Three‐Dimensional Visualization of the Extracellular Matrix During Murine Development

Abstract

Recent findings have suggested that the extracellular matrix (ECM) of developing tissues may better promote regeneration than the adult ECM, indicating that the characterization of embryonic tissues may yield parameters more suitable for engineered scaffold design than those derived from the adult. However, little is known about the spatiotemporal expression patterns and 3D structure of the ECM during embryonic development due to light scattering lipids and refractive index mismatches that limit the visualization of ECM within intact tissues. To better resolve the interactions of different ECM networks, we developed a novel decellularization method that removes signal interference from cellular components, enhances antibody penetration and maintains the geometry of fragile tissues. Murine embryos were incubated in an acrylamide‐based solution then polymerized into a hydrogel to create an interpenetrating framework that maintains the 3D geometry after detergent‐mediated decellularization. Initial experiments showed that the hydrogel polymerization did not disrupt tissue architecture and that 0.05% sodium dodecyl sulfate (SDS) was the best compromise between protein removal and maintenance of ECM networks. To confirm that our method maintains the independent architecture of independent ECM networks, control (whole‐mount) and decellularized embryos were stained for fibronectin (FN, red) and fibrillin‐2 (FBN2, white), imaged using confocal microscopy and rendered in 3D using ImageJ (Fig. 1A, B). Optimization of decellularization enabled a significant increase in visibility of the internal structure of E14.5 forelimbs, where FN and FBN2 maintained independent, interpenetrating networks in 3D. The skeletal elements within the forelimb could be resolved in 3D by staining with WGA (green) and tenascin‐C (red; Fig. 1C). Higher magnification imaging of the forelimb reveals fine WGA+ fibrils within the digit tip and maintenance of myotendinous junction architecture (Fig. 1D, E). Furthermore, we found that distribution of independent ECM networks in various developing tissues, including the eye and spinal cord, can be visualized in E12.5 embryos at multiple scales. Comparative analysis of the autopod of E12.5 and E14.5 forelimbs revealed continuous proteoglycan‐rich fibrils extended between the epidermis and cartilage, which remained present after the formation of the extensor tendons. The persistence of these connections between the epidermis and cartilage during tendon specification, in combination with previous studies showing that elastic fibrils appear to anchor developing tendons to the perichondrium in the developing chick limb, suggesting that the matrix also plays a role in regulating extensor tendon development in the autopod. By combining our new method with more finely resolved time points, a clearer picture should emerge regarding the role the ECM plays during forelimb musculoskeletal assembly. This knowledge regarding how the expression, structure, and localization of ECM proteins change over the course of development will be utilized to guide the design of regenerative scaffolds to repair damaged tissues of the musculoskeletal system.Support or Funding InformationThis work was supported by the National Institutes of Health [R03AR065201, R21AR069248, R01AR071359, DP2AT009833 to S.C.].This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.