Fig. 2

A population of vimentin-positive cells with a rounded morphology developed at the wound edge and associate with areas of collagen misalignment.

(A) Whole mount in situ hybridization for vimentin RNA in 4 dpf unwounded larvae. (B) Unwounded caudal fin and (C) 24 hpw caudal fin indicates increased vimentin expression following amputation, observed in two experiments. (D-G) Confocal microscopy of a live larval caudal fin showed that following amputation in the Tg(−2vim:egfp) line, in the wound region (box), a population of cells activated the vimentin promoter (arrow) by 26 hpw. (H,I) The activation of the vimentin promoter was regulated by ROS, as treatment with DPI reduced EGFP signal at the wound edge at 24 hpw as compared to control DMSO. (J) Ratio of the intensity of the vim:egfp expression at the central wound edge over background intensity of the fin in DPI and DMSO treated larvae at 24 hpw. (p=0.0282; n = 12 total fins over three replicates, with 2 to 6 fins per treatment per replicate; DMSO 95% CI = 1.18 to 1.33, DPI 95% CI = 1.07 to 1.21). (K) Spindle-shaped vim+ cells associate tightly with SHG fibers, as indicated by enface 3D reconstruction of SHG images of unwounded caudal fins in the Tg(−2vim:egfp) line. (L) End on view of a 3D surface rendered reconstruction of the association of vim+ cells and SHG fibers in unwounded caudal fins. (M) Spindle shaped vim+ cells associate with intact SHG fibers following amputation (Arrowhead), but a rounded vim+ cell population is observed in regions of collagen misalignment (Arrow) in the Tg(−2vim:egfp). (N) End on surface rendered reconstruction of the association of vim+ cells and SHG fibers in amputated caudal fins at 2 dpw, assessed in two replicates including 24 live larvae. Scale bar represents 100 µm in A-C, 50 µm in D-I, K and M and 30 µm in L and N. *p<0.05, error bars are standard deviation.