Cell-cell contact and signaling in the muscle stem cell niche

Abstract

Muscle stem cells (also called satellite cells, or SCs) rely on their local niche for regulatory signals during both homeostasis and regeneration. While a number of cell types communicate indirectly through secreted factors, here we focus on the significance of direct contact between SCs and their neighbors. During quiescence, SCs reside under a basal lamina and receive quiescence-promoting signals from their adjacent skeletal myofibers. Upon injury, the composition of the niche changes substantially, enabling the formation of new contacts that mediate proliferation, self-renewal, and differentiation. In this review, we summarize the latest work in understanding cell-cell contact within the satellite cell niche and highlight areas of open questions for future studies.

Background

Satellite cells (SCs) are the long-term adult stem cell population responsible for skeletal muscle’s ability to regenerate. Under homeostatic conditions, SCs are actively maintained in a dormant state known as quiescence; upon injury, SCs activate, proliferate, and differentiate to repair the damaged tissue.

The local niche is a critical regulator of SC function, providing a combination of biochemical and biomechanical signals to the stem cell [1,2]. During quiescence, SCs are located on the exterior of multinucleated skeletal myofibers, and each myofiber and its associated SCs are enwrapped by a basal lamina. This basement membrane physically separates SCs from other local niche cells, with myofibers being the only cells currently proven to make direct cell-cell contact with quiescent SCs (Figure 1). The basal lamina and interstitial extracellular matrix sequester various growth factors and signaling molecules associated with SC activation and proliferation [3].

Figure 1. The quiescent satellite cell niche.

Quiescent satellite cells (QSCs) reside between multinucleated myofibers and a surrounding basal lamina. QSCs adhere to the basal lamina through integrins (not shown), and mediate contact with myofibers through classical cadherins and Notch receptor-ligand interactions. Other niche cells (such as tissue-specific macrophages) are housed within an interstitial matrix, separated from QSCs. Blood vessels that make up the niche vasculature are found in close proximity to QSCs but have not been shown to penetrate through the basal lamina to form stable cell-cell contacts with SCs or myofibers. Various cells are not drawn to scale.

Muscle injury induces significant changes in the niche, including damage to myofibers and, often, disruption of the basal lamina, thereby exposing SCs to such signals [4] (Figure 2). A plethora of cell types in the niche have been shown to provide signaling cues during regeneration [5], but the formation and function of direct contacts between SCs and their neighboring cells has been understudied.

Figure 2. The regenerating satellite cell niche.

following muscle injury, damage to the niche enables the formation of new cell-cell contacts during regeneration. Perturbation of the basal lamina allows macrophages to interact with activated SCs through VCAM-1 and VLA-4. Proliferating, activated SCs communicate with each other through M-cadherin and Dll1/Notch 2 signaling, and activated SCs signal to their adjacent myofibers through Ephrin/Eph ligand-receptor interactions.

Many reviews have assessed cell-cell contact and adhesion during developmental myogenesis [6,7], and the SC niche as a whole [1,2]; here we aim to discuss the specific role of direct cell-cell interactions within the quiescent and regenerating SC niche.

Quiescence

The importance of myofibers in promoting SC quiescence has been understood for many years [8], but signaling pathways by which myofibers provide behavioral cues to SCs are only recently being elucidated. Direct binding of SCs to myofibers is facilitated through classical cadherins, a family of homophilic cell-cell adhesion molecules. There is likely redundancy in cadherin family members within the skeletal muscle lineage – at least three cadherins (N-, M-, R-cadherins) are expressed simultaneously during development [9] and at least three (N-, M-, VE-cadherins) are expressed by adult myofibers and SCs [10] and localized to the SC-myofiber junction [11]. Despite this potential redundancy, it appears that not all cadherins play equivalent roles. M-cadherin (Mcad) can be removed from the mouse germline with no discernable phenotype [12], but the removal of N-cadherin (Ncad) from adult SCs induces a break in quiescence in the absence of injury [11]. This phenotype is exacerbated by the further removal of Mcad, suggesting that some redundancy or compensatory function exists, but why Ncad is functionally more important than Mcad in promoting quiescence remains an open question. It’s possible that Mcad acts solely as a physical tether between myofiber and SC (and is therefore most easily compensated by other cadherins), while there are findings in other systems implicating a role for Ncad in growth factor signaling, mechanotransduction, and cell movement [13,14]. Importantly, removal of Ncad from SCs does not lead to exit from the niche or loss of cell polarity, indicating that Ncad has a function more directly related to maintenance of SC quiescence [11]. More work is needed to elucidate the exact functions of each cadherin within the SC niche.

A rapidly growing area of interest in SC biology is Notch signaling, a juxtacrine pathway in which signal-sending cells express transmembrane ligands from their surface (e.g., Delta-like 1/3/4, Jagged 1/2), and signal-receiving cells express transmembrane receptors (e.g., Notch1/2/3/4). Upon ligand binding, the receptor’s intracellular domain is proteolytically cleaved and translocates into the nucleus, where it associates with the DNA-binding protein RBPJ to activate target gene expression [15]. Active Notch signaling has been linked to maintenance of the quiescent SC state. Quiescent SCs display high levels of active Notch signaling, and satellite cell-specific removal of RBPJ induces a break in quiescence and depletion of the SC pool [16,17]. Notch signaling exerts its effects via multiple target genes. It induces expression of Collagen V which acts in an autocrine manner to promote SC quiescence, by serving as a ligand for Calcitonin receptor [18]. Notch signaling also drives expression of miR-708, a miRNA that targets Tns3 transcripts (encoding the focal adhesion component Tensin 3) to antagonize SC migration, thereby keeping them stationary in quiescence [19].

Recent work has attempted to parse which Notch ligands and receptors are involved in modulating pathway dynamics within the niche. Quiescent SCs express Notch receptors 1 and 3, and myofibers express Dll4 [10,20,21]. Recent work suggested that Dll4 may be provided to the SC by niche endothelial cells (ECs) [22]. While it is possible that ECs could penetrate through the basal lamina to make stable contacts with SCs, this interaction was assessed with cells co-cultured in vitro, and direct contact between ECs and SCs in vivo has not yet been observed. Another recent study has demonstrated that genetic removal of Dll4 from myofibers induced a break in SC quiescence [21]. It therefore appears more likely that the Dll4 required for SC quiescence is provided by the muscle fiber. The larger question of whether niche cells which reside outside the basal lamina can form stable, direct cell-cell contacts with quiescent SCs remains unclear.

Regeneration

During muscle regeneration, the SC niche experiences significant changes in both cell composition and signaling cascades. Many of the same juxtacrine pathways involved in the maintenance of SC quiescence are employed iteratively in regeneration, shifting from mediating contact between SCs and myofibers to SCs and their transit-amplifying progeny or other cells. One such example is Mcad. Under homeostatic conditions, Mcad functions alongside other cadherins to mediate adhesion between SCs and myofibers. During regeneration, however, Mcad’s role shifts to mediate contact between SC progeny and aid in cell-cell fusion [23]. Similar shifts in contact dynamics are seen in Notch signaling. Recent work has illustrated the importance of oscillations in expression of Hes1 (a transcription factor induced by Notch signaling) and the myogenic transcription factor MyoD in regulating proliferation vs. differentiation of activated SCs [24]. Single-cell RNA sequencing studies further supported these data and revealed a role for Dll1 and Notch2 in regeneration; differentiating SCs express Dll1, which feeds back to trigger Notch signaling associated with SC self-renewal [25**]. Zhang et al. elaborated on these findings and linked Dll1 activity to Hes1 expression, demonstrating that both the presence, and oscillatory dynamics, of Dll1 are critical for maintaining the proper balance between self-renewal and differentiation of muscle stem cells [26**]. Therefore, while Dll4 is presented to SCs by adjacent niche cells (myofibers) during quiescence, Dll1 required for self-renewal is derived from SC transit-amplifying progeny. The opposing roles of Dll4 and Dll1 seen with SCs are consistent with mechanistic work assessing differences between signaling dynamics induced by these two ligands – Dll1 activates Notch1 receptor in transient pulses driven by dense ligand-receptor clusters, whereas Dll4 can sustain Notch1 activation at lower ligand densities [27]. These ligand dynamics drive different downstream transcriptional responses, facilitating distinct cell fate decisions and demonstrating how a change in Notch ligand can modulate context-specific pathway signaling.

An area of SC biology that remains understudied is the relationship between SCs and myofibers during regeneration. Ex vivo live imaging revealed that activated SCs quickly acquire a motile phenotype [28]. Intravital imaging showed that SCs migrate along persisting basal laminae (“ghost fibers”) towards the area of injury [29]. This directional migration corresponds with dynamic expression within both myofibers [30] and SCs [31] of Eph receptor family tyrosine kinases and their ephrin ligands, molecules involved in classical cell guidance signaling pathways [32]. In vitro assays demonstrated that SCs are highly responsive to ephrin signaling; stripes of ephrin ligand affected both direction of cell migration and orientation of differentiated myotubes [30]. Only one other study has investigated the role of Eph-ephrin signaling in adult SCs, demonstrating that EphB1 regulates dynamics of proliferation and differentiation [31]. A more recent study assessed the role of EphA7 in muscle development, and SCs lacking EphA7 display delayed differentiation [33*]. The developmental community effect described in this report is likely to be conserved in some capacity during adult regeneration, underscoring the need for further study into the function of Ephs and ephrins in SCs and adult myogenesis.

In addition to SC progeny contacting each other and their myofibers during regeneration, injury-induced perturbation of the basal lamina may enable SCs to encounter other niche cells. Upon injury, immune cells are recruited to the wound in well-defined cascades [5]. The majority of studies on immune cells in regenerating muscle have focused on secreted factors and chemokines [34], but the adhesion molecule VCAM-1 has been implicated as a potential SC cell surface molecule that can bind to α4β1 integrin (VLA4) on macrophages [35]. Recent live-imaging work in zebrafish has addressed functions of resident macrophages in muscle regeneration, demonstrating their role in promoting SC proliferation [36**]. The focus of this report was a secreted cytokine, but the live-imaging data therein clearly showed prolonged, direct interactions between SCs and macrophages. More work will be needed to assess the role of this cell-cell contact and whether these interactions also occur in the mammalian niche.

Unlike macrophages, direct cell-cell interactions have not yet been demonstrated between SCs and nearby ECs during regeneration. SCs secrete VEGFA to recruit nearby blood vessels and maintain a properly vascularized niche [22]. The proximity of the two populations and the significance of direct stem cell-EC contact in other systems [37], however, suggests that there may be more of a direct role than we currently understand. Taken together, the above studies have laid the groundwork for exciting new exploration of cell-cell contact-based communication during both SC quiescence and regeneration.

Challenges and future approaches

Among the foremost challenges in the muscle stem cell field are: 1) maintaining stemness while expanding SCs in vitro for therapeutic approaches; and 2) developing techniques to allow discovery of novel SC biology. The easiest way to study cell biology is through isolation and in vitro culture of a target population, but SC stem cell properties are so dependent on maintenance of niche-derived signals and interactions that isolation induces activation and loss of efficient stem cell activity [38]. Many groups have attempted to incorporate known mechanisms of intercellular communication to maintain stem-like properties ex vivo, with varying degrees of success. Progress has been made in using biomaterials to modulate niche signals in vitro, both with and without added signaling molecules [39-43]. Reconstituting the complex cell-cell interactions described above has, however, proved to be significantly more difficult. Significant recent advances have used Notch ligands. Culturing primary myoblasts on affixed recombinant Notch ligands promoted expansion of SC-like cells in vitro, consistent with the in vivo role of Notch in maintaining SC quiescence, but proved insufficient to improve regenerative potential upon transplantation [44]. When SCs were cultured on a combination of Dll4 and PDGF-BB ligands, however, engraftment into host muscle was increased [45]. While these culture systems can initiate ligand/receptor presentation, feedback mechanisms provided from neighboring cells are impossible to reconstitute with such systems. In addition, there is emerging evidence that signaling is not only affected by the presence or absence of specific ligands, but also via mechanical sensation and spatiotemporal clustering [26,27,33]. Providing stem cells with dispersed lawns of tethered ligands ex vivo might induce different behaviors than discrete clusters of such ligands found in vivo at sites of cell-cell contact.

Even though their importance in translational biology is indisputable, in vitro systems cannot yet reliably incorporate all necessary niche signals (molecular, mechanical, or other), and therefore are limited in their ability to investigate novel aspects of SC biology. Live-imaging of stem cells remains the gold standard for assessing in vivo behavior, and 2-photon/multiphoton microscopy has been used in both mice [29] and zebrafish [36] to describe SC-niche dynamics. Despite these herculean efforts, significant challenges remain in live imaging approaches, particularly in the mouse. First, SCs are primarily quiescent, making population and cell-cell dynamics relatively slow in the absence of an injury. Second, live-imaging setups are technically challenging and costly. Lastly, live-imaging of animals allows characterization and descriptive analysis of stem cell behavior, but limits the ability to manipulate the stem cell niche. To circumvent some of these limitations, hindlimb explants have been used to study SC motility [19]. Explant systems are still a new and relatively challenging technique, but require less extensive setups than whole animal live-imaging and allow some experimental perturbations while still maintaining the stem cells in their direct niche.

The complexity of the SC niche exemplifies the importance of studying dynamics between cells. Some of the challenges associated with this may be evaded through the use of in silico studies, as well as large-scale transcriptomic and proteomic screens. Transcriptional profiling has already revealed insights into the earliest transcriptional signs of SC activation [46-48], control of SC establishment and maintenance [31], and stage-specific regulation of regeneration [49*]. Proteomic studies provide additional information about the cellular dynamics of both SCs and their niche cells during muscle repair [50*]. Combining these high-quality datasets with improved imaging systems will enable an unprecedented look at cell-cell dynamics. Future studies should focus on uncovering mechanisms of cell-cell contact-dependent niche regulation, generating novel perspectives into basic SC biology and therapeutic possibilities.