Fig. 2

Fig. 2

Fig. 2. Generation of col9a1c-knockout zebrafish and abnormal shape of the caudal fin in the knockout. (A) The col9a1c coding region map. The numbers in the boxes indicate exon number and the exon5 region is highlighted in white. The yellow and green box show the targeted region of CRISPR/Cas9. (B) Gene sequences around the target site (highlighted in yellow) and schematic of the structure of col9a1c protein in the wild-type (WT) and knockout alleles. The 10-bp deletion (del10; 505–514 bp; red hyphens) in the target 1 site and the 1-bp insertion (ins1; at 664 bp; highlighted in blue) in the target 2 site caused a frameshift at the position 169 aa and 222 aa, respectively. Both mutations deleted all the G-X-Y repeats (grey column), which is a common and unique structure of collagen. The del10 mutation also deleted half of the thrombospondin domain (blue column) in exon5. The red and bold letter in the target 1 sequence indicates the position of the prp mutation (Huang et al., 2009). (C–H) Phenotype of col9a1c(-/-) fish in the caudal fin. K and L show the caudal fin fold at 5 days post-fertilization (dpf) of the WT and col9a1c(-/-) fish, respectively. Both their shape and size were indistinguishable. C′ and D′ are the magnified images of the black dashed box in C and D. Radially arranged actinotrichia were visible in both fin folds. E and F are the caudal fins at 3 weeks post-fertilization (wpf) in WT and col9a1c(-/-) fish. The number in each image represents the average of fin length (N ​= ​5 each from control and col9a1c(-/-); t-test, ∗∗∗p ​< ​0.001). G and H are adult fins of WT and col9a1c(-/-) fish. The caudal fin of col9a1c(-/-) fish was slightly shrunken along the anterior-posterior (AP) axis, whereas the contraction in the dorsal-ventral (DV) was more pronounced and the fin-rays were curved towards the center. In addition, there was few bifurcations of the fin-ray. Scale bars: 100 ​μm in C, D; 25 ​μm in C′, D′; 300 ​μm in E, F; 500 ​μm in G, H.