CELL CYCLE AND GROWTH ARREST IN SKELETAL MUSCLE AND BEYOND 

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Myotome: the first skeletal muscle

As the somite matures, cells delaminate and migrate underneath to form a third compartment, called myotome (Fig. 1.4), which corresponds to the first differentiated skeletal muscle. The groups of Ordahl and Kalcheim came to contradictory results when trying to elucidate the starting points and movements of myotomal precursors [Brent & Tabin, 2002], but a more recent stepwise model described by Gros et al. [2004] helped resolve the controversy. According to these findings, at a first phase cells translocate from the dorso-medial lip to the myotome and once there, they elongate along the rostral-caudal axis. At a second phase, cells invade myotome starting from all four lips and elongate along the anterior-posterior axis. Elongation can be unidirectional (cells from rostral or caudal lip) or bidirectional (cells from dorso-medial or ventro-lateral lip).
A) Major factors controlling the consecutive steps of limb myogenesis. B) At E10.5 in the mouse embryo, Pax3-LacZ progenitors have migrated to the forelimb (up) and begin to migrate to the hindlimb (down). C,E) Cxcr4 expression in limb at E10.5 mouse embryo. D,F) Sdf1 expression in limb at E10.5 mouse embryo. G-J) Lbx1 expression in somites and limbs of chick embryos at HH18 and HH22 stages. K-L) Limb muscles (black arrows) are absent from and somites are fused and truncated (red arrowheads) in Pax3IRESnLacZ/Sp E11.5 mouse embryos as opposed to Pax3IRESnLacZ/+ control embryos at the same stage. M-P) PAX3-expressing cells do not colonize the forelimbs of Splotch mutants at 30- 33 somite stages. Q-R) Lack of muscle (identified with MyoD) in the forelimbs of c-Met mutant E11.5 mouse embryos, in contrast to controls. NC: notochord, NT: neural tube, SE: surface ectoderm. Adapted from: Bober et al., 1994; Maina et al., 1996; Mennerich et al., 1998; Buckingham et al., 2003; Relaix et al., 2003; Vasyutina et al., 2005. Once the primary myotome is formed, a second population of myogenic progenitor cells originating from the central dermomyotome is colonizing the underlying myotome, rendering its initial name “dermatome” erroneous (Fig. 1.4). At later stages of embryonic and fetal life, muscle growth was found to depend on progenitors originating from the central dermomyotome, rather than the lips (see session 1.2.1) [Ben-Yair & Kalcheim, 2005; Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005]. Of note, embryonic and fetal progenitors are mitotically active and have not engaged to the myogenic program. They maintain their proliferative status in embryonic and fetal muscles of trunk and limbs throughout development. They depend on transcription factors of the PAX family and they contribute to the forming muscles as well as their associated stem cells, as discussed in the following sections.

Migration of muscle progenitors to support limb myogenesis

Distant sites of myogenesis, such as the developing limb, depend on long-range migration of progenitors from the hypaxial dermomyotome to the limb buds, where they proliferate and subsequently commit to the myogenic lineage and undergo differentiation into skeletal muscle (Fig. 1.5 A) [Buckingham et al., 2003].
The transcription factor LBX1 is considered a bona fide marker of long-range migrating muscle precursors [Dietrich et al., 1998]. Lbx1 starts to be expressed in the dispersing dermomyotomal lips, meaning prior to delamination. It then follows the migrating population (Fig. 1.5 G-J) and declines only when these progenitors arrive at the target sites and start to differentiate [Jagla et al., 1995; Dietrich et al., 1998; Mennerich et al., 1998]. In its absence, migratory precursors manage to form and delaminate but they display defective routing, demonstrating that LBX1 is critical for migration [Schäfer & Braun, 1999; Brohmann et al., 2000; Gross et al., 2000].
The migratory behavior of muscle progenitors is also controlled by PAX3, which is essential for the initiation of their migration. Splotch and other PAX3 mutant embryos show a number of developmental phenotypes in dorsal neural regions, neural crest cells and derivatives and muscle tissues [Auerbach, 1954; Relaix et al., 2004]. Strikingly, PAX3-deficient embryos are devoid of myogenic migrating cells, leading to complete absence of muscular diaphragm, tongue and limb muscles (Fig. 1.5 K-P) [Mennerich et al., 1998; Relaix et al., 2004]. PAX3-expressing migrating progenitor cells also express LBX1 [Vasyutina et al., 2005].
Central in the genetic hierarchy controlling delamination and migration are the c-MET tyrosine kinase receptor – expressed by hypaxial muscle precursors – and its ligand scatter factor/ hepatocyte growth factor (SF/HGF) – lining the migratory route in the limb mesenchyme and other sites of migratory myogenesis [Birchmeier & Brohmann, 2000]. In their absence, migrating myogenic progenitors and, subsequently, muscle masses are missing from the limbs, tongue and diaphragm (Fig. 1.5 Q-R) [Bladt et al., 1995; Maina et al., 1996; Dietrich et al., 1999]. c-Met transcription depends on PAX3 [Epstein et al., 1996; Relaix et al., 2003]; c-Met expression as well as migratory progenitors and limb muscles are absent from Splotch embryos [Bober et al., 1994; Epstein et al., 1996; Yang et al., 1996; Tajbakhsh et al., 1997]. The c-Met promoter contains a PAX3 binding site [Epstein et al., 1996] and c-Met has been established as PAX3 target in vitro and in vivo [Epstein et al., 1996; Relaix et al., 2003]. Cxcr4 and Sdf1 constitute a further receptor-ligand pair affecting progenitor migration to the limbs [Vasyutina et al., 2005]. Similarly to c-MET and SF/HGF, CXCR4 receptor-expressing muscle progenitors are guided by a SDF1-paved route to the limb (Fig. 1.5 C-F). However, CXCR4/SDF1 seems to be required only for a subset of cells and to have a transient expression [Vasyutina et al., 2005].

Genetic hierarchies in head and body musculature establishment

A complex repertoire of transcription factor is crucial for the acquisition of the myogenic fate and skeletal muscle differentiation. Myogenic determination and differentiation rely on the Myogenic Regulatory Factors (MRFs), a family of basic helix-loop-helix (bHLH) transcription factors, including MYF5, MYOD, MRF4, and MYOGENIN. Upstream transcription factors act in the activation of MRFs as well as by regulating the proliferation and survival of progenitor cells. The upstream regulators differ between head/neck and trunk/limb muscles (Fig. 1.6) [Bismuth & Relaix, 2010; Braun & Gautel, 2011; Buckingham & Mayeuf, 2012].
In the body musculature PAX3/7 play a central role (see section 1.3), while a similar upstream role, linked to that of PAX3, was shown for the SIX homeodomain transcription factors and EYA cofactors [Buckingham & Rigby, 2014]. SIX1/4 or EYA1/2 deficient mice show a pronounced downregulation of MRFs and lacked limb and many trunk muscles [Grifone et al., 2005; Grifone et al., 2007]. Accordingly, SIX proteins were found to control Myf5 [Giordani et al., 2007], MyoD [Relaix et al., 2013] and Myogenin [Spitz et al., 1998] expression, the first two in synergy with PAX3.
Head muscle development follows a distinct program, not requiring PAX3/7 but depending on four transcription factors -MYOR, Capsulin, PITX2, and TBX1- acting on different head muscle groups. PAX3 is not expressed in mesodermal derivatives in the head, while PAX7 is expressed in some head muscles, but its absence does not cause any head muscle phenotype [Bismuth & Relaix, 2010]. MYOR and Capsulin are bHLH transcription factors that redundantly function in specifying masticatory muscles [Bismuth & Relaix, 2010]. PITX2 is central in the regulation of non-somitic myogenic progenitors, controlling the survival and differentiation of muscle progenitors from the first branchial arches as well as progenitors that will form extraocular muscles [Buckingham & Mayeuf, 2012]. Finally, TBX1 has been described as “genetically equivalent to PAX3 during branchial arch development”, as it is expressed in the mesodermal cores of branchial arches and it is involved in bilateral branchiomeric myogenesis [Bismuth & Relaix, 2010].

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 role in survival (Fig. 1.8) and cell cycle progression [Relaix et al., 2006]. Large scale analysis of PAX3 and PAX7 binding profiles revealed several factors that could account for these differences, such as a) differential binding affinities for paired (PAX3) versus homeobox (PAX7) motifs, b) PAX3 binding only a subset of PAX7 targets (~5K sites for PAX3 vs ~53K sites for PAX7, with ~3.5K common sites), c) PAX7 occupying in the adult sites bound by PAX3 in the embryo, d) unique PAX3 targets involved in embryonic myogenesis (i.e. enrichment in ontology terms of skeletal muscle morphogenesis and neural and epithelial tube formation) [Soleimani et al., 2012].
PAX3 and PAX7 expression begins early in the nascent myogenic lineage and their absence leads to a complete arrest of skeletal muscle development. Transcript and reporter analyses revealed that PAX3 expression initiates in the presomitic mesoderm prior to segmentation (around E8 in the mouse) and its expression is progressively confined to the dermomyotome covering the epaxial and hypaxial extremities, while PAX7 appears later (around E9 in the mouse) and is concentrated in the central dermomyotome [Murphy & Kardon, 2011]. In the limb, PAX3+ progenitors migrate to the limb buds, where they are transiently present from E10 to E12.5 [Bober et al., 1994], while PAX7 appears later (E11.5), in PAX3-expressing myogenic progenitors and persists until fetal/neonatal stages [Relaix et al., 2004]. Later on, PAX3 gets downregulated but PAX7 persists [Kassar-Duchossoy et al., 2005]. In adult muscles, PAX7 is a universal marker of satellite cells, the progenitor/stem cell population responsible for postnatal growth/regeneration (see section 1.3), while PAX3 is restricted to a subset of trunk and limb muscle satellite cells [Seale et al., 2000; Relaix et al., 2006; Calhabeu et al., 2013].

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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., 2014], while MYOD and MYOGENIN targets include chromatin remodeling factors [Cao et al., 2006].
The specific expression of MRFs transcripts is initiated early during muscle development and follow distinct spatio-temporal patterns (Fig. 1.10) [summarized in Murphy & Kardon, 2011; Singh & Dilworth, 2013]. Myf5 is the first MRF expressed, with its transcripts being observed from E8 in the epaxial dermomyotome and showing declining levels from recently formed (caudal) to mature (rostral) somites [Ott et al., 1991]. Myf5 expression decreases from E14 onwards [Ott et al., 1991]. Of note, some PAX7+ cells do not express MYF5 and represent progenitors with slower proliferation and earlier exit from the cell cycle [Picard & Marcelle, 2013]. Myogenin is found from E8.5, accumulating in the most rostral somites and coinciding with differentiating muscle cells [Sassoon et al., 1989; Ott et al., 1991]. MYOD appears at E10.25-E10.5 [Sassoon et al., 1989; Kablar et al., 1997; Zabludoff et al., 1998]. In the limb, Myf5 is detected in forelimb and hindlimb from E10.5 and E11, respectively, and gets downregulated by E11.5 when MYOD and MYOGENIN accumulate [Sassoon et al., 1989; Ott et al., 1991; Kablar et al., 1997]. Mrf4 shows a biphasic pattern, with its transcripts appearing from E9 until E12 and then again from E16 onwards [Bober et al., 1991]. Adult myonuclei will maintain the expression of MRF4, which becomes the predominant MRF in adult muscle [Hinterberger et al., 1991; Gayraud-Morel et al., 2007].
Genetic ablation during embryonic and fetal myogenesis established MYF5, MYOD, and MRF4 as myogenic determination factors and MYOD, MYOGENIN, and MRF4 as myogenic differentiation factor [Murphy & Kardon, 2011]. MYF5-null mice form myotome with a 2-day delay [Braun et al., 1992; Tajbakhsh et al., 1997; Kassar-Duchossoy et al., 2004] and a MYF5-driven LacZ reporter revealed the presence of progenitors which activated Myf5 in MYF5-null mice, but remained multipotent, failed to localize correctly and eventually differentiated into non-muscle derivatives according to their local environment [Tajbakhsh et al., 1996]. Despite the delayed myotome initiation in MYF5-deficient embryos, the myogenic program gets rescued around E11.5 by the delayed activation of MYOD [Braun et al., 1994] and muscles of MYF5 mutants become structurally and functionally normal until birth (Figs. 1.11-1.12) [Braun et al., 1992; Tajbakhsh et al., 1997]. It has been proposed that MYF5-independent MYOD- expressing myoblasts sustain myogenesis in the absence of a distinct, MYF5- dependent lineage [Haldar et al., 2008]. Conversely, mice lacking MYOD have morphologically normal muscles (albeit showing 2-day delayed differentiation in the limb [Kablar et al., 1997]) (Figs. 1.11-1.12) and maintain high levels of MYF5 [Rudnicki et al., 1992; Kablar et al., 1998]. In the absence of both factors, newborns are completely devoid of skeletal muscles in both trunk and limbs [Rudnicki et al., 1993]. However, this effect appears to depend on compromised Mrf4 expression, since skeletal muscle manages to differentiate in MYF5/MYOD double knockouts with functional MRF4 [Kassar-Duchossoy et al., 2004]. It has been proposed that the proximity of Mrf4 and Myf5 (the former residing 8kb 5’ of the latter) likely account for cis-regulatory interactions [Olson et al., 1996], that are diversely affected in different MYF5 nulls. Thus, Mrf4 was also identified as a determination gene, while genetic manipulation of the three factors placed both MYF5 and MRF4 upstream of MyoD [Kassar-Duchossoy et al., 2004]. Specific ablation of Myf5-expressing cells using Myf5Cre; R26RDTA/+, suggested the presence of distinct Myf5-dependent and Myf5-independent MyoD-expressing myoblasts [Gensch et al., 2008; Haldar et al., 2008]. However, when Myf5-expressing cells were eliminated in a MyoD null background, no muscles were formed, indicating that the previously observed Myf5-independent myoblasts were in fact MYF5+ escaper cells [Comai et al., 2014].
Myogenic differentiation was found to depend on MYOGENIN, MYOD, and MRF4 [Murphy & Kardon, 2011]. MYOGENIN did not overlap with MYOD or MYF5 in specification of the myogenic lineage [Rawls et al., 1995]. However, its deficiency led to compromised muscle-specific gene expression and differentiation, including a generalized fusion defect so that mutant mice presented with severely reduced muscle masses associated with lethality at birth [Hasty et al., 1993; Nabeshima et al., 1993; Rawls et al., 1995; Rawls et al., 1998]. These defects are phenocopied in MYOD/MRF4 double knockout mice [Rawls et al., 1998], implying that either MYOGENIN or MYOD and MRF4 need to be present to drive differentiation. Finally, single MRF4 loss-of-function overall did not jeopardize muscle development [reviewed in Olson et al., 1996], although different strategies for Mrf4 disruption resulted in phenotypes as different as ranging from perinatal lethality to normal survival [Braun & Arnold, 1995; Patapoutian et al., 1995; Zhang et al., 1995], again likely due to interrelated cis-regulatory interactions with Myf5 [Olson et al., 1996].

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
AIMS AND HYPOTHESES
ANTAGONISTIC REGULATION OF p57KIP2 BY HES/HEY DOWNSTREAM OF NOTCH SIGNALING AND MUSCLE
REGULATORY FACTORS REGULATES SKELETAL MUSCLE GROWTH ARREST
A p57 CONDITIONAL MUTANT ALLELE THAT ALLOWS TRACKING OF P57-EXPRESSING CELLS
DISTINCT REGULATION AND FUNCTION OF p21 AND p57 DURING MUSCLE STEM CELL ACTIVATION AND
DIFFERENTIATION
DISCUSSION
CDKIS, MRFS AND NOTCH SIGNALING INTERPLAY IN CELL CYCLE EXIT DURING DEVELOPMENT
CONDITIONAL p57 ABLATION FOR POSTNATAL STUDIES
CDKIS IN THE CONTROL OF SATELLITE CELLS
ANNEX
REFERENCES

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