QUANTIFICATION OF EXTRACELLULAR MATRIX DYNAMICS DURING MURINE FORELIMB DEVELOPMENT AND DISEASE

 Musculoskeletal injuries are one of the leading causes of human disability. Tissue engineers aim to restore damaged musculoskeletal tissues by creating scaffolds that promote cellular adhesion, proliferation, and eventual differentiation into functional tissue. It is known that the extracellular matrix (ECM) regulates cellular behavior and is often used as a basis for biological scaffolds; however, current scaffolds often mimic the ECM of adult, homeostatic tissue and frequently lead to poor tissue restoration. What is rarely taken into consideration is that the ECM undergoes extensive remodeling during development to facilitate growth.

In the musculoskeletal system, myogenic progenitors (Pax3+) and connective tissue cells (Prx1+) proliferate and differentiate into muscle, tendon, cartilage, and conjoining interfaces (e.g. myotendinous junction), while depositing and remodeling the ECM. As tissues mature, cells continue to refine ECM networks to withstand the functional demands to facilitate movement. The ECM composition and architecture of adult musculoskeletal tissues have been studied individually and are thought to be distinct; however, there has yet to be a comprehensive comparative analysis of the ECM in adult muscle, tendon, and the myotendinous junction (MTJ) in a single study. Additionally, how the matrisome of adult musculoskeletal system compares to the ECM dynamics during forelimb development, remain largely unknown due to lack of techniques to analyze embryonic matrisome composition and synthesis. 

To address these research gaps, we (1) used quantitative proteomics to map the matrisome composition in the mature murine MTJ, relative to the tendon and muscle; (2) adapted tissue fractionation and biorthogonal non-canonical amino acid tagging techniques to embryonic tissues as a method to quantify the global and nascent embryonic matrisome; and (3) subsequently used these techniques to establish a baseline of ECM dynamics during forelimb morphogenesis (embryonic day, E11.5-E14.5) and growth (postnatal day, P3 and P35). We hypothesized that proteomic evaluation of ECM composition and synthesis in developing and adolescent limbs would resolve differences between embryonic and growing tissues. Indeed, we saw significant differences in global and nascent matrisome composition between embryonic and adolescent forelimbs. Notably, the relative abundance and ratios of collagens associated with type I fibrillogenesis (I, III, and V) were significantly different as a function of development embryogenesis and across the adult muscle, MTJ, and tendon.

Type I collagen fibrils are critical for tissue architecture and function. Using genetic mouse models, the regulatory roles of COL5A1 in the initiation of type I collagen fibrillogenesis, and organization of subsequent fibrils, have been well characterized in tendons and ligaments; however, is it unknown which cell types contribute COL5A1 to the ECM in the forelimb. To identify the functional contribution of COL5A1 by myogenic or connective tissue cell populations, we generated conditional (cre-flox) knock-out mouse models to inactivate Col5a1 using Pax3- or Prx1-drivers, respectively. Haploinsufficiency of COL5A1 in humans is associated classical Ehlers-Danlos syndrome, characterized by skin fragility and join instability; similar, albeit more severe, phenotypes were present in Prx1Cre/+;Col5a1fl/fl mutants, but not in Pax3Cre/+;Col5a1fl/fl mutants or controls. Interestingly, THBS4+ and COL22A1+ networks at the MTJ were morphologically affected in Prx1Cre/+;Col5a1fl/fl limbs. Additional work needs to be conducted to characterize the systematic phenotypes observed in Prx1Cre/+;Col5a1fl/fl limbs.

Together, our results indicate that there are distinct, complex ECM dynamics, originating from distinct cell-types, that drive musculoskeletal morphogenesis in the forelimb. Further, the tools developed here will serve as a foundation for quantitative proteomic analyses of the matrisome composition in embryonic tissues. Collectively, this work provides a baseline of ECM protein dynamics during musculoskeletal morphogenesis, a helpful guide for tissue engineers in designing scaffolds to promote restoration of damaged tissues, with enhanced integration into the host tissue.