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.