(A-F) Lateral views of 20-24 hpf embryos stained with anti-phosphorylated (Tyrosine 861) FAK (pFAK; red) and anti-MHC (green). (A and B) pFAK localizes to the ends of early myofibers at wild-type somite boundaries. (C and D) Reduced pFAK levels in Tsp4b-deficient embryos. (E and F) pFAK levels are restored in Tsp4b-deficient embryos injected with full length tsp4b mRNA. (G) Fluorescence intensity measurements (arbitrary units [A.U.]) for pFAK staining along somite boundaries confirm significant reductions in Tsp4b-deficient embryos, and partial rescue by co-injection of full length tsp4b mRNA (t test: one tailed, unequal variance; p-value: wt and tsp4b-deficient <0.05; Tsp4b-deficient and Tsp4b-deficient + tsp4b RNA p<0.001). (H-M) 36 hpf (lateral views) stained with anti-pFAK (red), and anti-MHC (green). Insets show higher magnification images of white boxed areas. (H and I) pFAK localizes to muscle sarcolemma. (J and K) In Tsp4b-deficient embryos, pFAK is reduced/discontinuous at somite boundaries. pFAK associates with ectopic muscle attachments (arrowheads). (L and M) pFAK localization is restored in Tsp4b-deficient embryos injected with full length tsp4b mRNA. (N) Embryo percentages (N = 70 embryos) with detached muscles from an intercross between two ilk+/- heterozygotes, injected with sub-threshold amounts (0.16 ng) of tsp4b-MO and stimulated (30 V) (Chi squared test; p-value: wt+ tsp4b-MO (stimulated) and ilk+tsp4b-MO (stimulated) p<0.0001, ilk (stimulated) and ilk+tsp4b-MO (stimulated) p<0.0001, ilk+ tsp4b-MO and ilk+tsp4b-MO (stimulated) p<0.0001). Scale bar = 30 microns.
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