Figure 4: Organization of the fibronectin matrix precedes new myofibril assembly. Zebrafish embryos at 14 (a), 20 (b)–(e), (g), 24 (f), (h)–(j) hpf were immunolabeled for fibronectin (red) and slow-twitch myosin heavy chain (F59 antibody) (green). (a) Fibronectin organizes next to the notochord (NC) at nascent somite boundaries (arrowheads). Myosin heavy chain is still diffusely localized (arrows: (a) and inset) as the fibronectin matrix is organizing. (b) In a control embryo, the fibronectin matrix (arrowhead) is organized at the somite boundary before assembly of mature myofibrils in slow-twitch skeletal muscle of the younger somites at the right of the panel. (c) At higher magnification, continuous bands of myosin staining (arrow) are noted extending between the organized fibronectin matrix at either end of the myocytes before organizing into a striated pattern (arrowhead). (d)–(f) As development proceeds, MHC localization transitions from a diffuse distribution (arrows) along new myofibrils to a distinct restriction to the A band (arrowheads) and finally to a mature “double-banded” pattern ((f): arrowhead) with central clearing in the region of the future M band. By comparison, obscurin A depletion (g)–(j) affects fibronectin matrix organization and results in reduced organization of the MHC filaments. (g)–(j) Myofibril maturation can progress from immature myofibrils ((g), (h): arrowheads) to mature myofibrils ((h): arrow) even with a reduced and poorly organized fibronectin matrix (i). However, myofibrillogenesis proceeds more rapidly in regions with preserved fibronectin deposition ((j): arrowhead). Note the fibronectin organization at the terminal ends of the myofibrils (j). Lateral bundling of myofibrils appeared to be delayed in response to obscurin depletion as noted by the gaps between the A bands of neighboring but not yet fused myofibrils (inset of (j)). Scale bars are 50 μm (b), (h), 20 μm (a), and 10 μm (c), (d), (e), (f), (i), (j). Insets in (a) and (j) are 3x magnification of the larger image.