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Roles of MRFs during Embryogenesis
What do the MRFs do in the embryo?
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; Gilbert, 1997, pp.
341-348; Kalthoff, 1996, pp. 300-306; Shostak, 1991, pp. 648-651; Wolpert
et al., 1998, pp. 97-100)
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
- Describe the compartmentalization of the somites.
- Discuss the pattern of expression of the MRFs during development in
- Discuss the effects of the overexpression of XMyoD and XMyf-5 on early
Xenopus development in vivo.
- You had better review how knockout mice are made.
- Describe the effects of knocking out either Mrf-5 or MyoD.
- Describe the effects of knocking out both Mrf-5 and MyoD.
- Describe the effects of knocking out the myogenin gene.
- Derive a hierarchy of MRF gene expression based upon all the information
you have received.
Denetclaw, W.F. Jr, Christ, B. and Ordahl, C.P. 1997. Location and growth
of epaxial myotome precursor cells. Development 124: 1601-1610.
Myer, A., Wagner, D.S., Vivian, J.L., Olson, E.N. and Klein, W.H. 1997.
Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse
in vivo. Develop. Biol. 185: 127-138.
Yoon, J.K., Olson, E.N., Arnold, H.-H. and Wold, B.J. 1997. Different MRF4
knockout alleles differentially disrupt Myf-5 expression: cis-regulatory
interactions at the MRF4/Myf-5 locus. Develop. Biol. 188:
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
Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology.
Third edition. Saunders College Pub. Philadelphia.
Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer.
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.
Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill.
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.
Ordahl, C.P. and N.M. Le Douarin. 1992. Two myogenic lineages within the
developing somite. Development 114: 339-353.
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.
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.
Shostak, S. 1991. Embryology. An Introduction to Developmental Biology.
HarperCollins. New York.
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.
Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and
Meyerowitz, E. 1998. Principles of Development. Current Biology.