CELL CYCLE AND GROWTH ARREST IN SKELETAL MUSCLE AND BEYOND 

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PAX proteins and bHLH MRFs play a central role in the myogenic program

My PhD work focuses on limb and trunk musculature and, thus, this section will cover general aspects of the function of PAX and MRFs in body musculature (Fig. 1.7) as well as their essential participation in embryonic myogenesis. Their role in postnatal growth and adult regeneration will be included in the session presenting satellite cells, which are the stem cells providing muscle precursors after birth.

PAX3 and PAX7 as upstream myogenic regulators

PAX proteins control the development of many lineages during embryogenesis (Table 1.2), with PAX3 and PAX7 acting as key regulators in the muscle lineage [Buckingham & Relaix, 2007]. In mammals, nine PAX proteins have been described, structurally characterized by a common paired box domain offering sequence-specific DNA binding. Some of them (including PAX3/7) also possess an octapeptide motif and an entire or truncated homeodomain (Table 1.2) [Buckingham & Relaix, 2007; Olguín & Pisconti, 2012]. Pax genes encode transcription factors and both PAX3 and PAX7 were shown to act as transcriptional activators in vivo [Relaix et al., 2003; Relaix et al., 2004] and orchestrate various biological aspects of myogenic progenitors and stem cells, including survival, proliferation, migration, self-renewal and triggering the myogenic program [Buckingham & Relaix, 2015]. Apart from their essential role in the muscle tissue, they are also important for neural crest derivatives and the central nervous system [Buckingham & Relaix, 2007].
As early as in the somite, compartmentalization and lineage specification are accompanied by alterations in the expression patterns of Pax genes [Christ & Ordahl, 1995]. PAX3 is mainly functioning during early embryonic myogenesis and gets downregulated in most muscles after birth, while PAX7 prevails in the post-natal growth phase as well as during adult muscle regeneration [Buckingham & Relaix, 2015]. Genetic replacement of PAX3 by PAX7 rescues most of the phenotypes of PAX3 mutants, but also shows that PAX7 cannot fully substitute PAX3 function in delamination, migration and proliferation of limb muscle progenitors (Fig. 1.8) [Relaix et al., 2004]. Furthermore, despite some overlapping functions of PAX3 and PAX7 in triggering the adult myogenic program, PAX7 has a distinct A-D) PAX7 cannot rescue limb defects of Pax3-mutant embryos, when knock-in in the Pax3 locus. E) In the absence of PAX7, PAX3 cannot replace its antiapoptotic function.

MRFs play a central role in myogenic determination and differentiation

Skeletal muscle identity is conferred by the MRF family of transcription factors, which are expressed solely in skeletal muscle. In order to activate muscle-specific genes via direct binding to an E-box -a specific DNA sequence (CANNTG)-, MRFs heterodimerize with the ubiquitously expressed E proteins [Singh & Dilworth, 2013]. The MRF family consists of four members, MYOD [Davis et al., 1987], MYF5 [Braun et al., 1989], MRF4 [Rhodes & Konieczny, 1989], and MYOGENIN [Wright et al., 1989], which were originally identified by their ability to trigger conversion of non-muscle cell types into myogenic fate when ectopically expressed [Olson & Klein, 1994]. All four MRFs share a bHLH domain, mediating DNA binding as well as dimerization to form transcriptional complexes [Maroto et al., 2008]. The bHLH domain is characterized by ~80% amino acid identity among the four members, while limited sequence similarity is observed in the transcriptional activation domains, residing in the amino- and carboxyl-termini [Olson & Klein, 1994]. Target binding and expression profiling revealed shared targets between some members of the family and, in the case of MYOD and MYOGENIN, suggested a model whereby MYOD establishes an open chromatin structure at muscle-specific genes and MYOGENIN enhances transcription once chromatin is rendered accessible [Blais et al., 2005; Cao et al., 2006]. A further study implicated MYOD in chromatin loop dynamics regulation [Battistelli et al.

Satellite cells: the skeletal muscle stem cells

Originally described as “wedged between the plasma membrane of the muscle fiber and the basement membrane” by A. Mauro in 1961, satellite cells were named after their anatomical position in the periphery of the myofibers (Fig. 1.18A-B), under the basal lamina (Fig. 1.18C). They were predicted to represent dormant myoblasts ready to recapitulate the embryonic developmental myogenic program for myofiber repair [Katz, 1961; Mauro, 1961]. However, it was not until 2005 that their stem cell potential was proven, by virtue of a) providing differentiated progeny for muscleregeneration (Fig. 1.18E-F) and b) self-renewing their pool, upon engraftment of FACS-sorted satellite cells [Montarras et al., 2005] or single myofibers [Collins et al., 2005] into the muscles of mdx mice that lack dystrophin and undergo continuous regeneration. Recently, human satellite cells were also shown to support regeneration and repopulation of the satellite cell compartment after transplantation [Marg et al., 2014]. Importantly, muscle fails to regenerate in the absence of satellite cells. DTA (diphtheria toxin fragment A)-mediated ablation of satellite cells demonstrated the absolute requirement of satellite cells for muscle regeneration [Lepper et al., 2011; McCarthy et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011]. The term stem cell often entails mutlipotency, although this is not an obligate criterion for stem cells [Tajbakhsh, 2003]. Indeed, satellite cells can be driven to adipogenic and osteogenic fates in culture [Asakura et al., 2001], arguing for multipotent nature. However, culture contamination by other lineages cannot be excluded and satellite cells are generally considered monopotent cells in physiological conditions [Relaix & Zammit, 2012].

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Satellite cells in the control of postnatal growth and homeostasis

Postnatal muscle growth depends on myofiber size increase, in the mouse (see section 1.4.2). Of note, the number of myonuclei per myofiber undergoes a 5-fold increase within the first three weeks of life [White et al., 2010]. Satellite cells are the main contributors to this hypertrophic phenomenon. They proliferate rapidly and extensively and are the source of new myoblasts that fuse with existing myofibers, while myonuclei have stopped dividing [Moss & Leblond, 1971; Lepper et al., 2009; White et al., 2010].
Satellite cell fusion reaches a plateau around three weeks postnatally [White et al., 2010]. Thereafter, satellite cells were hypothesized to contribute to adult skeletal muscle homeostasis even in the absence of injuries. Indeed, genetic lineage studies provided experimental evidence for the predicted low rate fusion that constantly occurs (Fig. 1.19) [Keefe et al., 2015; Pawlikowski et al., 2015]. It should be stressed, however, that in general adult myofibers persist throughout the life in the absence of injury or myopathies that trigger a regeneration response resulting to new muscle formation [Grounds & Shavlakadze, 2011].

Acquisition of quiescence for function preservation

At three weeks of age (i.e. P21) there is a critical period of change from juvenile muscle/satellite cells to their form observed in the adult. Postnatally, satellite cells undergo a progressive number diminution and loss of proliferative capacity, which culminates in entering into a quiescent, noncycling state around P21 [Lepper et al., 2009; White et al., 2010]. Long-standing efforts have described a series of markers (Fig. 1.20) to identify quiescent satellite cells, with PAX7 being central.
Active Notch is fundamental to maintain quiescence (see Chapter 3), while Angiopoieitin-1/TIE2 is a further signaling promoting this state [Abou-Khalil et al., 2009]. On the DNA level, the histone methyltransferase Suv4-20H1 was recently found to maintain satellite cell quiescence by promoting a heterochromatic state [Boonsanay et al., 2016]. Moreover, PAX7 has a rather unappreciated role in chromatin architecture modifications, with its loss leading to euchromatic morphology (Fig. 1.21) [Günther et al., 2013]. Quiescence preservation is also ensured by translation repression, via phosphorylation of the translation initiation factor eIF2a [Zismanov et al., 2016].

Table of contents :

ACKNOWLEDGMENTS
LIST OF ABBREVIATIONS
ABSTRACT
INTRODUCTION
CHAPTER 1. SKELETAL MUSCLE DEVELOPMENT, GROWTH, AND REGENERATION 
1.1 EMBRYONIC MYOGENESIS: FROM SOMITES TO THE FIRST MUSCLE MASSES
1.1.1 SOMITOGENESIS: FORMATION OF MULTIPOTENT MESODERMAL STRUCTURES
1.1.2 MYOTOME: THE FIRST SKELETAL MUSCLE
1.1.3 MIGRATION OF MUSCLE PROGENITORS TO SUPPORT LIMB MYOGENESIS
1.2 GENETIC HIERARCHIES IN HEAD AND BODY MUSCULATURE ESTABLISHMENT
1.3 PAX PROTEINS AND BHLH MRFS PLAY A CENTRAL ROLE IN THE MYOGENIC PROGRAM
1.3.1 PAX3 AND PAX7 AS UPSTREAM MYOGENIC REGULATORS
1.3.2 MRFS PLAY A CENTRAL ROLE IN MYOGENIC DETERMINATION AND DIFFERENTIATION
1.4 FROM EMBRYONIC MYOGENIC DEVELOPMENT TO POSTNATAL MUSCLE
1.4.1 EMBRYONIC AND FETAL WAVES OF MYOGENESIS
1.4.2 POSTNATAL MUSCLE GROWTH
1.4.3 ADULT MUSCLE: STRUCTURE & FUNCTION
1.5 SATELLITE CELLS: THE SKELETAL MUSCLE STEM CELLS
1.5.1 ESTABLISHMENT DURING DEVELOPMENT
1.5.2 SATELLITE CELLS IN THE CONTROL OF POSTNATAL GROWTH AND HOMEOSTASIS
1.5.3 ACQUISITION OF QUIESCENCE FOR FUNCTION PRESERVATION
1.5.4 SATELLITE CELL NICHE
1.5.5 SATELLITE CELLS IN THE CONTROL OF REGENERATION
1.5.6 SATELLITE CELL HETEROGENEITY
1.5.7 AGING EFFECT IN MUSCLE AND SATELLITE CELLS
CHAPTER 2. CELL CYCLE AND GROWTH ARREST IN SKELETAL MUSCLE AND BEYOND 
2.1 CELL CYCLE OVERVIEW
2.2 CDK-CYCLIN COMPLEXES: CELL CYCLE PROGRESSION
2.3 THE POCKET PROTEIN- E2F NETWORK: DOWNSTREAM EFFECTORS OF CDK/CYCLINS
2.4 CDKIS: MAJOR NEGATIVE REGULATORS OF CDK-CYCLIN ACTIVITY
2.5 P57 – “KI P”LAYER IN CELL PHYSIOLOGY AND PATHOLOGY
CHAPTER 3. NOTCH SIGNALING PATHWAY: PLEIOTROPIC ROLE OF A MASTER CELL FATE REGULATOR IN MYOGENESIS 
RESULTS

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