Cell Determination and Differentiation:
The Muscle Paradigm

Much of the current research in developmental biology is focused on identifying the genes that are involved in determinative events in development and unraveling the roles of the proteins they encode. Our understanding of the molecular events leading to functional cells and tissues remains quite sketchy. A major discovery that has facilitated progress in understanding cell determination and cell differentiation came with the discovery of a family of myogenic regulatory factors (MRFs), which are a group of transcription factors involved in switching on the muscle cell lineage during development. This mechanism serves as a model for the gene control of cell determination and differentiation.

The initial member of the MRF family to be discovered was MyoD, which was identified by its ability to convert cultured fibroblasts into skeletal myoblasts (Davis et al., 1987). This fascinating story should be reviewed. See Browder et al. (1991), pages 731-737, especially Figs. 18.4-18.7. The other members of this family in vertebrates are Myf-5, myogenin and MRF4. Each of these factors has the potential to turn tissue culture cells into myoblasts, which can - in turn - fuse with one-another and differentiate into muscle. Myoblast fusion occurs when growth factors become limiting and the myoblasts cease dividing (Olsen, 1992).

The genes encoding the MRFs are thought to be master regulatory genes, whose expression initiates a cascade of events that lead to muscle cell differentiation. They are expressed in a hierarchical fashion during myogenesis. Myf-5 and MyoD are expressed in cultured myoblasts (and continue to be expressed after muscle differentiation). Myogenin is expressed after myoblast fusion. It is an essential intermediate as shown by the prevention of myoblast differentiation by inhibition of myogenin expression with antisense oligonucleotides (Fig. 1, Florini and Ewton, 1990). MRF4 is expressed only after muscle differentiation.

The MRFs share a region of homology with two functionally significant domains: the helix-loop-helix (HLH) domain, which facilitates dimerization, and the basic region, which contains positively charged amino acids that mediate binding to DNA. These characteristics define a large family of proteins that function primarily as transcriptional activators. These are the basic helix-loop-helix (bHLH) proteins. MRFs form functional entities that bind to DNA by dimerizing with a member of the ubiquitously-expressed E protein family. This family includes E12, E47, ITF1 and ITF2. The most prevalent heterodimers in myotube extracts contain E12, but any of the E proteins can pair with the MRFs to form a functional heterodimer.

Dimerization is essential for bHLH protein function, but their specificity of binding to DNA is due to the basic region. Modifications to this region can either abolish the DNA binding capability of MyoD or eliminate its ability to activate transcription of muscle-specific genes (see Figs 2, 3 and 6, Davis et al., 1990). Thus, these mutants act like dominant-negative inhibitors of wild-type MyoD by competing with it for binding to its partners and inhibiting its activity. Nature has produced its own dominant-negative inhibitor of the MRFs. The interactions of MRFs with DNA can be prevented by members of of family of HLH factors called "Id", which stand for "inhibitor of binding". Id proteins lack a basic region. When they bind to MRF proteins, they impede their ability to bind to DNA and activate transcription of target genes (Fig. 6, Benezra et al., 1990). The inhibitory role of Id proteins is supported by the observations that:

(1) Id proteins are expressed in proliferating myoblasts in culture, but disappear when the myoblasts differentiate to form myotubes;
(2) overexpression of Id protein in cultured myoblasts prevents their differentiation into myotubes (Jen et al., 1992);
(3) Id transcripts are detected during the gastrula stage of mouse development before MRF transcripts first appear and are downregulated before MRFs are expressed (Wang et al., 1992).

Although the roles of Id in embryonic development are uncertain, the evidence suggests that it is initially an inhibitor of myogenesis and its downregulation then permits myogenesis to proceed by allowing MRFs to bind DNA of target genes.

MRF proteins bind to a sequence in the promoter of target genes called the E box. E boxes contain the sequence CANNTG (where N is any nucleotide). The genes encoding the MRFs contain an E box, which suggests that these proteins may regulate their own and one-another's transcription. Each MRF presumably owes its functional distinctiveness to unique sequences outside the bHLH domain.

The roles of MRFs in promoting myogenesis in cultured cells suggest that they may also play a role in muscle development during embryogenesis. Most of the skeletal muscle in vertebrates originates from progenitor cells in the somites. The somites are condensations of paraxial mesoderm that later become compartmentalized into the dermamyotome dorsally and the sclerotome ventrally. The dermamyotome subdivides into the dermatome and the myotome. The medial myotomal cells form the axial musculature, and the lateral cells migrate to the limbs to form limb muscle. (See Browder et al., 1991, pp. 293-298, especially Figs. 8.2-8.11.)

A role for MRFs in promoting muscle development during embryonic development is suggested by the location and timing of their expression during development. The MRFs are expressed sequentially in the somites, although details vary somewhat between species. In mice, the first MRF protein detected in trunk somites is Myf-5, which is first seen in medial somite cells (Fig. 1, Smith et al., 1994). Myogenin expression follows shortly after the initial detection of Myf-5, and MRF4 is expressed next. MyoD appearance is delayed and is first localized to the lateral portion of the somites. Initially, Myf-5 and MyoD expression is mutually exclusive and later overlaps (Fig. 3, Smith et al., 1994). Myf-5 and MyoD may be involved in establishing the two distinct subdomains of muscle: back musculature and limb musculature (Fig. 12, Ordahl and Le Douarin, 1992).

Data from knockout mice have helped to clarify the roles of the MRF genes in murine development. The initial null mouse experiments produced quite unexpected results: Mice that were null for either Myf-5 or MyoD genes developed normal amounts of skeletal muscle (Rudnicki et al., 1992; Braun et al., 1992). In homozygous MyoD null newborn mice, there was a 3- to 4-fold increase in Myf-5 expression. This gene is normally down-regulated after day 14 of development. The prolongation and enhancement of Myf-5 expression suggests that Myf-5 compensated for the lack of MyoD. In the Myf-5 knockouts, muscle development was delayed until MyoD was expressed, and then it proceeded (Braun et al., 1994). These observations suggest that MyoD and Myf-5 may be redundant. If so, does elimination of expression of both genes eliminate muscle development? The Myf-5 and MyoD mutant mice were interbred; the progeny that lacked both of these early-acting MRF genes were unable to initiate myogenesis, produced no myogenin and were devoid of skeletal muscle (Figs. 1, 2 , and 4, Rudnicki et al., 1993).

If myogenin is an essential intermediate in myogenesis, one would predict that myoblasts would form in myogenin knockout mice, but that skeletal muscle formation would be impaired. This is what has been observed, as shown in Figure 3 (Hasty et al., 1993) and Figure 3 (Nabeshima et al., 1993). The myogenin knock-out mice had deficient accumulation of transcripts for a number of muscle-specific proteins, including muscle creatine kinase, myosin heavy chain, the alpha and gamma subunits of the acetylcholine receptor and MRF4. However, normal amounts of MyoD transcripts were present, consistent with the hypothesis that MyoD acts upstream of myogenin (Fig. 5, Hasty et al., 1993). During development of myogenin knock-outs, somites developed normally and compartmentalized into myotome, dermatome and sclerotome (Fig. 1, Venuti et al , 1995). They even initiated muscle mass differentiation, but myosin heavy chain protein expression was attenuated, and myofibers were diffuse. The disparity between mutant and wild-type embryos widened as development continued (Fig. 4, Venuti et al , 1995). Large numbers of myoblasts that failed to differentiate appeared to be present in the mutant muscle masses.

The picture of myogenesis that is emerging is that MyoD and Myf-5 are redundant and initiate myogenesis in the myoblasts. They control expression of myogenin, which - in turn - controls myotube differentiation and may control expression of MRF4. MRF4 may be responsible for events in fully-differentiated myofibers. possibly by maintaining the differentiated state (Fig. 5, Rudnicki et al., 1993). According to this scheme, transcription of distinct sets of genes at each stage are regulated by MRFs, which also control the expression of the MRF that initiates the next stage of differentiation (Venuti et al., 1995).

How is expression of the MRFs themselves initiated? Transplantation experiments with chick embryos have shown that the somites are induced by the neural tube/notochord complex to form muscle, although the identities of the inducing molecules are unknown (Rong et al., 1992; Buffinger and Stockdale, 1994). Recent work in mice has led to the identification of a gene that encodes a basic helix-loop-helix protein called Paraxis, which is expressed in the unsegmented paraxial mesoderm immediately before somite formation (see Figs. 2 and 3, Burgess et al., 1995). Its expression precedes that of the MRF genes. Its expression becomes downregulated in the myotomes after somite compartmentalization, although it persists in the dermatome and sclerotome. A related bHLH protein, Scleraxis, is co-expressed with Paraxis in the sclerotome. The expression of Scleraxis later increases in the sclerotome (see Figs. 5 and 7, Burgess et al., 1995). It will be exciting to learn what role these two proteins play in establishment and compartmentalization of the somites and whether they are involved in mediating the response to the inductive signals from the dorsal midline in activating expression of the MRF cascade.

Myogenic determination and differentiation occur through a complex cascade of events involving a network of factors whose interaction ensures that muscle forms in the right places and at the right times to facilitate orderly embryonic development. Muscle development serves as a valuable paradigm for the understanding of determination and differentiation of other tissue-types, whose development likely involves networks of factors similar to those described here.


References

This material is based substantially upon a recent review article on this subject (Browder, 1996). Readers should also see the recent review by Ludolph and Konieczny (1995).

Benezra, R., R.L. Davis, D. Lockshon et al. 1990. The protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell 61: 49-59.

Braun, T., M.A. Rudnicki, H.-H. Arnold et al. 1992. Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell 71: 369-382.

Braun, T., E. Bober, M.A. Rudnicki et al. 1994. MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice. Development 120: 3083-3092.

Browder, L.W. 1996. Gene expression, cell determination and differentiation. In Textbook of Tissue Engineering, edited by R. Lanza, R. Langer, and W. Chick. R.G. Landes, Austin, Texas. In press.

Browder, L.W., C.A. Erickson and W.R. Jeffery. 1991. Developmental Biology. Third ed. Saunders College Publishing. Philadelphia.

Buffinger, N. and F.E. Stockdale. 1994. Myogenic specification in somites: induction by axial structures. Development 120: 1443-1452.

Burgess, R., P. Cserjesi, K.L. Ligon and E.N. Olson. 1995. Paraxis: a basic helix-loop-helix protein expressed in paraxial mesoderm and developing somites. Develop. Biol. 168: 296-306.

Davis, R.L., H. Weintraub and A.B. Lassar AB. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.

Davis, R.L., P.-F. Cheng, A.B. Lassar and H. Weintraub. 1990. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell 60: 733-746.

Florini, J.R. and D.A. Ewton. 1990. Highly specific inhibition of IGF-I-stimulated differentiation by an antisense oligodeoxyribonucleotide to myogenin mRNA. J. Biol. Chem. 265: 13435-13437.

Hasty, P., A. Bradley, J.H. Morris et al. 1993. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364: 501-506.

Jen, Y., H. Weintraub and R. Benezra. 1992. Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins. Genes & Dev. 6: 1466-1479.

Ludolph, D.C. and S.F. Konieczny. 1995. Transcription factor families: muscling in on the myogenic program. FASEB J. 9: 1595-1604.

Nabeshima, Y., K. Hanaoka, M. Hayasaka et al. 1993. Myogenin gene disruption results in perinatal lethality because of severe muscle defect. Nature 364: 532-535.

Olson, E.N. 1992. Interplay between proliferation and differentiation within the myogenic lineage. Develop. Biol. 154: 261-272.

Ordahl, C.P. and N.M. Le Douarin. 1992. Two myogenic lineages within the developing somite. Development 114: 339-353.

Rong, P.M., M.-A. Teillet, C. Ziller et al. 1992. The neural tube/notochord complex is necessary for vertebral but not limb and body wall striated muscle differentiation. Development 115: 657-672.

Rudnicki, M.A., T. Braun, S. Hinuma et al. 1992. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell 71: 383-390.

Rudnicki, M.A., P.N.J. Schnegelsberg, R.H. Stead et al. 1993. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75: 1351-1359.

Smith, T.H., A.M. Kachinsky and J.B. Miller. 1994. Somite subdomains, muscle origins, and the four muscle regulatory proteins. J. Cell Biol. 127: 95-105.

Venuti, J.M., J.H. Morris, J.L. Vivian et al. 1995. Myogenin is required for late but not early aspects of myogenesis during mouse development. J. Cell Biol. 128: 563-576.

Wang, Y., R. Benezra and D.A. Sassoon. 1992. Id expression during mouse development: a role in morphogenesis. Dev Dynamics 194: 222-230.



Reference for Student Seminar

Cossu, G., R. Kelly, S. Tajbakhsh, S. Di Donna, E. Vivarelli and M. Buckingham. 1996. Activation of different myogenic pathways: myf-5 is induced by the neural tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm. Development 122: 429-437.


Browder, L.W. 1996. Cell determination and differentiation: the muscle paradigm. In L.W. Browder (Ed.), Advanced Developmental Biology, <http://www.ucalgary.ca/~browder>.

Copyright © 1996 Leon W. Browder. This material may be reproduced for educational purposes only provided credit is given to the original source.

April 1, 1996

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