Organized laminin deposition is required for Fn downregulation. a Hypothesis that laminin deposition regulates Fn downregulation. Upper panel: laminin is polymerized at the MTJ and Fn is downregulated. Lower panel: laminin deposition/organization is disrupted and Fn perdures at the MTJ. b Western blot of whole embryo homogenates at 48 hpf using anti-Fn antibody. Laminin gamma1 mutants have approximately 2.5-fold more Fn compared to siblings. Alpha-tubulin was used as a loading control. (C–F; H–K) Anterior left, dorsal top, side-mounted embryos. Fn antibody (red), F59 antibody to label slow-twitch fibers (dark blue), laminin-111 antibody (green), phalloidin staining (light blue). c Medial focal plane of 32 hpf sibling. Note Fn (red) has been downregulated adjacent to fast-twitch fibers. d Medial focal plane of 32 hpf laminin beta1 mutant (n = 16 embryos). Note that Fn persists adjacent to medial, fast-twitch fibers. e Medial focal plane of 32 hpf sibling. Note Fn (red) has been downregulated adjacent to fast-twitch fibers. f Medial focal plane of 32 hpf laminin gamma1 mutant (n = 28 embryos). Note that Fn persists adjacent to medial, fast-twitch fibers in laminin beta1 and gamma1 mutant zebrafish. g Relative mRNA abundance of fn1a and fn1b in laminin gamma1 mutants compared to siblings at 26 hpf (n = 3 biological replicates of at least five embryos each). Fn1a expression is unchanged whereas fn1b expression is upregulated in laminin gamma1 mutants. h Focal plane of laminin-111 antibody staining in a 24-hpf control embryo. i Focal plane of laminin-111 antibody staining in a 24-hpf nrk2b morphant embryo. Note that laminin-111 protein is present, but that the organization of laminin-111 at MTJs is disrupted. j Medial focal plane of Fn antibody staining in a 32-hpf control embryo. k Medial focal plane of Fn antibody staining in a 32-hpf nrk2b morphant embryo (n = 9 embryos). Fn is present at the MTJ adjacent to MPs and fast-twitch fibers in nrk2b morphants. Scale bars are 50 μm
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