Table of Contents

List of figures

ii

List of tables

Summary

Muscle stem cell transplantation possesses great potential for long-term repair of dystrophic muscle. However expansion of muscle stem cells ex vivo significantly reduces their engraftment efficiency since the myogenic potential is dramatically lost in culture.

The Notch signaling pathway has emerged as a major regulator of muscle stem cells (MuSCs) and it has recently been discovered that high Notch activity is crucial for maintaining stemness in MuSCs. This feature might be exploited and developed into a novel therapeutic approach.

Murine MuSCs were freshly isolated and seeded on culture vessels coated with Dll1-Fc, which fused Delta-like-1 extracellular domain with human Fc, to activate Notch signaling and with human IgG as a control. The rAAV gene delivery system was employed to express Dll1 in murine muscles. P3 mice were treated with AAV for 3 weeks and 6 weeks to investigate the effect of Dll1 during postnatal development. To investigate the regeneration process, AAV were injected into mdx muscles whereas wild-type mice were used as control.

Higher potential stemness (marked by Pax7 positivity) was observed in MuSCs growing on a Dll1-Fc surface as compared to their counterparts on the control surface, while their proliferation rate was reduced.

General introduction 1.1 Muscle tissue

About 640 skeletal muscles in the human body, which compose up to 40% of the total body mass, are vital to provide locomotive activity. Skeletal muscle is composed of individual components known as myocytes, also called "myofibers". As the basic cellular units of muscle, myofibers are syncytia. They are multinuclear cells in post mitotic state, which are normally formed during embryonic and fetal development [START_REF] Sabourin | The molecular regulation of myogenesis[END_REF]. Throughout life they maintain the capability of regeneration following muscle damage [START_REF] Charge | Cellular and molecular regulation of muscle regeneration[END_REF]. Myofibers are grouped into bundles (of up to 150 fibers) called fasciculi, which are surrounded by the perimysium (Figure 1). Several fasciculi form the skeletal muscle, which is covered by the epimysium and is attached to the periosteum of the bone through tendons. The ingenious arrangement of the muscle fibers allows highly efficient force transduction from the muscle to the bone.

Besides the myofibers, which are the contractile components, skeletal muscle also contains other components that support and regulate muscle function. The vascular system ensures the supply of nutrients, oxygen and the removal of toxic metabolites. Cells of the immune system defend against pathogenic organisms and remove the debris of apoptotic cells after damage. Nerves innervate the muscle via the neuromuscular junction, which is essential for voluntary muscle contraction. In addition, skeletal muscle contains a specific stem cell population, termed "satellite cells" [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF], which are important for regeneration. Further, fibroblasts provide the extracellular matrix, which contributes to the maintenance of the extracellular homeostasis [START_REF] Kuang | The emerging biology of satellite cells and their therapeutic potential[END_REF].

Muscle development

Muscle , 2005). There is convincing experimental evidence that satellite cells from different muscle types originate from the same embryonic source as the muscle in which they reside [START_REF] Gros | A common somitic origin for embryonic muscle progenitors and satellite cells[END_REF][START_REF] Kassar-Duchossoy | Pax3/Pax7 mark a novel population of primitive myogenic cells during development[END_REF][START_REF] Relaix | A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[END_REF][START_REF] Schienda | Somitic origin of limb muscle satellite and side population cells[END_REF].

In mice, at the initial stage of embryonic myogenesis (E10.5-E12.5 of limb myogenesis), embryonic myoblasts differentiate in order to generate primary muscle fibers.

Fetal myoblasts fuse with primary fibers and can also fuse amongst themselves to generate additional secondary fibers during the fetal phase of myogenesis (E14.5-P0). Notably, embryonic and fetal myoblasts have distinct features and can also be distinguished from postnatal myoblasts. Microarray analysis of purified embryonic (E11.5) and fetal myoblasts (E16.5) demonstrated unique gene expression signatures with differences in transcription factor and cell surface receptor expression signatures for both cell types [START_REF] Biressi | Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells[END_REF].

In mice, during the first weeks of postnatal life, muscle mass increases nearly threefold while the satellite cell number decreases from 30% to less than 5% of the total myonuclei. After P21, skeletal muscle reaches a steady-state of homeostasis and the satellite cells enter quiescence, which is essential to maintain the satellite cell pool throughout life. Following muscle injury, quiescent satellite cells are activated and give birth to one daughter cell, which will return to quiescence, while the other daughter cell will further divide and fuse with the myofiber hence contributing to muscle regeneration (This asymmetric cell division will be discussed in detail in section 1.2).

Although embryonic, fetal and adult myoblasts differ in many aspects of their cellular morphology, proliferation rate, growth requirements, response to extrinsic molecular signals and drug treatment, and even in the morphology of their differentiated myofibers [START_REF] Biressi | Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells[END_REF], many similarities exist. Similar mechanisms during the activation of satellite cells and the myogenesis in the somite have reinforced the idea that adult muscle regeneration recapitulates -at least to a certain extent -embryonic development via analogous, but not necessarily identical, mechanisms.

Pax3 and Pax7

The paired-homeobox family of transcription factors (Pax 1-9) play important roles in the regulation of development and differentiation of diverse cell lineages during embryogenesis [START_REF] Mansouri | Pax genes and their role in organogenesis[END_REF][START_REF] Walther | Pax: a murine multigene family of paired box-containing genes[END_REF]. The Pax7 and Pax3 genes are paralogs with almost identical amino acid sequence and partially overlapping expression patterns during mouse embryogenesis [START_REF] Goulding | Pax-3, a novel murine DNA binding protein expressed during early neurogenesis[END_REF][START_REF] Jostes | The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system[END_REF]. In skeletal muscle, Pax3 and Pax7 have overlapping yet redundant roles in myogenesis, as both of them target genes that promote proliferation and commitment to the myogenic lineage, while repressing genes that induce terminal myogenic differentiation. Recent genome-wide comparative binding site studies between Pax7 and Pax3 confirm that Pax7 plays a dominant role in regulating transcription in adult skeletal muscle cells. Pax7 and Pax3 exhibit differential binding affinities for paired homeobox motifs, suggesting that differences in DNA binding affinities and chromatin status may contribute to the distinct developmental and functional differences between these two paralogs [START_REF] Soleimani | Transcriptional Dominance of Pax7 in Adult Myogenesis Is Due to High-Affinity Recognition of Homeodomain Motifs[END_REF].

All adult quiescent satellite cells express Pax7 [START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF], while Pax3 is only expressed in a subset of satellite cells of certain muscles [START_REF] Relaix | Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[END_REF]. Pax3 plays a critical role during embryonic myogenesis. Most satellite cells down-regulate Pax3 before birth [START_REF] Horst | Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis[END_REF][START_REF] Kassar-Duchossoy | Pax3/Pax7 mark a novel population of primitive myogenic cells during development[END_REF]). Pax7 appears to be dispensable for embryonic myogenesis, however, it is uniquely required in satellite cells after birth as Pax7-null mice are viable, but lack functional satellite cells [START_REF] Kuang | Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[END_REF]. enhancer [START_REF] Bajard | A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb[END_REF]. Daubas and Buckingham revealed that Pax3 together with another factor (Six1/4) bind to an enhancer 111 kb upstream of the Myf5 gene, which subsequently directs its expression in some limb muscles [START_REF] Daubas | Direct molecular regulation of the myogenic determination gene Myf5 by Pax3, with modulation by Six1/4 factors, is exemplified by the -111 kb-Myf5 enhancer[END_REF].

Pax3 and

Pax7 has been shown to directly activate Myf5 by promoting recruitment of the Wdr5/ASH2-like (ASH2L)/MLL2 histone methyltransferase complex (which directs methylation of histone H3 lysine 4) to a regulatory sequence upstream of the Myf5 coding region [START_REF] Mckinnell | Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex[END_REF]. In coordination with FoxO3 (which belongs to the large forkhead family of transcription factors), Pax3/7 recruit RNA polymerase II to form the pre-initiation complex for activation of MyoD transcription in myoblasts [START_REF] Hu | Codependent activators direct myoblast-specific MyoD transcription[END_REF].

Pax7 specifies the satellite cell lineage. Extensive evidence from multiple laboratories has been gathered to elucidate the function of Pax7. In the early postnatal period Pax7 probably prevents the transition of muscle precursors into terminal differentiation while maintaining the myogenic identity by promoting Myf5 expression. Hence its expression correlates with an undifferentiated albeit committed myogenic state [START_REF] Olguin | Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination[END_REF].

In the adult muscle, the exact role of Pax7 is still under debate. Various groups confirmed Pax7 to be a crucial factor for maintenance of the undifferentiated state of adult satellite cells [START_REF] Günther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF][START_REF] Mourikis | Cell-autonomous Notch activity maintains the temporal specification potential of skeletal muscle stem cells[END_REF]. Lepper et al. suggested Pax7 to have a crucial function during postnatal muscle growth but might be entirely dispensable for normal satellite cell function beyond the juvenile period [START_REF] Lepper | Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[END_REF]. Using the same tamoxi- are responsible for the specification of satellite cells for the myogenic lineage as well as for the regulation of myogenic differentiation. The MRFs comprise myogenic factor 5 (Myf5), myoblast determination protein (MyoD), myogenin (MyoG), and musclespecific regulatory factor 4 (Mrf4, also known as Myf6). These proteins contain a conserved basic helix-loop-helix (HLH) DNA-binding domain that binds the E-box, which is a DNA motif containing the core E-box sequence CANNTG [START_REF] Weintraub | The myoD gene family: nodal point during specification of the muscle cell lineage[END_REF]. The HLH-domain mediates dimerization with other HLH-containing proteins, such as factors encoded by the E2-2 and E2-5 genes: E12, E47, HEB and ITF2 [START_REF] Barndt | Controlling lymphopoiesis with a combinatorial E-protein code[END_REF]. Depending on its dimerization partner, the activity of the transcription factor can be modulated.

Gene targeting and expression analysis have classified the MRFs into two functional groups: Myf5 and MyoD as the first group act as determination factors, and in the second group, myogenin and Mrf4 act as differentiation factors. Myf5 and MyoD expression are upregulated upon satellite cell activation [START_REF] Cooper | In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle[END_REF]. Following the generation of sufficient myogenic progenitor cells, Pax7 is down-regulated before terminal differentiation [START_REF] Olguin | Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal[END_REF]. Myogenin together with Myf4, initiates terminal differentiation, which entails the transition of myoblasts into elongated myocytes, fusion into myotubes, and finally their maturation into myofibers [START_REF] Rudnicki | The molecular regulation of muscle stem cell function[END_REF].

MyoD and Myf5:

The introduction of null mutations into the genes of the MRF-family in mice demonstrated that the MRF regulatory network exhibits a considerable functional overlap. Newborn mice lacking a functional MyoD gene do not display any overt abnormalities in muscle but express about fourfold higher levels of Myf5 [START_REF] Rudnicki | Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development[END_REF]. Newborn Myf5-deficient animals are also viable and have apparently normal muscle [START_REF] Braun | Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death[END_REF][START_REF] Kaul | Myf-5 revisited: loss of early myotome formation does not lead to a rib phenotype in homozygous Myf-5 mutant mice[END_REF]. Muscle development in the trunk of embryos lacking Myf5 is delayed until the onset of MyoD expression, which occurs somewhat later [START_REF] Braun | Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death[END_REF][START_REF] Tajbakhsh | Establishing myogenic identity during somitogenesis[END_REF]. In addition, newborn mice double-deficient in Myf5 and MyoD are entirely devoid of myoblasts and myofibers [START_REF] Kassar-Duchossoy | Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice[END_REF]. In conclusion, Myf5 and My-oD act upstream of myogenin and Mrf4 [START_REF] Rudnicki | MyoD or Myf-5 is required for the formation of skeletal muscle[END_REF] as determination factors for myogenesis and could functionally compensate for each other to a large extent.

Analysis of primary myoblasts expressing either MyoD or Myf5 indicated that Myf5

and MyoD are not functionally equivalent [START_REF] Sabourin | Reduced differentiation potential of primary MyoD-/-myogenic cells derived from adult skeletal muscle[END_REF]. The temporalspatial pattern of myogenesis in Myf5and MyoD-deficient embryos provides strong evidence for the roles of Myf5 and MyoD and clearly indicates that mere Myf5 expression in the limb is insufficient for normal progression of myogenic development [START_REF] Kablar | Development in the absence of skeletal muscle results in the sequential ablation of motor neurons from the spinal cord to the brain[END_REF]. Presently it is still under debate, how exactly these two transcription factors work and interact. The "two lineage" model suggests that two distinct lineages co-exist in myogenesis that are either regulated by Myf5 or by MyoD.

Conditional ablation studies in mice [START_REF] Gensch | Different autonomous myogenic cell populations revealed by ablation of Myf5-expressing cells during mouse embryogenesis[END_REF][START_REF] Haldar | Two cell lineages, myf5 and myf5-independent, participate in mouse skeletal myogenesis[END_REF] demonstrated that Myf5 determines a myogenic cell population and that its abrogation entails the loss of cranial and somatic mesoderm-derived muscles.

Myogenin (MyoG):

Mice lacking myogenin are immobile and die prenatally due to deficits of myoblast differentiation, as evidenced by the almost complete absence of myofibers [START_REF] Hasty | Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene[END_REF][START_REF] Nabeshima | Myogenin gene disruption results in perinatal lethality because of severe muscle defect[END_REF], while normal numbers of My-oD-expressing myoblasts are present, which are organized in groups similar to wildtype muscle. Myogenin-deficient embryos form primary myofibers normally, but appear unable to form secondary myofibers [START_REF] Venuti | Myogenin is required for late but not early aspects of myogenesis during mouse development[END_REF]. In proliferating myoblasts, MyoD represses transcription by recruiting Suv39h1 to promoters of genes including myogenin. Sur39h1 is a histone H3 lysine 9 (H3K9)-specific methyltransferase that silences genes by chromatin modification. Silencing of myogenin transcription requires sustained methylation of H2K9 on the myogenin promoter as well as a stable interaction between Sur39h1 and MyoD [START_REF] Mal | Histone methyltransferase Suv39h1 represses MyoD-stimulated myogenic differentiation[END_REF].

Mrf4: Mice carrying different targeted Mrf4 mutations display a range of phenotypes consistent with a role for Mrf4 in late myogenesis [START_REF] Rawls | Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice[END_REF]. Interestingly, mice lacking both MyoD and Mrf4 display a phenotype similar to the myogenin-null phenotype [START_REF] Rawls | Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice[END_REF]. Therefore, Mrf4 function might be substituted for by myogenin but only in the presence of MyoD. Notably, Mrf4 functions as a determination factor in a subset of myocytes in the early somite and as a differentiation factor in later muscle fibers [START_REF] Kassar-Duchossoy | Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice[END_REF].

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 [START_REF] Morrison | Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population[END_REF]. 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 [START_REF] Luz | Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells[END_REF]. 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 [START_REF] Sadeh | Chronic myopathy induced by repeated bupivacaine injections[END_REF]. Such results clearly 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 [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF]. The name of the "satellite" cell refers to mononucleated cells "wedged" between 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 adjacent 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 [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF].

In the late 1960s [ 3 H]thymidine labeling and electron microscopy studies indicated that satellite cells undergo mitosis, assume a cytoplasm-enriched morphology and contribute to myofiber nuclei [START_REF] Moss | Nature of dividing nuclei in skeletal muscle of growing rats[END_REF][START_REF] Reznik | Thymidine-3H uptake by satellite cells of regenerating skeletal muscle[END_REF][START_REF] Shafiq | Regeneration in skeletal muscle of mouse: some electron-microscope observations[END_REF]. Later, [ 3 H]thymidine tracing experiments demonstrated that satellite cells are mitotically quiescent in adult muscle but can quickly enter the cell cycle upon muscle injury [START_REF] Snow | Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. II. An autoradiographic study[END_REF]. The same study also demonstrated that satellite cells give rise to proliferating myoblasts, which were shown to form multinucleated myotubes in vitro [START_REF] Konigsberg | Clonal analysis of myogenesis[END_REF][START_REF] Yaffe | Cellular aspects of muscle differentiation in vitro[END_REF]. In vitro cultured single myofibers may become necrotic, which is accompanied by satellite cell outgrowth, clonal expansion, and later fusion with functionally regenerating myotubes [START_REF] Bischoff | Regeneration of single skeletal muscle fibers in vitro[END_REF]. These ex- periments support the view that it is the satellite cell, rather than the myonuclei, that contribute to postnatal muscle growth and repair.

By definition, stem cells in adult tissues do not only give rise to functional progeny (differentiation) but also replicate themselves (self-renewal). The first evidence for satellite cell self-renewal came from single myofiber transplantation experiments.

Researchers found out that transplantation of as few as seven satellite cells into irradiated regeneration-incapable mouse muscle gave rise to hundreds of satellite cells and thousands of myonuclei. Most remarkably, transplanted satellite cells from a single fiber were able to support subsequent rounds of muscle regeneration (Collins et al., 2005). Furthermore, using Myf5-Cre and ROSA26-YFP Cre-reporter alleles Kuang et al. showed that satellite cells are a heterogeneous population consisting of both stem cells (Pax7+|Myf5-) and committed progenitors (Pax7+|Myf5+) (Kuang et al., 2007). These observations demonstrated that satellite cells are bona fide muscle stem cells, which are quiescent in adult muscle under normal physiological conditions [START_REF] Schultz | Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF]. In response to stress such as exercise and injury, satellite cells have a remarkable ability to self-renew, expand, proliferate as myoblasts or undergo myogenic differentiation to fuse and restore damaged muscle (Figure 4) [START_REF] Chang | Satellite cells: the architects of skeletal muscle[END_REF][START_REF] Relaix | Muscle stem cells[END_REF].

Characterization of MuSCs

As the term "satellite" indicates, the satellite cells reside "around" a muscle fiber between the basal lamina and the sarcolemma. As mentioned in section 1. They are present in similar numbers throughout life and do not exhibit gender difference [START_REF] Neal | The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration[END_REF]. This hypothesis is further supported by the investigation of DNA-strand segregation in dividing satellite cells: during muscle regeneration a subpopulation of satellite cells is observed to produce distinct daughter cell fates by asymmetrically segregating the template strand to the stem cell and the newly synthesized strand to the further dividing satellite cells that contribute to muscle repair [START_REF] Rocheteau | A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division[END_REF].

Within the last years, studies have uncovered proteins that are enriched in satellite cells. These comprise M-cadherin [START_REF] Irintchev | Expression pattern of M-cadherin in normal, denervated, and regenerating mouse muscles[END_REF], Pax7 [START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF],

Human satellite cells

Comparison of ultra-structural data suggested that the percentages of satellite cells in adult mouse and human muscles are similar. In the mouse the percentage lies, dependent on the study, around 5% of all myonuclei: less than 5% [START_REF] Allbrook | Population of muscle satellite cells in relation to age and mitotic activity[END_REF], or 5-7% [START_REF] Neal | The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration[END_REF]. In humans, the percentage is reported to be about 2% [START_REF] Sajko | Frequency of M-cadherin-stained satellite cells declines in human muscles during aging[END_REF][START_REF] Verdijk | Satellite cells in human skeletal muscle; from birth to old age[END_REF], or about 4% [START_REF] Schmalbruch | The number of satellite cells in normal human muscle[END_REF]. It is evident that the number of satellite cells in mice depends on the type of muscle, in which they reside [START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF][START_REF] Zammit | All muscle satellite cells are equal, but are some more equal than others?[END_REF]. Similarly, human satellite cells in type II fiber are less abundant than in type I fibers [START_REF] Verdijk | Satellite cells in human skeletal muscle; from birth to old age[END_REF].

Presently our understanding of murine satellite cell biology is rapidly expanding due to the large body of data that can be obtained from transgenic mouse experiments.

Knowledge about human satellite cell biology, however, is still wanting and limited to comparatively few studies: such studies comprise human satellite cells gene profiling [START_REF] Bortoli | Gene expression profiling of human satellite cells during muscular aging using cDNA arrays[END_REF] and analyses about satellite cell content in different fiber types of 165 individuals from different age groups [START_REF] Verdijk | Satellite cells in human skeletal muscle; from birth to old age[END_REF].

The few experimental studies on human satellite cells were hampered by the lack of suitable antibodies. The first and now widely used marker for human satellite cells is CD56, also named "neural cell adhesion molecule" (NCAM) [START_REF] Schubert | Lymphocyte antigen Leu-19 as a molecular marker of regeneration in human skeletal muscle[END_REF].

CD56 is expressed by quiescent human [START_REF] Lindström | New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men[END_REF] and rat satellite cells, but not in mice. Murine satellite cells only express NCAM after being committed to differentiation [START_REF] Capkovic | Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation[END_REF]. Pax7, a reliable murine satellite cell marker, is also widely used for human satellite cells. However, some authors pointed out that Pax7 fails to identify all satellite cells and may also stain myonuclei [START_REF] Reimann | Pax7 distribution in human skeletal muscle biopsies and myogenic tissue cultures[END_REF]. Lindstrom et al. demonstrated that the majority of human satellite cells express both Pax7 and NCAM, but there are also small numbers of NCAM+|Pax7and NCAM-|Pax7+ cells, which might be in an activation or differentiation state [START_REF] Lindström | New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men[END_REF]. M-Cadherin has also been reported to be a reliable human satellite cell marker [START_REF] Boldrin | Human satellite cells: identification on human muscle fibres[END_REF][START_REF] Sajko | Frequency of M-cadherin-stained satellite cells declines in human muscles during aging[END_REF], but a wellfunctioning antibody is not commercially available and hence this marker is not widely used (More satellite cell markers are listed in Table 1).

It is generally assumed that murine and human satellite cell biology is basically identical. However, Bareja et al. only recently demonstrated that not all the mechanisms that regulate satellite cell activation in mice are conserved in humans. The authors compared murine and human satellite cells in culture and identified differences in the myogenic genetic program and in the sensitivity of the cells upon cytokine stimulation [START_REF] Bareja | Human and mouse skeletal muscle stem cells: convergent and divergent mechanisms of myogenesis[END_REF]. Hence, more studies on human satellite cells are needed to understand muscle regeneration in humans and exploit the satellite cells for therapeutic interventions.

The stem cell niche

Early in 1978, Raymond Schofield postulated a specific "environment" that might induce the unlimited proliferation capabilities and the failure of maturation of stem cells [START_REF] Schofield | The relationship between the spleen colony-forming cell and the haemopoietic stem cell[END_REF]. By coining the term "Niche" he postulated stem cells to be located at physical locations, which suppress their entry into the cell cycle and differentiation programs, determine the fate of daughter cells, integrate with signals that are coming from the organism (endocrine), circulation and surrounding tissue (paracrine) environment, and limit the "mutational errors". However, at this early time of satellite cell research the term "niche" was rather a hypothetic concept being in want of direct evidence rather than proof of its actual existence.

The concept of the "niche" is now well accepted. Mounting evidence shows that it seems to be correct [START_REF] Ohlstein | The stem cell niche: theme and variations[END_REF]: Studies of D. melanogaster oogenesis initially revealed that the niche influences stem cell behavior via adhesive interactions.

Germ stem cells adhere to cap cells, which are located between the basal lamina and germ stem cells. Daughter cells that lose cap cell contact become cytoblasts and are destined to differentiate into oocytes [START_REF] Song | DE-cadherin-mediated cell adhesion is essential for maintaining somatic stem cells in the Drosophila ovary[END_REF]. In mammals, adult stem cells and their corresponding niches have been well described in numerous tissues including neural stem cells in the brain [START_REF] Zhong | Mouse numb is an essential gene involved in cortical neurogenesis[END_REF], hematopoietic stem cells in the bone marrow [START_REF] Calvi | Osteoblastic cells regulate the haematopoietic stem cell niche[END_REF][START_REF] Zhang | Identification of the haematopoietic stem cell niche and control of the niche size[END_REF], stem cells in the crypts of intestinal villi [START_REF] Sancho | Live and let die in the intestinal epithelium[END_REF], in the skin [START_REF] Tumbar | Defining the epithelial stem cell niche in skin[END_REF], and in the heart [START_REF] Leri | Myocardial regeneration and stem cell repair[END_REF].

Further, satellite cells can also be located in highly specified niches, which may comprise components of the extracellular matrix (ECM), vascular and neural networks, various types of surrounding cells, and numerous diffusible molecules (Figure 5). The authors suggested that Wnt signaling promotes myogenic commitment and terminal differentiation in adult myogenesis [START_REF] Brack | A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis[END_REF], while Perez-Ruiz et al.

proposed that β-catenin promotes self-renewal of satellite cells and prevents them [START_REF] Yin | Satellite cells and the muscle stem cell niche[END_REF].

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 satellite 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 [START_REF] Perez-Ruiz | beta-Catenin promotes self-renewal of skeletal-muscle satellite cells[END_REF]. 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 [START_REF] Kim | Beta-catenin interacts with MyoD and regulates its transcription activity[END_REF]. However, in vivo evidence is required to further elucidate this mechanism. Otto et al.

carefully profiled the temporal progression of Wnt signaling in cells during myogenesis 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 [START_REF] Otto | Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration[END_REF]. 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. [START_REF] Brack | A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis[END_REF]. In this regard the different sources of Wnt ligands deriving, either from Wntexpressing cells or as a recombinant protein, might have an influence and should hence be taken into account.

Researchers discovered that satellite cells are under the regulation of the Wnt/PCP pathway as well. Wnt7a molecules activate the planar-cell-polarity pathway and drive the symmetric expansion of satellite stem cells via the Fzd7 receptor, which results in enhanced skeletal muscle repair (von [START_REF] Maltzahn | Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle[END_REF]. A recent study revealed synergistic functions between the Wnt/β-catenin and Wnt/PCP pathways in the oriented elongation of myocytes during embryonic muscle patterning [START_REF] Gros | WNT11 acts as a directional cue to organize the elongation of early muscle fibres[END_REF].

It is thus fascinating to further speculate that the activated Wnt/β-catenin pathway in regenerating myofibers might induce Wnt7a secretion and subsequent activation of the Wnt/PCP pathway in adjacent satellite stem cells, which in turn could direct their symmetric division and replenish the satellite cell pool.

Wnt signaling is also implicated in satellite cell related transdifferentiation. Rudnicki's group revealed that muscle Wnt/β-catenin is required for muscle regeneration and may increase the myogenic potential of CD45+/Sca1+ cells that reside in the muscle [START_REF] Polesskaya | Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration[END_REF]. Furthermore, recent studies demonstrated that Wnt signaling seems to be important for transdifferentiation of satellite cells into fibrogenic cells.

In favor of this hypothesis, an increase in Wnt ligands [START_REF] Brack | Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis[END_REF][START_REF] Liu | Augmented Wnt signaling in a mammalian model of accelerated aging[END_REF] or a decrease in Wnt inhibitors [START_REF] Kurosu | Regulation of fibroblast growth factor-23 signaling by klotho[END_REF] may account for the fibrogenic transformation in aged mice. Wnt signaling also controls the balance between myogenic and adipogenic potential of myoblasts in vitro [START_REF] Abiola | Activation of Wnt/beta-catenin signaling increases insulin sensitivity through a reciprocal regulation of Wnt10b and SREBP-1c in skeletal muscle cells[END_REF].

Notch signaling: Notch signaling regulates cell proliferation, differentiation and cell fate determination. Since it is the main focus of my dissertation, this pathway will be further discussed in detail in the "Notch signaling" section 1.4.

Sphingolipid signaling: Sphingolipids are a large group of naturally occurring glycolipids that are characterized by their sphenoid backbone. Sphingolipids have been recently discovered as important bioactive signaling molecules in the regulation of cell proliferation, migration, senescence, and death (reviewed in [START_REF] Hannun | Principles of bioactive lipid signalling: lessons from sphingolipids[END_REF]. It was found that sphingomyelin is enriched in the plasma membrane of quiescent satellite cells, becomes markedly diminished upon satellite cell activation, and reappears in some satellite cells after returning to their quiescent state [START_REF] Nagata | Sphingomyelin levels in the plasma membrane correlate with the activation state of muscle satellite cells[END_REF]. Pharmacological inhibition of the conversion of sphingomyelin to sphingosine-1-phosphate (S1P) reduced the number of activated satellite cells and impeded muscle regeneration. Evidence was found that S1P acts through S1P receptors on the satellite cells in an autocrine/paracrine fashion [START_REF] Calise | Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors[END_REF]. However, our current understanding of the influence of sphingolipids on satellite cells is still incomplete.

BMP signaling: Bone morphogenic proteins (BMPs) are members of the TGF-β superfamily and function through dedicated BMP receptors such as activin-receptor-like kinase 3 (ALK3) and BMP receptor type 2 (BMPR2). Besides their critical role in regulating adult muscle maintenance, growth and atrophy [START_REF] Sartori | BMP signaling controls muscle mass[END_REF], BMP signaling also plays an important role in "routine" satellite cell function. Wang et al. discovered that BMP signaling is active in a subpopulation of fetal progenitors at the extremities of muscles and regulates the number of muscle progenitors and satellite cells during development [START_REF] Wang | Bmp signaling at the tips of skeletal muscles regulates the number of fetal muscle progenitors and satellite cells during development[END_REF]. Further, BMP signaling is instrumental to coordinate the balance between proliferation and differentiation before activation of Noggin, which antagonizes BMPs thereby facilitating terminal differentiation [START_REF] Ono | BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells[END_REF].

Other signaling pathways:

A precise balance between physical microenvironmental cues and cell autonomous signals is required to preserve the capacity of satellite cells for self-renewal. In this complicated dynamic regulatory process additional signaling proteins are involved: e.g. the Yes-associated protein, a Hippo pathway member [START_REF] Judson | The Hippo pathway member Yap plays a key role in influencing fate decisions in muscle satellite cells[END_REF], Sprouty 1 (Spry 1), a receptor tyrosine kinase signaling inhibitor [START_REF] Shea | Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration[END_REF], as well as microRNAs [START_REF] Cheung | Maintenance of muscle stem-cell quiescence by microRNA-489[END_REF] and RNA-binding proteins [START_REF] Li | An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis[END_REF]. The understanding of such regulatory fac-tors will improve our understanding of the satellite cell biology and promote their potential therapeutic utility.

The satellite cell niche 1.3.2.1 The myofiber surface

Due to their direct contact with the satellite cells, the myofibers constitute a primary component of the satellite cell niche and influence satellite cell proliferation and quiescence. The selective destruction of myofibers with Marcaine largely increased the satellite cell numbers [START_REF] Bischoff | Interaction between satellite cells and skeletal muscle fibers[END_REF]. Recent studies revealed numerous satellite cell regulatory factors that are either present on the surface of the myofibers or secreted by them: SDF-1 is secreted by the myofiber and serves as ligand for the cell surface receptor CXCR4 on the satellite cells [START_REF] Sherwood | Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle[END_REF] to stimulate satellite cell migration [START_REF] Ratajczak | Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles[END_REF]. In addition to this, after injury myofibers up-regulate Delta-like 1, which activates the Notch signaling cascade in satellite cells and hence induces their proliferation [START_REF] Conboy | Notch-mediated restoration of regenerative potential to aged muscle[END_REF].

ECM and associated factors

The basal lamina on the other side of the satellite cell niche contains type IV collagen, laminin, entactin, fibronectin, perlecan, and decorin glycoproteins along with other proteoglycans. These ECM molecules are mainly synthesized and excreted by interstitial fibroblasts. Satellite cells locate between the basal lamina and the apical sarcolemma, which is covered with laminin (reviewed in [START_REF] Yin | Satellite cells and the muscle stem cell niche[END_REF].

The basal lamina offers a large number of binding sites for α7/β1-integrins; sites that anchor the actin cytoskeleton of satellite cells to the ECM. This physical tethering is critical for satellite cell activation as it allows the transduction of extracellular mechanical force into intracellular chemical signals (Figure 5). ECM is also associated with a suite of inactive growth factor precursors, such as hepatocyte growth factor/scatter factor (HGF) [START_REF] Tatsumi | HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells[END_REF], basic fibroblast growth factor (bFGF) [START_REF] Tatsumi | HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells[END_REF], epidermal growth factor (EGF) [START_REF] Golding | Skeletal muscle stem cells express antiapoptotic ErbB receptors during activation from quiescence[END_REF], insulin-like growth factor isoforms (IGF-I, IGF-II) [START_REF] Machida | Insulin-like growth factor 1 and muscle growth: implication for satellite cell proliferation[END_REF], and various Wnt glycoproteins (Le [START_REF] Grand | Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells[END_REF]. Those precursors originate either from satellite cells, myofibers, interstitial cells, or from serum. In resting muscle, the sequestering of inactive growth factor precursors forms local reservoirs, which upon activation facilitates a rapid response to muscle injury.

Notch signaling

The Notch locus was initially described in Thomas Hunt Morgan's laboratory about 100 years ago as sex-linked lethal mutations in D. melanogaster, where heterozygous females carriers had notches in their wings [START_REF] Bridges | Non-Disjunction as Proof of the Chromosome Theory of Heredity[END_REF]. The Notch cascade has been recognized as one of a few signaling pathways that are repeatedly used in multiple developmental processes in embryonic and adult tissue. The essential components of Notch signaling have been found to be highly conserved and function in a similar manner in all Metazoans (D' Souza et al., 2010).

Notch ligands and receptors

The There the NICD induces target gene transcription, via interactions with CSL. CSL, which is an acronym for the orthologous proteins CBF-1/Rbp-J (mammalian), Suppressor of hairless (D. melanogaster), and Lag (C. elegans), is the DNA binding transcription factor required for both activation and repression of Notch target genes [START_REF] Koch | Stem cells living with a Notch[END_REF][START_REF] Perdigoto | Sending the right signal: Notch and stem cells[END_REF].

The to transand cis-ligands to be strikingly different: the response to trans-Delta is graded, whereas the response to cis-Delta is sharp and occurs at a fixed threshold [START_REF] Sprinzak | Cis-interactions between Notch and Delta generate mutually exclusive signalling states[END_REF]. This mechanism constitutes, an ultrasensitive switch between signal-sending (high Delta/low Notch) and signal-receiving (high Notch/low Delta) states.

Notch determines asymmetric division

Early work in D. melanogaster established the importance of Notch pathway in selfrenewal 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 distribution of cell fate determinants results in the generation of daughter cells with two different fates or properties. A classical example was discovered in the peripheral nervous system of D. melanogaster. Once a sensory organ precursor cell has been generated 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 [START_REF] Bardin | Asymmetric localization and function of cell-fate determinants: a fly's view[END_REF]. During this developmental process, the fate choices are precisely regulat- [START_REF] Jan | Genetic control of cell fate specification in Drosophila peripheral nervous system[END_REF].

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 specifically 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 differentiate [START_REF] Dong | Intralineage directional Notch signaling regulates selfrenewal and differentiation of asymmetrically dividing radial glia[END_REF].

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 multifaceted. 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 [START_REF] Dezawa | Treatment of neurodegenerative diseases using adult bone marrow stromal cell-derived neurons[END_REF]. 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, sustained Notch signaling inhibits myogenic differentiation at the medial border of the dermomyotome [START_REF] Rios | Neural crest regulates myogenesis through the transient activation of NOTCH[END_REF]. 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 [START_REF] Cappellari | Dll4 and PDGF-BB Convert Committed Skeletal Myoblasts to Pericytes without Erasing Their Myogenic Memory[END_REF].

Notch signaling as an inhibitor of differentiation

Notch signaling inhibits myogenesis differentiation via suppressing MyoD expression [START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF] or by directly suppressing Myogenin promoter activity via Hey1 [START_REF] Buas | The Notch effector Hey1 associates with myogenic target genes to repress myogenesis[END_REF]. Consequently, activation of Notch signaling in cultured myogenic cells suppresses the differentiation progress, while inhibition of Notch signal-ing promotes the differentiation program. Constitutive expression of an active form of Notch 1 in myogenic cells cultured in vitro [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF][START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF] or the constitutive expression of NICD in progenitors [START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF] leads to the expansion of these cells through blocking of myogenic differentiation. Coculture of myogenic cells with cells overexpressing Notch ligands [START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF] inhibits myogenic differentiation, while shedding of Notch ligand Dll1 in a subset of cultured myoblasts facilitates differentiation in neighboring cells [START_REF] Sun | The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation[END_REF]. Enhanced myogenic differentiation is also observed by overexpression of Numb, a negative regulator of Notch [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF] or by inhibition of γsecretase activity [START_REF] Kitzmann | Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells[END_REF]. Furthermore, constitutive activation of Notch signaling in muscle cells by overexpression of Delta-like 1 in vivo inhibits myogenesis during chick limb development [START_REF] Delfini | Delta 1-activated notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis[END_REF][START_REF] Hirsinger | Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation[END_REF].

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) [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF][START_REF] Mourikis | Cell-autonomous Notch activity maintains the temporal specification potential of skeletal muscle stem cells[END_REF].

Asymmetric distribution of Notch components during cell division

Asymmetric division of stem cells is essential to produce daughter cells for both stem cell pool maintenance and regeneration. The opposite fates of two daughter cells are strictly regulated by various Notch components.

During satellite cell division, Numb (Notch antagonist) was asymmetrically segregated to one daughter cell [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF]: in the presence of Numb, Notch signaling is decreased, while during its absence high levels of Notch activity are maintained, hence determining opposite cell fates. Consequently, other Notch components, such as Delta-like 1 and Notch3 [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF] have also been reported to be exclusively either up or down regulated in the two opposite daughter cells.

Notch signaling is crucial for maintaining stemness

Two independent groups reported high Notch activity to be crucial for maintenance of quiescence in satellite cells through examination of their transcriptional signatures.

After down-regulation of Notch signaling upon injury, satellite cells undergo activation and proliferate while up-regulation leads to re-entry into quiescence [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF][START_REF] Mourikis | Cell-autonomous Notch activity maintains the temporal specification potential of skeletal muscle stem cells[END_REF]Mourikis, Sambasivan, et al., 2012). By different techniques two different groups abrogated Rbp-J, a downstream target of canonical Notch signaling. This led the satellite cells to transcend immediately from the G 0 phase to differentiation (G 0 -differentiation transition) without an intervening proliferative phase and severely depleted the satellite cell pool [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF][START_REF] Vasyutina | RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells[END_REF]. These results show that Notch signaling functions to maintain satellite cell quiescence in a Rbp-Jк dependent manner.

Satellite cells could be categorized according to their levels of Notch activity into upstream myogenic cells (Pax7 Hi ) with higher Notch activity and differentiating myogenic cells (Pax7 Lo ) with elevated Dll1 ligand expression. The importance of Notch signaling for satellite cell maintenance has also been confirmed in ageing animals.

Acute activation of the Notch signaling pathway by a Notch1 specific antibody, which directly binds to its extracellular domain, was able to restore the regenerative potential of satellite cells in aged muscle. This indicates that loss of regenerative capacity of ageing satellite cells might be secondary to their decline of Notch activity [START_REF] Conboy | Notch-mediated restoration of regenerative potential to aged muscle[END_REF].

In conclusion, Notch signaling is crucial for the maintenance of satellite cells in skeletal muscle tissue, regardless of age. However, the origin of Notch signaling to main-

Notch signaling is dispensable for satellite cell proliferation

It was initially thought that Notch signaling was essential for satellite cell proliferation, based on experimental findings of in vitro cultured progenitors: constitutive expression of NICD leads to the expansion of these cells, while inhibition of Notch signaling abolishes satellite cell activation and impairs muscle regeneration [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF]. However, inactivation of the Notch down-stream target CSL/RBP-Jк in satellite cells demonstrated that they still proliferate in vitro albeit at a lower rate [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF]Mourikis, Sambasivan, et al., 2012). Up to date a large body of data suggests that Notch activity inhibits differentiation in activated satellite cells, while it might be (at least partially) dispensable for their proliferation.

Notch signaling plays key role in regeneration.

In resting muscle, Notch activity is maintained at high levels in quiescent satellite cells as discussed before. Given the presence of Dll1-ligand on committed muscle progenitors [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF], it seems likely that the myofibers proper send the signal to uphold Notch activity in quiescent satellite cells.

Muscle injury triggers the regeneration. Multiple Notch components respond quickly to injury in order to initiate subsequent regeneration. For instance, Delta-like 1 is quickly up-regulated in a third of activated satellite cells [START_REF] Conboy | Notch-mediated restoration of regenerative potential to aged muscle[END_REF], while Hes1, Hey1, and HeyL are down-regulated in activated satellite cells. These events are accompanied by the exit of those cells from quiescence into a proliferation and differentiation program [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF]. On the opposite side, however, muscle failed to regenerate if Notch signaling was blocked globally via systemic administration of Jagged1-Fc [START_REF] Brack | A temporal switch from notch to Wnt signaling in muscle stem cells is necessary for normal adult myogenesis[END_REF][START_REF] Conboy | Notch-mediated restoration of regenerative potential to aged muscle[END_REF].

Other conflicting results, however, revealed that a high level of uncleaved Notch receptor 1 in quiescent satellite cells becomes activated in response to muscle injury, suggesting that Notch signaling was up-regulated in response to muscle injury [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF]. Together with the evidence that one Notch target, Hes6, is greatly upregulated upon muscle injury [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF], we should be aware that some Notch components might function differently from the canonical pathway, thus serving as negative feed-back/regulators or might be additionally involved in other pathways.

Multiple signaling pathways might be initiated by other cells involved in the complex coordinated process of muscle cell regeneration such as fibro/adipogenic progenitors [START_REF] Joe | Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis[END_REF] and infiltrating inflammatory cells [START_REF] Arnold | Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis[END_REF]. Such cell-to-cell interactions could be, at least in part, mediated by Notch signaling, which could serve as a principle mediator of intercellular communication.

Muscle stem cells for cell therapy

When Alexander Mauro coined the term "satellite cell" over 50 years ago, he suggested that these cells "might be pertinent to the vexing problem of skeletal muscle regeneration" [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF]. The capability of muscle stem cells to self-renew and to form new muscle tissue offers the potential for therapies against muscle disease or muscle loss (sarcopenia).

DMD model and treatment

The mdx mouse is the most widely used and first animal model of Duchenne muscular dystrophy (DMD) [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF]. This mouse line occurred spontaneously on a C57BL/10 background and was identified by chance during a screen for red blood cell defects in a C57BL/10 colony because their strikingly elevated serum creatine kinase levels suggested a dystrophic process [START_REF] Bulfield | X chromosome-linked muscular dystrophy (mdx) in the mouse[END_REF]. Dystrophin deficiency in the mdx muscle was subsequently identified [START_REF] Hoffman | Dystrophin: the protein product of the Duchenne muscular dystrophy locus[END_REF] and shown to result from a stop codon mutation in exon 23 [START_REF] Sicinski | The molecular basis of muscular dystrophy in the mdx mouse: a point mutation[END_REF] of the dystrophin gene.

However, the muscle regeneration capacity of the mdx mouse remains unaltered and muscle histopathology is very mild as compared to DMD in humans. In contrast to mdx mice, transgenic mdx/mTR mice which are dystrophin-deficient and additionally have a telomere dysfunction/shortening specifically in their muscle stem cells, develop a more severe dystrophic phenotype than mdx mice. Their clinical phenotype also rapidly worsens with age, due to the rapid depletion of their MPCs [START_REF] Sacco | Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice[END_REF]. Therefore, the mild phenotype of the 'classic' mdx mouse can be, at least partially, attributed to the fact that they do not exhaust their muscle stem cell pool.

Hence, treatment schemes that only aim to restore dystrophin expression in the mdx muscle fibers may not be sufficient for treating human DMD patients, especially if they are older [START_REF] Dilworth | Epigenetic regulation of satellite cell activation during muscle regeneration[END_REF][START_REF] Sacco | Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice[END_REF]. Therapeutic modulation of muscle stem cell activity could thus represent an alternative approach for alleviating muscle weakness in DMD [START_REF] Dilworth | Epigenetic regulation of satellite cell activation during muscle regeneration[END_REF].

Cell therapy

About four decades ago pioneering studies already found out that donor myoblasts were able fuse with host myoblasts [START_REF] Partridge | Evidence of fusion between host and donor myoblasts in skeletal muscle grafts[END_REF][START_REF] Snow | Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. II. An autoradiographic study[END_REF] or form myonuclei upon engraftment [START_REF] Lipton | Developmental fate of skeletal muscle satellite cells[END_REF]. Researchers thought that this might provide an opportunity for functional restoration of defective fibers. Ever since, cell therapy has been considered a potential therapy against various muscular disorders; especially the most frequent one: Duchenne Muscular Dystrophy (DMD). A number of clinical trials with DMD patients were done in the 1990s. Disappointingly, little or no functional improvement was observed after myoblasts engraftment (Huard, Bouchard, et al., 1992;Huard, Roy, et al., 1992;[START_REF] Mendell | Myoblast transfer in the treatment of Duchenne's muscular dystrophy[END_REF][START_REF] Morandi | Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer[END_REF].

After these failures a consensus was reached to use satellite cells for cell therapy instead of myoblasts, because of their capacity for self-renewal and regeneration.

Many laboratories worked on this issue, thus increasing our knowledge about satellite cell physiology and about the methods to deliver these in vivo for muscle regeneration [START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF][START_REF] Lipton | Developmental fate of skeletal muscle satellite cells[END_REF][START_REF] Snow | Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. II. An autoradiographic study[END_REF][START_REF] Snow | An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats[END_REF]. Ehrhardt et al. demonstrated that the contribution of transplanted human fetal muscle progenitor cells to both muscle fibers and to the stem cell pool in immunocompromised mice persisted for up to 6 months [START_REF] Ehrhardt | Human muscle precursor cells give rise to functional satellite cells in vivo[END_REF]. Moreover, transplanted single murine satellite cells were capable of extensive clonal expansion, proliferation and contribution to muscle fibers [START_REF] Sacco | Self-renewal and expansion of single transplanted muscle stem cells[END_REF]. Hence a manageable number of "pure muscle stem cells" would be sufficient for cell therapy.

The advent of induced pluripotent stem cells (iPSC) opened the opportunity to obtain muscle progenitor cells (MPCs) directly from autologous material of a patient via directional differentiation protocols. Such differentiation could be achieved by conditional expression of specific myogenic transcription factors, such as Pax7 [START_REF] Darabi | Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7[END_REF][START_REF] Filareto | An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells[END_REF] or MyoD [START_REF] Tanaka | Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro[END_REF]. On the other side, Hosoyama et al. developed a free-floating spherical culture protocol in which they were able to produce myogenic progenitors via stimulation with high concentrations of fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF)

without the necessity for MYOD or PAX7 gene transfer [START_REF] Hosoyama | Derivation of myogenic progenitors directly from human pluripotent stem cells using a sphere-based culture[END_REF]. The transplantation and in vivo expansion of autologous patient material after genetic repair did open a whole new field of investigation.

In addition to satellite cells, various non-satellite cell precursors with myogenic potential [START_REF] Tedesco | Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells[END_REF] have been described as potential source material for regeneration. These comprise bone marrow cells [START_REF] Ferrari | Muscle regeneration by bone marrow-derived myogenic progenitors[END_REF], CD34 + /45 -interstitial myo-endothelial cells [START_REF] Tamaki | Identification of myogenicendothelial progenitor cells in the interstitial spaces of skeletal muscle[END_REF], PW1 + /Pax7 -interstitial cells (PICs) [START_REF] Mitchell | Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development[END_REF], as well as vasculature associated mesangioblasts [START_REF] Sampaolesi | Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts[END_REF]. Although the inherent myogenic potential of those 'unor-thodox' cells remains to be fully understood, the fact that they can be manipulated and the possibility of their systemic delivery has made them a major target of interest for many researchers in the field.

Isolation of MuSCs

The discovery and identification of satellite cells had been made possible through electron microscopy (EM) [START_REF] Mauro | Satellite cell of skeletal muscle fibers[END_REF]. However, the material for EM has to be fixed and stained before imaging and only allows small sections of the muscle fiber to be imaged at once. In addition, since the imaging has to be performed in vacuum, unfortunately in vivo investigations are not possible. Hence, in order to promote the understanding of satellite cell biology, the establishment of protocols for their isolation and in vitro culture was crucial. At present there are several isolation protocols, each having its own strengths and weaknesses.

Richard Bischoff was one of the pioneer researchers on satellite cell biology [START_REF] Bischoff | Enzymatic liberation of myogenic cells from adult rat muscle[END_REF][START_REF] Bischoff | Regeneration of single skeletal muscle fibers in vitro[END_REF].

In his enzymatic isolation protocol [START_REF] Bischoff | Enzymatic liberation of myogenic cells from adult rat muscle[END_REF], satellite cells were isolated from muscle tissue by a long process that involved mincing the muscle, digestions with various enzymes and a series of filtrations and centrifugations. The released cells, consisting mainly of satellite cells and fibroblasts, were subsequently seeded into culture dishes. Different approaches such as 'pre-plating' or 'myoblast selection medium' were aimed to remove fibroblasts. However, none of these approaches were sufficient and contamination with other cell types remained a problem for a long time.

Myofiber isolation. In 1975 Bischoff observed for the first time living satellite cells on myofibers and he reported in his publication that "[. . .] a culture system utilizing single skeletal muscle fibers from adult rats was developed to study the origin and behavior of mononucleated myoblasts." [START_REF] Bischoff | Regeneration of single skeletal muscle fibers in vitro[END_REF]. This method has been further optimized by Rosenblatt [START_REF] Rosenblatt | Culturing satellite cells from living single muscle fiber explants[END_REF] and is now widely used as 'myofiber-associated satellite cell culture' [START_REF] Pasut | Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle[END_REF][START_REF] Wozniak | Single-fiber isolation and maintenance of satellite cell quiescence[END_REF]. The myofiber-associated culture provides a fiber milieu that mimics the in vivo environment and can thus be considered an ex vivo method. The whole process is time consuming and requires demanding manipulation. Unfortunately the yield of satellite cells from this method is quite low. However, engraftment of a single myofiber together with its associated satellite cells can generate many new satellite cells with the ability for self renewal in the host muscle [START_REF] Boldrin | Donor Satellite Cell Engraftment is Significantly Augmented When the Host Niche is Preserved and Endogenous Satellite Cells are Incapacitated[END_REF][START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF][START_REF] Marg | Human satellite cells have regenerative capacity and are genetically manipulable[END_REF]. Sca-1(-), and Mac-1(-) fraction contained only myogenic cells [START_REF] Sherwood | Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle[END_REF].

In the same year, Fukada et al. developed a new monoclonal antibody SM/C-2.6 [START_REF] Fukada | Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody[END_REF] and later established their protocol to isolate pure satellite cells in the SM/C-2.6(+), CD34(+), CD45(-), and Sca-1(-) fraction [START_REF] Ikemoto | Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice[END_REF]. In 2005, Montarras et al. published that satellite cells were highly enriched in the CD34(+), CD45(-), and Sca-1(-) fraction [START_REF] Montarras | Direct isolation of satellite cells for skeletal muscle regeneration[END_REF]. Syndecan 3/4 has also been used as a positive marker for satellite cell isolation [START_REF] Tanaka | Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration[END_REF]. In summary, the choice of positive markers may vary between different laboratories [START_REF] Motohashi | Isolation, culture, and transplantation of muscle satellite cells[END_REF][START_REF] Pisani | Isolation of a highly myogenic CD34-negative subset of human skeletal muscle cells free of adipogenic potential[END_REF][START_REF] Yi | Purification of progenitors from skeletal muscle[END_REF], but the set of negative markers remains the same (Table 2).

In addition to these cell surface-based methods, genetically modified reporter mice allow direct isolation of quiescent satellite cells as well. The established mouse models harbor genes for green or yellow fluorescent protein that are transcribed under the control of a Pax3 promoter [START_REF] Montarras | Direct isolation of satellite cells for skeletal muscle regeneration[END_REF], a Pax7 promoter [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF] or a Myf5 promoter [START_REF] Biressi | Intrinsic phenotypic diversity of embryonic and fetal myoblasts is revealed by genome-wide gene expression analysis on purified cells[END_REF][START_REF] Kassar-Duchossoy | Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice[END_REF].

In contrast to all those well-established methods for murine satellite cell isolation, the set of surface markers for human satellite cells is still wanting. NCAM (CD56), as the so far only human satellite cell marker, is widely used for identification of human satellite cells. Some groups have tried the isolation of satellite cells using CD56 as a surface marker [START_REF] Bareja | Human and mouse skeletal muscle stem cells: convergent and divergent mechanisms of myogenesis[END_REF][START_REF] Scott | Large-scale isolation of human skeletal muscle satellite cells from post-mortem tissue and development of quantitative assays to evaluate modulators of myogenesis[END_REF]. However, such isolates of human satellite cells have not yet been confirmed as 'true" muscle stem cells by classical cell transplantation experiments. The major hurdle in developing these methods can be attributed to the limited access to human muscle tissue.

Adeno-associated virus (AAV)

The adeno-associated virus (AAV) is a single-stranded DNA virus that belongs to the Dependovirus genus of the Parvoviridae family. Humans are the primary host of AAVs, but so far no known pathology is associated with them. Eleven virus subtypes (AAV1-11) have been described; among those, AAV2 happens to be the most frequent subtype. The AAV is a small icosahedral virus with a single copy of a 4.7 kb genome, which comprises three functional regions: two open reading frames termed rep and cap, which encode a non-structural and a structural protein, as well as inverted termini repeats. In the absence of a helper virus (e.g. adenovirus or herpes simplex virus), AAV integrates itself into the human genome at a specific region (designated AAVS1) on chromosome 19 and persists in latent form. If accompanied by an infection with a helper virus, which provides factors essential for active replication and assembly, the AAV would enter a lytic cycle (Figure 8A).

The absent replication capabilities and the non-pathogenic nature of wild-type AAV triggered the rapid development of recombinant AAV (rAAV) as a tool for gene transfer. In the early 1980s Hermonat & Muzyczka published the first paper on the cloning of the AAV vector, which was capable of expressing foreign genes in mammalian cells [START_REF] Hermonat | Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells[END_REF]. Since then the interest in the AAV as a vector for gene therapy continues to grow and multiple variants of modified vectors have been described [START_REF] Riviere | Long-term expression and repeated administration of AAV type 1, 2 and 5 vectors in skeletal muscle of immunocompetent adult mice[END_REF][START_REF] Vasileva | Precise hit: adeno-associated virus in gene targeting[END_REF]. Since in the rAAV the rep and cap genes are replaced by the expression cassette for the 'gene of interest', such vector variants persist almost entirely in episomal form within the transduced tissues. This minimizes the risk of a potentially deleterious integration near oncogenes. For the vector production, the rep and cap products as well as other essential elements are supplied in trans (Figure 8B). The rAAV2-mediated gene transfer was initially described to be very efficient. However, since the AAV2 is the most common subtype found in humans, more than 80% of the people develop a humoral immune response against the wild-type AAV2 and are thus sero-positive for anti-AAV2 antibodies [START_REF] Chirmule | Immune responses to adenovirus and adeno-associated virus in humans[END_REF]. This curtailed the efficiency of this serotype as a widely usable vector for gene therapy and spurred the development of alternative serotypes. Now, rAAV vectors based on the serotypes 1, 5, 6 and 7 are able to transduce murine skeletal muscle much more efficiently than rAAV2, with a reported increase in expression levels ranging from 2-to 1,000-fold [START_REF] Rabinowitz | Cross-packaging of a single adenoassociated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity[END_REF][START_REF] Riviere | Long-term expression and repeated administration of AAV type 1, 2 and 5 vectors in skeletal muscle of immunocompetent adult mice[END_REF][START_REF] Xiao | Gene therapy vectors based on adeno-associated virus type 1[END_REF]. Although rAAV1 is superior to rAAV2 with respect to its transduction efficiency of muscle, its biology (e.g.

the entry mechanism and its primary attachment receptors) is largely unknown.

Aim of the Project

As previously discussed in section 1.5, the capability of MuSCs to both self-renew and differentiate qualifies them as ideal candidates for cell based therapies. After transplantation, donor-derived MuSCs are capable to successfully relocate into their niche in order to proliferate, engraft, and eventually reach quiescent state (Collins et al., 2005;[START_REF] Ehrhardt | Human muscle precursor cells give rise to functional satellite cells in vivo[END_REF][START_REF] Sacco | Self-renewal and expansion of single transplanted muscle stem cells[END_REF]. To increase their long-time regeneration capacity of transplanted MuSCs, it is crucial for them to maintain their "stemness" during the intermittent extra-corporal in vitro expansion. Under standard myoblast culture conditions, however, MuSCs become activated, proliferate and differentiate rapidly. This is paralleled by loss of their myogenic potential, which becomes a major obstacle against the use of MuSCs in cell based therapies.

Various cell culture conditions and additives have been described to have an influence on the myogenic potential. These include the presence of extracellular matrix components such as laminin or fibronectin [START_REF] Cosgrove | A home away from home: challenges and opportunities in engineering in vitro muscle satellite cell niches[END_REF][START_REF] Gilbert | Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture[END_REF][START_REF] Kuang | The emerging biology of satellite cells and their therapeutic potential[END_REF], growth under hypoxic conditions or growth on an elastic substratum as such as Matrigel ® [START_REF] Wang | An in vitro culture system that supports robust expansion and maintenance of in vivo engraftment capabilities for myogenic progenitor cells from adult mice[END_REF] or hydrogel [START_REF] Gilbert | Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture[END_REF].

These studies encourage the search for factors that preserve stemness through modification of cell culture conditions or by adding extrinsic cues.

As detailed in section 1.4, the Notch pathway is prominently involved in the determination of muscle stem cell fate. Based on several published in vitro experiments, it was reasonable to assume that Notch stimulation via Delta-like 1 (Dll1) might be capable to maintain stemness of MuSCs during ex vivo expansion.

We hence set out to investigate

Materials and methods

Materials

Chemicals and consumables

The standard chemicals and media were purchased from Sigma-Aldrich, Roth, Merck, Applichem and Gibco (

Plasmids and cell lines

The following plasmids were used for cloning: pGEM-T Easy (Promega); pTracer-CMV-DII1 (gift of Prof. Carmen Birchmeier, MDC, Germany); pcDNA3-D1Fc (gift of Prof. Gerry Weinmaster, UCLA, USA); pSMD2, pXX6, pRep-Cap (gift of Prof. Luis Garcia, Université de Versailles Saint-Quentin-en-Yvelines, UVSQ, France).

Animals

For rAAV injection, mdx mice (on a C57BL/10ScSn background) and wild-type C57BL/10ScSn control mice were bred in the animal facility of UVSQ according to institutional guidelines. Wild-type C57BL/6J mice for cell culture were purchased from Forschungseinrichtung fuer Experimentelle Medizin (FEM Charité) and kept according to institutional guidelines. The animal studies had been approved and were carried out under the license T0219/13 issued by the LaGeSo Berlin.

Antibodies

Methods

Molecular biological methods

All experiments were conducted with RNase-free/DNase-free plastic materials, filtered solutions, and buffers. The water for RNA experiments was filtered, treated with DEPC overnight and autoclaved.

Escherichia coli bacteria were cultured in a dry incubator at 37˚C. All experiments (biosafety level S1) involving recombinant DNA were carried out with the permission of the LaGeSo in a certified "Gentechnische Anlage" according to the German guidelines and regulations.

Cloning of plasmids

For cloning of the pSMD2-Dll1 plasmid, the 2.2kb Dll1 fragment was from BamH Idigested pTracer-CMV-DII1 plasmid, and inserted into the pSMD2 vector via the Bgl II restriction sites. The size of the fragments was confirmed on agarose gels stained with ethidium bromide [1 µg/ml] and photographed under UV illumination. The corrected Dll1 fragment was cut from the agarose gel using a clean scalpel under UV illumination, and extracted with QIAquick Gel Extraction Kit (Qiagen). The correct sequence of DNA was confirmed by automatic Sanger Sequencing.

Extraction of plasmid DNA

Typically, a single colony from a LB agar plate was picked and cultured in LB medium (with corresponding antibiotics) at 37˚C overnight with 225 rpm shaking. In order to obtain the bacterial cell pellet, the LB medium was centrifuged (x 10,000 g) at 4˚C.

The plasmid DNA was extracted from the pellet using a Mini-or Midi-prep Kit (Qiagen) according to the manufacturer's instructions.

Preparation of RNA and cDNA synthesis

Total RNA was extracted from cell pellets with the RNeasy Mini Kit (Qiagen) or from sectioned muscle using TRIzol ® reagent (Invitrogen) according to the manufacturer's instructions. RNA quality and quantity were quantified using a NanoDrop ® spectrophotometer (Thermo Scientific). After DNA removal with the TURBO DNA-free™ Kit (Ambion), 5 µg RNA were reverse transcribed with SuperScript ® II reverse transcriptase (Invitrogen) using random hexamers as primers.

Quantitative PCR

Real-time qPCR was performed in triplicates for each cDNA sample on an ABI PRISM 7700 sequence detection system (Applied Biosystems), using SYBR ® green PCR master mix (Applied Biosystems) in a 20 µl reaction system. Samples were held for 10 min at 94˚C and then subjected to 40 cycles consisting of denaturation at 94˚C

for 5 sec and annealing/extension at 61˚C for 60 sec with a final extension step at 72˚C for 30 sec. Gapdh and Timm17b were used as references; all the primers are listed in the Table 13.

Cell biology Methods

All experiments with eukaryotic cells were conducted under a laminar flow bench, with sterilized glass or plastic materials; autoclaved or sterile-filtered solutions and media. The cells were cultured in humid incubator in 20% O 2 , 5% CO 2 at 37˚C.

Culture of cell lines

The C 2 C 12 cell line was purchased from ATCC and handled according to the ATCC protocol. When the cells reached 70~80% confluence, they were washed twice with PBS, treated with 0.05% Trypsin-EDTA for 5 min until most of cells had detached and then sub-cultured at a ratio of 1:10. Cells were changed to differentiation medium (DMEM supplemented with 10% horse serum) when they had reached over 90% confluence, and were grown further to achieve fusion into myotubes.

Human myoblasts were sub-cultured at a ratio of 1:6, when they had reached 70% confluence.

293T cells were grown in standard DMEM medium supplemented with 10% FBS.

When cells reached 80~90% confluence, the culture surface was rinsed once with PBS, trypsinized with 0.05% Trypsin-EDTA for less than 1 min and sub-cultured at ratios ranging between 1:8 to 1:10.

293T-D1Fc cells were grown in DMEM medium containing Hygromycin B and subcultured at ratios ranging from 1:8 to 1:10. When the cells reached >80% confluence, fresh DMEM medium without FBS and Hygromycin B was added to produce the conditioned medium. The conditioned medium was harvested after 4 days in culture [START_REF] Wang | Notch Receptor Activation Inhibits Oligodendrocyte Differentiation[END_REF].

Culture of murine myogenic progenitor cells

Isolation of murine myogenic progenitor cells. Muscles from wild-type C57BL6J (age 3-5 weeks) hind limbs were mechanically minced and enzymatically dissociated in 0.3 mg/ml NB4 collagenase (Serva) at 37˚C for 80 min with shaking. The mixture was filtered through serial 100 µm, 70 µm and 40 µm cell strainers prior to centrifugation (1,300 x g) for 10 min. Subsequently two different protocols were applied to iso-late murine MuSCs from the cell pellets: fluorescence-activated cell sorting (FACS)

as well as immune-magnetic cell sorting (MACS ® ).

The FACS protocol was adapted from Prof. Birchmeier's laboratory [START_REF] Brohl | Colonization of the satellite cell niche by skeletal muscle progenitor cells depends on Notch signals[END_REF]. After treatment with red blood cell lysis solution (Miltenyi), the pellet was washed once with staining buffer [20 mM HEPES, 2 mM EDTA, 1% w/v BSA, 100 µg/ml Gentamycin in PBS] and stained with appropriate cell-surface markers at 4˚C

for 15 min. The following materials were used: anti-goat VCAM, ImaGene Green (Gibco), PE-conjugated anti-CD31, PE-conjugated anti-CD45, PE-conjugated anti-Sca-1. After washing twice with staining buffer, cells were labeled with secondary Alexa488 antibodies at 4˚C for 45 min. PE-conjugated murine IgG (BD Pharmingen)

was used to control the staining process. Propidiumiodide was used to exclude the dead cells and side-scatter plots were used to exclude dead cells and debris from the histographic analysis. FACS-sorting was performed with a BD FACS Aria II machine at the Flow Cytometry Core Facility of the Deutsches Rheuma-Forschungszentrum supported by the staff of the core facility. Data were collected with the BD FACSDiva software (version 6.1.3) and saved as FCS 3.0 data files. The data analysis including sequential gating was performed using FlowJo v9.8 (Tree Star, USA). The two methods, FACS and MACS, are based on distinct principles in order to mechanically separate targeted cells from the total population. The FACS method suspends the cells in a fluid stream and passes them along an optic detection apparatus and eventually sorts one cell at a time based upon their specific light scattering and fluorescent features. By contrast, MACS separates targeted cells that are specifically marked with magnetic beads that bind to specific cell surface proteins. If passed through a strong magnetic field, cells labeled with magnetic beads are retained in the magnet while unwanted cells are gathered in the flow-through. This procedure can be conducted as positive or negative selection, depending on the specificity of the magnetic bead coupled antibodies used. Due to the comparatively low throughput of the FACS procedure, several successive runs have to be performed to obtain sufficient cells for the experiment. The bulk processing in the MACS procedure is easier, because many samples can be processed in parallel. Since both procedures rely on the interaction between conjugated antibody and various MuSCs cell surface markers, the intrinsic features of the sorted cells are more or less the same and only depend on the marker/antibody used.

Culture. Isolated satellite cells were seeded on either Dll1-Fc coated or IgG coated control surface (Figure 13) in satellite cell medium at 20, 000-50, 000 cells/cm 2 . The cultured cells from then on are referred to as myogenic progenitor cells (MPCs).

Notch signaling inhibition by DAPT

DAPT, a γ-secretase inhibitor, blocks the cleavage and activation of the intracellular Notch domain by inhibiting γ-secretase activity. Hence it has been widely used as a Notch signaling inhibitor in variety of experiments [START_REF] Sun | The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation[END_REF]. To suppress Notch signaling in Dll1-Fc cultured cells, 5 µg/ml DAPT (Sigma) were added to the culture medium, whereas DMSO [5 µg/ml] served as control.

Proliferation assays

In order to determine the growth characteristics of cells under various experimental conditions I used two different assays:

The MTT assay is a colorimetric assay for assessing cell viability based on the concept that the total activity of NAD(P)H-dependent cellular oxidoreductases may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4, 5-dimethylthiazol-2-yl)-2 plus 5diphenyltetrazolium bromide to insoluble formazan, which can be detected spectrophotometrically via its purple color. Hence the measured OD after a defined time period correlates with the amount of these enzymes. The MTT assay was performed with the Vybrant® MTT Cell Proliferation Kit (LifeTechnologies) according to the manufacturer's instructions. Culture plates were frozen at -80°C after incubation with MTT substance. After all samples had been collected they were defrozen, dissolved with SDS-HCl, and the OD was measured in a microplate reader (Thermo scientific) using the SkanIT ® software.

Using a different biological principle, the CellTiter-Glo ® Assay (Promega) assesses the cell number based on ATP quantification, which serves as an indicator of metabolically active cells. This assay was performed with CellTiter-Glo ® Luminescent Cell Viability Kit (Promega) and measured with the GloMax ® 96 Microplate Luminometer (Promega).

The Ki-67 protein is expressed during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0). As a result of its strict association with cell proliferation, the Ki-67 immunosignal is widely used for proliferation assays.

After fixation, an anti-Ki-67 antibody (1:200; Dako) was used for the standard immunofluorescence assay (see section 2.2.4.1). For image analysis, the percentage of Ki-67 positive cells over total cells (DAPI) in the same visual field was compared between the Dll1-Fc and control IgG surface.

Single muscle fiber culture

Single muscle fibers were enzymatically dissociated from the extensor digitorum longus muscle (EDL) of adult wild-type mice according to a classic protocol [START_REF] Rosenblatt | Culturing satellite cells from living single muscle fiber explants[END_REF]. The EDL muscle was carefully dissected through cutting the tendons at both ends and digested in 0.2% collagenase type | (Worthington) at 37˚C for 120 min with agitation. Isolation and purification of single muscle fibers was carried out by repeatedly flushing the muscle samples with a fine-mouthed Pasteur pipette under stereoscopy. Isolated intact fibers were selected, placed in a 5% BSA-coated 6 cm

Petri dish containing 5 ml of DMEM and maintained in a cell culture incubator for further procedures.

For floating fiber culture, single fibers were transferred into 5% BSA coated 6 cm Petri dishes and incubated in plating medium [DMEM supplemented with 10% v/v horse serum, 0.5% chick embryo extract (US Biological), 2% L-glutamine, 1% Pen-Strep]. After different culture periods, fibers were transferred into a 2 ml Eppendorf tube and fixed with 4% paraformaldehyde (PFA) for 10 min before being processed for DAPI and immunostainings.

For fiber-derived satellite cell culture, EDL fibers were individually plated on Matrigel ® (BD Bioscience)-coated Petri dishes. Fibers were incubated for 3 days in plating medium to allow cells to migrate from the fiber and attach to the Petri dish / Matrigel ® . If enough cells had migrated, the fibers were removed with a fine Pasteur pipette. The remaining attached cells were cultured for different lengths of time and fixed with PFA for immunostainings.

Live cell imaging

For live cell imaging, isolated fibers were individually plated on Matrigel ® coated glass-bottom µ-dish (ibidi GmbH). The dish was kept and monitored on an inverted Leica (DMI4000) microscope equipped with a 37˚C, 5% CO 2 humidified culture chamber (Pecon).

Biochemical methods

Affinity chromatography

Recombinant Dll1-Fc protein was purified from the conditioned medium of the stable cell line 293T-Dl1Fc [START_REF] Hicks | A secreted Delta1-Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling[END_REF]. Purification was done by affinity chromatography with the ÄKTA Purifier FPLC system (GE Healthcare) through binding of the Fc tag to a HiTrap ® Protein G column (GE Healthcare).

For that, the conditioned medium from 293T-D1Fc cells was centrifuged (1,300 x g)

for 10 min and filtered through a 0.45 µm PMSF filter to remove the debris. Subsequently the pH value of medium was adjusted to ≈7.0. The conditioned medium was then pumped with a flow rate of 1 ml/min over protein G column to allow maximal binding of the Fc fusion protein. After washing, bound protein was eluted with 0.1 M glycine-HCl (pH 2.7) and subsequently neutralized with 1 M Tris (pH 9.0). The whole process was controlled by Unicorn Software 5.11 (GE Healthcare). The fractions containing Dll1-Fc were identified by Western blot using an HRP conjugated anti-human Fc antibody.

Each production batch was separated by 10% SDS-PAGE and visualized via Coomassie staining. The concentration of the produced recombinant Dll1-Fc was determined by the BCA-protein assay using a Helios ® photometer (Thermo Fisher Scientific). Sample were loaded and run in 10% SDS-PAGE gel.

Surface coating for cell culture

Culture dishes were coated with 1 ml/cm 2 of a mixture containing 3 ng/ml Fibronectin (Sigma) in PBS plus either Dll1-Fc [3 ng/ml] or IgG [3 ng/ml] (Sigma) and incubated overnight at 4˚C. The coated surfaces were then washed five times with PBS and blocked with 2% BSA at 37˚C for 1 hour followed by thorough rinsing with PBS.

Western blot

Total protein was extracted from cells or muscle tissue using lysis buffer mixed with a cocktail of proteinase inhibitors (Table 7). After clearing insoluble debris by high speed centrifugation (11,000 x g, 10 min at 4˚C), protein concentration was determined by the BCA-protein assay. Lysates were adjusted with loading buffer, denatured at 95˚C for 5 min followed by 2 min chill-down on ice. Each lysate was loaded onto a 10% or 12% SDS-PAGE gel, electrophoresed and then transferred onto nitrocellulose membranes (Whatman) using transfer buffer and a Fastblot ® apparatus (Biometra) at 2 mA per cm 2 for 150 min. Blots were blocked in 5% milk-TBS for 1 hour, washed once with TBS, followed by incubation with the primary antibody overnight at 4°C. After a triple wash with TBS, the blots were incubated for 2 hours with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibodies (1:2,000 dilution; Calbiochem). After a triple wash with TBS, the protein bands were visualized using an enhanced chemiluminescence (ECL) reaction mixture for 5 min at room temperature, and exposed to an X-ray film in the dark room.

Primary antibodies used in this assay were anti-GAPDH (1:10,000 dilution; Abcam), anti-Flag M2 (1:1,000 dilution; Sigma); anti-Dll1 (1:1,000 dilution; Santa Cruz), and anti-Tubulin (1:10,000; Invitrogen). Tubulin and GAPDH bands were used as loading controls. The bands were quantified by measuring their integrated density within a rectangle that covered the entire individual band and subtracting the integrated density of an empty rectangle of exactly the same size in the vicinity using the Image J software.

Histological methods

Immunofluorescent staining of cultured cells

Cells were grown on glass cover slips. At the desired time point they were fixed in 4%

PFA in PBS for 10 min at room temperature (RT), rinsed with PBS twice and permeabilized with 0.2% TritonX-100/PBS for 10 min. Cover slips were then incubated with primary antibodies in PBS at 4˚C overnight, washed twice with PBS and then incubated with secondary fluorescent antibodies for 60 min at RT. 4,6-diamidino-2phenylindole (DAPI) staining was done to visualize the nuclei prior to mounting the cover slips on glass slides with Mowiol ® to prevent photo bleaching. Primary and secondary antibodies used are listed in Table 4.

Immunofluorescent staining of single fibers

Isolated single fibers were collected in a 2 ml round bottom Eppendorf tube. For each washing step, the tube was kept standing for 8-10 min to allow all fibers to settledown to the bottom. The liquid was carefully removed with a 1 ml pipette in multiple steps until only ≈0.5 ml remained. After washing with PBS twice, fibers were permeabilized with 0.5% TritonX-100/PBS for 8 min. Primary antibodies were incubated in PBS at 4˚C overnight, washed twice with PBS and incubated with secondary fluorescent antibodies for 60 min at RT. After DAPI staining and two PBS washes, the fibers were poured onto a glass slide, where excessive PBS was carefully removed prior to mounting on Mowiol ® .

Immunofluorescent staining of muscle cryosections

The muscles were harvested, embedded in optimum cutting temperature (O.C.T.) compound (Tissue-Tek ® , Electron Microscopy Sciences) and frozen in liquid nitrogen cooled isopentane (Merck). Muscles were cut into 10 µm sections on a cryostat and fixed to poly-L-lysine coated slides. After fixation with 4% PFA and two rinses with PBS, muscle sections were permeabilized in methanol for 6 min at -20˚C. In order to prevent unspecific staining, sections were blocked in 10% normal goat serum in PBS for 60 min and in anti-mouse F ab fragments (1:50 dilution) in PBS for 30 min. This was followed by incubation with primary antibodies (e.g. anti-Pax7, anti-Laminin, anti-BrdU etc.) at 4˚C overnight and with secondary antibodies at RT for 60 min. After 5 min DAPI staining the sections were washed with PBS and mounted with Mowiol ® .

Muscle integrity and the presence of inflammatory cells were evaluated by Haematoxylin-Eosin (H&E) staining and subsequent light microscopic inspection.

BrdU incorporation assay. BrdU (5-bromo-2'-deoxyuridine), a nucleotide analog of thymidine can be incorporated into DNA during active DNA synthesis. The BrdU as-say is hence widely used to directly estimate active DNA synthesis or S-phase synthesis during the cell cycle. Growing cells were incubated with BrdU [100 µM] 3 h prior to fixation; living animals were i.p. injected with BrdU for different lengths of time prior to sacrifice. After permeabilization samples were treated with 2 M HCI for 30 min, washed with PBS, followed by 10 min incubation with 0.1 M sodium borate (pH 8.5) in order to expose the incorporated BrdU for detection. Samples were then washed twice with PBS and subjected to immunofluorescent staining with anti-BrdU antibodies as described above.

Haematoxylin and Eosin staining

The cryo-slides were thawed and hydrated in PBS for 2 min, stained with Haematoxylin solution for 5 min, rinsed, and incubated with Eosin (1% w/v in water) for about 1 min and washed again. Water was then removed by a serial ethanol bath (30%, 50%, 70%, 85%, and 100%). Finally, slides were incubated twice in Xylene for 10 min and mounted in Vecta ® Mount medium.

Imaging acquisition and analysis

Microscopic images were acquired using the microscope software Image-Pro Plus ® 7.0 (Media Cybernetics) and a cooled b/w CCD camera (RT3 SPOT, Visitron Systems) mounted on a Leica DMI4000 system equipped with a spinning disk confocal scan head (Carv II, BD Bioscience). All image-related works were either performed with the Image-Pro Plus ® 7.0 or with the ImageJ software.

Transgenic animal experiments

rAAV production

Full-length Dll1 was inserted into the AAV2/1 backbone vector pSMD2. Recombinant adeno-associated virus (rAAV) was generated using the classic three-plasmid transfection protocol [START_REF] Grimm | Novel tools for production and purification of recombinant adenoassociated virus vectors[END_REF][START_REF] Xiao | Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus[END_REF]. Briefly, human embryonic kidney 293T (HEK293T) cells were triple-transfected using polyethylenimine transfection reagent with adenovirus helper plasmid pXX6 [START_REF] Xiao | Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus[END_REF], a pAAV packaging plasmid expressing the rep and cap genes encoding the serotype 1 capsid (from Penn Vector Core, University of Pennsylvania, USA), and a pAAV cis-plasmid vector, pSMD2-Dll1, encoding full length murine Dll1 under the transcriptional control of the Cytomegalovirus (CMV) promoter and flanked by two inverted terminal repeats (ITR) originating from AAV2. rAAV was released from cells 72 h after transfection by repetitive freeze-thaw cycles and purified by ultracentrifugation (500,000 x g) through iodixanol isopycnic density gradients [START_REF] Zolotukhin | Recombinant adenoassociated virus purification using novel methods improves infectious titer and yield[END_REF], followed by diafiltration through an Amicon 15 ultracentrifugation unit with 100 kDa molecular weight cut off (Millipore). Titration of viral particles was done by quantitative PCR as described (Rivière et al, 2006) and expressed as viral genomes per milliliter (vg/ml).

Intramuscular viral vector delivery

3-week-old female wild-type and mdx mice were anesthetized through injection of 100 mg/kg BW ketamine and of 10 mg/kg BW xylazine. Tibialis anterior muscles were injected with 40 µl of rAAV-Dll1 into the left side and with rAAV-U7-scramble into the right side as control. The viral dosage used for each injection was >10 10 vg.

3-days-old newborn wild-type mice were injected with 3 µl of rAAV-Dll1 into the left calf and with rAAV-U7-scramble into the right calf.

Prior to sacrifice, BrdU (Invitrogen) was injected intraperitoneally at a dosage of 1 ml per 100 g body weight daily for 3 days. Tibialis anterior muscles were frozen in liquid nitrogen cooled isopentane and processed for histological investigation. Extensor digitorum longus (EDL) muscles were used for single fiber isolation.

Determination of transduction efficiency

Muscle cryo-sections were collected from both injected sides for RNA extraction, cDNA reverse transcription, and subsequent qPCR using a standard SYBR green ® protocol on an ABI PRISM 7700 Sequence Detection system. For qPCR we used a primer pair, which specifically anneals to the exogenous Dll1Flag gene. This was used in parallel with two other primer pairs that detect Dll1 cDNA.

Statistical analysis

Typically the data were stored or directly exported from the experimental instrument into an Excel sheet. The GraphPad Prism ® software was used for statistical computa-

Results

Fiber-associated satellite cell culture

As satellite cells are located in their niche and are attached to the muscle fiber, I started my experiments to isolate single muscle fibers from mouse EDL muscle using an enzymatic digestion and trituration protocol. The features of the EDL fibers vary, with a length between 2-5 mm; 200 to over 300 myonuclei, and 0-14 satellite cells per fiber (Figure 15C).

In the floating fiber culture, the fibers were cultured in medium without attachment to the culture dish. Typically 6-8 h after a single fiber was placed in the culture medium, the satellite cells exited from their quiescent state and began to be activated, which could be monitored via life cell imaging by their active movement along the fiber. Typically the first cell division occurred after 24 h in culture; both symmetric and asymmetric cell divisions have been observed (Figure 16). lite cells on Matrigel ® move as well (Figure 18). Interestingly, one cell division of a satellite cell in the culture dish takes a much shorter time as compared to their fiberassociated counterparts (30 min in Figure 18 versus 60 min and 90 min in Figure16).

As a consequence, detached satellite cells in the culture dish produced larger numbers of daughter cells.

In summary, in this section I have isolated single muscle fibers in order to culture fiber-associated satellite cells. Those fibers were either cultured free-floating in medium or attached to a cell culture dish coated with Matrigel ® matrix. The features and velocity of cell division were examined in detail under both culture conditions.

Recombinant Dll1-Fc protein production

The recombinant Dll1-Fc protein, in which the extracellular Delta-like-1 (Dll1) domain is fused to the human Fc-domain (Figure 19), was produced at larger scale for subsequent experiments. Dll1-Fc protein was obtained from a stable cell line 293T-Dl1Fc and purified by Affinity chromatography using G-protein columns. Each batch was investigated and documented separately (Table 10).

The identity and molecular size of the Dll1-Fc protein was confirmed using an antihuman-Fc antibody on Western blot. The molecular weight of Dll1-Fc is ≈90 kDa (Figure 19).

The biological effect of Dll1-Fc on cultured myoblasts

In order to confirm the biological effect of our recombinant Dll1-Fc protein, we immobilized Dll1-Fc on the culture dish surface and tested its effect on cultured murine and human myoblasts. The rate of cell proliferation was assessed through Ki-67/DAPI staining. Since the growth rates between human and murine myoblasts differ, we allowed 2 days for the 'short culture period' and 4 days for the 'long culture period' in murine myoblasts while we allowed 4 days and 6 days in human myoblasts, respectively. In murine C 2 C 12 myoblasts, we observed comparable levels of proliferating cells on the Dll1-Fc surface as compared to the control surface after 2 and 4 days of culture.

In human myoblasts, the proliferation rates of both culture surfaces were at comparable levels, measured both by Ki-67 positivity and by BrdU incorporation.

Total RNA was extracted from the cells of all experimental groups; cDNA synthesis and qPCR experiments were performed simultaneously. As expected, known Notch target genes were expressed at higher levels in cells cultured on the Dll1-Fc surface as compared to cells on the control IgG surface, which applied for both murine and human myoblasts. Amongst the several Notch target genes, in human myoblasts HES1 appeared to be most sensitive upon Dll1-mediated stimulation (increase of 1,500±13% at day 4 and of 4,000±16% on day 6), whereas in murine myoblasts Hey2 appeared to be most sensitive (increase of >2,000% on day 2 and day 4).

In addition to Notch target genes, we also examined the expression levels of genes related to myogenesis. For human myoblasts cultured on the Dll1-Fc surface, expression levels of MYOD and MYOGENIN were constantly lower as compared to those on the control surface, whereas MYF5, an early marker of myogenesis, did not exhibit such difference. In murine myoblasts, Myf5 is detected at higher levels (157±25% at day 2 and 666±47% at day 4) when Dll1-Fc is present. However, the expression level of Myogenin (MyoG) on Dll1-Fc surface was lower on day 2 and higher on day 4 as compared to the counterparts growing on the IgG surface.

In order to investigate the Myogenin expression in more detail, murine myoblast cultures were examined with immunofluorescent staining: tiled images from cells cultured on both surfaces were acquired and counted. With high Notch activity stimulated by Dll1, we found less than 5% myogenin-positive cells on the Dll1-Fc surface, while on the control surface more than 15% of cells were Myogenin positive.

The effect of Dll1-Fc on muscle progenitor cells (MPCs)

Once the biological effect of our recombinant Dll1-Fc was confirmed, we proceeded to check its effect on ex vivo cultured muscle stem cells, also termed muscle progenitor cells (MPCs).

Dll1-Fc impairs proliferation of MPCs

In contrast to the effect of Dll1-Fc on myoblasts, we noticed a reduced growth rate of

Dll1-Fc promotes Pax7 expression in MPCs

Although growth on the Dll1-Fc surface impaired MPC proliferation, it increased Pax7-positivity both in α7-integrin as well as VCAM sorted cells. In order to confirm the the effect of Dll1 stimulation would be indeed the consequence of increased Notch signaling, we added DAPT, a Notch inhibitor, to the culture medium. In the presence of DAPT, the percentage of Pax7 + cells on the Dll1-Fc surface dropped from 84.8±5.0% to 49.8±5.8%, whereas the percentage on the control surface changed from 56.5±6.1% to 54.8±5.9%. Hence, the addition of DAPT decreased the Left panel: Numbers of muscle progenitor cells per cm 2 culture dish surface. Freshly isolated cells were seeded at identical densities on the Dll1-Fc or IgG surface. Three biological replicates were prepared, and cell numbers were investigated after 2 and 4 days. The p-value (p=0.003) was calculated via MANOVA with repeated measurements. Between days 5 and 6 cells began to fuse and to form myotubes; therefore counting of single cells by the used method became unreliable beyond day 4.

Right panel:

The proliferation rate of MPCs as determined by the percent of positive staining for BrdU. Three biological replicates were prepared; at least three random visual field images were acquired from each culture plate; the p-values were calculated with Mann-Whitney U test). percentage of Pax7+ cells on both surfaces to the comparable levels (51.8±2.5% versus 55.8±3.0%; p=0.85, n=3 experiments). Furthermore, the number of cells per surface area, hence the growth rate, were similar between Dll1-Fc and IgG surface in the presence of DAPT (p=0.350) We further investigated the distribution of two different subpopulations of Pax7 + cells: Pax7+|MyoD-cells corresponding to quiescent MuSCs, while Pax7+|MyoD+ cells corresponding to MPCs committed to proliferation and differentiation [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF]. As described above, Dll1-Fc impairs proliferation of MPCs thus fewer cells were growing in the presence of Dll1. However, the percentage of cells with higher stemness potential largely increased (Figure 25C). If the cells were cultured for a prolonged period of time, MPCs kept proliferating, hence the total cell number increased while the percentage of Pax7 + cells decreased (at day 6: 53.3±5.6% on Dll1-Fc surface versus 35.6±6.5% on control surface; p=0.057, n=3 experiments, Figure 26B). Phase contrast microscopy revealed different morphologies of those long-time cultured MPCs depending on the surface coating: MPCs growing on the Dll1-Fc surface were able to preserve a more roundish -was significantly higher on the Dll1-Fc surfaces (60.9±6.7% versus 21.3±7.2%; p<0.001, one-way ANOVA test) shape and did not fuse so easily as compared to MPCs growing on the IgG surface.

In the presence of DAPT cells on both surfaces elongated, fused and formed myotubes (Figure 26A).

In parallel, I quantified the expression levels of Notch target genes as well as of genes related to the myogenic differentiation by real time qPCR. In comparison to the IgG control surface, the Dll1-Fc surface was more capable to stimulate expression of Notch target genes as well as of the stemness marker Pax7 (≈3-4 fold). However, after 2 weeks of in vitro growth, such differences in Pax7 expression levels disappeared despite persisting high expression levels of Notch target genes.

Transduction efficiency

After intramuscular injection of rAAV, we harvested the muscles from both sides and examined the transduction efficiencies on the mRNA level. A specific primer pair binding only to the exogenous Dll1Flag gene showed exogenous Dll1 to be over expressed by 60 fold. Two other primer pairs, which annealed to the wild-type murine Dll1 cDNA, also demonstrated higher Dll1-mRN expression levels (40-fold, 76-fold) in the rAAV injected muscle as compared to contralateral control muscles.

Figure 29: Expression levels of the Dll1 transgene examined by RT-qPCR

Gapdh mRNA levels were used as reference. Three muscle samples were collected and each mRNA-quantification was performed with three technical replicates, error bar=SD. Relative expression levels were normalized to the expression levels of the muscles that had been injected with AAV2/1-U7-scramble.

The effect of Notch overstimulation on postnatal muscle growth

In order investigate the effect of Notch overstimulation during the immediate postnatal period of muscle growth, we injected rAAV2/1-Dll1 into muscles of newborn mice and investigated the effect after 2, 3, and 6 weeks (Figure 14A).

Dll1 overexpression does not lead to pathologic changes in the muscle

Although we observed some myofibers with centralized nuclei (as a possible reaction to the needle injection), the muscles from both injected sides did not show any further pathologic features on HE staining. The muscle tissue was intact with normal distribution of connective tissue, polygonally shaped myofibers with peripheral nuclei, intact sarcolemma, unfragmented sarcoplasm and the absence of muscle fiber atrophy.

Dll1 impairs postnatal growth of muscle fibers

In the mice dissected on P17, 2 weeks after Dll1 treatment, the muscle mass of the Dll1 treated side and scramble side did not exhibit any significant differences. Furthermore, there was no significant difference of the fiber size (minimum Feret's diameter) distribution in the histograms of muscles from both sides. While after 2 weeks of treatment no differences in fiber size and muscle weight were observed, significant differences appeared after 3 and 6 weeks of treatment. The mass of Dll1-overexpressing TA muscles was significantly reduced as compared to control muscles (Figure 32A,B). For EDL muscles, a significant difference was only observed after 6, but not after 3 weeks of treatment (Figure 32C,D). Histograms of the fiber diameters revealed a shift towards smaller fiber sizes after 3 (Figure 32C) and 6 weeks of Dll1 exposure (Figure 32D). The effect was more pronounced after 6 weeks of treatment.

Dll1 overexpression does not affect the number of myofibers

In parallel to the analysis of fiber diameters, I also quantified the total number of myofibers per muscle cross section in the tibialis anterior (TA) muscle of the rAAV-Dll1 treated and control side, but did not find any significant differences in both the 3 and 6 weeks treatment groups.

Dll1 overexpression does not affect the accretion of myonuclei into the muscle fiber

Further, I quantified the total number of myonuclei per EDL fiber in order to investigate whether the fusion of the MuSCs with their myofiber, also called 'myonuclear accretion', would be affected by Dll1 overexpression. As detailed in Figure 34, these numbers did not differ between Dll1 overexpressing and control muscles thus verifying that the fusion of the activated myoblasts with their respective myofiber would not be affected by Dll1 overexpression on the myofiber. The number of fibers per TA section from newborns with 3-week treatment (A) and from newborns with 6-week treatment (B). TA sections were acquired from 4 animals for the measurement; each TA muscle was counted 2-4 x times at different levels. One dot represents a number that was counted in one entire TA cross section; the red line depicts the median. There is no significant difference between two sides (the p-value was calculated using the non-parametric Mann-Whitney U test). + MuSCs were present in Dll1overexpressing muscle after 3 weeks of treatment, an effect not seen after 6 weeks of treatment any more; p-values were calculated with the one-tailed Wilcoxon matched-pairs signed rank tests, because we already knew rAAV-Dll1 treated muscles to have significant lower weights. Four muscles from each side were used for analysis.

The effect of Dll1-overexpression during muscle regeneration

In order to gauge the effect of Dll1-overexpression on muscle regeneration, we chose a genetic model, the mdx mouse model of Duchenne Muscular Dystrophy (DMD), which is known to exhibit a highly accelerated degeneration/ regeneration cycle in the early phase of disease. 3-week-old mdx and wild-type mice were injected with rAAV-Dll1 and rAAV-scramble vector (as control) and assessed 4 weeks later (Figure 14B).

Overexpression of Dll1 does not induce or alter the histopathological features in adult muscle

Overexpression of Dll1 in wild-type adult muscle did not cause obvious histopathological changes. On the other side, both Dll1-overexpressing as well as control muscles of mdx mice displayed typical dystrophic features such as myofibers with central nuclei, an increase of small-sized myofibers, fiber splitting, and an increase in connective tissue (Figure 36).

Dll1-overexpression increases the stem cell pool without affecting the regeneration process

While we did not seen any difference in the number of Pax7 + cells between rAAV-Dll1 treated and control muscle in wild-type mice, the percentage of Pax7 + cells was significantly higher in rAAV-Dll1 treated mdx muscle as compared to controls (Figures 38,39). This hints to the fact that not only during development, but also in states + nuclei in Dll1-overexpressing mdx muscles was significantly increased, while such difference was not seen in wild-type muscle (the p-values were calculated with the Kruskal-Wallis test) (C) The percentage of BrdU + nuclei in the stem cell pool of mdx mice. Each dot represents one muscle; Pax7 + cells were counted from ≈30 EDL fibers (the p value was calculated with the one-tailed Wilcoxon matched-pairs signed rank tests, since we already knew rAAV-Dll1 treated muscles had significant lower weights. (D) The percentage of regenerated fibers with central nuclei was not significantly different in mdx muscles of the rAAV-Dll1 treated versus the control side. Each dot represents the percentage from one muscle cross section. Four cross sections of each mouse were analyzed (the p-value was calculated with the Kruskal-Wallis test).

Discussion

In

5.1

Immobilization of Dll1-Fc and its effect in myoblasts.

The recombinant production of the secreted extracellular domain of proteins from the Delta/Serrate/Lag2 (DSL) family has provided a powerful tool to study Notch signaling in various models [START_REF] Han | A soluble form of human Delta-like-1 inhibits differentiation of hematopoietic progenitor cells[END_REF][START_REF] Hicks | A secreted Delta1-Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling[END_REF]. However, conflicting evidence has been obtained regarding the function of soluble DSL ligands. On one hand they may suppress differentiation in hematopoietic precursors [START_REF] Han | A soluble form of human Delta-like-1 inhibits differentiation of hematopoietic progenitor cells[END_REF], while failing to repress differentiation of myoblasts [START_REF] Varnum-Finney | Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling[END_REF].

Different mechanisms were proposed to explain the molecular basis for such antagonistic and agonistic effect of soluble DSL ligands. Varnum-Finney et al. demonstrated that only immobilized Dll1 ligand was capable to stimulate Notch signaling in myoblasts, whereas the soluble ligand was unable to do so [START_REF] Varnum-Finney | Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling[END_REF].

Hence they stipulated the Dll1 ligand to be immobilized in order to stimulate Notch signaling [START_REF] Varnum-Finney | Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling[END_REF]. Along these lines, Hicks et al. demonstrated that only an artificially clustered form of the ligand, but not its free form was capable to induce Notch signaling [START_REF] Hicks | A secreted Delta1-Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling[END_REF]. They proposed that multimerization of ligands within the plasma membrane might be an important event in ligand-induced Notch signaling. Hence, oligomerization of ligands is required for biological activity [START_REF] Hicks | A secreted Delta1-Fc fusion protein functions both as an activator and inhibitor of Notch1 signaling[END_REF].

More insight was eventually provided by Sprinzak et al. who discovered, that Notch receptors show a strikingly different response to trans or cis ligands [START_REF] Sprinzak | Cis-interactions between Notch and Delta generate mutually exclusive signalling states[END_REF]. As soluble ligands might activate the receptors via transor cis-interaction, their effect is unpredictable, or opposite effects might antagonize each other. In order to achieve consistent trans-activation it is a prerequisite to immobilize the extracellular ligand domain. We therefore immobilized the Dll1-Fc protein for our experiments via Fibronectin at the culture dish surface.

Indeed we were able to see a substantial upregulation of Notch downstream target genes such as Hes1 and HeyL in cultured human and murine myoblasts (Figure 21).

Due to the different growth dynamics we investigated murine myoblasts at day 2 and 4 and human myoblasts at 4 and 6 days of culture. The upregulation of Hes1 and HeyL was sustained over several days. These results clearly demonstrate that our experimental system of Dll1-Fc immobilization was biologically active and was functioning as intended.

Next we wanted to investigate the effect of Notch signaling on the (i) differentiation and (ii) proliferation of human and murine myoblasts.

Dll1-Fc stimulation suppresses myoblast differentiation.

As the degree of myogenic differentiation of myoblasts can be estimated by their expression of certain maker proteins (Figure 2) we first chose Myogenin as a comparatively late marker of myogenic differentiation. As seen on Figure 21, Myogenin mRNA expression is suppressed at the early time point of culture on the Dll1 surface (2 days in murine, 4 days in human myoblast cultures). This effect is maintained also until the later time point (6 days) in human myoblast culture. However, in murine myoblasts Myogenin-mRNA expression at day 4 even surpassed the mRNA expression levels on the control surface. Hence we investigated in parallel Myogenin expression on the protein level by immunostaining and still found a significantly lower number of myoblasts expressing Myogenin on the Dll1-Fc surface as compared to the control IgG surface (Figure 22B) at day 4. The discrepant results between immunostaining and qPCR might be explained by the fact that the murine myoblasts at day 4 had already started to fuse. In this case a low number of myotubes in advanced differentiation with a very high Myogenin mRNA expression might have significantly increased the average mRNA expression levels. Since the qPCR method only detects average mRNA expression levels in a pool of millions of cells, immunostaining is more reliable to gauge the effect of Dll1 on the single cell level.

We hence were able with our experimental system to confirm the findings of other researchers, who described an inhibitory effect of Notch signaling on myogenic differentiation [START_REF] Buas | The Notch effector Hey1 associates with myogenic target genes to repress myogenesis[END_REF][START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF][START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF][START_REF] Sun | The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation[END_REF][START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF]. The constitutive expression of an active form of Notch1 in myoblasts [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF][START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF] or co-culture with cells overexpressing the Notch ligand Jagged [START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF] led to cell expansion through blocking myogenic differentiation. Such inhibitory effect on myogenic differentiation might have been achieved (i) through preventing the binding of Mef2C (Myocyte-specific enhancer factor 2C) to the DNA, (ii) by suppressing Myogenin promoter activity via Hey1 [START_REF] Buas | The Notch effector Hey1 associates with myogenic target genes to repress myogenesis[END_REF], (iii) by directly suppressing MyoD expression [START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF], or (iv) by reducing the MyoD activity on gene transcription [START_REF] Reynaud | Stabilization of MyoD by Direct Binding to p57Kip2[END_REF] via HES1 mediated downregulation of p57 expression [START_REF] Zalc | Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest[END_REF].

5.1.2

The effect of Dll1-Fc on myoblast proliferation.

Notch over-stimulation via Dll1-Fc in cultured murine and human myoblasts did not significantly alter their proliferation rates, as investigated by BrdU and Ki67 staining.

The overall percentage of BrdU+ cells in human myoblasts was lower than in murine myoblasts, which is consistent with our observation that the growth rate of human myoblasts is lower.

Our findings differ from a previous report, which demonstrated that overexpression of a constitutive active form of Notch1 increased myoblast proliferation, while coexpression of Numb, a negative regulator of the Notch pathway, abrogated this effect [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF]. Such deviating results might be explained by the magnitude of Notch signaling brought about by different experimental conditions. The expression of a constitutive active form of Notch1 constitutes the maximum "stimulus" available since it is independent of the binding of Notch ligands to its receptor. In our experimental system we stimulated the Notch-receptors on the myoblast surface.

Since it is known that differentiating myogenic cells already express Dll1 at their surface [START_REF] Mourikis | Distinct contextual roles for Notch signalling in skeletal muscle stem cells[END_REF] they might have inactivated their own Notch receptors through cis-inhibition (Figure 7) [START_REF] Sprinzak | Cis-interactions between Notch and Delta generate mutually exclusive signalling states[END_REF] thus rendering them insensitive to external cues.

In this section, we confirmed our recombinant Dll1-Fc protein to effectively stimulate Notch activity in cultured murine and human myoblasts. Consistent with previously published data [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF][START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF], we demonstrated that Dll1-stimulated Notch suppresses myogenic differentiation in cultured myoblasts.

5.2

The ex vivo cultivation of muscle stem cells.

The capability of muscle stem cells to self-renew and to differentiate qualifies them as ideal candidates for cell-based therapies. In contrast, committed myoblasts have yielded disappointing results in the past because they did not repopulate the stem cell niche (Huard, Bouchard, et al., 1992;Huard, Roy, et al., 1992;[START_REF] Mendell | Myoblast transfer in the treatment of Duchenne's muscular dystrophy[END_REF][START_REF] Morandi | Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer[END_REF]. As alternative for myoblast-based therapies, muscle stem cells could be used, which have the capability to relocate into the stem cell niche, proliferate, contribute to muscle regeneration, and finally reach quiescent state [START_REF] Collins | Isolation and grafting of single muscle fibres[END_REF][START_REF] Ehrhardt | Human muscle precursor cells give rise to functional satellite cells in vivo[END_REF][START_REF] Sacco | Self-renewal and expansion of single transplanted muscle stem cells[END_REF].

A great disadvantage of muscle stem cells, however, is the fact that freshly isolated donor-derived muscle stem cells rapidly lose their 'stemness' potential (as indicated by Pax7-positivity) during in vitro culture and thus cannot be sufficiently multiplied ex vivo. Hence, the main obstacles of MuSCs in cell-based therapies would be the problem to generate the large numbers of autologous cells extra corporally, which would be needed to treat an entire human body. Therefore, a culture method would be very welcome, which stimulates cell division of MuSCs while preserving their myogenic stem cell potential.

We investigated whether Notch stimulation through Delta-like 1 (Dll1) might contribute to the in vitro maintenance of stemness of muscle progenitor cells through cultivation on surface-immobilized Dll1-Fc. Since MuSCs have to be separated from a pool of heterogeneous embryonic cells (e.g. mainly from mouse embryonic fibroblasts) [START_REF] Collins | Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche[END_REF], we made use two well-accepted cell surface markers (VCAM and α7-Integrin) for MuSCs isolation via either Fluorescent (FACS) or Magnetic Cell Sorting (MACS) techniques.

Notch stimulation impairs proliferation of cultured MPCs

As described above, we have observed that Notch overstimulation through Dll1 does not affect the proliferation later stage of myoblasts. However, in contrast to these findings, Notch stimulation in cultured early myogenic precursor cells (MPCs) largely reduced their proliferation rate (Figure 23).

To explain the effect of Notch signaling on the proliferation rate we have to consider its effect(s) on the control of cell cycle progression. Two regulatory protein complexes, which act in opposite directions, determine cell cycle progression. Cyclin and cyclin dependent kinases (CDKs) promote cell cycle progression, while the CDK inhibitors (CDKI) repress cell cycle progression. The Notch signaling pathway might interfere with the Cyclin pathway via Hes1, which is able to suppress the expression of CDKIs, such as p27 Kip1 [START_REF] Monahan | The notch target gene HES1 regulates cell cycle inhibitor expression in the developing pituitary[END_REF], p57 Kip2 [START_REF] Riccio | Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2[END_REF]. Alternatively, since it is known that MyoD directly activates p21 Cip1 [START_REF] Halevy | Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD[END_REF] and p57 Kip2 [START_REF] Zalc | Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest[END_REF], while Notch directly suppresses MyoD expression [START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF], the Notch influence on the Cyclin pathway might also be exerted through more complex signaling loops.

We hypothesize that such various, sometimes even antagonistic effects of Notchligands depend on (i) the context dependent state of myogenic differentiation of the muscle progenitor cell (e.g. muscle stem cell versus differentiating myoblasts) which receive the signal or (ii) on the preexisting levels of the active Notch-receptor Intracellular Domain (NICD).

Myoblasts are committed cells that are prone to fuse into myotubes and express myogenic regulatory factors (e.g. Myf5, MyoD) at very high levels (Figure 2), which cannot be suppressed by Notch any more. In this case, high Notch activity in myoblasts may predominantly suppress their differentiation [START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF][START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF][START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF], which keeps the myoblasts in proliferative state for a longer time thus increasing the absolute number of cells.

In contrast to proliferating myoblasts, our freshly isolated muscle stem cells were to a large extent still in quiescent state (Pax7+|Myf5-|MyoD-) in vivo (Figure 2). At this stage Notch can still be effective to suppress or delay MyoD expression [START_REF] Shawber | Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway[END_REF] thereby preventing exit from quiescence [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF] and entry into the proliferation / differentiation program. In this case the Notch-overstimulated MPCs would hence not proliferate effectively.

Notch stimulation via Dll1-Fc preserves stemness in cultured

MPCs.

Notch stimulation increased the percentage of Pax7+|MyoD-cells to ≈60% after 4 days in culture, whereas only ≈20% of such cells were seen on the control surface (Figure 25). The distinction between Pax7+|MyoD-and Pax7+|MyoD+ cells is important, because only Pax7+|MyoD-cells constitute the genuine muscle stem cell pool. Pax7+|MyoD+ cells have already been primed to exit quiescence and enter into a proliferation / differentiation program in order to become proliferating myoblasts [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF]. The Dll1-Fc mediated stimulation of the Notch-signaling pathway was again verified through detection of upregulated downstream Notch target genes Hes1, HeyL and Hey2 (Figure 27). The specificity of the effect was further confirmed by application of DAPT, which is a Notch inhibitor that blocks the activation of intracellular Notch by inhibiting γ-secretase activity [START_REF] Sun | The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation[END_REF]. Since DAPT suppresses all Notch activity (e.g. the naturally occurring as well as the surplus activity via stimulation with Dll1-Fc) the effect on the maintenance of the Pax7+|MyoDstate was abolished as no significant differences were observed between the Dll1-Fc and IgG coated surfaces (Figure 25B).

The main findings from this investigation are in agreement with previous reports, which suggest that Dll1 has an inhibitory effect on myogenic differentiation [START_REF] Sun | The role of Delta-like 1 shedding in muscle cell self-renewal and differentiation[END_REF][START_REF] Varnum-Finney | Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling[END_REF] and enriches the expression of early myogenic markers such as Pax7 [START_REF] Parker | Activation of Notch signaling during ex vivo expansion maintains donor muscle cell engraftment[END_REF]. In the current work, we demonstrated that Notch stimulation via Dll1 ligand inhibited the exit of quiescence and entry to proliferation of ex vivo cultured MuSCs. The similar effects of enhance Notchsignaling were reported in targeted NICD overexpression in MuSCs in vivo [START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF]. Since Notch signaling plays an important role in maintaining stem cells in quiescence [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF]Mourikis, Sambasivan, et al., 2012), it is possible that early Notch simulation prevents cells from proliferation and entering into the myogenic program via down-regulation of the cyclin kinase inhibitor (CKI) p57 kip2

through the Notch target genes Hes1/Hey1 [START_REF] Zalc | Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest[END_REF]. Dll1-induced high Notch activity, are unable to exit from quiescence [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF] and hence do not proliferate effectively. The positive effect of keeping the cells in stemness state is thwarted by their largely reduced proliferation rate. Only those cells that escape this quiescence become Pax7+|MyoD+ myoblasts, rapidly proliferate, and ultimately overtake the culture after some days. However, as already shown previously, such myoblasts are unable to populate the stem cell niche (Huard, Bou-chard, et al., 1992;Huard, Roy, et al., 1992;[START_REF] Mendell | Myoblast transfer in the treatment of Duchenne's muscular dystrophy[END_REF][START_REF] Morandi | Lack of mRNA and dystrophin expression in DMD patients three months after myoblast transfer[END_REF].

More investigations have to be done to determine, whether modifications of Dll1 dosage, seeding density of the cells and time in culture might be able to tip the balance towards a cell population that expands while maintaining the capability to contribute to muscle repair.

In life cell imaging experiments (Figure 16, Figure 18) we observed that muscle stem cells which were still associated with the myofiber exhibited different dynamics of cell division and maintenance of Pax7 positivity as compared to their counterparts grown on Matrigel ® . The first asymmetric or symmetric division of myofiber-associated muscle stem cells normally occurred at ≈24 h after isolation and took ≈60 minutes to complete. Conversely, cells seeded directly on Matrigel ® started to proliferate soon after seeding and a cell division took only ≈40 minutes. Due to the limited number of observations we still cannot conclude that we deal with a general principle. However, it hints towards other factors in the stem cell niche that influence the dynamics of cell division.

It would thus be important to identify further components and cues of the stem cell niche with an influence on the fine tuning of satellite cell behavior in their progression from quiescence => proliferation => differentiation. Of special interest would be, whether this was a unidirectional cascade or whether it could be reversed. Indeed, the re-entry of proliferating cells into quiescent state is achieved rapidly in injured muscle in vivo (reviewed in section 1.2). This rapid re-entry indicates that the quiescent state is highly regulated. Microarray analysis revealed that 500 quiescent stem cell-associated genes were up-regulated in quiescent muscle stem cells in comparison to committed myoblasts [START_REF] Fukada | Molecular signature of quiescent satellite cells in adult skeletal muscle[END_REF][START_REF] Liu | Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging[END_REF]. Among those genes, several negative cell cycle regulators were identified, which comprised CKIs (Cdkn1b p27 Kip1 ; Cdkn1c p57 Kip2 ), the retinoblastoma tumor suppressor protein (Rb1), and Spry1 (the negative regulator of FGF signaling sprouty 1) [START_REF] Fukada | Molecular signature of quiescent satellite cells in adult skeletal muscle[END_REF][START_REF] Liu | Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging[END_REF].

However, the re-entry of proliferating cells into quiescence has not yet been successfully achieved in vitro. Studies of senescent MuSCs, whose self-renewal and regenerative capabilities are known to be defective, might provide some insight as it has been shown that these cells can indeed be pushed back into quiescence via silencing p16 INK4a (Sousa-Victor et al., 2014), or inhibiting p38α/β [START_REF] Cosgrove | Rejuvenation of the muscle stem cell population restores strength to injured aged muscles[END_REF].

In summary, we examined the effect of Dll1-Fc on cultured muscle progenitor cells. In agreement with previous findings [START_REF] Parker | Activation of Notch signaling during ex vivo expansion maintains donor muscle cell engraftment[END_REF][START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF], Dll1stimulated Notch and maintained Pax7-positivity. However, this came at the cost of a largely reduced proliferative capacity by restraining exit from quiescence. At present, these data do not support the possibility of using this ex vivo culturing technique to generate sufficient cells for cell-based therapies of genetic muscle diseases.

Overexpression of Dll1 in vivo

In During the period of postnatal muscle growth in mice, the number of myonuclei keeps rising rapidly until P21 by the addition of over 13 myonuclei per fiber per day between P3 and P14, followed by 5.5 myonuclei per day between P14 and P21 [START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF]. This rapid rise of myonuclei is achieved by adding new myonuclei that derive from muscle stem cells. We thus injected the rAAV-Dll1 virus soon after birth and defined 3 observation time points to investigate the effects of Dll1overexpression during postnatal muscle growth at 2, 3 and 6 weeks of treatment.

In mdx mice, the onset of myofiber necrosis and muscle regeneration goes in parallel with the exit from MuSC-dependent fiber growth at around postnatal day 21 (P21) [START_REF] Partridge | The mdx mouse model as a surrogate for Duchenne muscular dystrophy[END_REF]. We hence injected the rAAV vector into 3-week-old mdx mice to observe the effect of Dll1 overexpression during the peak of degeneration and regeneration cycles at around postnatal week 7.

To deliver the Dll1 molecule into muscle tissue, we employed an effective recombinant Adeno-Associated Viral (rAAV) vector developed by our group (see section 3.2.5), which has high efficiency in skeletal muscle tissue and has been successfully applied in previous experiments (Le [START_REF] Hir | AAV Genome Loss From Dystrophic Mouse Muscles During AAV-U7 snRNA-mediated Exon-skipping Therapy[END_REF][START_REF] Sartori | BMP signaling controls muscle mass[END_REF]. The main aim of this investigation was to transfect mature myofibers with the rAAV in order to overexpress Dll1. It was our aim to specifically overexpress Dll1 in muscle fibers, but not in muscle stem cells. A recent study has shown that AAV preferentially targets post-mitotic multinucleated myotubes and to a much lesser degree mononuclear myoblasts or myocytes [START_REF] Arnett | Adeno-associated viral (AAV) vectors do not efficiently target muscle satellite cells[END_REF]. AAV is thus able to effectively transduce mature myofibers in vivo, while quiescent MuSCs are refractory to AAV infection [START_REF] Arnett | Adeno-associated viral (AAV) vectors do not efficiently target muscle satellite cells[END_REF]. Furthermore, even if MuSCs had been transduced, their subsequent proliferation would dilute the replication-deficient AAV, which would not be the case for the terminally differentiated myofibers. These findings imply that the MuSCs in our experimental system were the recipients and not the senders of Notch signals.

It is also noteworthy that previous studies by our group have shown that foreign genes transferred via a rAAV vector (driven by the same CMV promoter) are expressed in muscle tissue several hours post injectionem (unpublished data). We thus consider the injection day as the initial day of Dll1 treatment.

Overexpression of Dll1 in wild-type mice during muscle development

The aim of this experiment was to investigate whether our experimental system would also function efficiently in an in vivo mouse model, and whether Dll1 overexpression in muscle fiber might have an effect on MuSCs.

The rAAV transgenic system

At first I examined whether our experimental system worked successfully in vivo. The rAAV transduction was efficient if examined at the mRNA gene expression level (Figure 29), despite the failure of detecting Dll1 signal at the protein level (data not shown). For lack of appropriate antibodies, other researchers had also failed to detect the Dll1 protein directly on resting muscle fibers via Western blot or immunostaining [START_REF] Conboy | Notch-mediated restoration of regenerative potential to aged muscle[END_REF][START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF][START_REF] Mourikis | Distinct contextual roles for Notch signalling in skeletal muscle stem cells[END_REF], although they speculated that muscle fibers expressed Dll1 in order to explain the high Notch activity in MuSCs.

The difficulty to detect the Dll1 protein on the surface of the muscle fiber might arise from the small amount of the active Dll1-ligand or from an altered three-dimensional structure of the extracellular domain, which is not recognized by antibodies generated against peptides or recombinant proteins.

rAAV transduced muscle does not exhibit gross pathological changes

Secondly, by morphological analysis (H&E staining) I was able to rule out that Dll1overexpression might have damaged the myofibers or have led to any pathological changes in the muscle tissue, such as myofiber degeneration, inflammation, or fibrosis (Figure 30).

The question remains, whether the Dll1-intracellular domain per se might have an effect on the muscle fiber. The Dll1 protein is composed of an extracellular domain that interacts with the Notch receptor and an intracellular domain, which contains a carboxyl terminal PSD-95/Dlg/ZO (PDZ) -1-ligand motif (D 'Souza et al., 2010) (reviewed in Figure 6). This motif mediates interactions with other PDZ-containing scaffold / adaptor proteins and is involved in the effects of ligands on cell adhesion, migration, and oncogenic transformation, which is independent of Notch signaling (D 'Souza et al., 2010). Overexpression of the Dll1 intracellular domain in ES cells and in mouse embryos did not exhibit any signaling activity, and the normal development of the mouse embryos remained unaffected [START_REF] Redeker | Normal development in mice over-expressing the intracellular domain of DLL1 argues against reverse signaling by DLL1 in vivo[END_REF].

In non-mammalian muscle, as observed in D. melanogaster, Delta ligand can be proteolytically cleaved from the cell surface to become a soluble extracellular protein, which is capable to bind to Notch receptors of distant cells and elicit paracrine biological activity [START_REF] Qi | Processing of the notch ligand delta by the metalloprotease Kuzbanian[END_REF]. In mammals such a paracrine effect of the Dll1 ligand has not been described (D 'Souza et al., 2010;[START_REF] Perdigoto | Sending the right signal: Notch and stem cells[END_REF].

In summary, in this part of my dissertation I confirmed that our rAAV-mediated transgenic gene delivery system functioned and led to an overexpression of Dll1 in the transduced muscle but did not cause any pathological changes.

The time window for Dll1-overexpression

For technical reasons we were not able to inject the rAAV before postnatal day 3 (P3), which we hence considered the initial day of treatment. However, after 2 weeks of treatment no effect, neither on muscle weight nor on muscle fiber size could be seen.

Such effect only manifested after 3 weeks and more pronouncedly after 6 weeks. We suppose that this time lag of 2 weeks between initiation of treatment and measurable effect might come about only as a cumulative effect on the signaling of MuSCs over a certain time period (Figure 32).

Effect of Dll1-overexpression on muscle stem cells

In parallel to the effects seen in vitro, Dll1-induced overstimulation of Notch activity in vivo enlarged the muscle stem-cell pool only during period of rapid muscle growth (at the age of 3 and 6 weeks), but not in adult mice. This implicates that such effect is dependent on ongoing MuSC proliferation (and their return to quiescence), which can only be seen in adolescent mice before postnatal muscle development is completed [START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF] or in states of ongoing muscle regeneration in mdx mice.

The finding of a reduction of muscle weight and fiber diameter in Dll1-overexpressing muscles led to the assumption that the fusion of the muscle progenitor cells might be impeded. In this case, the fibers of rAAV-Dll1 treated muscles would contain less myonuclei than their control counterparts. However, unexpectedly, we did not find Since we do not deal with an 'all-or-nothing" principle, but with a quantitative phenomenon, not all the cells are kept at quiescent state. Those cells that had 'escaped' such return into quiescence went on to express MyoD and proliferate, albeit at lower numbers but were ultimately able to fuse with the muscle fibers in sufficient numbers.

The finding that myonuclear accretion remained unaffected also during late postnatal muscle development suggests that increased Notch activity has no influence on myoblast proliferation beyond a certain state of differentiation.

In summary, we demonstrate that Dll1-induced high Notch activity enlarged the stemcell pool by preventing a subpopulation of MuSCs from proliferation or by pushing them back into quiescent state after cell division. Future work should aim to identify and distinguish the molecular markers of quiescent and proliferating muscle stem cells in order to uncover the mechanisms that redirect proliferating muscle stem cells into quiescence.

Dll1-overexpression on the sarcolemma restrains muscle hypertrophy in the developing muscle.

In parallel with the profound effect on the muscle stem-cell pool, the weight of Dll1overexpressing muscles (TA as well as EDL) was significantly reduced in 3-week and 6-week-old mice (Figure 36). Muscle fiber diameters also exhibited a clear shift towards smaller sized fibers. These data suggest that DII1 overexpression levels the physiological hypertrophic progression during muscle development.

Myofiber hypertrophy is mainly achieved by (i) myonuclear addition from MuSCs (accretion) and (ii) by the expansion of the myonuclear domain (hypertrophy).The postnatal muscle growth in mice is mainly caused by hypertrophy of muscle fibers and not via addition of new fibers (hyperplasia) [START_REF] Ontell | Myosatellite cells, growth, and regeneration in murine dystrophic muscle: a quantitative study[END_REF][START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF]. We observed comparable numbers of fibers in Dll1-treated and control TA, which is in agreement with previous studies that the number of fibers is set in utero [START_REF] Ontell | Myosatellite cells, growth, and regeneration in murine dystrophic muscle: a quantitative study[END_REF][START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF]. During physiological development the number of myonuclei rapidly increases only until P21, while the myonuclear domain continues to expand until P56 [START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF]. Since we did not observe that overexpression of Dll1 affected myonuclear accretion, we hypothesize that overexpression of Dll1 prevents muscle fiber enlargement via its influence on the increase of the myonuclear domain.

As previously discussed in 1.4.1, Notch ligand cis-inhibits Notch activity in the same cell and trans-activates Notch activity in neighboring cells [START_REF] Sprinzak | Cis-interactions between Notch and Delta generate mutually exclusive signalling states[END_REF]. It is likely that our Dll1-overexpressing muscle fibers might inhibit their own Notch signaling (cis-inhibition). However, very little is known on the cell autonomous effect of Notch signaling on the myofiber proper so that we can only speculate: Investigators from cancer research already demonstrated that Notch is able to stimulate the AKT/mTOR/PI3K pathway [START_REF] Chan | Notch signals positively regulate activity of the mTOR pathway in T-cell acute lymphoblastic leukemia[END_REF], thus having a direct influence on cell anabolism and size. Consequently, stimulation of the AKT/mTOR/PI3K pathway leads to muscle hypertrophy [START_REF] Bodine | Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[END_REF], while abrogation of its effectors (PI3K) causes muscle wasting [START_REF] Ohanna | Atrophy of S6K1|[minus]|/|[minus]| skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control[END_REF]. In our study, Dll1overexpression might hence down-regulate the mTOR pathway via cis-inhibition of Notch and ultimately restrain the size of the growing muscle fibers.

However, given the fact that the hypotrophic effect of Dll1-overexpression is only seen during muscle development or regeneration (e.g. states of muscle progenitor proliferation), and not in resting adult muscle (see below in 5.3.2.3) such cell autonomous effect seems to be more complicated and may depend on additional factors that are only active during satellite cell proliferation. More detailed investigations are needed that should aim to independently dissect the effects of Notch stimulation on the MuSCs and the myofiber proper. This could be achieved through conditional skeletal muscle specific knockout of the Notch1 receptor via the Cre/loxP system using a diver mice that express Cre-recombinase under the human alpha-skeletal actin (ACTA1) promoter, which is only active in adult striated skeletal muscle (http://www.jax.org; FVB.Cg-Tg(ACTA1-cre)79Jme/J.), but not in muscle progenitor cells.

In summary, our main findings confirmed that rAAV-mediated Dll1 overexpression in muscle has an influence on muscle stem cells. We show that the difference in muscle fiber size if compared with the control side cannot be attributed to impaired myoblast fusion, as Dll1-overexpression does not affect the process of myonuclear accretion.

We thus conclude that Dll1 overexpression must also have an influence on the size of the nuclear domain and subsequent fiber hypertrophy. Further studies should aim to investigate the mechanism of how Notch influences fiber size and to identify the interactions between the Notch pathway and other anabolic pathways, such as the mTOR pathway.

Overexpression of Dll1 in mdx muscles

Since the Dll1-overexpression had a profound effect in wild-type newborn animals, we proceed further to investigate its effects in the mdx model, where MuSCs are in constant proliferative state. From the various models of muscle injury via injection of noxious substances (e.g. cardiotoxin (CTX) [START_REF] Couteaux | Regeneration of muscles after cardiotoxin injury. I. Cytological aspects[END_REF], and BaCl 2 [START_REF] George | Numb-deficient satellite cells have regeneration and proliferation defects[END_REF] or gene mutations we had opted for the mdx mouse model, because it is known that in mdx mice muscle is in a constant state of myofiber dam-age and regeneration peaking between 3-6 weeks of age [START_REF] Duddy | Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia[END_REF][START_REF] Partridge | The mdx mouse model as a surrogate for Duchenne muscular dystrophy[END_REF].

rAAV transgenic system

We injected the rAAV vectors into 3-week-old mdx mice because this time window represents the phase of highest regeneration activity in mdx muscle leading to the replacement of almost the entire musculature by the age of 6-7 weeks [START_REF] Duddy | Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia[END_REF][START_REF] Partridge | The mdx mouse model as a surrogate for Duchenne muscular dystrophy[END_REF]. When Dll1-mRNA levels were examined at 7 weeks, the rAAV transduction appeared to be less efficient in mdx mice in comparison to wildtype controls (Figure 29). This phenomenon might be due to the fact that the multiple degeneration / regeneration cycles in the mdx muscle had diluted the titer of the replication deficient rAAV. Nevertheless, 4 weeks of treatment after a one time injection seemed to be sufficient to produce an effect.

Further, we demonstrated that the injection of rAVV into the mdx muscle did not alter its dystrophic histopathological image.

In summary, I confirmed that our rAAV-mediated transgenic system functioned in adult mdx muscle although with lower efficiency due to subsequent viral titer dilution while Dll1-overexpression did not lead to any additional pathological changes in the mdx muscle beyond the normal characteristics of muscle dystrophy.

Effect of Dll1-stimulated Notch on muscle stem cells

In accordance with the findings from the developing muscle, Dll1-overexpression increased the percentage of quiescent MuSCs in mdx mice, while it did not have an effect on wild-type age-matched control mice (Figure 38). This could be explained by the fact that the MuSCs in young mdx muscle are in a state of hyperactive proliferation and regeneration [START_REF] Duddy | Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia[END_REF][START_REF] Partridge | The mdx mouse model as a surrogate for Duchenne muscular dystrophy[END_REF], while MuSCs in wild-type resting muscle are already at quiescent state with high resting Notch activity [START_REF] Fukada | Molecular regulation of muscle stem cells by 'quiescence genes[END_REF][START_REF] Relaix | Muscle stem cells[END_REF]. Thus further overexpression of a Notch ligand in resting muscle would barely have an effect on them.

In order to contribute to muscle repair, quiescent stem cells with high Notch activity need to temporarily down-regulate their Notch activity to be able to exit from quiescence and to re-enter the cell cycle for proliferation and differentiation [START_REF] Bjornson | Notch signaling is necessary to maintain quiescence in adult muscle stem cells[END_REF]. Therefore, overstimulation of Notch1 via the Dll1 ligand might impede muscle regeneration by increasingly pushing the MuSCs back into cell cycle arrest (quiescence) via repression of CDKIs, such as p27 Kip1 [START_REF] Monahan | The notch target gene HES1 regulates cell cycle inhibitor expression in the developing pituitary[END_REF] or p57 Kip2 [START_REF] Riccio | Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2[END_REF][START_REF] Zalc | Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest[END_REF]. Such arrest of the MuSCs in quiescence and subsequent impediment of muscle regeneration had been demonstrated by Wen et al., who had over-expressed the constitutively active NICD selectively in satellite cells [START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF].

In our experimental system of external Dll1-ligand mediated overstimulation of the Notch pathway, analysis of the effect of Dll1-overstimulation on the regenerating muscle revealed on one hand an increase of the satellite cell pool, as expected, but unexpectedly a normal number of myonuclei in the regenerated fibers. Hence also during regeneration Dll1-overexpression did not affect myonuclear accretion. However, despite the fact that myonuclear accretion obviously proceeded unimpeded, resulting myofiber diameters were smaller as compared to the control side suggesting again a decline of the myonuclear domain (see section 5.2.3.2).

In this regard our data are not completely congruent with the results of Wen et al. [START_REF] Wen | Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells[END_REF], which might be explained by the different experimental systems. Despite this debate, Notch signaling, as an important cell-to-cell signaling pathway, seems to be clearly involved in the dysfunction of MuSCs in mdx mice [START_REF] Jiang | Notch signaling deficiency underlies agedependent depletion of satellite cells in muscular dystrophy[END_REF][START_REF] Mu | The role of Notch signaling in muscle progenitor cell depletion and the rapid onset of histopathology in muscular dystrophy[END_REF][START_REF] Turk | Muscle regeneration in dystrophin-deficient mdx mice studied by gene expression profiling[END_REF]. Using Rbpj-GFP reporter mice Jiang et al.

were able to demonstrate reduced Notch-activity in MuSCs from mdx mice leading to a deficiency of self-renewal, which could be rescued by expression of the constitutively active NICD [START_REF] Jiang | Notch signaling deficiency underlies agedependent depletion of satellite cells in muscular dystrophy[END_REF].

In summary, we demonstrate that Dll1-induced high Notch activity enlarged the stemcell pool in regenerating in mdx muscle as well. Such knowledge could be exploited to develop therapeutic interventions to preserve the stem cell pool in patients with Duchenne Muscular Dystrophy (DMD), which is known to be depleted at around one third of the normal life span. In contrast, the disease process in mdx mice, the murine model of DMD, is much slower thanks to a better regenerative capacity of the mdx muscle. Further investigations should be directed to find out the best time window to treat mdx mice and to investigate whether such increase in the stem cell pool would translate into clinical benefit and functional improvement, or at least slower decline of the mdx muscle.

Dll1-overexpression on the sarcolemma restrains muscle hypertrophy in mdx muscle.

In accordance with the previous findings about the developing muscle, effects of Dll1overexpression on muscle mass and muscle fiber diameter were more profound in mdx muscles. Interestingly, no such changes were seen in the age-matched wildtype animals that had been treated in parallel in exactly the same manner (Figure 37).

The reason for this striking difference might lie in the time point of the rAAV-Dll1 injection. It is well known that murine MuSCs have mostly returned to quiescence after the final number of nuclei in the myofiber is established and the proliferation/fusion process of rapid postnatal muscle growth has been terminated [START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF].

Therefore, Dll1-mediated Notch overstimulation might have little effect on the quiescent MuSCs which are already in a state of high Notch activity.

As already observed in the developing muscle, we found an increased number of small myofibers in the Dll1-overexpressing mdx muscle. Such left shift of the fiber size histogram was not seen in rAAV-Dll1 treated wild-type muscle (Figure 37E, F).

Again this difference in myofiber size cannot be accounted for by a decreased ten-dency of myoblasts to fuse with the damaged muscle fibers. Since the myonuclei of the fusing myoblasts are generally not located at the periphery of the myofiber but at its center (Figure 36), the number of the myofibers exhibiting such central nuclei can be taken as a measure for regenerative activity of the mdx muscle. No difference between rAAV-Dll1 treated and control side were found with respect to the percentage of myofibers with central nuclei (Figure 38D), pointing to the fact that myonuclear accretion remained unaffected also in the mdx muscle.

Again, as already seen in the developing muscle, such left-shift of muscle fiber size seems to be linked to the ongoing process of muscle precursor cell proliferation and fusion, which is present in adult mdx muscle, but not in wild-type animals (see discussion in section 5.3.1.5).

Outlook

The overarching aims of my investigations were (i) to investigate how Notch stimula-

Acknowledgement

"路漫漫其修远兮, 吾将上下而求索."

"The road ahead is still long, I shall search high and low."

  List of figures ................................................................................................................................... i

Figure 1 :

 1 Figure 1: Schematic illustration of the various components of a muscle ...................................

Figure 2 :

 2 Figure 2: Transcriptional hierarchy regulating myogenic development .....................................

Figure 3 :

 3 Figure 3: A schematic drawing of a muscle fiber .......................................................................

Figure 4 :

 4 Figure 4: Two models explaining the self-renewal and differentiation of satellite cells ...........

Figure 5 :

 5 Figure 5: The satellite cell niche...............................................................................................

Figure 6 :

 6 Figure 6: Structure of canonical Notch receptors and ligands .................................................

Figure 7 :

 7 Figure 7: Notch signaling between cells ..................................................................................

Figure 8 :

 8 Figure 8: Biology of wild-type and recombinant adeno-associated virus (AAV). .....................

Figure 9 :

 9 Figure 9: Vector maps ..............................................................................................................

Figure 10 :

 10 Figure 10: FACS sorting of VCAM (FITC) positive cells ............................................................

Figure 11 :

 11 Figure 11: MACS procedure ......................................................................................................

Figure 12 :

 12 Figure 12: Affinity chromatography ............................................................................................

Figure 13 :

 13 Figure 13: Schematic illustration of the surface coating ............................................................

Figure 14 :

 14 Figure 14: Schematic illustration of the rAAV injection experiments .........................................

Figure 15 :

 15 Figure 15: Satellite cells on freshly isolated muscle fibers ........................................................

Figure 16 :

 16 Figure 16: Divisions of satellite cells ..........................................................................................

Figure 17 :

 17 Figure 17: Floating fibers after 4 days in culture ........................................................................

Figure 18 :

 18 Figure 18: Satellite cell proliferation on Matrigel ® ......................................................................

Figure 19 :

 19 Figure 19: Production of recombinant Delta-like-1 protein ........................................................

Figure 20 :

 20 Figure 20: Percentage of Ki-67 positive cells in cultured myoblasts..........................................

Figure 21 :

 21 Figure 21: Gene expression levels measured by qPCR ............................................................

Figure 22 :

 22 Figure 22: Expression of Myogenin in cultured C 2 C 12 myoblasts ..............................................

Figure 23 :

 23 Figure 23: Growth curves and proliferation rate of MPCs ..........................................................

Figure 24 :

 24 Figure 24: VCAM-selected murine MPCs in culture for 4 days .................................................

Figure 25 :

 25 Figure 25: Subpopulation of 4day cultured Pax7+ MPCs ..........................................................

Figure 26 :

 26 Figure 26: MPCs cultured for 6 days. ........................................................................................

Figure 27 :

 27 Figure 27: Gene expression levels in cultured MPCs measured by qPCR. ..............................

Figure 28 :

 28 Figure 28: The pSMD-Dll1 plasmid ............................................................................................

Figure 29 :

 29 Figure 29: Expression levels of the Dll1 transgene examined by RT-qPCR .............................

Figure 30 :

 30 Figure 30: HE staining of muscle sections from the newborn group .........................................

Figure 31 :

 31 Figure 31: Muscles of newborn mice treated for 2 weeks .........................................................

Figure 32 :

 32 Figure 32: Muscles of newborn mice treated for 3 and 6 weeks ...............................................

Figure 33 :

 33 Figure 33: Number of fibers per TA muscle cross section .........................................................

Figure 34 :

 34 Figure 34: Number of myonuclei per EDL fiber ..........................................................................

Figure 35 :

 35 Figure 35: Dll1 overexpression enlarges the stem cell pool ......................................................

Figure 36 :

 36 Figure 36: HE staining of rAAV-injected mdx and wild-type TA muscles ..................................

Figure 37 :

 37 Figure 37: Effect of Dll1-overexpression during muscle regeneration in mdx mice ...................

Figure 38 :

 38 Figure 38: Dll1-overexpression increases the muscle stem cell pool in mdx muscle ................

Figure 39 :

 39 Figure 39: Representative images of mdx EDL fibers ................................................................

  During postnatal development, overstimulation of Notch signaling by Dll1 on the muscle fibers was able to enlarge the Pax7+ cell pool, while also resulting in decreased muscle mass and smaller muscle fibers without affecting the accretion of myonuclei into the fiber. In quiescent (wild-type) MuSCs, overstimulation of Notch signaling did not have any discernible effect. Overexpression of Dll1 in mdx muscle decreased the muscle mass and enlarged the muscle stem cell pool, while muscle regeneration remained unaffected. By investigating Notch stimulation in MuSCs both in vitro and in vivo, we demonstrate that high Notch activity preserves stemness via inhibition of MuSCs proliferation and myogenic differentiation. Our findings point out that the Dll1 molecule, as a canonical Notch ligand, might have a therapeutic potential in cell-based therapies against muscular dystrophies. vi Zusammenfassung Die Muskelstammzelltransplantation hat ein großes Potential zur Heilung oder Verbesserung der Funktion dystrophen Muskels. Hierfür müssten die vom Patienten gewonnen Muskelstammzellen (Satellitenzellen) molekulargenetisch repariert, ex vivo vermehrt und dann retransplantiert werden. Letzteres stellt ein großes Problem dar, da die Muskelstammzellen in der Kultur schnell ihre Stammzelleigenschaften verlieren. Der Notch Signalweg ist ein wichtiger Regulator der Satellitenzellen und man hat kürzlich herausgefunden, dass die Aktivität dieses Signalweges wichtig für die Erhaltung der Stammzelleigenschaften ist. Diese Erkenntnis könnte zur Entwicklung neuer Therapieansätze genutzt werden. Satellitenzellen der Maus wurden frisch aus Muskel isoliert und in Kulturschalen vermehrt, die mit Dll1-Fc, einem Stimulator des Notch-Rezeptors beschichtet waren. Im lebenden Tier wurde die Dll1-Expression an der Oberfläche der Muskelfasern durch Infektion mit einem i.m. injizierten rAAV-Dll1 erreicht. Die Injektion erfolgte in P3 Jungtiere, welche entweder nach 3 oder nach 6 Wochen untersucht wurden. In einem dritten Versuchsmodell wurden erwachsene P21 mdx Mäuse mit dem o.g. Virus injiziert, um den Effekt einer Überstimulation des Notch Signalweges auf den sich regenerierenden Muskel zu untersuchen. Die Beschichtung der Kulturschalen mit Dll1-Fc bewahrte die Stammzelleigenschaften der Satellitenzellen, welche eine höhere Pax7 Positivität aufwiesen. Gleichzeitig war aber die Proliferationsrate und Differenzierung dieser Zellen erniedrigt. Während der postnatalen Muskelentwicklung führte die Notch Überstimulation zu einer Vergrößerung des Satellitenzell-Pools, aber auch zu einer reduzierten Muskelmasse, welche auf einer Verkleinerung der nukleären Domäne, nicht aber auf einer verminderten Verschmelzung ("Accretion") der myogenen Progenitorzellen mit der Muskelfaser beruhten. Im adulten Muskel fanden sich eine Erhöhung des Stammzell-Pools und eine Verringerung der Muskelmaße nur bei mdx Mäusen, hingegen nicht bei gesunden Kontrollmäusen. Zusammenfassend führt eine in vitro und in vivo Stimulation des Notch Signalweges zu einer verbesserten Aufrechterhaltung der Stammzellfunktion, inhibiert aber die Proliferation und Differenzierung. Die Erhöhung des Stammzellpools und somit des regenerativen Potentials könnte therapeutisch zur Behandlung der Muskeldystrophie genutzt werden. vii Résumé La transplantation de cellules souches de muscle possède un grand potentiel pour la réparation à long terme du muscle dystrophique. Cependant, la croissance ex vivo des cellules souches musculaires réduit de manière significative l'efficacité de leur greffe puisque le potentiel myogénique est considérablement réduit lors de la mise en culture. La voie de signalisation Notch a émergé comme un régulateur majeur des cellules souches musculaires (MuSCs) et il a également été décrit que la sur-activation de Notch est crucial pour le maintien du caractère souche des MuSC. Cette découverte pourrait être traduite comme un bénéfice thérapeutique potentiel. Des MuSCs murines ont été fraîchement isolées et ensemencées sur des boîtes de culture recouverte de Dll1-Fc, le domaine extracellulaire de Delta-like-1 est fusionné au fragment Fc humain, afin d'activer la voie de signalisation Notch et avec un IgG humain comme contrôle. Nous avons utilisé le rAAV afin d'exprimer le Dll1 spécifiquement dans les muscles de souris. Les souris P3 ont été traitées avec de l'AAV pendant 3 semaines et 6 semaines afin d'étudier l'effet de Dll1 au cours du développement postnatal. Afin d'étudier le processus de régénération, l'AAV a également été injecté dans les muscles de souris mdx alors que les souris de type sauvage ont été utilisées comme contrôle. Un potentiel caractère souche supérieur (marquée avec le Pax7) est observé dans les cultures des MuSCs qui sont recouverte de Dll1-Fc par rapport à leurs homologues contrôles, par contre le taux de proliférer est réduit. Au cours du développement postnatal, la sur-activation de la voie de signalisation Notch par Dll1 sur les fibres musculaires a été en mesure d'élargir le pool des cellules Pax7+, cependant elle entraîne une diminution de la masse musculaire avec réduction de la taille des fibres et ceci sans affecter l'accumulation des myonuclei. Dans les MuSCs quiescentes (de type sauvage), la sur-activation de la voie de signalisation Notch ne présente pas de réel effet. La surexpression de Dll1 dans le muscle mdx a diminué la masse musculaire et agrandit le pool de cellules souches musculaires, cependant le taux de régénération n'a pas été affecté. L'augmentation des MuSCs est attribuée à une différenciation entravée des cellules souches musculaires. En étudiant la stimulation de la voie de signalisation Notch dans les MuSCs à la fois in vitro et in vivo, nous démontrons que sur-activation de Notch préserve le caractère souche des cellules via l'inhibition de la prolifération et de la différenciation myogénique

Figure 1 :

 1 Figure 1: Schematic illustration of the various components of a muscle Figure by Karima Relizani, adapted from (Baechle and Earle, 2008)

  development occurs in mammals in three distinct stages: embryonic, fetal and postnatal. All skeletal muscles originate from the mesoderm, one of the three primary germ layers established in the early embryo. The majority of skeletal muscles arise from somites, which are segmented structures that derive from the paraxial mesoderm. The somites are later compartmentalized into dermomyotomes, which in turn develop into skeletal muscle and parts of the dermis as well as sclerotomes that give rise to bones and ribs. Expression of the transcription factors Pax3 and Pax7 marks myogenic stem cells and progenitor cells. These Pax3/7-positive cells initiate expression of muscle regulatory factors (MRFs). Subsequently, the muscle progenitors exit the cell cycle, differentiate into myoblasts and myocytes and fuse into multinucleated myotubes (Figure 2). During this process a population of "uncommitted" Pax3/7-positive cells, which do not express any of the MRFs, persists in the developing embryonic muscles and are thought to give rise to satellite cells (Kassar-Duchossoy et al.

Figure 2 :

 2 Figure 2: Transcriptional hierarchy regulating myogenic development Progenitors of satellite stem cells originate from the somite as Pax3 and/or Pax7-expressing progenitors. Satellite stem cells express Pax7, whereas satellite myogenic cells additionally express Myf5, as revealed by expression of Myf5-lacZ and Myf5-cre knock-in alleles (Rudnicki et al., 2008). Following activation and entrance into the cell cycle, myogenic precursor cells express Myf5 and MyoD. Induction of MyoD and then of Myogenin mark the withdrawal from the cell cycle and entrance into the terminal differentiation program.

  Pax7 specify myogenic progenitors. Both Pax3 and Pax7 have been shown to directly bind proximal promoters of MyoD and distal enhancer elements of Myf5, thereby regulating their expression. Pax3 binds at approximately 57.5 kb upstream of the transcriptional start of the Myf5 gene and functions via this 145 bp-Myf5

  fen-induced Pax7 ablation allele generated byLepper et al., von Maltzahn et al. demonstrated that continuous tamoxifen treatment resulted in a pronounced deficit of muscle regeneration. Further, Pax7-deficient progenitors exhibit cell-cycle arrest in cell culture and are incapable of expansion in vivo. Hence the authors concluded that Pax7 is an absolute requirement for satellite cell function in adult skeletal muscle[START_REF] Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF]. The two controversial results may arise from a different approach to tamoxifen application.Lepper et al. injected tamoxifen at multiple time points, yet von Maltzahn et al. supplemented multiple tamoxifen injections with an oral tamoxifen application in between the injections. A tamoxifen pulse might not be sufficient to completely abrogate Pax7 function in the satellite cell pool in vivo. 1.1.1.2 Myogenic regulatory factors Downstream of the Pax transcription factors, the myogenic regulatory factors (MRFs)

Figure 3 :

 3 Figure 3: A schematic drawing of a muscle fiber One muscle fiber contains hundreds of myonuclei, which are all located just beneath sarcolemma. Satellite cells reside between the basal lamina and the sarcolemma.

Figure 4 :

 4 Figure 4: Two models explaining the self-renewal and differentiation of satellite cells (A) In a first model, Pax7+|Myf5-satellite 'stem' cells co-exist with Pax7+|Myf5+ 'committed' satellite cells. Pax7-only cells undergo symmetric or baso-apical cell division to amplify or maintain, the stem cell pool. Wnt7a and Frizzled7 (Fzd7), through the Wnt Planar Cell Polarity (PCP) pathway, stimulate the symmetric division of satellite cells, thus promoting the expansion of the satellite stem cells pool. Notch signaling favors the self-renewal of satellite stem cells. Committed Pax7+|Myf5+ satellite cells, probably through planar cell division, preferentially undergo terminal differentiation. (B) In a second model, when muscles are injured, quiescent (Pax7+) satellite cells go through an activated, proliferative (Pax7+|MyoD+) stage. From this transitory proliferating population, most cells undergo terminal differentiation, while a few return to a Pax7+|Myf5-stage to renew the quiescent satellite cell pool. Figure adapted from (Relaix and Marcelle, 2009) and re-published with permission by License 3554290028418.

  Vice versa, satellite cells also influence other niche components by means of cell-cell interaction and autocrine or paracrine signals. The dynamic interactions between satellite cells and their niche specifically regulate satellite cell quiescence, selfrenewal, proliferation and differentiation. By these mechanisms muscle mass is regulated to prevent either atrophy or excessive growth, while the satellite cell pool is maintained during regeneration. A clear understanding of the various interactions within the niche is of paramount importance for the development of therapies to treat both age-related skeletal muscle atrophy and skeletal muscle diseases.

Figure 5 :

 5 Figure 5: The satellite cell niche 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[START_REF] Yin | Satellite cells and the muscle stem cell niche[END_REF]. 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 satellite 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.

  majority of Notch signaling is induced by a family of DSL ligands, which are characterized by the presence of a DSL (Delta, Serrate, and Lag3) domain. Both the ligands and the Notch receptors are type I transmembrane proteins. The mammalian DSL ligands are classified as either Delta-like (Dll1, Dll3, and Dll4) or Serrate (Jagged)-like (Jagged1 and Jagged2) based on their homology to the D. melanogaster prototypes Delta and Serrate.

Figure 6 :

 6 Figure 6: Structure of canonical Notch receptors and ligands (A) The extracellular domain of Notch receptors is composed by EGF repeats (the principal EGF repeats for interaction with the ligand are depicted in dark blue) and the Notch1-negative Regulatory Region (NRR). The intracellular domain contains motifs that mediate localization to the nucleus and stability of the Notch Intracellular Domain (NICD). (B) The extracellular domains of canonical ligands are characterized by the presence of an N-terminal (NT) domain followed by a Delta/Serrate/Lag2 (DSL) domain and multiple in-tandem arranged EGF-like repeats. The DSL domain together with the flanking NT domain and the first two EGF repeats containing the Delta and OSM-11-like proteins (DOS) motif are required for canonical Notch binding. Serrate/Jagged ligands contain an additional cysteine-rich region, which is absent in Delta-like ligands. The intracellular domains of some canonical ligands contain a carboxy-terminal PSD-95/D1g/ZO-1-ligand (PDZL) motif that has a role independent of Notch signaling. Figure from (Perdigoto and Bardin, 2013) re-published with permission under license Nature Publishing Group CCC 3554281361180.

  Notch receptors and DSL ligands are widely expressed in various cell types during development. The majority of interacting cells express both ligands and receptors; however, they have different fates because Notch signaling is consistently activated in only one of the two interacting cells. This finding indicates that, the signaling polarity between two interacting cells is highly regulated. It is achieved via the dual roles of the ligands, which may either act as activator or as inhibitor of Notch signaling. It has been proposed that trans-ligand binding induces conformational changes in the Notch Regulatory Region, leading to relaxation of the interaction of the Lin-12-Notch motifs with the heterodimerization domain and hence expose the cleavage site (Gordon et al., 2007). By analyzing Notch-Delta signaling dynamics in individual cells by quantitative time-lapse microscopy, Sprinzak et al. revealed the responses of Notch

Figure 7 :

 7 Figure 7: Notch signaling between cells (A) Canonical Notch signaling in cells; (B) DSL ligand trans-activation and cis-inhibition of Notch signaling

  tain satellite cells in quiescence has not yet been fully understood. Researchers think that muscle fibers are the signal "sending" cells taking into account the (i) physiological closeness between satellite cells and muscle fiber and (ii) the elevation of Dll1 ligand in committed progenitor cells during satellite cell division[START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF]. However, so far researchers have failed to detect Delta-like 1 protein on muscle fibers (e.g. the sarcolemma) through immunostaining.Conboy et al. demonstrated immunohistological staining of Delta-like 1, Notch1, and Numb only on the mononucleated satellite cells of explanted myofibers, but not on the myofiber proper[START_REF] Conboy | The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis[END_REF]. So far, various anti-Dll1 antibodies (H-20, H-265, sc-12531, SantaCruz) have failed to detect a Dll1 signal on the muscle fiber. Such difficulty might arise from the relatively low expression levels of Dll1, as a signaling molecule, or from a conformational change, which cannot be recognized by the available antibodies. We hypothesize that a trace amount of Dll1 on the muscle fiber would be sufficient to uphold high Notch activity in the satellite cells. It would hence be interesting to address the question about how satellite cells might be affected by an alteration of the Dll1 expression levels (via up-or down-regulation) on the muscle fiber.

Table 2 :

 2 List of satellite markers direct cell isolationDirect cell sorting is a method used in succession to enzymatic digestion in order to obtain Muscle Satellite Cells (MuSCs) with higher purity. Direct sorting is based on the binding of surface proteins of MuSCs either to fluorescently labeled antibodies for FACS sorting or to magnetic bead-conjugated antibodies for magnetic sorting (MACS). FACS can further characterize intrinsic features of the satellite cells such as their size, cellular granularity, and relative frequency. Direct sorting provides enough cells for subsequent studies such as cell transplantation and in vitro expansion experiments.The successful isolation of living quiescent satellite cells relies on the development of specific antibodies that recognize the extracellular domain of molecules at the satellite cell surface. The group of Helen M. Blau reported for the first time direct isolation of myogenic cells using the H36 antibody against the extracellular epitope of integrin[START_REF] Blanco-Bose | Purification of mouse primary myoblasts based on alpha 7 integrin expression[END_REF] although their purity was not satisfactory enough. Later in 2004,Sherwood et al. showed that an Integrin α7/β1(+), Cxcr4(+), CD34(+), CD45(-),

Figure 8 :

 8 Figure 8: Biology of wild-type and recombinant adeno-associated virus (AAV). (A) The structure of the wild-type adeno-associated virus (AAV). A single-stranded DNA genome is framed by palindromic inverted terminal repeats (ITRs). The rep open reading frame (ORF) encodes proteins that are involved in viral replication, and the cap ORF encodes proteins that are necessary for viral packaging. AAV integrates into the human genome at a specific locus on chromosome 19 (red) and persists in latent form. It can exit this stage only if the cell is co-or super-infected with a helper virus, such as adenovirus (Ad) or herpes simplex virus (HSV), which provides factors necessary for active AAV replication. (B) The generic gene-delivery vector based on AAV. The viral genome is replaced by an expression cassette, which usually consists of a promoter, transgene and polyA (pA) tail. For production of the recombinant virus (rAAV), rep and cap proteins as well as Ad or HSV elements (Ad E1, E2 and E4orf6) have to be provided in trans. Examples of intracellular forms of the delivery vector that are responsible for transgene expression following transduction with rAAV (doublestranded circular episomes and randomly integrated vector genomes) are depicted in red.Figure from (Vasileva and Jessberger, 2005) re-published with permission under license 3554281033105.

  Bacterial strains: JM109 (Promega), XL10 Gold (Stratagene) were incubated on LB Agar plates (Roth) with 125 µg/ml Ampicillin or grown in shaker flasks filled with LB medium (Roth) with corresponding antibiotics.Eukaryotic cell lines:The 293T cell line (Human Embryonic Kidney cells) is routinely used in Prof. Gracia's laboratory for rAAV production. The 293T-D1Fc cell line for Dll1-Fc production, which is stably transfected with the pcDNA3-D1Fc plasmid, was a kind gift from Prof. Gerry Weinmaster. The murine myoblast cell line C 2 C 12 (ATCC ® CRL-1772 TM ) was purchased from ATCC ® . The immortalized human myoblast cell line called Clone24673 is a kind gift from Prof. Simone Spuler[START_REF] Mamchaoui | Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders[END_REF].

Figure 9 :

 9 Figure 9: Vector maps Left panel: Vector map of pcDNA3-D1Fc. The extracellular domain of rat Delta1 (amino acid 1-487, GeneBank accession number U7889) was fused in-frame with human IgG-Fc lacking the hinge region (encodes 1-217 amino acids of the Fc domain) using a PCR-overlap strategy. The Delta-Fc cDNA sequences were subcloned into the mammalian expression vector pcDNA3 (Invitrogen). Right panel: Vector map of pSMD2.

  MACS were performed according to the manufacturer's instructions. After centrifugation, the cell pellets were washed with staining buffer [2 mM EDTA, 1% w/v BSA, 100 µg/ml gentamycin in PBS] and enriched in muscle stem cells by two-step magnetic cell separation protocol: (i) first depletion of cells (e.g. fibroblasts) labeled with the Satellite Cell Isolation Kit (Miltenyi) and (ii) subsequent enrichment of cells labeled with anti-α7-integrin antibody coated magnetic beads (Miltenyi).

Figure 10 :

 10 Figure 10: FACS sorting of VCAM (FITC) positive cells Data were collected using the BD FACSDiva software (BD Bioscience, version 6.1.3) and saved as FCS 3.0 data files. The data analysis including sequential gating was performed by using FlowJo 9.8 (Tree Star, USA).

Figure 11 :

 11 Figure 11: MACS procedure Upper lane: Processing of mouse muscle, comprising hind limb dissection, removal of tendons and fat tissue, mincing of muscle tissue, enzymatic and mechanic digestion and finally lysis of red blood cells. Lower lane: Schematic illustration of MACS sorting procedure

Figure

  Figure 12: Affinity chromatography (A) Schematic illustration of the principle of affinity chromatography. (B) The workflow of one automated run.

Figure 13 :

 13 Figure 13: Schematic illustration of the surface coating

Figure 14 :

 14 Figure 14: Schematic illustration of the rAAV injection experiments (A) Experimental design to determine the effect of Dll1 during postnatal muscle development; (B) Experimental design to determine the effect of Dll1 during muscle regeneration in mdx mice.

Figure 15 :

 15 Figure 15: Satellite cells on freshly isolated muscle fibers (A) A single muscle fiber isolated from mouse EDL muscle. Scale bar = 1 mm; (B) A single nucleus being Pax7 positive belongs to a satellite cell that is located on a freshly isolated muscle fiber. Scale bar = 50 µm. (C) Histogram of satellite cell numbers per fiber. Each line represents one EDL fiber (n=65), isolated from an 8-week-old male C57BL6J mouse.

Figure 18 :

 18 Figure 18: Satellite cell proliferation on Matrigel ® A series of time-lapse images showing proliferating muscle progenitor cells with a faster division rate than cells retained on the muscle fiber. Scale bar = 10µm.

Figure 19 :

 19 Figure 19: Production of recombinant Delta-like-1 protein Left panel: Schematic illustration of the protein domain structure; right panel: Purified Dll1-Fc protein and human IgG were blotted and detected with an anti-human Fc antibody.

Figure 20 :

 20 Figure 20: Percentage of Ki-67 positive cells in cultured myoblasts (A, C) The proliferation rate of total murine myoblasts after two days and four days in culture, measured by Ki-67 positivity (A) and BrdU incorporation (C). Five random fields of three different experiments were counted; error bar = SD. The one -way ANOVA test was used and no significant differences were found between the two different culture dish coatings. (B, D) The proliferation rate of total human myoblasts after two days and four days in culture, measured by Ki-67 (B) or BrdU (D). Five random fields of three different experiments were counted; error bar = SD. The one-way ANOVA test was used and no significant differences were found between the two different culture dish coatings.

Figure 21 :

 21 Figure 21: Gene expression levels measured by qPCR (A, B) Murine myoblasts were cultured on two different surface coatings for 2 days (A) and for 4 days (B). The Gapdh gene expression levels were used as reference. Three biological replicates were collected and each qPCR was performed with three technical replicates; error bar = SD. Gene expression on the Dll1 surface was normalized to the expression levels on the IgG surface, which were set to 100% (C, D) Human myoblasts were cultured on two different surfaces for 4 days (C) and for 6 days (D). The TIMM17B gene expression levels were used as reference. Three biological replicates were collected and each qPCR was performed with three technical replicates; error bar = SD. Gene expression on the Dll1 surface was normalized to the expression levels on the IgG surface, which were set to 100%

Figure 22 :

 22 Figure 22: Expression of Myogenin in cultured C2C12 myoblasts (A-D) Representative images of C2C12 cells cultured for 2 days on both Dll1-Fc (left) and IgG-Fc (right) surfaces and stained with anti-myogenin antibodies (upper panels) and with DAPI (lower panels). Scale bar = 100 µm. (E) The percentage of myogenin positive cells in the total cell population after 4 days of culture. Each dot represents a different biological replicate; the red line depicts the median; the p value was calculated using the non-parametric Mann-Whitney U test.

  freshly isolated MPCs growing on the Dll1-Fc as compared to the control IgG surface, especially at day 4. Furthermore, as indicated by BrdU positivity, the percentage of proliferating cells was much lower on the Dll1-Fc surface (29.1±2.6% on the Dll1-Fc surface versus 40.0±5.1% on the control IgG surface; n=3 experiments).

Figure 23 :

 23 Figure 23: Growth curves and proliferation rate of MPCs Left panel: Numbers of muscle progenitor cells per cm2 culture dish surface. Freshly isolated cells were seeded at identical densities on the Dll1-Fc or IgG surface. Three biological replicates were prepared, and cell numbers were investigated after 2 and 4 days. The p-value (p=0.003) was calculated via MANOVA with repeated measurements. Between days 5 and 6 cells began to fuse and to form myotubes; therefore counting of single cells by the used method became unreliable beyond day 4.Right panel:The proliferation rate of MPCs as determined by the percent of positive staining for BrdU. Three biological replicates were prepared; at least three random visual field images were acquired from each culture plate; the p-values were calculated with Mann-Whitney U test).

Figure 24 :

 24 Figure 24: VCAM-selected murine MPCs in culture for 4 days The same number of VCAM-selected cells was seeded, grown, and stained with stem the cell marker Pax7 after 4 days in culture. Scale bar = 100 µm.

Figure 25 :

 25 Figure 25: Subpopulation of 4day cultured Pax7+ MPCs (A) After 4 days in culture, cells were stained with anti-Pax7 and anti-MyoD antibodies. Scale bar = 100 µm. (B) The percentage of Pax7 + nuclei of α7-integrin selected MPCs after 4 day culture under different conditions. Three biological replicates were prepared; from each sample five random field images were acquired and analyzed. error bar = SD. The MPCs cultured on the Dll1-Fc surface without inhibition of DAPT maintained the highest percentage of Pax7-positivity (p<0.001, one-way ANOVA test). After blockade with DAPT, the difference in Pax7-positivity between Dll1-Fc and IgG control surface was abolished (p=0.350, one-way ANOVA test). (C) The percentage of MyoD + MPCs amongst the Pax7 + population. Three biological replicates were cultured for 4 days; from each sample five random field images were acquired and analyzed. The percentage of Pax7 + |MyoD-was significantly higher on the Dll1-Fc surfaces (60.9±6.7% versus 21.3±7.2%; p<0.001, one-way ANOVA test)

Figure 26 :

 26 Figure 26: MPCs cultured for 6 days. (A) Phase contract microscopy images. Scale bar = 50 µm. (B) The percentage of Pax7 positive cells after culture under different conditions for 6 days. Three different biological samples were prepared; from each three random field images were acquired. Error bar = SD. The p-value was calculated with the nonparametric Man-Whitney U test.

Figure 30 :

 30 Figure 30: HE staining of muscle sections from the newborn group Scale bar = 50 µm

Figure 31 :

 31 Figure 31: Muscles of newborn mice treated for 2 weeks (A) Muscle mass of the TA and EDL in mice after 2 weeks of treatment. One dot represents one muscle; the red line represents the median. There is no significant difference in the muscle mass of Dll1-treated muscles as compared to controls (p-values were calculated with the Wilcoxon matchedpairs signed rank test). (B) The histogram of the muscle fiber diameters of the Dll1 treated versus scramble side from the group of newborn mice after 2 weeks of treatment does not reveal any difference in fiber diameter (p=0.982 was calculated with Chi 2 test for trend). At least 4 muscles from each side were used for measurement, error bar=SD.

Figure 33 :

 33 Figure 33: Number of fibers per TA muscle cross section (A, B)The number of fibers per TA section from newborns with 3-week treatment (A) and from newborns with 6-week treatment (B). TA sections were acquired from 4 animals for the measurement; each TA muscle was counted 2-4 x times at different levels. One dot represents a number that was counted in one entire TA cross section; the red line depicts the median. There is no significant difference between two sides (the p-value was calculated using the non-parametric Mann-Whitney U test).

Figure 35 :

 35 Figure 35: Dll1 overexpression enlarges the stem cell pool (A, B) The percentage of Pax7 + cells per total number of myonuclei (epimysium excluded) in TA muscles after 3-week (A) and 6-week treatment (B). Each dot represents one entire muscle cross section; from each mouse 3 cross sections were investigated; p-values were calculated with the nonparametric Kruskal-Wallis tests. (C, D) The percentage of BrdU + nuclei per total Pax7 + cells after 3-week (C) and 6-week (D) treatment. Each dot represents the results of one EDL muscle from which around 30 single fibers were analyzed. Less proliferating BrdU+ MuSCs were present in Dll1overexpressing muscle after 3 weeks of treatment, an effect not seen after 6 weeks of treatment any more; p-values were calculated with the one-tailed Wilcoxon matched-pairs signed rank tests, because we already knew rAAV-Dll1 treated muscles to have significant lower weights. Four muscles from each side were used for analysis.

Figure 36 :

 36 Figure 36: HE staining of rAAV-injected mdx and wild-type TA muscles Scale bar = 50µm

Figure 37 :

 37 Figure 37: Effect of Dll1-overexpression during muscle regeneration in mdx mice (A, B) Muscle mass of the TA muscles of the mdx (A) and the wild-type group (B). Each dot represents one muscle; the lines connect the muscles from both sides of the same mouse. (C, D) Muscle mass of the EDL muscles of the mdx (C) and wild-type group (D). rAAV-Dll1 treated TA and EDL muscles of mdx mice had a significantly lower weight as compared to controls, whereas rAAV-Dll1 treated TA and EDL muscles of control wild-type animals had comparable weights (p-values were calculated using the Wilcoxon matched-pairs signed rank test). (E, F) Histogram of the fiber diameters of the rAAV-Dll1 treated mdx TA muscle (E) and wild-type TA muscles in comparison to the control side. The muscle fibers of the rAAV-Dll1 treated mdx group show a clear shift towards smaller fiber diameters. At least 4 muscles from each side were analyzed, error bar=SD. (p=0.004 for mdx group, p=0.608 for wild-type group, Chi 2 test for trend).

  of (secondary) MuSC activation due to ongoing degeneration/regeneration cycles in adult mdx muscle the muscle stem cell pool is increased. Conversely, the percentage of proliferating BrdU+|Pax7+ proliferating cells was again decreased in rAAV-Dll1 treated mdx muscle indicating a reduced proliferation rate of MuSCs. The percentage of fibers with central nuclei, as a measure of regenerated muscle fibers remained equal in mdx muscles irrespective of Dll1-overexpression, indicating that muscle regeneration itself was not affected by rAAV-Dll1 treatment.

Figure 38 :

 38 Figure 38: Dll1-overexpression increases the muscle stem cell pool in mdx muscle (A, B) The percentage of Pax7 + nuclei per total nuclei in mdx TA (A) and wild-type TA muscle (B). The number of Pax7+ nuclei in Dll1-overexpressing mdx muscles was significantly increased, while such difference was not seen in wild-type muscle (the p-values were calculated with the Kruskal-Wallis test) (C) The percentage of BrdU + nuclei in the stem cell pool of mdx mice. Each dot represents one muscle; Pax7 + cells were counted from ≈30 EDL fibers (the p value was calculated with the one-tailed Wilcoxon matched-pairs signed rank tests, since we already knew rAAV-Dll1 treated muscles had significant lower weights. (D) The percentage of regenerated fibers with central nuclei was not significantly different in mdx muscles of the rAAV-Dll1 treated versus the control side. Each dot represents the percentage from one muscle cross section. Four cross sections of each mouse were analyzed (the p-value was calculated with the Kruskal-Wallis test).

Figure 39 :

 39 Figure 39: Representative images of mdx EDL fibers Upper panel: Fiber of mdx EDL muscle from the rAAV-Dll1 treated side; Lower panel: Fiber of mdx EDL muscle from the control side. Green dots are DAPI stained myonuclei. Pax7-positive cells indicate myofiber-associated stem cells (satellite cells). Scale bar = 1 mm.

  my dissertation work, I demonstrate that the Delta-like 1 (Dll1) molecule is capable to stimulate Notch signaling both ex vivo and in vivo. If included into the coating of culture dishes, Dll1 contributes to the ex vivo maintenance of stemness in proliferating muscle progenitor cells (MPCs), but, on the other hand, suppresses their proliferative capacity. rAAV-mediated in vivo overexpression of Dll1 on the muscle fiber enlarges the muscle stem cell pool but diminishes muscle enlargement. This effect can be seen during normal postnatal muscle development as well as during states of increased muscle regeneration in adult mdx mice.

  Regarding the experimental design (growth of MuSCs on Dll1-Fc coated surfaces) and the interpretation of its results we come to different conclusions as Parker et al[START_REF] Parker | Activation of Notch signaling during ex vivo expansion maintains donor muscle cell engraftment[END_REF], who demonstrated that muscle stem cells cultured on a Dll1-Fc surface (i) retained expression of MuSC markers (Pax7, Myf5) up to culture day 8, (ii) were able to engraft into the stem cell niche and (iii) to contribute to muscle regeneration. Here we show, however, that the maintenance of MuSC status (Pax7+|MyoD-) comes at the cost of a reduced proliferative capacity of these cells since Notch signaling largely reduces their proliferation rate. In contrast to Parker et al. we hence take a much more pessimistic view on the possibility to use this ex vivo culturing technique to generate sufficient cells for cell-based therapies of genetic muscle diseases. MuSCs are arrested in their stemness state by

  the first part of my dissertation I have observed that the Dll1 molecule had a profound effect on the maintenance of stemness of MuSCs in culture. Since our ultimate goal, however, was to contribute to the establishment of a treatment for patients with muscle diseases we continued to investigate the effect of Dll1 overexpression in vivo. Patients with Duchenne Muscular Dystrophy (DMD) suffer from loss of functional muscle mass which is associated with a severe depletion of the satellite cell pool (Partridge, 2013), which makes it possible that the modulation of Notch-signals might have a therapeutic effect. We therefore investigated the effect of Dll1-overexpression during two phases of rapid stem cell proliferation: (i) the phase of rapid postnatal muscle growth and (ii) during regeneration upon muscle fiber damage (as exemplified by the genetic mdx mouse model of DMD).

  any difference in the number of myonuclei between rAAV-Dll1 and control injected muscle demonstrating that the accretion of myoblasts into the myofiber had remained unaffected by Dll1-overexpression. On the other hand, Dll1-overexpression led to an increase of the number of quiescent muscle stem cells. This finding was verified by two different methods: (i) by counting the numbers of Pax7+ satellite cells on isolated muscle fibers (Figure35, Figure39) and (ii) by determining the number of Pax7+ satellite cells per muscle fiber on cryo-cross sections of the TA muscle (Figure35)In line with our findings is a study, in which the authors investigated a Dll1hypomorphic mouse model, which revealed that Dll1 is needed to prevent uncontrolled differentiation and for maintenance of the satellite cell pool. In the absence of sufficient functional Dll1-ligand Pax3/Pax7+ progenitors were initially formed but were lost and almost absent at embryonic day P14.5, whereas muscle growth declined starting around embryonic day P12, leading to subsequent severe muscle hypotrophy in hypomorphic Dll1 fetuses. The authors concluded that Dll1 provides essential signals to prevent uncontrolled premature differentiation and to ensure sustained periods of muscle differentiation during development[START_REF] Schuster-Gossler | Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants[END_REF].The increase of the satellite cell pool might have been achieved via (i) a stricter maintenance of MuSC quiescence in the first place (i.e. a conservation of the MuSC pool) or (ii) via an increased proliferation and subsequent re-entry into quiescence (i.e. an active increase of the MuSC pool). Our results of an increase of total cell numbers coinciding with a higher percentage of Pax7+|MyoD-cells under Dll1stimulation in vitro would imply that the MuSCs indeed divided, but were immediately forced back into quiescence through high Notch activity without expressing MyoD.

( i )

 i As Wen et al. had conditionally knocked-in the constitutively active Notch Intracellular Domain, the muscle progenitor cells were subjected to constant high Notch activity, which would be unresponsive to external stimuli through the Notch1 receptor (e.g. via modulation of the Notch-ligands or via regulation of the Notch-receptor density on the cell surface). In contrast to this 'constitutive' approach, we chose to overexpress the Notch ligand Dll1 on the muscle fibers. This stimulated the Notch receptor on the cell membrane of muscle stem cells, while leaving its regulatory system intact. As demonstrated by our in vitro experiments, a certain number of myogenic precursor cells always managed to 'escape' the push towards quiescence in order to become myoblasts, which tended to be unresponsive to Notch-stimulation (Figure20).

(

  ii) Furthermore, Wen et al. investigated the regenerative capacity in CTX-injured muscles, where the intrinsic features of MuSCs are basically unaltered, while we used the genetic mdx mouse model where the capability of MuSCs for self-renewal declines over time. It is still under debate, whether the reduction of the self-renewal capability of mdx MuSCs is triggered exclusively by the muscle environment or might be as well governed by intrinsic factors of MuSCs (Boldrin et al., 2014; Yablonka-Reuveni and Anderson, 2006). Boldrin et al. discovered that MuSCs isolated from aged mdx muscle were as functional as those derived from young or aged wild-type donors upon engraftment, indicating that the host environment of the mdx muscle causes MuSC dysfunction (Boldrin et al., 2014). In contrast, Yablonka et al. demonstrated that cultured mdx MuSCs differentiated in an accelerated manner in compari-son to wild-type MuSCs, indicating that intrinsic features of mdx MuSCs might have been altered (Yablonka-Reuveni and Anderson, 2006).

  tion via Dll1 contributes to the maintenance of stemness in proliferating MuSCs under cell culture conditions and (ii) to identify how artificial up-regulation of Dll1 signaling on muscle fibers might affect MuSCs in vivo.Our results demonstrate that overexpressing the Notch ligand Dll1 in order to induce Notch activity preserves stemness ex vivo and enlarges the stem-cell pool in vivo.This finding could be both attributed to impaired exit of the satellite cells from quiescence through upkeep of the Notch signaling cascade when it is supposed to be down regulated in developing and regenerating muscle. Modulating the Notch pathway by its ligands might hence offer a way to externally control the muscle stem cell pool even in states of increased regeneration such as in Duchenne muscular dystrophy. The question would be when and how to apply and how do dose the Notch ligands.Since the clinical phenotype of dystrophin related disorders strongly depends on the potential of the organism to regenerate damaged muscle fibers, the size of the stem cell pool will be a crucial factor. Future work should thus explore the effect of Dll1 overexpression in aged mdx mice with regard to the following aspects: (i) Can the depletion of the muscle stem cell pool be ameliorated by Notch stimulation or would a shift of the balance between quiescence and proliferation even have a disadvantage for the whole organism. (ii) Would such treatment have any clinical benefit and translate into longer life span and improved muscle function? (iii) Would an increase of Notch signaling also benefit other muscular dystrophies beyond DMD?

-

  -屈原 Qu Yuan First of all, I would like to thank Markus Schuelke for the opportunity to prepare my doctoral thesis in his laboratory. I am really grateful for the financial support, intellectual and scientific guidance of my projects, and encouragements whenever I encountered difficulties during the past 5 years. I also want to thank Luis Garcia for supervising my project and offering me the opportunity to work in his laboratory in Paris for six months during my Ph.D. I want to thank Helge Amthor for helping me to thoughtfully design the experiments and mentoring me during my whole Ph.D. I want to thank Rachid Benchaouir for his great help with the AAV experiments and kind advice. I also want to thank Guillaume Précigout for technical support, Karima Relizani and Gabriella Dias for fruitful discussions and helping me with the life in France. It was a pleasure to work in Paris. I want to thank all members of the Schuelke laboratory for their help, interest, and valuable advice, their continuous support, and the great working atmosphere. I especially want to thank Cora Beckmann and Franziska Seifert for helping me with my experiments. I also want to thank our technical assistants: Barbara Lucke, Susanne Morales Gonzalez, Angilika Zwirner and Esther Gill for teaching or helping me with various lab techniques and all the other small big things in everyday lab life. I want to thank Mina Petkova for her support and advice during experiments and other times in life; thank you. I also want to thank Dominik Seelow, Jana-Marie Schwarz, Ioanna Polydorou, Rodina Acharya, Henrik Sadlowski, Ellen Knierim, Gudrun Schottmann for their discussion and fruitful comments and advice on my medical career. I am grateful that I could be part of the MyoGrad program and get to know so many excellent young scientists. I appreciate the stimulating work environment, the excellent scientific training, and the great technical and financial framework Most of all, I like to thank my parents for supporting me throughout my studies. I am grateful for the support I received from Kun, thank you for listening, caring and tolerating.

Table 1 :

 1 Markers of satellite cells............................................................................................

Table 2 :

 2 List of satellite markers direct cell isolation...............................................................

Table 3 :

 3 List of chemicals and kits ..........................................................................................

Table 4 :

 4 List of primary and secondary antibodies .................................................................

Table 5 :

 5 Universal buffers and solutions .................................................................................

Table 6 :

 6 Buffers and solutions for Affinity Chromatography ...................................................

Table 7 :

 7 Buffers and solutions for SDS-PAGE and Western Blot ...........................................

Table 8 :

 8 Buffers, solutions and Media for cell culture .............................................................

Table 9 :

 9 List of software ..........................................................................................................

Table 10 :

 10 Production of Dll1-Fc protein ....................................................................................

Table 11 :

 11 Production of recombinant viral particles (rAAV) ......................................................

	293T cells Human Embryonic Kidney 293 cells
	AAV	Adeno-associated virus
	Ab	Antibody
	bp	base pair
	bFGF	bovine Fibroblast growth factor
	BCA	bicinchoninic acid
	BrdU	5-bromo-2'-deoxyuridine
	BSA	Bovine serum albumin
	cDNA	Complementary DNA
	DAPI	4',6-diamidin-2-phenylindol
	DAPT	N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester
	DEPC	Diethylpyrocarbonate
	Dll1	Delta-like 1 protein
	DMD	Duchenne muscular dystrophy
	DMEM	Dulbecco's Modified Eagle's Medium
	DMSO	dimethylsulfoxid
	DNA	desoxyribonucleic acid
	E. coli	Escherichia coli
	EDL	Extensor digitorum longus muscle
	EDTA	ethylendiamin-tetraacetat
	et al.	and others (lat: et alterae)
	FACS	Fluorescence-activated cell sorting
	FBS	fetal bovine serum
	Fc	fragment crystallizable region
	GAPDH	glycerinaldehyd-3-Phosphat-Dehydrogenase
	H&E	Hematoxylin and eosin stain
	HEPES	4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
	Hes	hairy and enhancer of split
	Hey	hairy/enhancer-of-split related with YRPW motif
	HRP	horseradish peroxidase
	LB	Lysogeny broth medium
	IgG	Immunoglobulin G
	iPS	Induced pluripotent stem (cells)
	Ki-67	marker of proliferation
	LIF	leukemia inhibitory factor
	mdx	Muscular dystrophy X-chromosome linked
	mRNA	Messenger ribonucleic acid
	Myf	Myogenic factor
	MyoD	Myoblast determination protein
	NICD	Notch-receptor intracellular domain
	Pax	Paired box transcription factor
	PBS	Phosphate buffered saline
	PCR	Polymerase chain reaction

Table 12: List of suppliers ....................................................................................................... Table 13: List of primers for real-time qPCR .......................................................................... iii List of abbreviations

  In this case a pool of MyoD+ progenitors that did not express Myf5 by lineage tracing criteria was proposed to compensate. Against this model,Wood et al. have shown that MyoD ablation caused complete loss of muscle at all anatomical locations without being compensated for[START_REF] Wood | MyoD-expressing progenitors are essential for skeletal myogenesis and satellite cell development[END_REF]. Additionally, Comai et al. conducted Myf5 Credriven cell ablation and pointed out that Myf5-expressing cells cannot be eliminated for 100%. Therefore, the "independent" MyoD+ population demonstrated by the studies ofGensch et al. and Haldar et al. is actually composed of "Myf5+ escaper cells"[START_REF] Comai | Variations in the Efficiency of Lineage Marking and Ablation Confound Distinctions between Myogenic Cell Populations[END_REF]. Hence, Comai et al. proposed a serial model, in which Myf5 functions upstream of MyoD in a gradual manner.

1.3.1 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.

  Table 12) if not stated otherwise. The consumables for molecular genetic and biochemical experiments, such as pipettes, pipette tips, plastic vials, and Eppendorf tubes, were obtained from Eppendorf, BD Biosciences, Roth and Brand. Sterilized disposable materials for cell culture, such as culture flasks and dishes, cryotubes were purchased from Nunc, BD Bioscience and TPP. Primers for DNA-Sequencing, Cloning and qPCR were purchased from MWG Biotech AG. The sequences of those primers are listed in Appendix Table 13.

Table 3 : List of chemicals and kits

 3

		Name				Catalog Number Supplier
	DNA Ladder	TrackIt	TM 100bp	10488-058	Invitrogen
		TrackIt	TM 1 Kb	N3232L	New England BioLab
		Smart Ladder	MW-1700-10	Eurogentec
	Protein Ladder	PageRuler	TM prestained	SM0671	Fermentas
		PageRuler	TM Plus prestained SM1819	Fermentas
		Name				Catalog Number Supplier
	Restriction en-	Bam HI		R0136T	New England BioLab
	donucleases	BgI II				R0144L	New England BioLab
		EcoR I-HF		R3101T	New England BioLab
		Name				Catalog Number Supplier
	Polymerases	SYBR	® Green PCR Master	4312704	Applied Biosystems
		Mix
		GoTaq ® PCR Core System I	M7660	Promega
		SuperScript II® reverse tran-	18064-071	Invitrogen
		scriptase
		Name				Catalog Number Supplier
	Kits	QIAprep Spin Miniprep Kit	27106	Qiagen
		QIAquick Gel Extraction Kit	28704	Qiagen
		NucleoBond® Xtra Midi	740410.10	Macherey-Nagel
		RNeasy Mini Kit	74106	Qiagen

Table 4 : List of primary and secondary antibodies

 4

		Antigen	Cat#	IgG type	IF Dilution	Supplier
	Primary	BrdU	OBT0030G	rat	1:100	AbD
	antibodies	Delta-like 1	SC-9102	rabbit	1:150	Santa Cruz
		Flag M2	200471	murine IgG	1:200	Stratagene
		Dystrophin	Dys	murine IgG	1:4	DSHB
		anti-	AM4300	murine IgG		Applied Biosys-
		GAPDH				tems
		Laminin	L9393	rabbit	1:400	Sigma
		Ki-67	M7240	murine IgG1	1:200	Dako
		MyoD	D7F2	murine IgG1	1:2	DSHB
		MyoD	SC-304	rabbit	1:200	Santa Cruz
		Myogenin	F5D	murine IgG1	1:4	DSHB
		Myogenin	N-20	goat	1:200	Santa Cruz
		myosin	MF20	murine IgG2b	1:6	DSHB
		Myf5	SC-302	rabbit	1:200	Santa Cruz
		Tubulin	A11126	mouse		Invitrogen
		Pax7	Pax7	murine IgG1	1:2	DSHB
		Pax3	Pax3	murine IgG2a	1:2	DSHB
		VCAM	AF643	goat	1:100	R&D systems
		antibodies	Cat#	IgG type	Dilution	Supplier
	Conjugated	PE-CD31	553373	rat	1:100	BD Pharmingen
	primary antibodies	PE-CD45 PE-Sca1	01115A 553336	rat rat	1:100 1:100	BD Pharmingen BD Pharmingen
		RD1-CD56	6603067	murine IgG1	1:40	Dako
			Reaction	Absorption/	Dilution	Supplier
				Emission
				[nm]
	Secondary	Alexa 488	anti-mouse	495/519	1:400	LifeTechnologies
	antibodies	Alexa 594	anti-mouse	594/628	1:400	LifeTechnologies
		Alexa 488	anti-rat	495/519	1:400	LifeTechnologies
		Alexa 488	anti-rabbit	495/519	1:400	LifeTechnologies
		Alexa 647	anti-rabbit	650/668	1:400	LifeTechnologies
		Alexa 488	anti-goat	495/519	1:400	LifeTechnologies
		Alexa 594	anti-goat	594/628	1:400	LifeTechnologies
		HRP	anti-mouse	--	1:2000	Calbiochem
		HRP	anti-rabbit	--	1:2000	Calbiochem
		HRP	anti-human	--	1:2000	Calbiochem

Table 5 : Universal buffers and solutions

 5

	Buffer	Composition
	PBS (10x), pH 7.4	1.4 M NaCl, 27 mM KCl, 81 mM Na 2 HPO 4 x 2 H 2 O, 15 mM KH 2 PO 4
	TE (1x), pH 7.4	10 mM TRIS, 1 mM Na 2 -EDTA
	TBE (5x), pH 8.0	0.5 M TRIS-HCl, 0.5 M boric acid, 10 mM Na 2 -EDTA

Table 6 : Buffers and solutions for Affinity Chromatography

 6

	Buffer	Composition
	Binding buffer	0.1 M sodium phosphate, pH 7.0
	Elution buffer	0.1 M glycine-HCl, pH 2.7
	Neutralize buffer	1 M TRIS-HCl, pH 9.0
	Storage buffer	20% (v/v) ethanol in water

Table 7 : Buffers and solutions for SDS-PAGE and Western Blot

 7

	Buffer/Solution	Composition
	SDS-stacking gel	1 x Upper chamber buffer, 5% (v/v) polyacrylamide (29:1, Bio-
		Rad), 0.1% (v/v) APS*, 0.1% (v/v) TEMED** (Merck)
	SDS-separation gel	1 x Lower chamber buffer, 12% (v/v) polyacrylamide (29:1), 0.1%
		(v/v) APS, 0.1% (v/v) TEMED
	Upper chamber buffer	0.5 M TRIS-HCl (pH 6.8), 0.4% (w/v) SDS
	Lower chamber buffer	1.5 M TRIS-HCl (pH 8.7), 0.4% (w/v) SDS
	Laemmli buffer (10x)	0.25 M TRIS, 1.9 M glycin, 1% (w/v) SDS
	TBST buffer (10x)	0.2 M TRIS, 1.4 M NaCl, 1% (v/v) Tween 20, pH 7.6
	Lysis buffer	50 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 1% NaPO 4 , 0.5% so-
		dium desoxycholate, 0.1% (w/v) SDS, 0.1% (v/v) β-
		mercaptoethanol, 1 mM PMSF***, 1 mg/ml aprotinin, 10 mg/ml
		leupeptin, 1 mg/ml pepstatin
	Loading buffer (2x)	62.5 mM TRIS-HCl (pH 6.8), 20% (v/v) glycerin, 4% (w/v) SDS,
		1% (v/v) β-mercaptoethanol, 0.025% (w/v) bromphenol blue
	Transfer buffer	70% 1 x Laemmli buffer, 30% (v/v) methanol
	Blocking buffer	5% (w/v) milk powder in 1 x TBST buffer
	ECL-solution A	0.1 M Tris (pH 8.3), 0.4 mM coumaric acid, 2.5 mM luminol
	ECL-solution B	0.1 M Tris (pH 8.3), 0.018% (v/v) H 2 O 2
	Ponceau staining solu-	2% (w/v) Ponceau red S, 30% (w/v) trichloroacetic acid, 30%
	tion (20x)	(w/v) sulfosalicylic acid
	Hematoxylin buffer	5% (w/v) aluminium potassium sulfate, 0.02% (w/v) sodium io-
		date, 0.1% (w/v) hematoxylin
	Coomassie staining	0.25% (w/v) Brillant blue, 45% (v/v) methanol, 10% (v/v) acetic
	solution (1x)	acid
	1st Coomassie decolori-	10% (v/v) acetic acid, 30% methanol
	zation solution
	2nd Coomassie decolor-	10% (v/v) acetic acid
	ization solution

*APS, ammonium per sulfate; **TEMED, tetramethylendiamin; ***PMSF, phenylmethylsulfonylfluorid

Table 8 : Buffers, solutions and Media for cell culture

 8

	Solutions/ Media	Composition
	DMEM cell culture	500 ml DMEM medium supplemented with L-glutamin, 4000 mg/L
	medium	glucose and sodium pyruvate (GibcoBRL)
	293T-D1Fc cell growth	500 ml DMEM medium supplemented with 55 ml FBS, 5.5 ml
	medium	Pen/Strep [100 U/ml penicilline G, 100 µg/ml Streptomycin final
		solution], 200 µg/ml Hygromycin B (GibcoBRL)
	Human skeletal mus-	500 ml skeletal muscle cell growth medium (Provitro) supplement-
	cle cell growth medi-	ed with 50 µg/ml fetuin, 10 ng/ml EGF, 1 ng/ml bFGF, 10 µg/ml
	um	insulin, 400 ng/ml dexamethasone (Provitro), 75 ml FBS, 50 ng/ml
		amphotericin B, 50 µg/ml gentamycin
	Satellite cell medium	500 ml DMEM/F12 medium supplemented with 75 ml FBS, 100
		µg/ml gentamycin, 10 ng/ml LIF (BioChrom), 2.5 ng/ml bFGF (Sig-
		ma), 12 ml B27 supplement w/o vitamin A (GibcoBRL)
	Freezing medium	90% (v/v) FBS, 10% (v/v) DMSO (Applichem)
	Trypsin/EDTA	0.05% Trypsin, 0.02% EDTA without Ca	2+ and Mg	2+
	Trypsin (10x)	2.5% Trypsin
	PBS (1x)	without Ca	2+ and Mg	2+ (PAA)
	3.1.6 Software

Table 9 : List of software

 9

	Program	Supplier	Application
	7500 system SDS v1.4	Applied Biosystems	real-time PCR
	ImageJ	National Institutes of Health	image processing and
			analysis
	BioDoc Analyzer	Biometra	agarose gel documentation
	Microsoft Office	Microsoft	Word processing
	Vector NTI	Invitrogen	viewing and constructing
			plasmids
	FileMaker	FileMaker, Inc	database
	GraphPad Prism 6.0	GraphPad Software, Inc.	producing graphs, statistics
	Lasergene	DNASTAR, Inc	sequence analysis
	MutationSurveyor v3.10	Softgenetics	sequence analysis
	ImagePro Plus 7.0	Mediacy	fluorescent image acquisition
			and analysis
	VISIONlite	Thermo Scientific	spectrophotometric analysis
	NCBI Primer3	National Institutes of Health	primer design
	Unicorn Software 5.11	GE healthcare	affinity chromatography
	SPSS	IBM	statistical analysis
	R Studio	Open Resource	statistical analysis

Table 10 : Production of Dll1-Fc protein

 10

	Batch/date	Protein	Sample volume	Total yield [µg]	Concentration [µg/µl]
	22.05.2013	Dll1-Fc	120 ml	205.74	0.3562
	23.05.2013	Dll1-Fc	130 ml	194.76	0.3964
	06.06.2013	Dll1-Fc	120 ml	313.20	0.5884
	07.06.2013	Dll1-Fc	100 ml	152.34	0.3168
	15.06.2013	Dll1-Fc	135 ml	333.36	0.7706

Table 11 : Production of recombinant viral particles (rAAV)

 11

	Batch	Virus	Titration (vp)
	n~2012_088	AAV2/1-U7-scramble	2.25E+12
	n~2012_089	AAV2/1-Dll1	7.50E+11
	n~2012_095	AAV2/1-Dll1	1.27E+12
	n~2014_007	AAV2/1-Dll1	1.43E+11

Table 13 : List of primers for real-time qPCR

 13

	Gene	Forward primer	Reverse primer	Product
				length
	mouse/human	GCATGGCCTTCCGTGTTCC	GGGTGGTCCAGGGTTTCTTACTC bp
	Gapdh
	human HEYL	GAAACAGGGCTCTTCCAAGC	GGCACTCCCGAAAACCAATG	bp
	human HES1	GAAAGATAGCTCGCGGCATT	TACTTCCCCAGCACACTTGG	bp
	human	TGGACCAGATTGGGGAGTTC	GCACACTCGTCTGTGTTGAC	bp
	NOTCH1
	human	CCTTCCACTGTGAGTGTCTGA	CACACCTTTGAAACCTGGCAT	bp
	NOTCH2
	human	AGGTGATCGGCTCGGTAGTA	CAACGCTCCCAGGTAGTCAG	bp
	NOTCH3
	mouse/human	GCTACAGGGGGTAAAGGCTA	GAGATGAGAGACAAGGCGCA	bp
	Hey2
	human MYOD	CGACGGCATGATGGACTACA	AGGCAGTCTAGGCTCGACAC	bp
	human MYOG	CCAGCGAATGCAGCTCTCAC	GCAGATGATCCCCTGGGTTGG	bp
	human DTX4	TGGGAATGGCTGAACGAGCA	CGGAGAGTTCCCGTGTCTTGG	bp
	human	GGCACATATAGGAATTCGG-	ACAGTCGATGGTGGAGAACAG	bp
	TIMM17b	CAC
	human PAX7	TCCAAGATTCTTTGCCGCTAC	GGTCACAGTGCCCATCCTTC	bp
	human MYF5	TCCAACTGCTCTGATGGCATG	AGCAATCCAAGCTGGATAAGGA	bp
	human	GCGTCTCAGTGCCCTTTTGT	TGAAGTAGTGGGCTAAGGGC	bp
	CXCR4
	human CD34	CAACGGTACTGCTACCCCAG	TGACTGTCGTTTCTGTGATGT	bp
	human RBPJ	TCCATTATGGACAAACAG-	ATGCGGTCTGCTTATCAACTTTC	bp
		TCAAACTT
	mouse Hes1	CAACACGACACCGGACAAAC	GGAATGCCGGGAGCTATCTT	bp
	mouse HeyL	GAAGCGCAGAGGGATCATAG	GGCATGGAGCATCTTCAAGT	bp
	mouse Hey1	CGGACGAGAATGGAAACTTGA	CCAAAACCTGGGACGATGTC	bp
	mouse R bpj	GGTCCCAGACATTTCTGCAT	GGAGTTGGCTCTGAGAATCG	bp
	mouse Pax3	ACGCTGTCTGTGATCGGAAC	AGGCTCGCTCACTCAGGAT	bp
	mouse Pax7	TCTCCAAGATTCTGTGCCGAT	CGGGGTTCTCTCTCTTATACTCC	bp
	mouse Myf5	GGATGGCTCTGTAGACGTGA	GCAGCTTTGACAGCATCTACT	bp
	mouse MyoG	GTGAATGCAACTCCCACAGC	CGCGAGCAAATGATCTCCTG	bp
	mouse Notch1 GGTCGCAACTGTGAGAGTGA	TCGCAGAAGGCTGTGTTGAT	bp
	mouse Notch2 GCAGGAGCAGGAGGTGATAG	GCGTTTCTTGGACTCTCCAG	bp
	mouse Notch3 GTCCAGAGGCCAAGAGACTG	TTGACATCCACTCCGTCTGC	bp
	mouse MyoD	TGCTCTGATGGCATGATGGAT	CACTGTAGTAGGCGGTGTCG	bp
	mouse MyoD	CACTACAGTGGCGACTCAGA	GCCGCTGTAATCCATCATGC	bp
	Dll1Flag	GTGGGGAGATTCCTGACAGA	GCCGCTCATTTATCGTCATC	bp
	mouse Dll1	CTGCAGGAGTTCGTCAACAA	ATACGCGAAAGAAGGTCCTG	bp
	mouse Dll1	GCACCTCACTGTGGGAGAAG	GGCAGACAGATTGGGTCAGT	bp

After 3-4 days, clusters of multiple cells deriving from the same initial satellite cell could be observed along the fiber. One cell cluster normally contained 5-10 nuclei, indicating that the satellite cell had undergone 2-3 rounds of cell division. However, within one cell cluster, less than half of the total number of nuclei were Pax7 + (Figure 17).

When fibers were placed on a Matrigel ® -coated surface, the satellite cells became activated and migrated from the fiber into the Matrigel ® within 24 h, giving birth to daughter cells on the culture surface rather than forming cell clusters along the myofibers. Similar to the movement of activated fiber-associated satellite cells, the satel- Again, the presence of DAPT suppressed the expression levels of Notch target genes on the Dll1-Fc surface. For instance, HeyL mRNA-levels decreased from 287.0±2.2% to 170.1±6.1% in the 7-day culture. In parallel Pax7 mRNA-levels dropped from 391.2±4.0% to 187.3±7.5%. All gene expression levels were normalized to the gene expression levels on the IgG+DMSO surface, which were set to 100%.

In summary, in this section I investigated the effect of immobilized Dll1-Fc on the mRNA and protein levels of several Notch target genes and myogenic markers.

Hence, Dll1-stimulation has a significant effect on maintaining MPCs in a more stemness state, an effect that could be antagonized by DAPT. However, in the presence of Dll1, the proliferation rate of MPCs was severely reduced.

4.5

The in vivo effect of Notch overstimulation

The viral gene transfer system

In order to investigate the in vivo effect of Notch overstimulation, we used the AAVmediated gene delivery system to deliver Dll1 to murine muscle cells.

Construction and production of the viral particles

The full-length murine Delta-like 1 coding sequence (a 2.2 kb fragment from the

Production

The three plasmids were co-transfected into the 293T virus producing cell line to generate either the AAV2/1-Dll1 virus or the control AAV2/1-U7 scramble virus. The rAAV virus particles were harvested, concentrated and confirmed by real-time qPCR according to an established protocol in Luis Garcia's laboratory. Each batch was controlled separately (Table 11).

Overexpression of Dll1 enlarges the muscle stem-cell pool

The percentage of Pax7 + cells per total number of myonuclei (epimysium excluded)

was significantly larger in Dll1-overexpressing muscle than in scramble muscle (Figure 35A, B), hinting towards an increase of the Pax7 + MuSC pool around Dll1overexpressing myofibers. Further, using BrdU-labeling we demonstrated that the proliferation rate of the MuSCs was reduced after 3 weeks of treatment (Figure 35C)

indicating that more MuSCs maintain their quiescence in the presence of Dll1. The reduction of the proliferation rate was not seen any more after 6 weeks of treatment (Figure 35D), possibly due to the fact that most of the MuSC have returned to quiescence by this time. The number of myonuclei per fiber in EDL from newborns with 3-week treatment (A) and from newborns with 6-week treatment (B). Fibers were acquired from 4 animals for measurement; one dot represents one fiber; the red line depicts the median. There is no significant difference between two sides (the p-value was calculated using the non-parametric Mann-Whitney U test).

Appendix