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).
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Reference for Student Seminar
Cossu, G., R. Kelly, S. Tajbakhsh, S. Di Donna, E. Vivarelli and M.
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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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