Fig. 5

Laminin-111 at MTJs in not affected by Mmp11 knockdown or overexpression. A–F1, H–K1 Anterior left, dorsal top, side-mounted embryos stained for laminin-111 (green), nuclei (light blue), actin (light or dark blue), beta-dystroglycan (orange), or showing Mmp11-GFP (purple). A–B laminin-111 antibody and nuclei staining in 26 hpf embryos. A Control embryo. B mmp11 morphant embryo. C–D laminin-111 antibody and phalloidin staining in 48 hpf embryos. C Control embryo. D mmp11 morphant embryo (n = 8 embryos). Note that laminin-111 appears normal in mmp11 morphants compared to controls at 26 and 48 hpf. E–F1 Beta-dystroglycan antibody and phalloidin staining in 27 hpf embryos. E, E1 Control embryo showing merged panel E and beta-dystroglycan antibody staining alone (E1). F, F1 mmp11 morphant embryo showing merged panel F and beta-dystroglycan antibody staining alone (F1). Normal beta-dystroglycan antibody staining in mmp11 morphants indirectly supports that laminin-111 is unaffected by Mmp11 knockdown. G Relative mRNA abundance of lama1, lama2, lamb1, and lamc1 in mmp11 morphants compared to controls at 26 hpf. The expression of the laminin genes assayed is unchanged by Mmp11-GFP overexpression (n = 2 biological replicates of at least five embryos each). H–K1 laminin-111 antibody and phalloidin staining in heat-shocked AB controls and Tg(HSP:mmp11-GFP) embryos. H Control embryo at 26 hpf showing laminin-111 and laminin-111 merged with phalloidin (inset). I, I1 26 hpf Tg(HSP:mmp11-GFP) embryo showing laminin-111 (I), laminin-111 merged with phalloidin and Mmp11-GFP (inset in I), Mmp11-GFP (I1), and Mmp11-GFP merged with laminin-111 (inset in I1). J Control embryo at 48 hpf showing laminin-111 and laminin-111 merged with phalloidin (inset). K, K1 48 hpf Tg(HSP:mmp11-GFP) embryo showing laminin-111 (K), laminin-111 merged with phalloidin and Mmp11-GFP (inset in K), Mmp11-GFP (K1), and Mmp11-GFP merged with laminin-111 (inset in K1). Note that the timing of Mmp11-GFP MTJ localization recapitulates that of native Mmp11 protein, Mmp11-GFP and laminin-111 co-localize at MTJs, and laminin-111 appears normal in Tg(HSP:mmp11-GFP) embryos (n = 5 embryos). Scale bars are 50 μm