Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

Abstract

The development of a functional musculoskeletal system requires the combination of contractile muscle and extracellular matrix (ECM)-rich tendons that transmit muscle-generated force to bone. Despite the different embryologic origins, muscle and tendon integrate at the myotendinous junction (MTJ) to connect across this interface. While the cell-cell signaling factors have received considerable attention, how the ECM links these tissues remains unclear. Here, we show the 3D distribution of ECM during forelimb development in wildtype (WT) and muscle-less Pax3^Cre/Cre mice. At E12, prior to MTJ integration, an aligned ECM is present at the presumptive insertion of the long triceps into the ulna. Tendon-like and muscle compartmentalization structures still form when muscle is knocked out; however, MTJ-specific ECM is not observed when muscle is absent. Our results show that the architecture of the muscle-tendon unit is established independent of muscle, but muscle is needed for the proper assembly of ECM at the MTJ.

Fig. 1. ECM alignment during murine triceps development.

A–D’ To visualize the 3D ECM structure of the triceps (green, A), murine forelimbs (B) were decellularized (decell) in 0.05% sodium dodecyl sulfate (SDS; C) or optically cleared using SeeDB (D). Samples were imaged using confocal microscopy and the organization of the ECM (COL5; magenta) was consistent after both decell (C’) and SeeDB (D’). Representative images from E13.5-E13.75 forelimbs. E–E”’ Aligned COL1⁺ (magenta) and WGA⁺ fibers (green) inserted into the lateral triceps near the ulna (U) in decellularized wildtype (WT) E12 forelimbs. F–H In SeeDB-cleared E11.5-12 forelimbs, Pax3GFP⁺ cells (green, *) aligned along the proximal (p) - distal (d) axis (arrow) with TNC (magenta), FBN2 (magenta), and COL5 (grey). Insets show aligned ECM networks distal to Pax3GFP⁺ cells (G-d, H-d) and around Pax3GFP⁺ cells (F-p, G-p, H-p) (arrowheads). I–I” COL1⁺ fibers (magenta, arrowheads) extended between ScxGFP⁺ tendon (green) and MY32⁺ muscle (blue) in a wholemount E13.75 zeugopod. Magnification 10× (C’), 20× (E–E”), 25× (D’, F–H), 63× (I–I”), z-projection: z = 40 µm (B’, C’) and 11.4 µm (F–H-d), 3D rendering: z = 12.7 µm, scale bars = 1 mm (B–E); 100 µm (C’–I”). Representative images from N = 3 independent biological replicates.

Fig. 2. Forelimb ECM composition was partially disrupted by the absence of muscle.

A Left: log₁₀(combined LFQ intensity value) heat map of matrisome components manually grouped via ECM protein classification. Combined LFQ intensity values for different fractions were generated following and proteins identified in n ≥ 2 biological replicates were included. White boxes signify zero intensity values. Right: C1 Fluidigm single-cell RNA-sequencing (scRNA-seq) data of the matrisome identified in E10-E15.5 limbs generated by He et al., as previously reported in ref. . Color indicates log₂(average expression +1) and circle size represents percentage of cells expressing that specific transcript. Cell types: 1 mesenchymal; 2 muscle 1; 3 muscle 2; 4 muscle 3; 5 chondrocyte; 6 perichondrial; 7 neural crest; 8 endothelial; 9 erythro-myeloid progenitors; 10 macrophage; 11 epithelial. Heat map and scRNA-seq data of affiliated proteins can be found in Fig S2. B Pax3^Cre/Cre forelimb lacked limb musculature (MY32, myosin; magenta) at E13.5. The shoulder girdle begins below the line. Magnification 10×. Scale bar = 200 μm. C Two-way ANOVA revealed there were no significant differences in matrisome composition between genotypes at E13.5 (p > 0.05, Supplementary Data 1). D Volcano plot comparison of log₂(LFQ) values for Pax3^Cre/Cre and WT E13.5 forelimbs, significance based on p < 0.05. E Matrisome components exclusively identified in either WT or Pax3^Cre/Cre embryos for all tissue fractions. F Select GO terms identified as significant when comparing ECM components that were exclusive to or enriched in Pax3^Cre/Cre or WT E13.5 forelimbs. N = 3 independent biological replicates were analyzed.

Fig. 3. Matrisome distribution is altered in developing forelimbs lacking muscle.

A, B’ EMILIN1⁺ and WGA⁺ fibers (arrowheads) were observed in the tendons of both WT and Pax3^Cre/Cre embryos at E13.75. C, D’ Forelimb tendons from WT, but not Pax3^Cre/Cre, forelimbs were POSTN⁺ (arrow). E, F”’ FBN2 was still present in the ScxGFP⁺ area in Pax3^Cre/Cre forelimbs, whereas THBS4 was not. Dotted line delineates region of triceps muscle. Magnification 25× (A–D), 10× (E, F), 3D rendering of decellularized limbs z = 110 µm (A–D), cryosections (E–F”’), scale bars = 100 µm. U ulna, Hu humerus. Representative images from N = 3 independent biological replicates.

Fig. 4. Tendon and epimysial-like ECM formed in the absence of muscle.

A–F” Decellularized E12.5-E14.5 WT and Pax3^Cre/Cre forelimbs were rich in WGA⁺ fibers at the insertion of the long triceps into the ulna (U; arrowheads; A’–F’) and origin of the long triceps at the scapula (S; arrowhead; A”–F”). However, the fiber bundles were less dense in Pax3^Cre/Cre compared to WT forelimbs. G, H For the long triceps of both E13.5 WT and Pax3^Cre/Cre embryos (dotted outline), WGA⁺ fibers formed epimysial-like structures. I, J In Pax3^Cre/Cre forelimbs, laminin (LAM⁺) blood vessels (magenta) were still present (arrowheads). Magnification 20×, z-projection: z = 230 µm (G, H), 138 µm (I), and 181 µm (J), scale bars = 1 mm (A–F), 100 µm (G–J). Representative images from N = 3 independent biological replicates.

Fig. 5. Fibrillar ECM persisted in the muscle belly of Pax3^Cre/Cre mutants but MTJ-specific ECM did not.

A–F WT or Pax3^Cre/Cre decellularized E13.5 triceps tendon (arrowheads) and muscle belly fibers were COL1⁺, COL5⁺, and FBN2⁺. G–I’ In WT decellularized E13.75 forelimbs, the COL22A1⁺ MTJ (green) and WGA⁺ ECM fibers (magenta) elongated under tension and were restored to original configuration when tension was removed, as indicated by the fiducial markers (dotted outline). * denotes an unresolved fiducial marker. G’–I’ The MTJ exhibited elongation in under tension (dotted line outline). J Quantification of average COL22A1⁺ MTJ fiber length revealed MTJ length significantly increased when stretched under tension and returned to the relaxed configuration for N = 3 independent limbs *: P-value < 0.05 Tukey’s post-hoc test. K, L The MTJ of decellularized WT but not Pax3^Cre/Cre forelimbs was marked by COL22A1⁺ (green) at the end of the WGA⁺ tendons (magenta, arrowheads). Magnification = 20× (A–F), 25× (G–K). Z-projection: z = 230 µm (A), 122 µm (B), 24 µm (C), 23.6 µm (D), 230 µm (E), 115 µm (F), 96.3 µm (G, G’, H, H’), 96.5 µm (I, I’), 3D rendering: z = 110 µm (J, K), scale bars = 100 µm (A–K), 25 µm (G’–I’). Representative images from N = 3 independent biological replicates.