Myogenesis Markers Proteins


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 Myogenesis Markers Proteins Background

Adult myogenesis collectively refers to the process of satellite cell mediated muscle formation. In humans, satellite cell nuclei comprise 4-6% of all basal lamina incapsulated nuclei within muscle, and are able to remain quiescent for years. Satellite cells based on their physiology during the adult myogenic process are grouped into four classes, 1) quiescent, 2) activated, 3) proliferating and 4) differentiating.

Skeletal muscle in the embryo originates from paraxial mesoderm, located laterally to the neural tube. As development progresses, the paraxial mesoderm becomes segmented into epithelial block-like structures known as somites in a rostral to caudal pattern. Somitogenesis occurs in a strict periodic fashion in response to morphogen gradients. FGF and Wnt in the posterior portion of the presomitic mesoderm (PSM) antagonize somitogenesis while retinoic acid (RA) in the anterior PSM promotes epithelisation and somite segmentation. Within the PSM, FGF and Wnt are synthesized only in the most posterior region, such that as the body axis elongates posteriorly, the concentration of these ligands decreases in more anterior regions, resulting in downregulation of their downstream targets. This transition from exposure to high FGF/Wnt to high RA results in differentiation. Indeed, inappropriate exposure of PSM cells to continuously high FGF levels blocks somite segmentation. The region of the PSM where these two gradients meet is referred to as the determination front. The determination front progresses posteriorly as the axis elongates and FGF/Wnt mRNA undergoes progressive decay, and cells located anterior to this position undergo a mesenchymal to epithelial transition.

However, the actual segmentation of the somite from the PSM also relies on the action of a molecular clock that is triggered by Notch and Wnt signaling. Many members of the Notch pathway, including delta, the Hairy and Enhancer of split (HES) transcription factors, hes1 and hes7, and the glycosyl-transferase lunatic fringe exhibit temporal pulses of expression pattern throughout the PSM. The Wnt signalling inhibitor axin2 also cycles in the PSM, albeit out of phase with Notch pathway members. These temporal pulses represent the oscillations of a molecular clock, the output of which is integrated with the determination front to position somite boundaries. Specifically, when the determination front reaches a given position within the PSM, the cells become competent to respond to the stimulus of the ‘clock’. During somitogenesis, the determination front progresses posteriorly by the length of one somite, in the time it takes for one oscillation of the clock, thus positioning the somite boundary at the point where the determination front and the oscillation meet. This is referred to as the clock and wavefront model of somite segmentation.

 

Transcriptional Regulation of Myogenesis

The myogenic regulatory factors (MRF) family is composed of four basic helix loop helix (bHLH) transcription factors, Myf5, MyoD, MRF4 and myogenin, whose potent myogenic properties are evidenced by their ability to convert other cell types into skeletal muscle. The MRFs bind to E-box target sequences as monomers or as heterodimers in complex with E-proteins and activate muscle gene transcription by coordinating chromatin rearrangements in concert with transcriptional coactivators. The functions of the four MRF proteins have been extensively examined using gene targeting and these studies have revealed a complex and somewhat redundant network controlling myogenesis.

Myf5 is the first MRF to be expressed in the developing embryo and it can be detected as early as stage 3 in the PSM of developing chick embryos or 8.0 days post coitum(d.p.c.) in the mouse somite. The somitic expression domain of Myf5 includes the dermomyotome and both the dorsal-medial lip and ventral-lateral lip regions where it plays a role in progenitor migration into the myotome and specification of the myogenic fate. In the absence of Myf5, a subset of progenitor cells migrate abnormally to sites within the sclerotome and dermomyotome where they ultimately adopt fates specific to these regions. In spite of this, Myf5 is dispensable for normal myotomal muscle development and activation of MyoD expression, which is thought to rescue the myogenic program in the absence of Myf5. Myf5 null mice do however exhibit defects of epaxial muscle.

Like Myf5, MyoD is dispensable for normal embryonic muscle development, however, MyoD null mice exhibit elevated levels of Myf5, which is thought to compensate and rescue myogenesis in this context. Interestingly, loss of MyoD does impact on adult muscle regeneration as MyoD mutant mice exhibit impaired regeneration in response to injury, which results from a propensity of MyoD(-/-) satellite cells to undergo self-renewal at the expense of terminal differentiation. A recent high-throughput study aimed at identifying MyoD binding sites revealed, unexpectedly, that MyoD was in fact enriched at thousands of sites within the genome and that binding was associated with increased histone acetylation at these sites. This implies that MyoD has the potential to exert wideranging changes to the epigenome of a cell, resulting in reprogramming to the myogenic lineage.

Until recently, Mrf4 was regarded as being important primarily for terminal differentiation. The Mrf4 knockout mouse exhibited only minimal muscle defects, likely due to compensation by elevated levels of myogenin. However, retrospective analysis of the original Myf5/MyoD compound mutant mouse, which exhibited a complete lack of skeletal muscle, revealed that Mrf4 expression was also disrupted as a result of the targeting strategy and the proximity of Mrf4 and Myf5. Further analysis of the relationship between these three genes revealed that Mrf4 was sufficient to maintain myogenesis in the absence of Myf5 and MyoD. This challenged the traditional view of MyoD and Myf5 as determination genes functioning upstream of Mrf4 and myogenin driving terminal differentiation and revealed a role for Mrf4 in the specification of the myogenic fate.

Myogenin is the second MRF to be activated in the embryo, after Myf5, which is thought to regulate its expression. Interestingly, although myogenin mRNA is detected as early as 8.5 d.p.c. in the mouse, the protein product is not detected until 10.5 d.p.c., implying some level of post-transcriptional regulation or protein instability. Despite its early expression pattern, myogenin is thought to be primarily important for the terminal differentiation of skeletal muscle. Myogenin null mice form myocytes normally but exhibit a fusion defect whereby myotubes are not formed and mice die perinatally. Interestingly, when myogenin was knocked in to the Myf5 locus in the original Myf5/MyoD compound null mouse (which is in fact also deficient in Mrf4), it was able to compensate to some extent for the loss of these factors, indicating the myogenin has some ability to direct cells into the myogenic lineage, albeit less efficiently than Myf5. This is in contrast to rib development, for which Myf5 and myogenin were functionally interchangeable.

 

The Pax family in Myogenesis

Upstream of the MRFs in skeletal myogenesis are members of the Pax family of transcription factors, which have also been implicated in many other developmental processes. They are characterized by their paired box domain and paired-type homeodomain and are classified into groups based on structural characteristics and similarity of expression patterns. Pax3 and Pax7 are expressed in developing muscle, and are involved in the formation of both trunk and limb muscles, in addition to satellite cell specification.

 

The Meox Family: Meox1 and Meox2

Meox1 and Meox2 are homeodomain transcription factors that are expressed in the developing somite. Meox1 expression is initiated in the PSM and later becomes segregated to the dermomyotome whereas Meox2 expression is initiated later in the developing limb bud. Consistent with this, Meox1 null mice exhibit sclerotomal defects in the form of fused ribs and vertebrae while Meox2 deficient mice exhibit defective limb muscle development accompanied by a loss of Pax3 and Myf5 expression. Meox1/2 compound null mice exhibit a much more severe phenotype characterized by a loss of almost all skeletal muscles, implying that, like Pax3/7, some level of compensation exists between these two factors. A role for Meox factors in myogenesis was further supported by work in P19 embryonal carcinoma cells, which showed that a dominant negative Meox factor abolished skeletal myogenesis in this system. Interestingly, Meox factors appear to exert their influence in concert with Pax factors. Yeast two hybrid studies revealed that Meox1 and Meox2 can interact directly with Pax1 and Pax3 respectively via the Meox homeodomain. Although these interactions were demonstrated in vitro, it is tempting to speculate that Pax3 and Meox2 may cooperate to direct limb muscle development in vivo given the requirement of each factor for this process.