Expression and distribution of zebrafish col22a1 mRNA and protein. (A) RT-PCR analysis of zebrafish col22a1 at different developmental stages. β-Actin was used as a loading control. (B) Whole-mount in situ hybridization with col22a1 antisense probe in the trunk of 24 hpf (a), 48 hpf (b) and 72 hpf (c) embryos. Lateral view of a 72 hpf whole embryo (d) and zoomed image of a somite boundary (e). (a-e) Lateral views with anterior to the left; (a-c) somites in the posterior region of yolk extension are shown. Scale bars: 50 μm in a-c; 200 μm in d; 10 μm in e. (C) Western blot with anti-COLXXII of protein extracts from animals at different stages. COLXII and myosin heavy chain antibodies were used as collagen and late muscle differentiation marker controls, respectively. Antibodies to GAPDH and acetylated tubulin were used as loading controls. Number on the left indicate sizes (in kDa) of protein standard markers. hpf, hour post-fertilization. (D) Lateral views of whole-mount immunofluorescence staining with anti-COLXXII of 26 hpf (a) and 48 hpf (b) embryos. (a) Arrows indicate COLXXII staining that concentrated along myosepta. (c-e) Double immunostaining of a 72 hpf embryo with anti-COLXXII (c, red), dystrophin (d, green) and merge (e). Anterior is towards the left. Scale bars: 50 μm in a; 150 μm in b; 20 μm in c-e.
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