiation

Muscle

▲ EXPERIMENTAL FIGURE 22-19 In situ hybridization and injection experiments demonstrate that neurogenin acts before NeuroD in vertebrate neurogenesis. (a) Sections of rat neural tube were treated with a probe specific for neurogenin mRNA (left) or neuroD mRNA (right). The open space in the center is the ventricle, and the cells lining this cavity are in the ventricular layer. All the neural cells are born in the ventricular layer and then migrate outward. As illustrated in these micrographs, neurogenin mRNA is produced in proliferating neuroblasts in the ventricular layer (arrow), whereas neuroD mRNA is present in migrating neuroblasts that have left the ventricular zone (other arrow). (b) One of the two cells in early Xenopus embryos was injected with neurogenin mRNA (inj) and then stained with a probe specific for neuron-specific mRNAs encoding (3-tubulin (left) or NeuroD (right). The region of the embryo derived from the uninjected cell served as a control (con). The neurogenin mRNA induced a massive increase in the number of neuroblasts expressing neuroD mRNA and neurons expressing jj-tubulin mRNA in the region of the neural tube derived from the injected cell. [From Q. Ma et al., 1996, Cell 87:43; courtesy of D. J. Anderson.]

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