col22a1−/− mutant adults exhibit hemorrhages. (A,B) Generation of col22a1 zebrafish mutant line using TALEN. Sanger sequencing data of homozygous col22a1 mutant show that there is a 5 bp deletion TCTTC (boxed; arrow in the right panel) that results in a frame shift (A) and a premature stop codon in the protein sequence (B). (C,D) Immunostaining against zebrafish Col22a1 at 3 dpf in col22a1+/− and col22a1−/− embryos. Staining is absent at the myoseptae in the col22a1−/− mutant (arrows) compared with the col22a1+/− embryo, suggesting that the mutation is likely null. Selected sections from Z-stack were merged using the Extended Focus feature within the Axiovision software (Zeiss). (E) The frequency of hemorrhages increases with age in col22a1−/− mutants, while exercise caused col22a1−/− mutants to exhibit the phenotypes at an earlier age. (F,G) A 6-month-old col22a1−/− adult experiences visible blood accumulation in the eyes after a 3-week course of intermittent forced exercise (black arrow, G), whereas the WT adult does not (F). (H) Without exercise, a 2-year-old col22a1−/− adult also exhibits visible blood accumulation in the eyes (white arrow).
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