nautilus
Nautilus homologs: transcriptional targets Muscle cell differentiation causes a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels.
A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drives transcriptional activity in
myoblasts, and differentiation causes a decrease in transcription. The fragment -99/-33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced
transcription after muscle differentiation. Sp1 (Drosophila homolog: Buttonhead) protein binds to the fragment
-102/-37 in the myoblast state but not in myotubes, and Sp1 is found to transactivate the GLUT1 promoter. Sp1 is drastically down-regulated during myogenesis. The forced over-expression of MyoD in
C3H10T1/2 cells mimics the effects observed during myogenesis: Sp1 down-regulation and reduced transcriptional activity of the
GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the
down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in
GLUT1 expression and glucose transport (Vinals, 1997).
The Notch signaling cascade is involved in many developmental decisions: one paradigm involving Notch has been the selection between epidermal and
neural cell fates in both invertebrates and vertebrates. Notch has also been implicated as a regulator of myogenesis, although its precise function
there has remained controversial. The muscle-determining factor MyoD is a direct, positive regulator of the Notch ligand
Delta-1 in prospective myoblasts of the pre-involuted mesoderm in Xenopus gastrulae. Injection of a dominant MyoD repressor variant ablates
mesodermal Delta-1 expression in vivo. Furthermore, MyoD-dependent Delta-1 induction is sufficient to activate transcription from promoters of
E(spl)-related genes in a Notch-dependent manner. These results indicate that a hallmark of neural cell fate determination, i.e. the feedback loop
between differentiation promoting basic helix-loop-helix proteins and the Notch regulatory circuitry, is conserved in myogenesis, supporting a
direct involvement of Notch in muscle determination (Wittenberger, 1999).
The Fgf4 gene encodes an important signaling molecule that is expressed in specific developmental stages, including the
inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic
approach, overlapping but distinct enhancer elements have been identified in the Fgf4 3' untranslated region necessary
and sufficient for myotome and AER expression. The hypothesis that Fgf4 is a target of myogenic
bHLH factors has been investigated in this study. By mutational analysis it has been shown that a conserved E box located in the Fgf4 myotome enhancer is required
for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like
activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene
expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring
Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can
sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to
have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial
myotomes. Expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the
epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4
activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a
cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a
spatial- and temporal-specific manner (Fraidenraich, 2000).
Members of the MEF2 family of transcription factors are upregulated during skeletal muscle differentiation and cooperate with the MyoD family of myogenic basic helix-loop-helix (bHLH) transcription factors to control the expression of muscle-specific genes. To determine the mechanisms that regulate MEF2 gene expression during skeletal muscle development, the mouse Mef2c gene was analyzed for cis-regulatory elements that direct expression in the skeletal muscle lineage in vivo. A skeletal muscle-specific control region is described for Mef2c that is sufficient to direct lacZ reporter gene expression in a pattern that recapitulates that of the endogenous Mef2c gene in skeletal muscle during pre- and post-natal development. This control region is a direct target for the binding of myogenic bHLH and MEF2 proteins. Mutagenesis of the Mef2c control region shows that a binding site for myogenic bHLH proteins is essential for expression at all stages of skeletal muscle development, whereas an adjacent MEF2 binding site is required for maintenance but not for initiation of Mef2c transcription. These findings reveal the existence of a regulatory circuit between these two classes of transcription factors that induces, amplifies and maintains their expression during skeletal muscle development (Wang, 2001).
Expression arrays and chromatin immunoprecipitation assays were used to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter
expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and
promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not
for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease (Bergstrom, 2002).
A simple cascade model of gene activation would make MyoD a positive transcription factor at an initial set of genes, then the products of these genes would regulate the next tier, and so forth. In contrast, MyoD binds to the regulatory regions of all the muscle genes studied, whether expressed early or late in myogenesis. Using ChIP assays to detect in vivo MyoD binding, it was found that for early MyoD targets, MyoD is recruited at the earliest times examined. However, for later MyoD targets, such as Ckmm, Desmin, and
Mylf, MyoD binding is not detected until 12-24 hr after induction. Further, the binding of MyoD is prevented entirely at E boxes in genes not expressed in skeletal muscle. Therefore, delaying MyoD recruitment,
and perhaps that of other transcriptional activators, until the appropriate time is a key mechanism for determining the temporal regulation and specificity of gene expression during myogenesis. Instead of a simple cascade model, it has been demonstrated that the promoter-specific regulation of MyoD binding is a critical mechanism for patterning gene expression (Bergstrom, 2002).
Signal transduction pathways can modulate the expression mediated by MyoD at restricted sets of promoters. In studying the p38-dependent genes, loci were identified at which MyoD was stably bound but did not activate transcription without p38 signaling. p38 activity increases during myogenesis: this provides a means of selectively activating some targets only when there is sufficient p38 activity. It is possible that p38 is acting through a well-characterized mechanism of phosphorylating the activation domain of Mef2 and thereby increasing its activity. However, other groups have found that p38 can activate MyoD independent of Mef2, and thus it is also possible that p38 is acting through factors other than Mef2 to initiate transcription. Desmin and Mylf requires p38 activity for full expression, based on both S1 protections and array studies; however, ChIP assays also demonstrate delayed MyoD binding at these genes. Therefore, these mechanisms are not mutually exclusive, and it is concluded that promoter-specific regulation of MyoD binding and promoter-specific signal transduction cooperate to pattern MyoD-mediated gene expression (Bergstrom, 2002).
This paper addresses the molecular mechanisms that regulate the transcriptional activation of the myogenic regulatory factor XmyoD in the skeletal muscle lineage of Xenopus laevis. Using antisense morpholino oligonucleotide-mediated inhibition, it has been shown that the signaling molecule embryonic fibroblast growth factor (eFGF), which is the amphibian homolog of FGF4, is necessary for the initial activation of XmyoD transcription in myogenic cells. eFGF can activate the expression of XmyoD in the absence of protein synthesis, indicating that this regulation is direct. These data suggest that regulation of XmyoD expression may involve a labile transcriptional repressor. In addition, eFGF is itself an immediate early response to activin, a molecule that mimics the endogenous mesoderm-inducing signal. A model is proposed for the regulation of XmyoD within the early mesoderm. It is suggested that a maternal factor such as VegT induces the expression of a TGFß family member(s), which acts as the endogenous mesoderm inducing factor; this is likely a nodal related factor (Xnr1 and/or Xnr2). This endogenous mesoderm inducing factor is mimicked by activin, which induces the expression of eFGF directly. eFGF protein directly induces the expression of XmyoD, possibly acting through inhibition of a repressor. XmyoD is crucial in the specification of the myogenic cell lineage (Fisher, 2002).
The homeobox protein Barx2 is expressed in both smooth and skeletal muscle and is up-regulated during differentiation of skeletal myotubes. Antisense-oligonucleotide inhibition of Barx2 expression has been used in limb bud cell culture to show that Barx2 is required for myotube formation. Moreover, overexpression of Barx2 accelerates the fusion of MyoD-positive limb bud cells and C2C12 myoblasts. However, overexpression of Barx2 does not induce ectopic MyoD expression in either limb bud cultures or in multipotent C3H10T1/2 mesenchymal cells, and does not induce fusion of C3H10T1/2 cells. These results suggest that Barx2 acts downstream of MyoD. To test this hypothesis, the Barx2 gene promoter was isolated and DNA regulatory elements were identified that might control Barx2 expression during myogenesis. The proximal promoter of the Barx2 gene contains binding sites for several factors involved in myoblast differentiation including MyoD, myogenin, serum response factor, and myocyte enhancer factor 2. Co-transfection experiments showed that binding sites for both MyoD and serum response factor are necessary for activation of the promoter by MyoD and myogenin. Taken together, these studies indicate that Barx2 is a key regulator of myogenic differentiation that acts downstream of muscle regulatory factors (Meech, 2003).
Genome-wide transcription factor binding and expression profiling has been used to assemble a regulatory network controlling the myogenic differentiation program in mammalian cells. A cadre of overlapping and distinct targets of the key myogenic regulatory factors (MRFs) -- MyoD and myogenin -- and Myocyte Enhancer Factor 2 (MEF2) have been identified. MRFs and MEF2 regulate a remarkably extensive array of transcription factor genes that propagate and amplify the signals initiated by MRFs.
MRFs play an unexpectedly wide-ranging role in directing the assembly and usage of the neuromuscular junction. Interestingly, these factors also prepare myoblasts to respond to diverse types of stress. Computational analyses identified novel combinations of factors that, depending on the differentiation state, might collaborate with MRFs. These studies suggest unanticipated biological insights into muscle development and highlight new directions for further studies of genes involved in muscle repair and responses to stress and damage (Blais, 2005).
One of the most striking observations was that transcription factors represent
the largest cluster of MRF targets. Although consistent with a cascade model of
gene activation, the markedly high number of transcription factors regulated by
MRFs and MEF2 suggests that the cascade may be more extensive than expected. These
analyses suggest the existence of new nodal points from which the
transcriptional output of MyoD is relayed, greatly expanding the repertoire of
indirect targets of MyoD. The role of MRFs in differentiation is contrasted with
E2F4, a repressor that plays a role in cell cycle exit:
only a handful of transcription factor genes are bound by E2F4,
suggesting that gene regulatory programs involved in cell cycle control (and
cell cycle exit) may be wired in fundamentally different ways from terminal
differentiation (Blais, 2005).
It is proposed that transcriptional regulators (Eya1 and TEAD4/TEF-3) relay the
differentiation signal initiated by MyoD. Several
biochemical, computational, and genetic observations suggest that the Eya1/Six1
pathway is associated with MRF function. (1) ChIP-on-chip results
indicate that Eya1 is a direct target of MyoD in growing myoblasts. Eya1 has the
ability to switch the activity of Six1, a homeobox transcriptional regulator,
from repressor to activator. (2) The MEF3
PWM, a binding site for Six1, is specifically enriched among myogenin target
genes that are induced during differentiation.
(3) Mice lacking Six1 display defects in embryonic
myogenesis that are exacerbated when Eya1 function is also ablated (Blais, 2005).
TEAD4 is closely related to TEAD1 (TEF-1), the founding member of a family of
transcriptional regulators that bind M-CAT DNA elements (GGAATG).
By binding M-CAT sites, TEAD4 participates in
muscle-fiber-type switching and mediates in part the transcriptional effects of
hypoxia and alpha-adrenergic-stimulated muscular hypertrophy. Together with the
observation that the M-CAT sequence is enriched among MRF targets, this suggests
that, besides regulating additional genes during the muscle hypertrophic
response, TEAD4 propagates the myogenic
signal originating from MyoD and cooperates with MRFs to induce the expression
of their targets (Blais, 2005).
These studies identified other transcription factors likely to be involved in
propagating gene expression cascades during myogenesis. These MRF targets
include Naca (skNAC), a muscle-specific transcription factor involved in
muscle repair, and Ankrd1 and
Ankrd2, muscle-specific transcriptional modulators involved in
myofibril-based hypertrophic response signaling. Identifying their as yet
unknown targets will be essential to elucidate their role in response to
activation signals originating from MyoD (Blais, 2005).
The co-factor Vestigial-like 2 (Vgl-2), in association with the Scalloped/Tef/Tead transcription factors, has been identified as a component of the myogenic program in the C2C12 cell line. In order to understand Vgl-2 function in embryonic muscle formation, Vgl-2 expression and regulation were analyzed during chick embryonic development. Vgl-2 expression was associated with all known sites of skeletal muscle formation, including those in the head, trunk and limb. Vgl-2 was expressed after the myogenic factor MyoD, regardless of the site of myogenesis. Analysis of Vgl-2 regulation by Notch signalling showed that Vgl-2 expression was down-regulated by Delta1-activated Notch, similarly to the muscle differentiation genes MyoD, Myogenin,Desmin, and Mef2c, while the expression of the muscle progenitor markers such as Myf5, Six1 and FgfR4 was not modified. Moreover, it was established that the Myogenic Regulatory Factors (MRFs) associated with skeletal muscle differentiation (MyoD, Myogenin and Mrf4) were sufficient to activate Vgl-2 expression, while Myf5 was not able to do so. The Vgl-2 endogenous expression, the similar regulation of Vgl-2 and that of MyoD and Myogenin by Notch signalling, and the positive regulation of Vgl-2 by these MRFs suggest that Vgl-2 acts downstream of MyoD activation and is associated with the differentiation step in embryonic skeletal myogenesis (Bonnet, 2010).
The calcineurin/NFAT (nuclear factor of activated T-cells, see Drosophila NFAT homolog) signaling pathway is involved in the modulation of the adult muscle fiber type, but its role in the establishment of the muscle phenotype remains elusive. This study shows that the NFAT member NFATc2 cooperates with the basic helix-loop-helix transcription factor MyoD to induce the expression of a specific myosin heavy chain (MHC) isoform, the neonatal one, during embryogenesis. This cooperation is crucial, as Myod/Nfatc2 double-null mice die at birth, with a dramatic reduction of the major neonatal MHC isoform normally expressed at birth in skeletal muscles, such as limb and intercostal muscles, whereas its expression is unaffected in myofibers mutated for either factor alone. Using gel shift and chromatin immunoprecipitation assays, NFATc2 was found bound to the neonatal Mhc gene, whereas NFATc1 and NFATc3 would preferentially bind the embryonic Mhc gene. Evidence is provided that MyoD synergistically cooperates with NFATc2 at the neonatal Mhc promoter. Altogether, these findings demonstrate that the calcineurin/NFAT pathway plays a new role in establishing the early muscle fiber type in immature myofibers during embryogenesis (Daou, 2013)
Coactivators for Nautilus homologs p300 and CBP are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors, CREB, c-Jun, C-Myb, c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by not only direct but also indirect interactions through cofactors. p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononucleosomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).
p300 and CBP, two related molecules that act as transcriptional adapters, coactivate the myogenic basic-helix-helix-loop (bHLH) proteins. Coactivation by p300 induces novel physical interactions between p300 and the amino-terminal activation domain of MyoD. In particular, disruption of the FYD domain, a group of three amino acids coserved in the activation domains of other myogenic bHLH proteins, drastically diminishes the transactivation potential of MyoD and abolishes both p300-mediated coactivation and the physical interaction between MyoD and p300. Two domains of p300, at its amino and carboxy terminals, independently function to both mediate coactivation and physical interaction with MyoD. A truncated segment of p300, unable to bind MyoD, acts as a dominant negative mutation and abrogates both myogenic conversion and transactivation by MyoD, suggesting that endogenous p300 is a required coactivator for MyoD functions. The p300 dominant negative peptide forms multimers with intact p300. p300 and CBP serve as coactivators of another class of transcriptional activators critical for myogenesis, known as myocyte enhancer factor 2 (MEF2). In fact, transactivation mediated by the MEF2C protein is potentiated by the two coactivators; this phenomenon is associated with the ability of p300 to interact with the MADS domain of MEF2. These results suggest that p300 and CBP may positively influence myogenesis by reinforcing the transcriptional autoregulatory loop established between the myogenic bHLH and the MEF2 factors (Sartorelli, 1997).
The nuclear phosphoprotein p300 is a new member of a family of 'co-activators' (which also includes the CREB binding protein CBP) that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the
adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. In differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix-loop-helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle-specific transcription, it induces permanent cell cycle arrest by up-regulating the cylin-dependent kinase inhibitor p21. p300 is involved in both these activities. Indeed, E1A mutants lacking the
ability to bind p300 are greatly impaired in the repression of E-box-driven transcription, and p300 overexpression rescues the wild-type E1A-mediated repression. Moreover, p300 potentiates MyoD- and
myogenin-dependent activation of transcription from E-box-containing
reporter genes. p300 is also required for MyoD-dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD-converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the postmitotic state of myotubes (Puri, 1997).
MyoD modification by acetylation - effects of p300/CBP, P/CAF, and histone deacetylase The molecular mechanism(s) that are responsible for suppressing MyoD's transcriptional activities in undifferentiated skeletal muscle cells
have not yet been determined. MyoD associates with a histone deacetylase-1 (HDAC1) in these cells and this interaction is responsible for silencing MyoD-dependent transcription of endogenous p21 as well as muscle-specific genes. Specifically, evidence is presented that HDAC1 can bind directly to MyoD and HDAC1 can use an acetylated MyoD as a substrate in vitro, whereas a mutant
version of HDAC1 (H141A) can not. Furthermore, this mutant also fails to repress MyoD-mediated transcription in vivo, and unlike wild-type HDAC1, it can not inhibit myogenic conversion, as judged by confocal microscopy. An endogenous
MyoD can be acetylated upon its conversion to a hypophosphorylated state and only when the cells have been induced to differentiate. These results provide for a
model that postulates that MyoD may be co-dependent on HDAC1 and P/CAF for temporally controlling its transcriptional activity before and after the
differentiation of muscle cells (Mal, 2001).
p300/CBP may lack the potential to acetylate MyoD in vivo. If true, the acetylation
of MyoD, which is noticeably restricted to differentiated muscle cells, is most likely then a direct consequence of P/CAF's activities. Therefore, to
investigate whether MyoD might be interacting with P/CAF as muscle cells begin to differentiate, whole-cell extracts were prepared from C2 cells cultured for the indicated times. Immunoprecipitates were examined for the presence of P/CAF by Western blot analysis, using antibodies
specific for P/CAF. There is hardly any association between MyoD and P/CAF in proliferating myoblasts, whereas an interaction between these two proteins is clearly observable once the cells were placed into differentiation medium. What is highly relevant to this result is that the steady-state levels of both P/CAF and MyoD protein remain nearly constant under these conditions. Immune
complexes of MyoD recovered from these same extracts were also examined for the presence of p300/CBP, and no evidence was found for the appearance of this protein. Perhaps the simplest explanation for this result is that the epitopes that would otherwise be recognized by the MyoD antibody have become inaccessible because of the binding of p300/CBP. MyoD-p300/CBP complexes are presumed to exist in muscle cells only because others have shown that an in vitro translated p300 can bind directly to a purified GST-MyoD fusion protein (Mal, 2001).
A key point apparent from the data presented here is a role for HDAC complexes that may go beyond the deacetylation of histones and involve the direct deacetylation of MyoD, at least in undifferentiated muscle cells. This hypothesis is compatible with the demonstration that MyoD can bind directly to HDAC1 and act as a substrate for this enzyme following its acetylation in vitro. Interestingly, the site on MyoD with which HDAC1 appears to interact is the bHLH domain, a region necessary for converting 10T1/2 fibroblasts into muscle cells and which includes lysines that can be acetylated. Equally important is the observation that MyoD can be found in association with histone deacetylase activity in undifferentiated rather than in differentiated muscle cells, and this finding, together with the aforementioned results, is in excellent accord with MyoD's inability to activate transcription in proliferating myoblasts. It is worth noting that the histone deacetylase activity that was found in association with MyoD turned out to be sensitive to treatment with the TSA inhibitor, indicating therefore that the enzymatic activity is being contributed by HDAC, and not by any other factor. The likelihood of HDAC1 specifically mediating this activity is supported by the fact that this protein can stably interact with MyoD in undifferentiated cells; nonetheless, the possibility of HDAC2 contributing to this activity as well cannot be excluded (Mal, 2001).
Acetylation plays a role in regulating MyoD's transcriptional activities with respect to myogenic conversion. These results strongly indicate that the histone acetyltransferase P/CAF may be responsible for this modification. For instance, although P/CAF is relatively abundant in proliferating myoblasts, it chooses not to interact with MyoD until the cells have been induced to differentiate, and this association continues throughout the myogenic process. Perhaps more importantly, the kinetics of the MyoD-P/CAF interaction coincide with the time when MyoD begins to show evidence of acetylation, providing further proof of P/CAF's role in modifying this protein during myogenesis (Mal, 2001).
Together, the functional and biochemical data obtained are consistent with the proposal that HDACs may act to sustain MyoD in a deacetylated and transcriptionally repressed form until muscle cells are induced to differentiate. Once this occurs, MyoD is converted to an acetylated and transcriptionally active form, a process most likely mediated by P/CAF. In this respect, therefore, it appears that acetylation and deacetylation may be functionally linked
in controlling the transcriptional activities of MyoD, with consequences having either a negative or a positive effect on genes that are specific to the myogenic process (Mal, 2001).
c-Ski, originally identified as an oncogene product, induces myogenic differentiation in nonmyogenic fibroblasts through transcriptional activation of muscle regulatory factors. Although c-Ski does not bind to DNA directly, it binds to DNA through interaction with Smad proteins and regulates signaling activities of transforming growth factor-β. c-Ski has been shown to activate the myogenin promoter independently of regulation of endogenous TGF-β signaling. Expression of myogenin is regulated by a transcription factor complex containing proteins of the MyoD family and the myocyte enhancer factor 2 (MEF2) family. c-Ski acts on the MyoD-MEF2 complex and modulates the activity of MyoD in myogenin promoter regulation. Interestingly, histone deacetylase (HDAC) inhibitors up-regulated basal activity of transcription from a MyoD-responsive reporter, although c-Ski failed to further augment this transcription in the presence of HDAC inhibitors. c-Ski is observed both in the cytoplasm and in the nucleus, but its nuclear localization is required for myogenic differentiation. It is concluded that c-Ski induces myogenic differentiation through acting on MyoD and inhibiting HDAC activity in the nucleus of myogenic cells (Kobayashi, 2007).
Nautilus homologs and neural development Myf5 is a key basic Helix-Loop-Helix transcription factor
capable of converting many non-muscle cells into muscle.
Together with MyoD it is essential for initiating the skeletal
muscle program in the embryo. Unexpected restricted domains of Myf5 transcription have been identified in the
embryonic mouse brain. These Myf5 expressing neurons have been further characterized. Retrograde
labeling with diI, and the use of a transgenic mouse line
expressing lacZ under the control of Myf5 regulatory
sequences, show that Myf5 transcription provides a novel
axonal marker of the medial longitudinal fasciculus (mlf)
and the mammillotegmental tract (mtt), the earliest
longitudinal tracts to be established in the embryonic
mouse brain. Tracts projecting caudally from the
developing olfactory system are also labelled.
lacZ expression persist in the adult brain, in a few ventral
domains such as the mammillary bodies of the
hypothalamus and the interpeduncular nucleus, potentially
derived from the embryonic structures where the Myf5
gene is transcribed.
To investigate the role of Myf5 in the brain, Myf5 protein accumulation was monitered by immunofluorescence and
immunoblotting in neurons transcribing the gene.
Although Myf5 is detected in muscle myotomal cells, it
is absent in neurons. This would account for the lack of
myogenic conversion in brain structures and the absence of
a neural phenotype in homozygous null mutants. RT-PCR
experiments show that the splicing of Myf5 primary
transcripts occurs correctly in neurons, suggesting that the
lack of Myf5 protein accumulation is due to regulation at
the level of mRNA translation or protein stability.
In the embryonic neuroepithelium, Myf5 is transcribed
in differentiated neurons after the expression of neural
basic Helix-Loop-Helix transcription factors. The
signaling molecules Wnt1 and Sonic hedgehog, implicated
in the activation of Myf5 in myogenic progenitor cells in the
somite, are also produced in the viscinity of the Myf5
expression domain in the mesencephalon.
Cells expressing Wnt1 can activate
neuronal Myf5-lacZ gene expression in dissected head
explants isolated from E9.5 embryos. Furthermore, the
gene encoding the basic Helix-Loop-Helix transcription
factor mSim1 is expressed in adjacent cells in both the
somite and the brain, suggesting that signaling molecules
necessary for the activation of mSim1 as well as Myf5 are
present at these different sites in the embryo. This
phenomenon may be widespread and it remains to be seen
how many other potentially potent regulatory genes, in
addition to Myf5, when activated, do not accumulate protein
at inappropriate sites in the embryo (Daubas, 2000).
Forced expression of the bHLH myogenic factors, Myf5 and MyoD, in various
mammalian cell lines induces the full program of myogenic differentiation.
However, this property has not been extensively explored in vivo. Advantage was taken of the chick model to investigate the effect of
electroporation of the mouse Myf5 and MyoD genes in the
embryonic neural tube. Misexpression of either mouse
Myf5 or MyoD in the chick neural tube leads to ectopic
skeletal muscle differentiation, assayed by the expression of the myosin heavy chains in the neural tube and neural crest derivatives. The endogenous neuronal differentiation program is inhibited under the
influence of either ectopic mouse Myf5 or MyoD. This
new system of in vivo analysis, was used to study the transcriptional regulation between the
myogenic factors. MyoD and Myogenin expression
can be activated by ectopic mouse Myf5 or MyoD, while
Myf5 expression cannot be activated either by mouse MyoD or
by itself. The transcriptional regulation was analyzed between the
myogenic factors and the different genes involved in myogenesis, such as
Mef2c, Pax3, Paraxis, Six1, Mox1, Mox2 and FgfR4. The existence has been established of an unexpected regulatory loop between
MyoD and FgfR4. The consequences for myogenesis are
discussed. These results support the idea that Myf5 is the first myogenic regulatory factor to specify the skeletal muscle program in vertebrates; MyoD has been associated with later function. MyoD can induce muscle differentiation in the absence of Myf5, Paraxis, Six1, Mox1 and Mox2 in vivo, since the conversion of neural tissues into skeletal muscle cells can occur in the absence of the corresponding transcripts (Delfini, 2004).
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