Tethered Jagged-1 Synergizes with Culture Substrate Stiffness to Modulate Notch-Induced Myogenic Progenitor Differentiation
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Notch signaling is amongst the key intrinsic mechanisms regulating satellite cell fate, promoting the transition of activated satellite cells to highly proliferative myogenic progenitor cells and preventing their premature differentiation. Although much is known about the biochemical milieu that drives myogenic progression, less is known about the spatial cues providing spatiotemporal control of skeletal muscle repair in the context of Notch signaling.
Using a murine injury model, we quantified in vivo biophysical changes that occur within the skeletal muscle during regeneration. Employing tunable poly(ethylene glycol)—based hydrogel substrates, we modeled the measured changes in bulk stiffness in the context of Notch ligand signaling, which are present in the regenerative milieu at the time of injury.
Following injury, there is a transient increase in the bulk stiffness of the tibialis anterior muscle that may be explained in part by changes in extracellular matrix deposition. When presented to primary myoblasts, Jagged-1, Jagged-2, and Dll1 in a tethered format elicited greater degrees of Notch activity compared to their soluble form. Only tethered Jagged-1 effects were tuned by substrate stiffness, with the greatest Notch activation observed on stiff hydrogels matching the stiffness of regenerating muscle. When exposed to tethered Jagged-1 on stiff hydrogels, fewer primary myoblasts expressed myogenin, and pharmacological inhibitor studies suggest this effect is Notch and RhoA dependent.
Our study proposes that tethered Jagged-1 presented in the context of transient tissue stiffening serves to tune Notch activity in myogenic progenitors during skeletal muscle repair and delay differentiation.
KeywordsSkeletal muscle Regeneration Biophysical cues Biochemical cues Spatiotemporal Niche Compression testing Extracellular matrix Hydrogel Ligand presentation
Basic fibroblast growth factor
Immunoglobulin G subtype 1
Notch intracellular domain
Phosphate buffered saline
Rho-associated, coiled-coil containing protein kinase
Tris-buffered saline plus Tween-20
We are grateful to Dr. Yasuhiro Matsumura (National Cancer Center Hospital, Kashiwa City, Japan) for providing us with his custom antibody to analyze fibrin clot formation in our study.
HS, MAB, CAS, and PMG conceived the study and designed experiments. HS, SD, RYC, AJM, and EWL performed experiments, analyzed data, performed statistical analyses, and prepared figures. HS, MAB, SD, RYC, AJM, EWL, CAS, and PMG wrote, assembled, and revised the manuscript. All authors reviewed and approved the submission.
This study was funded by the Natural Sciences and Engineering Research Council (USRA fellowship to H.S., CREATE ToEP fellowship to S.D., RGPIN 327627-06 to C.A.S., RGPIN 435724-13 and Canada Research Chair 950-231201 to P.M.G.); Toronto Musculoskeletal Centre (Graduate Scholarships to M.A.B. and R.Y.C.); Barbara and Frank Milligan Foundation (R.Y.C.); Ontario Provincial Government (OGS-visa to M.A.B., 31390 and ER15-11-073 to P.M.G.); Canada Foundation for Innovation (31390 to P.M.G.); Krembil Foundation (Scholarship to M.A.B.); Toronto Western Arthritis Program (to P.M.G.); and Canadian Institutes of Health Research (MOP-302041 to C.A.S. and ONM-137370 to P.M.G.)
Conflict of interest
All authors declared that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.
- 11.Faux, C. H., A. M. Turnley, R. Epa, R. Cappai, and P. F. Bartlett. Interactions between growth factors and Notch regulate neuronal differentiation. J Neurosci. 21:5587–5596, 2001.Google Scholar
- 28.Quarta, M., J. O. Brett, R. DiMarco, A. De Morree, S. C. Boutet, R. Chacon, M. C. Gibbons, V. A. Garcia, J. Su, J. B. Shrager, S. Heilshorn, and T. A. Rando. An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy. Nat. Biotechnol. 34:752–759, 2016.CrossRefGoogle Scholar
- 30.Tierney, M. T., and A. Sacco. Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium Chloride. Methods in Molecular Biology, Clifton: Springer, 2016, pp. 53–60.Google Scholar
- 35.Varnum-Finney, B., L. Wu, M. Yu, C. Brashem-Stein, S. Staats, D. Flowers, J. D. Griffin, and I. D. Bernstein. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J. Cell Sci. 113(Pt 23):4313–4318, 2000.Google Scholar