Author response: Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mechanical forces between cells and extracellular matrix (ECM) influence cell shape and function. Tendons are ECM-rich tissues connecting muscles with bones that bear extreme tensional force. Analysis of transgenic zebrafish expressing mCherry driven by the tendon determinant scleraxis reveals that tendon fibroblasts (tenocytes) extend arrays of microtubule-rich projections at the onset of muscle contraction. In the trunk, these form a dense curtain along the myotendinous junctions at somite boundaries, perpendicular to myofibers, suggesting a role as force sensors to control ECM production and tendon strength. Paralysis or destabilization of microtubules reduces projection length and surrounding ECM, both of which are rescued by muscle stimulation. Paralysis also reduces SMAD3 phosphorylation in tenocytes and chemical inhibition of TGFβ signaling shortens tenocyte projections. These results suggest that TGFβ, released in response to force, acts on tenocytes to alter their morphology and ECM production, revealing a feedback mechanism by which tendons adapt to tension. https://doi.org/10.7554/eLife.38069.001 eLife digest Tendons – the fibrous structures that attach muscles to bones – must withstand some of the strongest forces in the body. Little is known about how tendons develop or adapt to withstand these forces. Studies have shown that muscles respond actively to force, as seen during exercise. Do tendons respond in similar ways? Tendons consist of collagen fibers surrounded by a ‘matrix’ of proteins. Also embedded in the matrix are specialized cells called tenocytes, which regulate the production of the different components of the tendon. A genetic modification allows tenocytes to be tracked using a fluorescent gene product that can be viewed using a microscope. Subramanian et al. have now used this technique in zebrafish to watch how the behaviors of the tenocytes change in response to forces applied to the tendon. Subramanian et al. show that at the start of muscle contraction, tenocytes put forth long projections from their cell bodies that extend perpendicular to the muscle fibers. This suggests that the projections act as force sensors. Consistent with this idea, paralyzing the muscle causes the projections to shrink. This shrinkage correlates with changes in how the tendon matrix proteins are organized. Further investigation reveals a force-responsive signaling pathway in the tenocytes that controls how these cells grow and produce key tendon matrix proteins. Subramanian et al. believe this pathway is central to how tendons adapt to the forces applied during muscle contraction. A better knowledge of how force affects tendon structure could ultimately help to improve treatments for tendon injuries and tendon atrophy. In particular, understanding how force affects how tenocytes develop could help researchers to develop new ways to regenerate and repair tendons. https://doi.org/10.7554/eLife.38069.002 Introduction Cells in all multicellular organisms are exposed to mechanical forces through adhesions to neighboring cells and to the extracellular matrix (ECM), as well as the ebb and flow of the environment. Force has been shown to influence cellular processes such as cell division, survival, migration, and differentiation (Behrndt et al., 2012; Culver and Dickinson, 2010; Hamada, 2015; Keller et al., 2008; Roman and Pekkan, 2012). Cellular responses to force include the activation of cell surface receptors such as integrins (Itgs), G-protein-coupled receptors (GPCRs), transient receptor potential (TRP) ion channels, and Piezo channels (Busch et al., 2017; Chachisvilis et al., 2006; Maartens and Brown, 2015; Mederos y Schnitzler et al., 2008; Popov et al., 2015; Wu et al., 2017). Despite recent insights into the nature of such responses, few in vivo studies have investigated how cells adapt to force and alter the ECM landscape to strengthen or weaken it accordingly (Maeda et al., 2011; Ng et al., 2014). The musculoskeletal system bears among the strongest forces experienced by any tissue, such as the tensional forces exerted upon tendons and ligaments (Heinemeier et al., 2013; Wang, 2006). Tendons can withstand such forces due to the specialized organization of collagen (Col) fibers and proteoglycans within each tendon fibril. Tendon injuries are extremely common and debilitating, especially in athletes, the elderly, and patients with neuromuscular diseases such as muscular dystrophy (Bönnemann, 2011; Walden et al., 2017). Despite their prevalence, little is known about how tendon fibroblasts (tenocytes) respond in vivo to tensional force at muscle attachments, or how they adapt to changes in mechanical load. Tendons form a variety of attachment sites - connecting muscles to cartilages and bones as well as other muscles and soft tissues. A myotendinous junction (MTJ) is a specialized ECM-rich region at the interface of muscle-tendon attachment sites that functions as the primary sources of force transmission. Each type of attachment bears varying levels of force, which correlates with distinct composition and organization of its tendon ECM (Ker et al., 2000; Wang, 2006). While extensive research has been conducted to evaluate the effects of exercise on size and strength of muscle fibers, less is known about how it effects tendon morphology and function. Understanding this is key to gaining insights into the causes of tendon defects and developing new treatments for tendon injuries or atrophy. Previous studies in vitro have suggested that tenocytes actively respond to changes in force in their environment by modulating ECM composition and organization (Maeda et al., 2010; Rullman et al., 2009). Excised tendons stretched in collagen gels, as well as tissue samples from chronic Achilles tendonitis patients, upregulate various collagens and ECM-modulating proteins, particularly Col3, Matrix Metalloproteinase 9 (MMP9) and MMP13 (Ireland et al., 2001; Pingel et al., 2014). In addition, collagen fibril size decreases and fibril packing increases in tendinopathies, likely due to increased ECM turnover (Pingel et al., 2014). These studies have suggested ECM modifications and morphological changes in tendinopathies but they have largely been limited to cultured tendons or tendon fragments. In vivo, several growth factor signaling pathways and transcription factors have been implicated downstream of mechanical force in tendon development and repair in mice. These include several members of the Transforming Growth Factor (TGF) superfamily, including TGFβ and Bone Morphogenetic Proteins (BMPs), as well as Fibroblast Growth Factors (FGF) (Gumucio et al., 2015; Nourissat et al., 2015). Mice lacking the transcription factor Scleraxis (Scx) show severe defects in force-transmitting and load-bearing tendons, suggesting that Scx is essential for maintenance of tendon ECM in response to mechanical force (Murchison et al., 2007). In addition, Scx directly regulates transcription of tendon ECM components, including Col1a1 (Havis et al., 2014; Subramanian and Schilling, 2015). Our studies of the ECM protein Thrombospondin 4b (Tsp4b) in zebrafish have shown that it is an essential scaffolding protein for tendon ECM assembly, required to maintain muscle attachments subjected to mechanical force via muscle contraction, and able to strengthen attachments when overexpressed (Subramanian and Schilling, 2014). Here, we show that mechanical force causes remarkable morphological changes in tenocytes in zebrafish, which form a dense curtain of projections at MTJs, and in their surrounding ECM. Tenocyte projections have been reported in electron micrographs of mammalian tendon fascicles yet how they form and their functions in tendon development remain largely unexplored (Kalson et al., 2015; Knudsen et al., 2015). Our results suggest that tenocytes play an active role in sensing force and thereby regulating ECM composition and overall tendon strength. In addition, we show that the force of muscle contraction regulates the growth and branching of tenocyte projections via TGFβ signaling. Such feedback between tenocytes and ECM may be a common mechanism for force adaptation within the musculoskeletal system. Results Tenocytes elongate with the onset of muscle contraction Tenocytes in zebrafish express two Scx orthologues, scxa and scxb (Chen and Galloway, 2014). Using a bacterial artificial chromosome (BAC) transgenic line that expresses mCherry under the control of regulatory elements for scxa, Tg(scxa:mCherry), we examined the morphogenesis of tenocytes during embryonic (20 hr post fertilization (hpf) to 72 hpf) and early larval (72 hpf to 5 dpf) zebrafish development. Expression of scxa:mCherry was first detected at 20 hpf in muscle and tendon progenitors of the somites. In a developing zebrafish embryo, muscles in the trunk establish attachments at bilateral, ‘chevron’ shaped somite boundaries that subdivide each muscle segment forming the vertical myoseptum (VMS). In addition, dorsal and ventral compartments within each somite are subdivided by a horizontal myoseptum (HMS), which extends laterally from the notochord (NC), along which oblique myofibers attach. By 24 hpf, as the first myofibers differentiated, scxa:mCherry expression in muscle progenitors diminished and became progressively restricted to scattered tendon progenitors along the HMS and VMS (~24 cells per VMS) (Figure 1A,D,G) (Figure 1—video 1). Cells with the highest levels of scxa:mCherry expression were located laterally, adjacent to the HMS, while more medial cells expressed lower levels (Figure 1A’, D’, G’). By 36 hpf, scxa:mCherry+ cells doubled in number (~44/VMS) and became increasingly localized to the HMS and VMS at future MTJs (Figure 1B,E,H) (Figure 1—video 1). At this stage, cells with the highest scxa:mCherry expression that were located medially and in the ventral somites began to extend projections laterally along the VMS, perpendicular to the orientation of muscle fibers (Figure 1B’, E’, H’). By 48 hpf these projections extended 70–80 μm (Figure 1C,F,I). 3D-reconstructions of confocal stacks at 60 hpf revealed that this polarized network of tenocyte projections covered the entire VMS (Figure 1C’, F’, I’; Figure 1—figure supplement 1). Time-lapsed videos of Tg(scxa:mCherry) embryos capturing images at 20 min intervals from 48 to 60 hpf showed that tenocyte projections are dynamic and constantly changing in length and branching pattern (Figure 1—video 2). Tenocytes along the HMS near the NC have shorter, more convoluted projections than tenocytes along the VMS (Figure 1I’; Figure 1—figure supplement 1B). Thus, during the period in which axial muscles in the trunk begin to contract and embryos become motile, tenocytes align along future MTJs and undergo dramatic changes in cell shape that correlate with the establishment and strengthening of muscle attachments. Figure 1 with 4 supplements see all Download asset Open asset Axial tenocyte morphogenesis. (A–C) Lateral views of live Tg(scx:mCherry) embryos showing developing tenocytes (A - 24 hpf, B - 36 hpf, C - 48 hpf). (A’–C’) Transverse views from 3D projections showing the positions of developing tenocytes in relation to the notochord (NC) and neural tube (NT) along the horizontal (HMS) and vertical myosepta (VMS) (arrows). Tenocytes form projections at 36–48 hpf (B’ and C’). (D–F) Diagrams of lateral views showing the morphology of tenocytes in the developing somites. (D’–F’) Diagrams of transverse views from 3D projections of live Tg(scx:mCherry) embryos show the development of tenocyte projections (E’ and F’). (G–I) Lateral views of co-immunostained Tg(scx:mCherry) embryos showing developing tenocytes (anti-mCherry - white) and muscle fibers (anti-MHC - green) (G – 24 hpf, H – 36 hpf, I – 48 hpf). (G’–I’) Transverse views from 3D projections of live Tg(scx:mCherry) embryos showing the positions of developing tenocytes (arrowheads in G’ and H’) in relation to the myotome. Scale bars = 20 microns. https://doi.org/10.7554/eLife.38069.003 Cranial tendons also undergo dramatic morphological changes during the onset of muscle attachment and contractility. A cluster of scxa:mCherry+ tenocyte progenitors is first observed at 36 hpf in the ventral midline near the future attachment sites of the sternohyoideus (SH) and adductor mandibulae (AM), which are among the earliest muscles to differentiate at 53 hpf (Schilling and Kimmel, 1997). By 48 hpf, three major clusters of scxa:mCherry+ tenocytes are visible ventrally, one anterior that forms ventral mandibular, hyoid, and oculomotor muscle tendons and two posterior clusters associated with each SH (Figure 1—figure supplement 2A). The anterior cluster subdivides over 14 hr into separate attachment sites for mandibular (IMA, IMP, AM) and hyoid (IH) muscles (Figure 1—figure supplement 2B). Double labeling with anti-MHC and anti-mCherry antibodies revealed a tight correlation between the timing of the onset of muscle contraction and tenocyte morphogenesis in each of these clusters (Figure 1—figure supplement 2A,B,D,E,G,H). Cranial myofibers remain immotile at 48–62 hpf and their corresponding tenocytes form clusters of rounded cells at future attachment sites. These tenocytes then undergo compaction and elongation as contractions begin at 72 hpf (Figure 1—figure supplement 2C,F,I). Tenocyte elongation requires muscle contraction Based on the close correlation between the onset of muscle contraction and tenocyte morphogenesis, we hypothesized that mechanical force serves as a cue for tenocytes to elongate and form projections. To test this idea, we first injected full-length mRNA encoding codon-optimized α-bungarotoxin (αBtx), a specific irreversible antagonist of acetylcholine receptors that blocks neuromuscular synapses and prevents skeletal muscle contractions (Swinburne et al., 2015; Westerfield et al., 1990). Embryos injected with αBtx mRNA at the one-cell stage were completely paralyzed until 60 hpf, after which they gradually recovered motility as αBtx activity declined. Depth-coded, 3D-reconstructed images of living trunk tenocytes along the MTJs of somites 16–17 at 48 hpf revealed an average reduction of 13 μm (18%) in axial tenocyte projection length in αBtx-injected embryos compared to uninjected controls (Figure 2A,B,E). Paralyzed embryos also showed reduced branching complexity in their projections (Figure 2F) and projection density along the VMS (Figure 2—figure supplement 1). To restore mechanical force, we electrically stimulated αBtx-injected embryos to induce muscle contractions, as described previously (Subramanian and Schilling, 2014). Stimulation at 48 hpf for 2 min at 20V caused no visible muscle damage or significant change in tenocyte projection lengths compared to controls (Figure 2C,E) while the same stimulation of αBtx-injected embryos rescued both tenocyte projection length and density along the VMS almost completely (Figure 2D,E; Figure 2—figure supplement 1). The observed reductions in projection length and density were caused by paralysis rather than any unanticipated effect of αBtx, since homozygous mutants paralyzed due to lack of a functional voltage-dependent L-type calcium channel subtype beta-1 (Cacnb1), necessary for excitation-contraction coupling in muscle, showed similar (10–15 μm) reductions in projection length (Figure 2—figure supplement 2) (Zhou et al., 2006). Tenocytes in cacnb1 mutant embryos fail to compact and elongate. Since αBtx-injected embryos recover from paralysis at 65 hpf, prior to cranial muscle contractions, we compared cranial tenocyte patterning in immunostained 4 dpf cacnb1 mutant embryos with their siblings. We observed both a failure of cranial tenocytes to compact and elongate, as well as reduced projections and frayed myofibers (Figure 2—figure supplement 3). These results indicate a strong correlation between mechanical force from muscle contraction and tenocyte morphogenesis, suggesting that force stimulates the dynamic growth and branching of tenocyte projections. Figure 2 with 3 supplements see all Download asset Open asset Tenocyte projection length and branching density is regulated by mechanical force. Lateral views of live Tg(scx:mCherry) embryos (48 hpf) showing tenocyte projections. Images are pseudocolored by depth from medial (red) to lateral (blue). Control embryos were imaged without stimulation (A) and after stimulation (B), and the length of tenocyte projections was compared with embryos injected with αBtx and imaged without (C) and with stimulation (D). Dot plot shows individual data points of tenocyte projection length under different conditions (E). The data points from each embryo are connected by a vertical line. NS – Not Stimulated, S – Stimulated. (n > 50 data points/embryo in three embryos/sample, p value was determined through ANOVA 1-way analysis ***<0.00001, **<0.0001). Histogram shows quantification of branch points along tenocyte projections per tenocyte in 36 hpf control and αBtx injected embryos for every level of branching (1o – primary, 2o – secondary, 3o – tertiary, 4o – quaternary). (n = 4, p value was determined through ttest *<0.01, ***<0.00001). The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 2—source data 1 and Figure 2—source data 2. https://doi.org/10.7554/eLife.38069.008 Figure 2—source data 1 Measurements of tenocyte projection length along VMS. https://doi.org/10.7554/eLife.38069.014 Download elife-38069-fig2-data1-v2.csv Figure 2—source data 2 Measurement of tenocyte projection branching complexity along VMS. https://doi.org/10.7554/eLife.38069.015 Download elife-38069-fig2-data2-v2.csv ECM organization at MTJs requires muscle contraction We previously showed that Tsp4b secreted by tenocytes is essential for ECM organization at MTJs and strengthens muscle attachments (Subramanian and Schilling, 2014). We hypothesized that force stimulates tenocytes to secrete Tsp4b from the projections they extend into the tendon ECM. Consistent with this, injection of tsp4b-gfp full length mRNA into Tg(scxa:mCherry) embryos produced Tsp4b-GFP protein that localized to MTJs along the attachment sites at 48 hpf (Figure 3A–C,I). This exogenous Tsp4b-GFP protein was dramatically reduced in αBtx-injected embryos, particularly around projections, and became diffuse compared to uninjected controls (Figure 3D–F,I). Likewise, immunohistochemical staining for Tsp4b at 48 hpf in αBtx-injected embryos showed dramatic reductions along the attachment sites compared to controls (Figure 3G,H,J). In contrast, other ECM proteins such as laminin (Lam) at 48 hpf and and fibronectin (Fn) at 24 hpf showed no significant changes at the MTJ in αBtx-injected embryos at 48 hpf (Figure 3—figure supplement 1). Defects in Tsp4b distribution were due to the lack of mechanical force, since restoring force in paralyzed embryos through electrical stimulation rescued both local levels and the overall area of Tsp4b protein localization along the VMS (Figure 3—figure supplement 2). To test the hypothesis that changes in Tsp4b localization were due to reduced tsp4b gene expression in response to lack of force, we performed real-time PCR and found a significant reduction in tsp4b expression at 48 hpf in αBtx-injected embryos, while no significant change in expression was observed at 24 hpf (Figure 3—figure supplement 3). These results suggest a role for mechanical force in both assembly of tendon ECM and expression of key MTJ ECM genes during development and demonstrate that muscle contractions regulate the composition and organization of the tendon ECM. Figure 3 with 3 supplements see all Download asset Open asset Tsp4b localization to VMS and tenocyte projections requires mechanical force. Lateral views of live control (A–C) and αBtx injected (D–F) Tg(scx:mCherry) embryos (48 hpf), injected with tsp4b-gfp mRNA showing localization of Tsp4b-GFP (green) (arrowheads) along the VMS and tenocyte projections (red). (I) Histogram shows the percentage of embryos with Tsp4b-GFP localized to VMS (n = 27, p value calculated by chi-squared test <0.05). (G–H) Lateral views of immunostained embryos showing Tsp4b protein localization detected immunohistochemically along VMS in control (G) and αBtx injected (H) embryos. (J) Dot plot shows individual data points of the fluorescent intensity of localized Tsp4b along the VMS in control and αBtx injected embryos. Three VMSs/embryo were sampled in control and αBtx-injected embryos. (n = 9, p value calculated by Wilcoxon Rank Sum Test - < 0.0001). Scale bars = 20 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure 3—source data 1 and Figure 3—source data 2. https://doi.org/10.7554/eLife.38069.016 Figure 3—source data 1 Count of embryos showing localized or diffuse Tsp4b-GFP. https://doi.org/10.7554/eLife.38069.023 Download elife-38069-fig3-data1-v2.csv Figure 3—source data 2 Measurements of Tsp4b fluorescence intensities along VMS. https://doi.org/10.7554/eLife.38069.024 Download elife-38069-fig3-data2-v2.csv Microtubules maintain tenocyte projections and their with tendon ECM Cellular projections in and cells are in microtubules and in some while of cells are more et al., 2015; et al., To the structure of tenocyte projections we injected full-length mRNA encoding and found that this protein localized to along the length of tenocyte projections (Figure et al., of encoding to show in the projections. To are for projections, we embryos with which caused to (Figure staining of embryos for Tsp4b showed scattered Tsp4b localized at MTJs along the VMS and reduced Tsp4b protein levels in the VMS (Figure These results suggest that are the key components of tenocyte projections required to the organization of tendon ECM. Figure 4 Download asset Open asset tenocyte projections control tendon ECM Lateral views of live 48 hpf Tg(scx:mCherry) embryos injected with mRNA (A–C) showing localization of along the length of projections with mCherry to in Transverse views of live 60 hpf embryos showing tenocyte projections in and embryos. Transverse of 60 hpf embryos immunostained for Tsp4b showing localization of Tsp4b in and (G) of Tsp4b localization intensity in VMS (H) and distribution of Tsp4b in VMS (I) of and embryos. value calculated by for samples with Scale bars = 20 microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure data 1 and Figure data 2. Figure data 1 of Tsp4b fluorescence intensities along VMS. Download Figure data 2 Count of Tsp4b along VMS. Download TGFβ signaling is required for tenocytes to extend projections in response to force Previous studies from primary of tenocytes and on tendons in vitro have a role for TGFβ signaling (Gumucio et al., 2015; et al., et al., TGFβ secreted by muscles or in the ECM of the MTJ could be released in response to force and thereby regulate both tenocyte morphogenesis and ECM To this we Tg(scxa:mCherry) embryos with a chemical of TGFβ signaling – which blocks TGFβ for hr from 24 to 36 hpf (Chen and Galloway, 2014). This reduced signaling in both muscle fibers and tenocytes as by for SMAD3 in embryos compared to controls (Figure In addition, tenocyte projections were reduced in length by an average of μm in embryos (Figure similar to the effects of αBtx (Figure embryos injected with αBtx, embryos to These results suggest that acts downstream of muscle contraction to growth and branching of tenocyte projections. To muscle contraction is essential for activation of TGFβ we control and Tg(scx:mCherry) embryos with While control embryos showed strong localization in the of muscles and tenocytes, staining was reduced in the of tenocytes in αBtx-injected embryos (Figure Here, in to embryos with (Figure paralysis reduced in tenocytes and in muscle This with the reduction in length of tenocyte projections (Figure These results suggest that mechanical force from muscle contraction serves as a cue for TGFβ signaling in tenocytes to control their morphogenesis and Figure 5 Download asset Open asset TGFβ signaling regulates tenocyte morphogenesis. Lateral views of immunostained Tg(scx:mCherry) control and embryos showing tenocytes and (I) of was as fluorescent intensity of by and as a plot showing data points (n = 9, p value was calculated by test 3D projections show tenocyte cell projections in control and embryos (J) Dot plot shows individual data points tenocyte projection lengths (n = 50 data points/embryo in embryos/sample, p value was calculated by Wilcoxon Rank Sum test muscle are by a Scale bars = microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure data 1 and Figure data 2. Figure data 1 Measurements of fluorescence intensities in tenocyte along VMS. Download Figure data 2 Measurements of tenocyte projection length along VMS. Download Figure with 3 supplements see all Download asset Open asset TGFβ signaling in tenocytes requires mechanical force. Lateral views of 48 hpf immunostained Tg(scx:mCherry) control (A–C) and αBtx injected (D–F) embryos showing tenocytes and by (G) of was as fluorescent intensity of and as a plot (n = 4, p value was calculated by (H) Dot plot shows individual tenocyte projection lengths value was calculated by muscle are by a Scale = microns. The measurements used for quantitative analysis and creation of the plots can be accessed from Figure data 1 and Figure data 2. Figure data 1 Measurements of Tenocyte projection length along VMS. Download Figure data 2 Measurements of tenocyte fluorescence intensity along VMS. Download To that mechanical force has a role in of responses in tenocytes, we control and αBtx-injected embryos with or without electrical with to localization of in of tenocytes could be αBtx-injected embryos stimulated with showed increased localization in tenocyte