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Discuss the role of transcription factor Pax7 in satellite cells, the muscle endogenous cells
Due to its remarkable ability to rapidly regenerate after injury, research into skeletal muscle has quickly come to the forefront of scientific discovery over the past few years. Multiple studies involving myogenic satellite cells have demonstrated the importance of muscle progenitor cell development and function in the specification of muscle formation and
Skeletal muscle is one of the most dynamic and fundamental tissues within the human body, it serves a variety of functions that include facilitating voluntary movement, and providing structural support through a series of contractions and relaxations that work to move bones that are attached to the muscle via tendons (Lieber, 2002). Unlike smooth muscle, it is easily characterized by the notable striations that arise through the formation of uniformly aligned, long multinucleated myofibers which make up repeated sarcomere units (Shadrin et al., 2016).
During development in vertebrates, there are multiple successive stages of embryonic myogenesis that eventually lead to the growth of skeletal muscles, this process is regulated by transcription factors, proteins that bind to specific DNA sequences and regulate the expression of genes (Songdej & Rao, 2017). Transcription factors function in a combinatorial manner as both repressors and activators, they control the rate of transcription of DNA to mRNA in cells by binding to DNA at certain target sequences and determining whether RNA polymerase can bind to the promoter site of the DNA. By doing this it can be ensured that the right genes are being expressed in the correct cell at any moment in an organism’s life (Eeckhoute et al., 2009). In particular, a family of box paired transcription factors called Pax genes are becoming increasingly important in developmental biology, as they are essential transcriptional regulators and their function is highly conserved in vertebrates. They regulate cell proliferation, migration and play major roles during organogenesis and neural crest induction (Tremblay & Gruss, 1994). The focus of this essay will therefore be the transcription factor Pax7 which is an important regulator of muscle stem cell development, with the aim to discuss its role in the muscle endogenous cells called satellite cells and their postnatal maintenance (Monsoro-Burg, 2015).
Satellite cells are mononucleated muscle progenitor cells that are located in between the sarcolemma and basement membrane of terminally-differentiated muscle fibres (Asakura et al., 2002). Satellite cells initially provide myoblasts for muscle growth, before becoming mitotically quiescent as the muscle matures, where they can be activated in response to injury and triggered into proliferation for self-renewal and differentiation into myogenic cells, to mediate the postnatal growth and regeneration of muscle (Morgan & Partridge, 2003). Recent studies have also identified a population of pluripotential stem cells in adult skeletal muscle, called side-population (SP) cells. Although the function of these cells is not fully understood, it is speculated that they are involved in some part of myogenesis, yet this may differ from satellite cells (Penton et al., 2013).
Figure 1: Electron microscopy showing the position of quiescent satellite cells (white). It sits below the basal lamina expressing laminin (red) and on top of the muscle fibre sarcolemma expressing dystrophin (green). Anatomical location of the satellite cell within its niche is depicted by immunofluorescence microscopy
(Adapted from Feige et al., 2018).
Muscle regeneration and repair
The exact mechanism behind this rapid regeneration starts when the skeletal muscle experiences trauma through exercise or physiological stress (Figure 2). The satellite cells move from their sublaminar location into the interstitium between muscle fibres, re-enter the cell cycle and proliferate to expand their numbers (Asakura et al., 2002). The progeny can undergo multiple rounds of cell division before myogenic differentiation where they can fuse with existing myofibers to contribute new nuclei and facilitate repair (Tidball, 2011). Some of these activated cells then leave the cell cycle and return to their quiescent state, the number of satellite cells over several regenerations usually stay constant which suggests that they also possess the ability for self-renewal (Birbrair & Delbono, 2015). One interesting characteristic of muscle fibres during the later stages of regeneration is that their nuclei move from the periphery to the centre of the myotube, this implies that nuclei positioning may be important in muscle formation and function, although more research is needed to confirm this (Cadot et al., 2015).
Figure 2: Schematic diagram of the muscle repair cycle following muscle injury. Arrow “A” represents the activation and proliferation of satellite cells. After this stage, activated cells can either differentiate into myoblasts or become quiescent to maintain the population. Arrow “B” represents this early differentiation stage, existing muscle fibres can be repaired, or new fibres formed during this process. Arrow “C” represents the approach towards the terminal differentiation stage of regeneration, after which the central nuclei migrate to the surface of the muscle fibre (Tidball, 2011).
Research into Pax7 and its significance
Research into the transcription factor Pax7 ever since its discovery has revealed that it is intrinsically linked to and uniformly expressed in satellite cells while in their quiescent state (Feige et al., 2018). Another closely related paired box transcription factor called Pax3 acts in conjunction with Pax7 during the specification and maintenance of skeletal muscle progenitors as shown in Figure 3 (Goljanek-Whysall et al., 2011). These two Pax genes possess near identical amino acid sequences and partial overlapping expression patterns throughout mouse embryogenesis (Péault et al., 2007). Genetic analyses in mice demonstrated that Pax3 is critical for the delamination and migration of somitic muscle precursor cells to the limbs (Kuang et al., 2006). In contrast, Pax7 does not affect early muscle development, but is crucial for the development of satellite cells in the perinatal period (Figure 4) (Martin, 2003).
Figure 3: Skeletal muscle development illustration showing Pax3 and Pax7 are initially activated in embryonic progenitors. With the continuous development of embryos, certain embryonic progenitor cells differentiate into muscle precursor cells termed myoblasts (Ju et al., 2015).
Figure 4: Timeline of the embryonic and postnatal periods of muscle development with the distribution of skeletal muscle in the developing embryo shown in blue. Dominance of embryonic and foetal muscle progenitor cells during myogenesis is indicated in red, whereas the times when satellite cells show dominance is emphasized in yellow. The dynamic expression of Pax7 (green) and Pax3 (blue) over this period is shown, along with the time points in which each Pax gene and its function is required by foetal muscle progenitor and satellite cells. Signifying the importance of Pax genes on the myogenic stem cell population (Relaix & Zammit, 2012).
Over the past 20 years, several preliminary studies have suggested that Pax7 is necessary for satellite cell biogenesis and self-renewal. These reports stated that without Pax7, further muscle development is hindered and only the early embryonic muscle of the myotome forms (Relaix et al., 2005). One pivotal investigation using in situ hybridization to localize Pax7 mRNA in skeletal muscle revealed that Pax7 was prominently expressed in regenerating areas of muscle, and in a frequency and location that matched specific expression in satellite cells. Furthermore, mice deficient in Pax7 were found to be significantly smaller than their wildtype counterparts, with mutant mice exhibiting muscle weakness and reduced fibre diameter as distinguished by their splayed hind limbs and thinned diaphragms. This apparent decrease in muscle mass and reduced muscle fibre diameter in Pax7 mutants suggests that the postnatal growth of skeletal muscle is not only dependent on satellite cells, but also on the expression of Pax7 within them, as it is believed that the cells fail to proliferate in the absence of Pax7 (Seale et al., 2000). By using techniques such as the Northern blot and representational difference analysis, Pax7 can be used as a tool to help analyse and thus enhance our understanding of the relationship between satellite cells, the Pax gene and other myogenic precursors.
On the other hand, an in vivo study of Tamoxifen-induced deletion of Pax7 in satellite cells was carried out by Lepper et al. (2009) using the Cre-loxP system to generate tissue-specific knockout in mice models. The results showed that after 3 weeks of age there was no deficiency in muscle regeneration or satellite cell number, they could reoccupy the sublaminar niche and continue to replicate, and Pax7 is only required up to the juvenile period when progenitor cells shift into quiescence, suggesting that Pax7 is not as important to satellite cell function as many people assume, and is in fact not needed in adult life or for muscle regeneration to take place. A difference between the Pax dependency on the neonatal progenitors in comparison to the adult satellite cells was also discovered. The contradicting conclusions of this experiment only goes to prove how little we know about the requirements of adult and embryonic stem cells and establishes the need to carry out more investigations into this subject.
The Cre-loxP recombination system is growing to be an essential device for generating animal models that could open up avenues into the study of gene function (Kos, 2004). It enables the control of specific gene activity in a restricted period of mouse’s lifetime, in a particular cell type or tissue (Bouabe & Okkenhaug, 2013). Mice with a targeted gene flanked by two loxP sites (34 bp DNA sequence) is usually crossed with another strain of mice expressing the enzyme Cre recombinase in a given tissue or cell type, this results in the floxed DNA sequence being excised from between the loxP sites and inactivated in the cell types or tissues where Cre is expressed (Schwenk et al., 1995). In Lepper’s experiment, to add inducibility to the system, ligand-dependent Cre recombinases that could be activated by administration of tamoxifen to the animal meant the excision of floxed chromosomal DNA could be controlled both spatially and temporally (Feil et al., 2009). This method is only one of the many new gene targeting technologies that have recently emerged to help advance research in this field.
A later experiment conducted by von Maltzahn et al. (2013) performed the same knockout mice procedure as Lepper et al. in 2009, however different conclusions were made regarding the importance of Pax7. The deletion of Pax7 in satellite cells resulted in extensive impairment in muscle regeneration with a 2.1-fold reduction in myofiber formation along with major fibrosis and adipogenesis. Additionally, satellite cells that were Pax7 deficient lost the ability to proliferate after repeated exposure to tamoxifen, leading to a significant reduction in muscle weight after two rounds of induced injury via cardiotoxin (CDX), muscle regeneration was also greatly affected and delayed due to diminished satellite cell capability. One explanation for the contrasting results between these experiments describes the possibility for satellite cells to escape tamoxifen-induced deletion of the floxed Pax7 gene sequence and consequently go on to repopulate a regenerating muscle and produce a partial regeneration phenotype. When comparing mice that received continuous doses of tamoxifen through their diet to mice that were injected with tamoxifen intraperitoneally, less than 40% of the remaining satellite cells in the former mice expressed Pax7. This further supports the notion that Pax7 is a key requirement for the normal progression of satellite cell function in myogenesis.
Role of other stem cells towards muscle development and their potential uses
It has been proposed that cells other than satellite cells have the potential to give rise to muscle, Péault et al found that when muscle SP cells were injected into diseased or injured muscle in vivo, it gave rise to Pax7 and Myf5-positive cells, which are markers expressed by activated or quiescent satellite cells, this identified muscle SP cells as a source of stem cells that can adopt a myogenic lineage. Different findings have confirmed the ability of bone marrow cells to give rise to myogenic cells that can participate in muscle regeneration (Ferrari et al., 2000). As more research is carried out, the prospects of using pluripotent adult stem cells isolated from numerous tissues in stem cell therapy for various degenerative diseases becomes more promising. Nevertheless, it is clear that a more thorough understanding of the mechanisms that regulate the development and function of muscle-derived stem cells and their transcription factors is needed before we can utilise them to our advantage.
Overall, these observations indicate that Pax7 is a central factor in the regulation and progression of skeletal muscle function ; it is, however, noteworthy that the aforementioned observations from lineage tracing and immunofluorescence labeling experiments cannot exclude the possibility that some adult satellite cells may originate from other sources during fetal and postnatal muscle development. For example, embryonic dorsal aorta explants, when cultured and disaggregated in vitro, can efficiently give rise to myogenic precursors (129). These myogenic precursors are similar to satellite cells in their gross morphology and expression of molecular markers. – YIN
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