Multiple roles of Notch in cells of the myogenic lineage

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Muscle stem cells (MuSCs)

Unlike many other tissues, skeletal muscle has a powerful capacity for regeneration throughout almost the entire life span and this potential is seen across many species (Morrison et al., 2006). Luz et al. showed that C57BL/10 mice regenerated muscle without loss of myofibers or gain of fibrotic tissue after 50 bupivacaine injections into the tibialis anterior (TA) muscle (Luz et al., 2002). Sadeh et al. demonstrated active regeneration cycles in rats that had received weekly injections of bupivacaine for 6 months. They did not find evidence for a reduction or exhaustion of the regenerative capacity of muscle fibers despite ongoing degeneration-regeneration cycles over a period lasting one fourth of the rat’s life span (Sadeh et al., 1985). Such results clear-ly support the existence of muscle stem cells and indicate the stem cell pool to be efficiently maintained and regenerated during multiple degeneration-regeneration cycles.
The muscle stem cell was discovered by Alexander Mauro in frog muscle (Mauro, 1961). The name of the “satellite” cell refers to mononucleated cells “wedged” be-tween the plasma membrane (sarcolemma) at the inside and the basal lamina at the outside (Figure 3). Within this niche, the satellite cell is commonly placed adja-cent to a myonucleus of their host muscle fiber and an endothelial cell of a nearby capillary. This association between satellite cells and myofibers immediately raised the hypothesis that these cells might have a role in muscle growth and regeneration (Mauro, 1961).

Regulatory signaling pathways

Wnt signaling: Wnt Signaling controls diverse biological processes, such as cell proliferation, cell fate determination, cell adhesion, cell polarity, and morphology. Wnt signaling is activated via binding of extracellular glycoproteins of the Wnt family to Frizzled receptors and to low-density lipoprotein receptor-related protein (LRP5, LRP6) pairs. The Frizzled receptor can initiate two distinct signaling pathways: the Wnt/β-catenin and Wnt/PCP pathways. The Wnt/β-catenin pathway is regulated via β-catenin stabilization and its entry into the nucleus, while the Wnt/PCP pathway is involved in planar cell polarization (PCP) and establishment of polarized cellular structures (Gao and Chen, 2010).
Accumulating evidence indicates that Wnt/β-catenin signaling is involved in satellite cell function during muscle regeneration, although its assumed role remains contro-versial. Brack et al. proposed that low Wnt signaling activity during early proliferation allows expansion of enough myoblasts by Notch signaling for later differentiation. The authors suggested that Wnt signaling promotes myogenic commitment and ter-minal differentiation in adult myogenesis (Brack et al., 2008), while Perez-Ruiz et al. proposed that β-catenin promotes self-renewal of satellite cells and prevents them Satellite cells reside between the basal lamina and the sarcolemma of adult skeletal myofibers and are influenced by structural and biochemical cues emanating from this microenvironment (Yin et al., 2013). A complex set of diffusible molecules (e.g. Wnt, Igf, and Fgf) are exchanged between the satellite cell and the myofiber in order to maintain quiescence or to promote activation. In addition, numerous ECM components and cellular receptors are present either on the surface of the sarcolemma, on the satel-lite cell proper, or on the basal lamina. These components comprise the immediate niche of the satellite cell and may dictate rapid changes in the satellite cell state.
from immediate myogenic differentiation (Perez-Ruiz et al., 2008). Additionally, Kim et al. discovered that β-catenin directly interacts with MyoD to enhance MyoD binding to E-box elements in order to initiate the myogenic program (Kim et al., 2008). How-ever, in vivo evidence is required to further elucidate this mechanism. Otto et al. carefully profiled the temporal progression of Wnt signaling in cells during myogene-sis and concluded that activating Wnt ligands (Wnt1, Wnt3a, and Wnt5a) function to promote satellite cell proliferation during muscle regeneration, whereas inhibitory Wnt ligands (Wnt4 and Wnt6) antagonize the proliferation by sequestering β-catenin to the cell membrane in quiescent satellite cells (Otto et al., 2008). To make the matter more complicated, the pro-proliferation activity of Wnt3a revealed by Otto et al. seems to oppose the pro-differentiation function reported by Brack et al. (Brack et al., 2008). In this regard the different sources of Wnt ligands deriving, either from Wnt-expressing cells or as a recombinant protein, might have an influence and should hence be taken into account.

Notch determines asymmetric division

Early work in D. melanogaster established the importance of Notch pathway in self-renewal and lineage specification, as well as its role in asymmetric cell division. In this context, asymmetric cell division defines a process, in which the unequal distribu-tion of cell fate determinants results in the generation of daughter cells with two dif-ferent fates or properties. A classical example was discovered in the peripheral nerv-ous system of D. melanogaster. Once a sensory organ precursor cell has been gen-erated within the proneural cluster in the ectoderm, it undergoes three rounds of asymmetric cell division to form the different cell types of a sensory bristle (Bardin et al., 2004). During this developmental process, the fate choices are precisely regulated by Notch signaling via asymmetric distribution of the Notch inhibitor Numb (Jan and Jan, 1994).
Asymmetric division is also found in regenerative processes. In this case the Notch pathway regulates fates between self-renewal versus differentiation in the cells of the same lineage. This has been demonstrated in radial glial cell division in zebrafish. In this process, Notch regulates the diverse fates of apical and basal daughter cells. The ubiquitin E3 ligase mindbomb (essential for ligand endocytosis) segregates spe-cifically to the apical daughter cell. Asymmetric localization of mindbomb and a Delta ligand in the apical daughter cell induces Notch signaling in the basal daughter cell. Basal cells with high Notch signaling undergo self-renewal, whereas apical cells dif-ferentiate (Dong et al., 2012).

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Multiple roles of Notch in cells of the myogenic lineage

Similar as in other cell lineages, the role of Notch signaling in myogenic cells is multi-faceted. It comprises multiple intermediate steps that can be regulated and hence may have opposite outcomes, which are still largely unknown.

Notch signaling as inducer of myogenesis

Transient Notch signaling functions as an inducer of myogenesis, for instance, leads to the transition from bone marrow stromal cells to myogenic cells (Dezawa et al., 2005). Rios et al. reported that neural crest-derived Dll1 transiently activates Notch signaling in cells located at the medial border of the dermomyotome and that this event is essential for the induction of both Myf5 and MyoD signaling. In contrast, sus-tained Notch signaling inhibits myogenic differentiation at the medial border of the dermomyotome (Rios et al., 2011). Cappellari et al. demonstrated that different Notch-ligands might have opposite effects on cell differentiation. As an example the authors demonstrated that stimulation with Dll4 and Platelet-derived Growth Factor homodimer down-regulated “myogenic” genes in myocytes, while up-regulating genes for pericyte markers. Such effect of “suppression of myogenesis” could not be found had the cells been subjected to Dll1 stimulation, which enhanced myogenesis (Cappellari et al., 2013).

Notch signaling as an inhibitor of differentiation

Notch signaling inhibits myogenesis differentiation via suppressing MyoD expression (Shawber et al., 1996) or by directly suppressing Myogenin promoter activity via Hey1 (Buas et al., 2010). Consequently, activation of Notch signaling in cultured my-ogenic cells suppresses the differentiation progress, while inhibition of Notch signaling promotes the differentiation program. Constitutive expression of an active form of Notch 1 in myogenic cells cultured in vitro (Conboy and Rando, 2002; Shawber et al., 1996) or the constitutive expression of NICD in progenitors (Wen et al., 2012) leads to the expansion of these cells through blocking of myogenic differentiation. Co-culture of myogenic cells with cells overexpressing Notch ligands (Shawber et al., 1996) inhibits myogenic differentiation, while shedding of Notch ligand Dll1 in a sub-set of cultured myoblasts facilitates differentiation in neighboring cells (Sun et al., 2008). Enhanced myogenic differentiation is also observed by overexpression of Numb, a negative regulator of Notch (Conboy and Rando, 2002) or by inhibition of γ-secretase activity (Kitzmann et al., 2006). Furthermore, constitutive activation of Notch signaling in muscle cells by overexpression of Delta-like 1 in vivo inhibits myo-genesis during chick limb development (Delfini et al., 2000; Hirsinger et al., 2001).

Notch signaling in satellite cells

Various researchers have identified high levels of various components of the Notch signaling pathway to be expressed in adult satellite cells, such as Notch ligands (Dll1, Jagged1), Notch receptors (Notch1, 2, and 3), Notch downstream targets (Hes1, Hey1, HeyL), as well as the regulators (Nrarp, Numb) (Bjornson et al., 2012; Mourikis, Gopalakrishnan, et al., 2012).

Table of contents :

List of tables
List of abbreviations
Summary
Zusammenfassung
Résumé
1. General introduction
1.1 Muscle tissue
1.1.1 Muscle development
1.2 Muscle stem cells (MuSCs)
1.2.1 Characterization of MuSCs
1.2.2 Human satellite cells
1.3 The stem cell niche
1.3.1 Regulatory signaling pathways
1.3.2 The satellite cell niche
1.4 Notch signaling
1.4.1 Notch ligands and receptors
1.4.2 Notch determines asymmetric division
1.4.3 Multiple roles of Notch in cells of the myogenic lineage
1.4.4 Notch signaling in satellite cells
1.5 Muscle stem cells for cell therapy
1.5.1 DMD model and treatment
1.5.2 Cell therapy
1.5.3 Isolation of MuSCs
1.5.4 Adeno-associated virus (AAV)
2. Aim of the Project
3. Materials and methods
3.1 Materials
3.1.1 Chemicals and consumables
3.1.2 Plasmids and cell lines
3.1.3 Animals
3.1.4 Antibodies
3.1.5 Buffers, solutions and media
3.1.6 Software
3.2 Methods
3.2.1 Molecular biological methods
3.2.2 Cell biology Methods
3.2.3 Biochemical methods
3.2.4 Histological methods
3.2.5 Transgenic animal experiments
3.2.6 Statistical analysis
4. Results
4.1 Fiber-associated satellite cell culture
4.2 Recombinant Dll1-Fc protein production
4.3 The biological effect of Dll1-Fc on cultured myoblasts
4.4 The effect of Dll1-Fc on muscle progenitor cells (MPCs)
4.5 The in vivo effect of Notch overstimulation
4.5.1 The viral gene transfer system
4.5.2 The effect of Notch overstimulation on postnatal muscle growth
4.5.3 The effect of Dll1-overexpression during muscle regeneration
5. Discussion
5.1 Immobilization of Dll1-Fc and its effect in myoblasts.
5.1.1 Dll1-Fc stimulation suppresses myoblast differentiation.
5.1.2 The effect of Dll1-Fc on myoblast proliferation.
5.2 The ex vivo cultivation of muscle stem cells.
5.2.1 Notch stimulation impairs proliferation of cultured MPCs
5.2.2 Notch stimulation via Dll1-Fc preserves stemness in cultured MPCs.
5.3 Overexpression of Dll1 in vivo
5.3.1 Overexpression of Dll1 in wild-type mice during muscle development
5.4 Outlook
6. Bibliography
7. Appendix
7.1 Copyright permissions
7.2 Own Publication
8. Acknowledgement

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