EGFP-Col9a1c localizes to collagen fibers called actinotrichia in caudal fin of zebrafish:

(A) Schematic illustration of the Col9a1c expression construct. The upper diagram shows the expression of EGFP-Col9a1c driven by the actinodin1 promoter that is specifically active only in the mesenchymal cells (and1mes pro, designed by Kuroda et al. (2020) based on the previous paper reported by Lalonde et al. (2016)), the lower diagram shows the EGFP-fusion Col9a1c. EGFP and V5 epitope tag were inserted between the 42nd and 43rd amino acids of wild-type Col9a1c. IRES-Hsa.HIST1H2BJ-mRFP (referred to as IRES-H2BRFP in this paper) was used to confirm that the introduced gene is expressed in the fins. (B) The phenotype of the transgenic zebrafish expressing an EGFP-Col9a1c fusion protein (Tg line name, ou2046Tg). The transgenic fish does not show any abnormalities in the shape of the body or the caudal fin. (C, D) Bright-field image of the caudal fin at 5 dpf (C) and 1 mpf (D). The grey line in C indicates the outline of the fin. The areas enclosed by black dotted lines in C and D represent near the center of the caudal fin and in the tip of the fin, respectively, and correspond to the observation areas of E and G, and F and H, described below. (E, F) Fluorescent images of EGFP-Col9a1c at 5 dpf (E) and 1 mpf (F). White dot lines indicate representative locations where the fluorescent signal was detected. The fluorescence indicated by the asterisks in E is the autofluorescence of xanthophores, not the Tg-specific signal. (G, H) Fluorescent images of actinotrichia visualized by And1-GFP (Tg line name, ou2106Tg (Kuroda et al., 2018)) at 5 dpf (G) and 1 mpf (H). At each stage, the localization of both EGFP-Col9a1c and And1-GFP is restricted to actinotrichia. (I) The transverse section of cryosection in the tip of an adult regenerating fin at 5 days post-amputation (dpa). Immunofluorescence staining for Collagen2 (magenta) was performed prior to cryosectioning to label the outline of actinotrichia in and1mes:EGFP-Col9a1c fish (EGFP-Col9a1c; green). (J–J’’­) Magnified image of the white dashed box in I. The fluorescence signals of EGFP-Col9a1c are specifically detected in actinotrichia. White arrows and arrowheads in J and J’ indicate the localization of EGFP-Col9a1c around actinotrichia surface and the inner center of actinotrichia, respectively. Scale bars: 3 mm in B; 100 µm in C­, E–H; 500 µm in D; 20 µm in I; 5 µm in J­–J’’.