Tsp4b is required for Laminin assembly at MTJs.(A-F) Lateral views of somites in 20 hpf embryos stained with anti-MHC (green) and anti-pan-Laminin (Lam, red) antibodies. Insets show higher magnification images of the areas marked by white boxes, where Lam localizes to developing myotendinous ECM (arrowheads). (A and B) Wild-type siblings. (C and D) tsp4b-MO injected embryos showing local loss of Lam at 20 hpf, and ectopic muscle attachments (arrowheads) at remaining Lam foci. (E and F) Lam localization was restored in Tsp4b-deficient embryos injected with full length tsp4b mRNA. (G) Fluorescence intensity measurements (arbitrary units [A.U.]) at somite boundaries of anti-Lam in 20-24 hpf wild type controls versus embryos injected with tsp4b-MO or co-injected with tsp4b-MO and tsp4b RNA. (t test: one tailed, unequal variance; p-value: wt and Tsp4b-deficient <0.01, Tsp4b-deficient and Tsp4b-deficient and tsp4b RNA <0.05) Scale bar = 30 microns. (H and I) Lateral views of somites in wild type embryos at 36 hpf stained with anti-pan-Lam (red), and anti-MHC (green). Insets show higher magnification images of white boxed areas. Lam localizes to myotendinous ECM (I, arrowheads). (J and K) In Tsp4b-deficient embryos, Lam is reduced/discontinuous at somite boundaries (K, arrowheads). (L and M) Lam localization is restored in Tsp4b-deficient embryos injected with full length tsp4b mRNA. (N) Embryo percentages (20 hpf embryos N = 30, 72 hpf embryos N = 50) with reduced/mislocalized Lam at 20 and 48 hpf in wild type and Tsp4b-deficient embryos. (Chi squared test; p-values: 20 hpf **<0.01, 48 hpf ***<0.001) Scale bar = 30 microns.
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