nautilus
Effects of mutation and ectopic expression of mammalian Nautilus homologs The MyoD family of basic helix-loop-helix (bHLH) proteins is required for myogenic determination and differentiation. The
basic region carries the myogenic code and DNA binding specificity, while the N terminus contains a potent transcriptional
activation domain. Myogenic activation is abolished when the basic region, bound to a myogenic E box, carries an Ala-114 mutation. It has been proposed that DNA binding of the MyoD basic region leads to recruitment of a recognition factor that
unmasks the activation domain. The A114N mutant exhibits an altered conformation in the basic
region and this local conformational difference can lead to a more global change affecting the conformation of the
activation domain. This suggests that the deleterious effects of this class of mutations may result directly from defective
conformation. Thus, the activation domain is unmasked only upon DNA binding by the correct basic region. Such a coupled
conformational relationship may have evolved to restrict myogenic specificity to a small number of bHLH proteins, from among the
many with diverse functions yet with DNA binding specificities known to be similar (Huang, 1998).
The myogenic progenitors of epaxial (paraspinal and intercostal) and hypaxial (limb and abdominal
wall) musculature are believed to originate in dorsal-medial and ventral-lateral domains, respectively, of
the developing somite. To investigate the hypothesis that Myf-5 and MyoD have different roles in the
development of epaxial and hypaxial musculature, myogenesis was characterized in Myf-5- and
MyoD-deficient embryos by several approaches. Expression of a MyoD-lacZ transgene was examined in Myf-5 and MyoD mutant embryos to characterize the temporal-spatial patterns of myogenesis in
mutant embryos. Immunohistochemistry was performed on sectioned Myf-5 and MyoD
mutant embryos with antibodies reactive with desmin, nestin, myosin heavy chain, sarcomeric actin,
Myf-5, MyoD and myogenin. While MyoD(-/-) embryos display normal development of paraspinal
and intercostal muscles in the body proper, muscle development in limb buds and brachial arches is
delayed by about 2.5 days. In contrast, Myf-5(-/-) embryos display normal muscle development in
limb buds and brachial arches, and markedly delayed development of paraspinal and intercostal
muscles. Although MyoD mutant embryos exhibited delayed development of limb musculature, normal
migration of Pax-3-expressing cells into the limb buds and normal subsequent induction of Myf-5 in
myogenic precursors is observed. These results suggest that Myf-5 expression in the limb is
insufficient for the normal progression of myogenic development. Taken together, these observations
strongly support the hypothesis that Myf-5 and MyoD play unique roles in the development of epaxial
and hypaxial muscle, respectively (Kablar, 1997).
MyoD is a muscle-specific transcription factor involved in commitment of cells to
myogenesis. MyoD mRNA levels differ between fast and slow muscles, suggesting
that MyoD may regulate aspects of fiber type. This study shows that detectable MyoD
protein becomes restricted to the nuclei during development of the fastest classes of
fibers in fast muscles. myoDml mice, in which the myoD gene has been disrupted,
show subtle shifts in fiber type of fast muscles toward a slower character, suggesting
that MyoD is involved in the maintenance of the fast IIB/IIX fiber type. In contrast,
slow muscle shifts to a faster phenotype in myoDm1. MD6.0-lacZ
transgenic mice with the myoD promoter driving lacZ, show highest
beta-galactosidase activity in the fastest fibers of fast muscles, but also express low
levels in slow fibers of slow, but not fast, muscles, suggesting distinct regulation of
gene expression in slow fibers of both fast and slow muscles. It is likely that MyoD acts at different concentrations or with different protein partners on different target genes (Hughes, 1997).
Either MyoD or Myf5 is required to form a committed myoblast, but myogenin is essential in either case for myoblast differentiation. In mice lacking the bHLH transcription factor myogenin,
myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This
indicates that myogenin is critical for the fusion process and the subsequent differentiation events of
myogenesis. Whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo was investigated
using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos
demonstrate that myogenin-null myoblasts readily fuse in the presence of wild-type myoblasts.
However, chimeric myofibers do not express wild-type levels of muscle-specific gene products, and
myofibers with a high percentage of mutant nuclei appear abnormal, suggesting that the wild-type
nuclei can not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast
fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific
subset of genes with diverse function. Thus, myogenin appears to control not only transcription of
muscle structural genes but also the extracellular environment in which myoblast fusion takes place.
It is proposed that myogenin regulates the expression of one or more extracellular or cell surface
proteins required to initiate the muscle differentiation program (Myer, 1997).
The myogenic basic Helix-Loop-Helix transcription factors (including Myf5, MyoD, myogenin (myg)
and MRF4) are crucial to skeletal muscle development. The phenotypes of mutant mice
deficient in one or another gene are different, suggesting that each gene may have a unique function in vivo. Myogenin can be targeted into the Myf5 locus by "knocking in" myogenin into the Myf5 locus to create (Myf5[myg-ki]). Myf5[myg-ki] rescues the rib cage
truncation in the Myf5-null mutant, hence demonstrating functional redundancy between Myf5 and
myogenin in skeletal morphogenesis. myogenin knock-in
(myg-ki) mice were crossed with either MyoD-null or myogenin-null mutants. The Myf5(myg-ki) allele rescues early
myogenesis, but Myf5(myg-ki/myg-ki);MyoD(-/-) mutant mice died immediately after birth owing to
reduced muscle formation. Therefore, myogenin, expressed from the Myf5 locus, is not able to
completely replace the function of Myf5 in muscle development although it is capable of determining
and/or maintaining myogenic lineage. Myf5(myg-ki/myg-ki);myg(-/-) mutant mice display the same
phenotype as myg(-/-) mutants. This indicates that the earlier expression of myogenin cannot promote
myogenic terminal differentiation, which is normally initiated by the endogenous myogenin. These
results are consistent with the notion that Myf5 and myogenin are functionally interchangeable in
determining myogenic lineage and assuring normal rib formation. This experiment reveals, however,
that some aspects of myogenesis may be unique to a given myogenic factor and are due either to
different regulatory sequences that control gene temporal and spatial expression or to different functional
protein domains (Wang, 1997).
The myogenic basic helix-loop-helix (bHLH) genes -- MyoD, Myf5, myogenin and MRF4 -- exhibit distinct, but overlapping expression patterns during development of the skeletal muscle lineage and
loss-of-function mutations in these genes result in different effects on muscle development. MyoD and
Myf5 have been shown to act early in the myogenic lineage to establish myoblast identity, whereas
myogenin acts later to control myoblast differentiation. In mice lacking myogenin, there is a severe
deficiency of skeletal muscle, but some residual muscle fibers are present in mutant mice at birth. Mice
lacking MRF4 are viable and have skeletal muscle, but they upregulate myogenin expression, which
could potentially compensate for the absence of MRF4. Previous studies in which Myf5 and MRF4 null mutations were combined suggested that these genes do not share overlapping myogenic functions in vivo. To determine whether the functions of MRF4 might overlap with those of myogenin or MyoD, double mutant mice were generated lacking MRF4 and either myogenin or MyoD. MRF4/myogenin double mutant mice contain a number of residual muscle fibers comparable to mice lacking myogenin alone; myoblasts from those double mutant mice form differentiated multinucleated myotubes in vitro as efficiently as wild-type myoblasts, indicating that neither myogenin nor MRF4 is absolutely essential for myoblast differentiation. Although mice lacking either MRF4 or MyoD are viable and do not show defects in muscle development, MRF4/MyoD double mutants display a severe muscle deficiency similar to that in myogenin mutants. Myogenin is expressed in MRF4/MyoD double mutants, indicating that myogenin is insufficient to support normal myogenesis in vivo. These results reveal
unanticipated compensatory roles for MRF4 and MyoD in the muscle differentiation pathway and suggest
that a threshold level of myogenic bHLH factors is required to activate muscle structural genes, with this
level normally being achieved by combinations of multiple myogenic bHLH factors (Rawls, 1998).
Genetic studies have demonstrated that MyoD and Myf5 establish the skeletal muscle
lineage, whereas myogenin mediates terminal differentiation. The molecular basis
for this distinction is not yet understood. MyoD can remodel chromatin at
binding sites in muscle gene enhancers and activate transcription at previously silent
loci. TGF-ß, basic-FGF, and sodium butyrate block MyoD-mediated chromatin
reorganization and the initiation of transcription. In contrast, TGF-ß and sodium
butyrate do not block transcription when added after chromatin remodeling has
occurred. MyoD and Myf-5 are 10-fold more efficient than myogenin at activating
genes in regions of transcriptionally silent chromatin. Deletion mutagenesis of the
MyoD protein demonstrates that the ability to activate endogenous genes depends on
two regions: a region rich in cysteine and histidine residues between the acidic
activation domain and the bHLH domain, and a second region in the carboxyl terminus
of the protein. Neither region has been previously shown to regulate gene transcription
and both have domains that are conserved in the Myf5 protein. These results establish a
mechanism for chromatin modeling in the skeletal muscle lineage and define domains
of MyoD (independent of the activation domain) that participate in chromatin
reorganization (Gerber, 1997).
A novel bHLH protein gene Mesp2 (for mesoderm posterior 2) has been isolated that
cross-hybridizes with Mesp1 expressed in the early mouse mesoderm. Mesp1 and Mesp2 are related the Twist and Nautilus protein families. Mesp2 is
expressed in the rostral presomitic mesoderm, but down-regulated immediately after
the formation of the segmented somites. To determine the function of MesP2 protein in somitogenesis, Mesp2-deficient mice were generated by gene targeting. The
homozygous Mesp2 (-/-) mice die shortly after birth and have fused vertebral columns
and dorsal root ganglia, with impaired sclerotomal polarity. The earliest defect in the
homozygous embryos is a lack of segmented somites. The disruption of the
metameric features, altered expression of Mox-1, Pax-1, and Dll1, and lack of
expression of Notch1, Notch2, and FGFR1, suggest that MesP2 controls sclerotomal
polarity by regulating the signaling systems mediated by notch-delta and FGF, which
are essential for segmentation (Saga, 1997).
During mouse development, the myogenic basic helix-loop-helix transcription factor myogenin plays an essential role in the
differentiation of skeletal muscle and, secondarily, in rib and sternum formation. However, virtually nothing is known about the quantitative requirements for myogenin in
these processes. Mice were generated carrying a hypomorphic allele of myogenin,
which expresses myogenin transcripts at approximately one-fourth the level of the wild-type myogenin
allele. The hypomorphic allele in combination with wild-type and myogenin-null alleles was used to
create an allelic series. Embryos representing the complete range of genotypes from homozygous wild
type to homozygous null were analyzed for their viability, ability to form normal ribs and sternum, and
extent of skeletal muscle differentiation. Embryos carrying the hypomorphic myogenin allele over a
wild-type allele are normal. In embryos bearing homozygous hypomorphic alleles, the sternum
develops normally and extensive skeletal muscle differentiation occurs. However, muscle
hypoplasia and reduced muscle-specific gene expression are apparent in these embryos, and the mice
are not viable as neonates. When the hypomorphic allele is placed over a myogenin-null allele, the
resulting embryos have sternum defects resembling homozygous myogenin-null embryos, and there is
severe muscle hypoplasia. These results demonstrate that skeletal muscle formation is highly sensitive to
the absolute levels of myogenin and that correct sternum formation, skeletal muscle differentiation, and
viability each require distinct threshold levels of myogenin (Vivian, 1999).
To gain insight into the regeneration deficit of MyoD-/- muscle, the growth and
differentiation of cultured MyoD-/- myogenic cells was investigated. Primary MyoD-/- myogenic cells exhibit a
stellate morphology distinct from the compact morphology of wild-type myoblasts, and express
c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD-/- myogenic cells do not
express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and
myogenic precursor cells in vivo. Northern analysis indicates that proliferating MyoD-/- myogenic cells
express fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain
transcription factor expressed in newly activated satellite cells. Under conditions that normally induce
differentiation, MyoD-/- cells continue to proliferate and with delayed kinetics yield reduced
numbers of predominantly mononuclear myocytes. Northern analysis reveals delayed induction of
myogenin, MRF4, and other differentiation-specific markers, although p21 is upregulated normally.
Expression of M-cadherin mRNA is severely decreased whereas expression of IGF-1 is
markedly increased in MyoD-/- myogenic cells. Mixing of lacZ-labeled MyoD-/- cells and wild-type
myoblasts reveals a strict autonomy in differentiation potential. Transfection of a MyoD-expression
cassette restores cytomorphology and rescues the differentiation deficit. These data are interpreted to
suggest that MyoD-/- myogenic cells represent an intermediate stage between a quiescent satellite cell
and a myogenic precursor cell (Sabourin, 1999).
The basic helix-loop-helix (bHLH) transcription factors -- MyoD, Myf5, myogenin, and MRF4 --- can each activate the
skeletal muscle-differentiation program in transfection assays. However, their functions during embryogenesis, as revealed
by gene-knockout studies in mice, are distinct. MyoD and Myf5 have redundant functions in myoblast specification,
whereas myogenin and either MyoD or MRF4 are required for differentiation. Paradoxically, myoblasts from myogenin
mutant or MyoD/MRF4 double-mutant neonates differentiate normally in vitro, despite their inability to differentiate in
vivo, suggesting that the functions of the myogenic bHLH factors are influenced by the cellular environment and that the
specific myogenic defects observed in mutant mice do not necessarily reflect essential functions of these factors.
Understanding the individual roles of these factors is further complicated by their ability to cross-regulate one anothers
expression. To investigate the functions of Myf5 in the absence of contributions from other myogenic bHLH factors, triple-mutant mice lacking myogenin, MyoD, and MRF4 were generated. These mice appear to contain a normal number of
myoblasts, but in contrast to myogenin or MyoD/MRF4 mutants, differentiated muscle fibers fail to form in vivo and
myoblasts from neonates of this triple-mutant genotype are unable to differentiate in vitro. These results suggest that
physiological levels of Myf5 are insufficient to activate the myogenic program in the absence of other myogenic factors and
suggest that specialized functions have evolved for the myogenic bHLH factors to switch on the complete program of muscle
gene expression (Valdez, 2000).
Satellite cells, the myogenic precursors in postnatal and adult skeletal muscle, coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the
recruitment of satellite cells. Moreover, the finding that muscle regeneration is compromised in MyoD-/- mice, has provided evidence for the role of MyoD during
myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, satellite cells from MyoD-/- and
wildtype mice were compared as these cells progress through myogenesis in single-myofiber cultures and in tissue-dissociated cell cultures (primary cultures). Satellite cells undergoing
proliferation and differentiation were traced immunohistochemically using antibodies against various regulatory proteins. In addition, an antibody against the
mitogen-activated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express
specific myogenic regulatory factor proteins. During the initial days in culture the myofibers isolated from both the MyoD-/- and the wildtype mice
contain the same number of proliferating, ERK+ satellite cells. However, the MyoD-/- satellite cells continue to proliferate and only a very small number of cells
transit into the myogenin+ state, whereas the wildtype cells exit the proliferative compartment and enter the myogenin+ stage. Analyzing tissue-dissociated cultures of
MyoD-/- satellite cells, numerous cells were identified whose nuclei were positive for the Myf5 protein. In contrast, quantification of Myf5+ cells in the wildtype
cultures was difficult due to the low level of Myf5 protein present. The Myf5+ cells in the MyoD-/- cultures are often positive for desmin, similar to the MyoD+
cells in the wildtype cultures. Myogenin+ cells have been identified in the MyoD-/- primary cultures, but their appearance is delayed compared to the wildtype cells.
These 'delayed' myogenin+ cells can express other differentiation markers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, these studies
suggest that the presence of MyoD is critical for the normal progression of satellite cells into the myogenin+, differentiative state. It is further proposed that the
Myf5+/MyoD- phenotype may represent the myogenic stem cell compartment, which is capable of maintaining the myogenic precursor pool in the adult muscle (Yablonka-Reuveni, 1999).
Coexpression of smooth and skeletal
differentiation markers that are not myogenic regulatory
factors (MRFs) has been observed in E16.5 mouse fetuses in
a small percentage of diaphragm level esophageal muscle
cells, suggesting that MRFs are not involved in the
process of initiation of developmentally programmed
transdifferentiation in the esophagus. To investigate
smooth-to-skeletal esophageal muscle transition, Myf5nlacZ knock-in mice, and MyoD-lacZ and
myogenin-lacZ transgenic embryos were examined with a panel of
antibodies reactive with myogenic regulatory factors
(MRFs) and smooth and skeletal muscle markers. lacZ-expressing myogenic precursors are
not detected in the esophagus before E15.5, arguing against
the hypothesis that muscle precursor cells populate the
esophagus at an earlier stage of development. Rather, the
expression of the MRFs initiate in smooth muscle cells in
the upper esophagus of E15.5 mouse embryos and is
immediately followed by the expression of skeletal muscle
markers. Moreover, transdifferentiation is markedly
delayed or absent only in the absence of Myf5, suggesting
that appropriate initiation and progression of smooth-to-skeletal
muscle transdifferentiation is Myf5-dependent.
Accordingly, the esophagus of Myf5-/-:MyoD-/- embryos
completely fails to undergo skeletal myogenesis and
consists entirely of smooth muscle. Lastly, extensive
proliferation of muscularis precursor cells, without
programmed cell death, occurs concomitantly with
esophageal smooth-to-skeletal muscle transdifferentiation.
Taken together, these results indicate that
transdifferentiation is the fate of all smooth muscle cells in
the upper esophagus and is normally initiated by Myf5 (Kablar, 2000).
myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD
to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene
expression. To distinguish between these two possibilities, the abilities of constitutively expressed myogenin
and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells were compared. Differentiated
embryoid bodies from wild-type embryonic stem (ES) cells make extensive skeletal muscle, but embryoid bodies from
myogenin (-/-) ES cells have greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to
generate muscle is independent of endogenous MyoD expression. Skeletal muscle is restored in myogenin (-/-) ES cells
by constitutive expression of myogenin. In contrast, constitutive expression of MyoD results in only marginal
enhancement of skeletal muscle, although myocyte numbers greatly increase. The results indicate that constitutive
expression of MyoD leads to enhanced myogenic commitment of myogenin (-/-) cells but also indicate that committed
cells are impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's
role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in
their expression properties (Myer, 2001).
Proper formation of the musculoskeletal system requires the coordinated development of the muscle, cartilage and tendon lineages arising from the somitic mesoderm. During early somite development, muscle and cartilage emerge from two distinct compartments, the myotome and sclerotome, in response to signals secreted from surrounding tissues. As the somite matures, the tendon lineage is established within the dorsolateral sclerotome, adjacent to and beneath the myotome. Interactions between the three lineages were examined by observing tendon development in mouse mutants with genetically disrupted muscle or cartilage development. Through analysis of embryos carrying null mutations in Myf5 and Myod1 (hence lacking both muscle progenitors and differentiated muscle), an essential role was identified for the specified myotome in axial tendon development, and it is suggested that absence of tendon formation in Myf5/Myod1 mutants results from loss of the myotomal FGF proteins, which depend upon Myf5 and Myod1 for their expression, and are required, in turn, for induction of the tendon progenitor markers. Analysis of Sox5/Sox6 double mutants, in which the chondroprogenitors are unable to differentiate into cartilage, reveals that the two cell fates arising from the sclerotome, axial tendon and cartilage are alternative lineages, and that cartilage differentiation is required to actively repress tendon development in the dorsolateral sclerotome (Brent, 2005).
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