Mmp11 is necessary and sufficient for Fn downregulation. A Western blot of whole embryo homogenates at 26 hpf using Mmp11 antibody. Injection of individual MOs (a or b) alone did not reduce Mmp11 protein levels compared to controls. Co-injection of both MOs (a?+?b) reduced Mmp11 levels by approximately 14-fold compared to controls. Alpha-tubulin was used as a loading control. B, C Focal planes, 3D projections (anterior left, dorsal top, side-mounted), and transverse views ((T) lateral left, dorsal top) of control embryo (B) and mmp11 morphant (C) at 27 hpf, fast muscle fibers are stained with phalloidin (light blue) and slow-twitch fibers are stained with F59 (white). D Western blot of whole embryo homogenates at 32 hpf (upper panel) or 23 hpf (lower panel) using anti-Fn antibody. Upper panel: Fn protein level is increased by approximately 4.7-fold in mmp11 morphants compared to controls. Lower panel: Fn protein level is decreased by approximately 4.2-fold in embryos overexpressing mmp11-EGFP (tTg) compared to controls. E Medial focal plane, anterior left, dorsal top, side-mounted embryos at 26 hpf stained with Fn antibody (red) and F59 antibody (blue). Overexpression of mmp11-EGFP is green. E1, E1? Control. (E2?E2?) mmp11 morphant (n?>?60 embryos). Note that Fn concentrates adjacent to MPs in both controls and mmp11 morphants (arrowheads in E1? and E2?) but only concentrates adjacent to fast-twitch muscle fibers in mmp11 morphants (arrow E2?). E3?E3? mmp11-EGFP transgenic. Note that Fn is downregulated adjacent to MPs in transgenics compared to controls. E4?E4? mmp11 morphant overexpressing mmp11-EGFP (n?>?50 embryos). Mmp11-EGFP overexpression reduced Fn levels throughout the MTJ in mmp11 morphants. Scale bars are 50 ?m
|
