Fig. 6

Fig. 6. The fin and fin-rays shape are not affected by the disorganization of blood vessel. (A, B) Fluorescent images of blood vessels at the adult fin tip visualized by fli1a:mCherry-CaaX in control (A) and col9a1c(-/-) (B). (C–H) Fluorescent images of blood vessels in regeneration fin at 14 dpa and 21 dpa in control (C and D), col9a1c(-/-) (E and F), and UV-irradiated control fin (G and H). As shown in G and H, UV-stimulated fin also showed the tangled vascular network at the fin tip (white arrows) and the blood vessels in inter-ray region (blue arrows) as seen in col9a1c(-/-) fin (white arrows in E and F, blue arrow in F). (D′, F′, H′) Fluorescent images of actinotrichia visualized actinodin1-GFP at 21 dpa in control, col9a1c(-/-), and UV- irradiated control fin, respectively. The white dashed box in each figure indicates the area in the magnified image surrounded by white line. Although the UV-stimulated fin had abnormal blood vessels as shown in G and H, the alignment of actinotrichia was intact and indistinguishable from the control fin (compare H′ with D′). (I) Analysis of the orientation angle of actinotrichia. The upper schematic diagram represents the analysis method. The angle between the midline of fin-ray and the straight line extended from actinotrichia was measured using D′, F′, and H′ (N ​= ​10 from each group). Actinotrichia are usually arranged radially with respect to fin-ray, so the angle is very small. Conversely, larger angles mean the direction of actinotrichia is disturbed. As the results show, actinotrichia in the knockout were distributed at various angles due to their mis-arrangement, while they in control and UV-irradiated fin were distributed between 0 and 30°. The result indicates that UV irradiation did not affect the actinotrichia orientation. (D″, F″, H″) The phenotype of fin and fin-rays in control, col9a1c(-/-), and UV-irradiated control fin including the area shown in D and D′, F and F′, H and H′, respectively. The col9a1c(-/-) fin shrunk in dorsoventrally and had the wavy fin-rays (white arrow), whereas UV-stimulated fin showed normal fin and fin-rays seen in control. (J) Measurement of segment length (N ​= ​15 each from group, i.e. each three individuals with 5 segments on the D3 fin-ray; t-test (vs. control), ∗∗∗p ​< ​0.001). The magnified view is the area surrounded by the white dashed box in D″, F″, and H″, respectively. As the result shown, the segment length was also unaffected by UV irradiation. Scale bars: 100 ​μm in A–H, D′, F′, H′; 500 ​μm in D″, F″, H″.