Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche.

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix1-3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells4-9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.

Dynamic binding of RBPJ is determined by Notch signaling status.

Notch signaling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighboring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and, together with the transcription factor moiety of Notch (NICD), regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole-genome binding profiles were generated for RBPJ; its coactivator, p300; NICD; and the histone H3 modifications H3 Lys 4 trimethylation (H3K4me3), H3 Lys 4 monomethylation (H3K4me1), and histone H3 Lys 27 acetylation (H3K27ac) in myogenic cells under active or inhibitory Notch signaling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300 and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signaling mediate their activities and consequently impact on cell fate decisions.

Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest.

A central question in development is to define how the equilibrium between cell proliferation and differentiation is temporally and spatially regulated during tissue formation. Here, we address how interactions between cyclin-dependent kinase inhibitors essential for myogenic growth arrest (p21(cip1) and p57(kip2)), the Notch pathway and myogenic regulatory factors (MRFs) orchestrate the proliferation, specification and differentiation of muscle progenitor cells. We first show that cell cycle exit and myogenic differentiation can be uncoupled. In addition, we establish that skeletal muscle progenitor cells require Notch signaling to maintain their cycling status. Using several mouse models combined with ex vivo studies, we demonstrate that Notch signaling is required to repress p21(cip1) and p57(kip2) expression in muscle progenitor cells. Finally, we identify a muscle-specific regulatory element of p57(kip2) directly activated by MRFs in myoblasts but repressed by the Notch targets Hes1/Hey1 in progenitor cells. We propose a molecular mechanism whereby information provided by Hes/Hey downstream of Notch as well as MRF activities are integrated at the level of the p57(kip2) enhancer to regulate the decision between progenitor cell maintenance and muscle differentiation.

Cell-autonomous Notch activity maintains the temporal specification potential of skeletal muscle stem cells.

During organogenesis, a continuum of founder stem cells produces temporally distinct progeny until development is complete. Similarly, in skeletal myogenesis, phenotypically and functionally distinct myoblasts and differentiated cells are generated during development. How this occurs in muscle and other tissues in vertebrates remains largely unclear. We showed previously that committed cells are required for maintaining muscle stem cells. Here we show that active Notch signalling specifies a subpopulation of myogenic cells with high Pax7 expression. By genetically modulating Notch activity, we demonstrate that activated Notch (NICD) blocks terminal differentiation in an Rbpj-dependent manner that is sufficient to sustain stem/progenitor cells throughout embryogenesis, despite the absence of committed progeny. Although arrested in lineage progression, NICD-expressing cells of embryonic origin progressively mature and adopt characteristics of foetal myogenic cells, including expression of the foetal myogenesis regulator Nfix. siRNA-mediated silencing of NICD promotes the temporally appropriate foetal myogenic fate in spite of expression of markers for multiple cell types. We uncover a differential effect of Notch, whereby high Notch activity is associated with stem/progenitor cell expansion in the mouse embryo, yet it promotes reversible cell cycle exit in the foetus and the appearance of an adult muscle stem cell state. We propose that active Notch signalling is sufficient to sustain an upstream population of muscle founder stem cells while suppressing differentiation. Significantly, Notch does not override other signals that promote temporal myogenic cell fates during ontology where spatiotemporal developmental cues produce distinct phenotypic classes of myoblasts.

Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myostatin.

Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-ß ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine.

Distinct contextual roles for Notch signalling in skeletal muscle stem cells.

Notch signalling acts in virtually every tissue during the lifetime of metazoans. Recent studies have pointed to multiple roles for Notch in stem cells during quiescence, proliferation, temporal specification, and maintenance of the niche architecture. Skeletal muscle has served as an excellent paradigm to examine these diverse roles as embryonic, foetal, and adult skeletal muscle stem cells have different molecular signatures and functional properties, reflecting their developmental specification during ontology. Notably, Notch signalling has emerged as a major regulator of all muscle stem cells. This review will provide an overview of Notch signalling during myogenic development and postnatally, and underscore the seemingly opposing contextual activities of Notch that have lead to a reassessment of its role in myogenesis.

The extracellular matrix niche of muscle stem cells.

Preserving the potency of stem cells in adult tissues is very demanding and relies on the concerted action of various cellular and non-cellular elements in a precise stoichiometry. This balanced microenvironment is found in specific anatomical "pockets" within the tissue, known as the stem cell niche. In this review, we explore the interplay between stem cells and their niches, with a primary focus on skeletal muscle stem cells and the extracellular matrix (ECM). Quiescent muscle stem cells, known as satellite cells are active producers of a diverse array of ECM molecules, encompassing major constituents like collagens, laminins, and integrins, some of which are explored in this review. The conventional perception of ECM as merely a structural scaffold is evolving. Collagens can directly interact as ligands with receptors on satellite cells, while other ECM proteins have the capacity to sequester growth factors and regulate their release, especially relevant during satellite cell turnover in homeostasis or activation upon injury. Additionally, we explore an evolutionary perspective on the ECM across a range of multicellular organisms and discuss a model wherein satellite cells are self-sustained by generating their own niche. Considering the prevalence of ECM proteins in the connective tissue of various organs it is not surprising that mutations in ECM genes have pathological implications, including in muscle, where they can lead to myopathies. However, the particular role of certain disease-related ECM proteins in stem cell maintenance highlights the potential contribution of stem cell deregulation to the progression of these disorders.

A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state.

Notch signaling plays a key role in virtually all tissues and organs in metazoans; however, limited examples are available for the regulatory role of this pathway in adult quiescent stem cells. We performed a temporal and ontological assessment of effectors of the Notch pathway that indicated highest activity in freshly isolated satellite cells and, unexpectedly, a sharp decline before the first mitosis, and subsequently in proliferating, satellite cell-derived myoblasts. Using genetic tools to conditionally abrogate canonical Notch signaling during homeostasis, we demonstrate that satellite cells differentiate spontaneously and contribute to myofibers, thereby resulting in a severe depletion of the stem cell pool. Furthermore, whereas loss of Rbpj function provokes some satellite cells to proliferate before fusing, strikingly, the majority of mutant cells terminally differentiate unusually from the quiescent state, without passing through S-phase. This study establishes Notch signaling pathway as the first regulator of cellular quiescence in adult muscle stem cells.

Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls.

Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies.

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

The Notch signaling network in muscle stem cells during development, homeostasis, and disease.

Skeletal muscle stem cells have a central role in muscle growth and regeneration. They reside as quiescent cells in resting muscle and in response to damage they transiently amplify and fuse to produce new myofibers or self-renew to replenish the stem cell pool. A signaling pathway that is critical in the regulation of all these processes is Notch. Despite the major differences in the anatomical and cellular niches between the embryonic myotome, the adult sarcolemma/basement-membrane interphase, and the regenerating muscle, Notch signaling has evolved to support the context-specific requirements of the muscle cells. In this review, we discuss the diverse ways by which Notch signaling factors and other modifying partners are operating during the lifetime of muscle stem cells to establish an adaptive dynamic network.

Notch signals control the fate of immature progenitor cells in the intestine.

The Notch signalling pathway plays a crucial role in specifying cellular fates in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in intestinal development. Here, by modulating Notch activity in the mouse intestine, we directly implicate Notch signals in intestinal cell lineage specification. We also show that Notch activation is capable of amplifying the intestinal progenitor pool while inhibiting cell differentiation. We conclude that Notch activity is required for the maintenance of proliferating crypt cells in the intestinal epithelium.

Stress relief: emerging methods to mitigate dissociation-induced artefacts.

The rapid progress of single-cell RNA-sequencing (scRNA-seq) at large scales has led to what seemed impossible until recently: the generation of comprehensive transcriptional maps of nearly all cells in multicellular tissues. We pinpoint three key elements as being critical to the production of these maps: scalability, spatial information, and accuracy of the transcriptome of the individual cells. Here, we discuss the ramifications of traditional cell-isolation protocols when capturing the transcriptional signature of cells as they exist in their native tissue context, the methods that have been developed to avoid these distortions, and the biological processes that have unraveled on account of these upgraded methodological approaches.

Extraction and sequencing of single nuclei from murine skeletal muscles.

Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).

Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells.

Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.

Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation.

Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations.

Modifiers of notch transcriptional activity identified by genome-wide RNAi.

BACKGROUND: The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription. RESULTS: Employing complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components. CONCLUSIONS: The genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.

Notch signalling: sensor and instructor of the microenvironment to coordinate cell fate and organ morphogenesis.

During development, stem cells give rise to specialised cell types in a tightly regulated, spatiotemporal manner to drive the formation of complex three-dimensional tissues. While mechanistic insights into the gene regulatory pathways that guide cell fate choices are emerging, how morphogenetic changes are coordinated with cell fate specification remains a fundamental question in organogenesis and adult tissue homeostasis. The requirement of cell contacts for Notch signalling makes it a central pathway capable of linking dynamic cellular rearrangements during tissue morphogenesis with stem cell function. Here, we highlight recent studies that support a critical role for the Notch pathway in translating microenvironmental cues into cell fate decisions, guiding the development of diverse organ systems.

Notch-Induced miR-708 Antagonizes Satellite Cell Migration and Maintains Quiescence.

Critical features of stem cells include anchoring within a niche and activation upon injury. Notch signaling maintains skeletal muscle satellite (stem) cell quiescence by inhibiting differentiation and inducing expression of extracellular components of the niche. However, the complete spectrum of how Notch safeguards quiescence is not well understood. Here, we perform Notch ChIP-sequencing and small RNA sequencing in satellite cells and identify the Notch-induced microRNA-708, which is a mirtron that is highly expressed in quiescent cells and sharply downregulated in activated cells. We employ in vivo and ex vivo functional studies, in addition to live imaging, to show that miR-708 regulates quiescence and self-renewal by antagonizing cell migration through targeting the transcripts of the focal-adhesion-associated protein Tensin3. Therefore, this study identifies a Notch-miR708-Tensin3 axis and suggests that Notch signaling can regulate satellite cell quiescence and transition to the activation state through dynamic regulation of the migratory machinery.

Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells.

Adult skeletal muscle maintenance and regeneration depend on efficient muscle stem cell (MuSC) functions. The mechanisms coordinating cell cycle with activation, renewal, and differentiation of MuSCs remain poorly understood. Here, we investigated how adult MuSCs are regulated by CDKN1c (p57kip2), a cyclin-dependent kinase inhibitor, using mouse molecular genetics. In the absence of CDKN1c, skeletal muscle repair is severely impaired after injury. We show that CDKN1c is not expressed in quiescent MuSCs, while being induced in activated and proliferating myoblasts and maintained in differentiating myogenic cells. In agreement, isolated Cdkn1c-deficient primary myoblasts display differentiation defects and increased proliferation. We further show that the subcellular localization of CDKN1c is dynamic; while CDKN1c is initially localized to the cytoplasm of activated/proliferating myoblasts, progressive nuclear translocation leads to growth arrest during differentiation. We propose that CDKN1c activity is restricted to differentiating myoblasts by regulated cyto-nuclear relocalization, coordinating the balance between proliferation and growth arrest.