Figure 3. Multiphoton microscopy of collagen and vimentin-expressing cells identify differences in tissue remodeling over time amongst the injuries.

(A) Z-projections of caudal fins imaged at different times after transection, thermal injury (burn) or infected transection showing the tissue wound edge in the bright field and corresponding SHG image. White dotted outline indicates wound edge from brightfield image. Scale bar is 100 microns. (B) Graph showing the area devoid of SHG fibers (from fiber ends to wound edge) following transection or thermal injury. Values are arithmetic means and SE from three biological replicates. p values obtained by analyzing ranks due to residuals not being normally distributed. Total N = 23–28 larvae per time point for each treatment. (C) Graph showing the angle of fiber segments relative to the wound edge, as quantified by CurveAlign software (see Materials and methods), within 50 microns of the wound edge with transection or thermal injury. 0^o is parallel to the wound edge while 90^o is perpendicular to the wound edge. Values are arithmetic means and SE from three experimental replicates. p values obtained by analyzing ranks due to residuals not being normally distributed. Total N = 14–25 larvae per time point for each treatment. (D) Graph showing the area devoid of SHG fibers (from fiber ends to wound edge) following uninfected or Lm-infected transection. Values are least square means and SE from three biological replicates, with associated p values. Total N = 28-32 larvae per time point for each treatment. (E) Graph showing the angle of fiber segments relative to the wound edge in uninfected or Lm-infected transections. Values are least square means and SE from three biological replicates, with associated p values. Total N = 17–31 larvae per time point for each treatment. **p<0.01, ***p<0.001, ****p<0.0001. (F) Z-projections of multiphoton microscopy images of vimGFP-positive cells at the wound edge in caudal fins at different times after simple transection, burn or Lm-infected transection. Scale bars are 100 microns. Merge of z-projections of vimGFP-positive cell images (green) with z-projections of corresponding SHG images (white) are shown in Figure 3—figure supplement 1B. (G) Graph showing the number of vimGFP-positive cells within a 50 × 100 micron box adjacent to the wound edge and centered vertically with the notochord (Figure 4—figure supplement 1A). Values are least square means and SE from three biological replicates, with associated p values. Total N = 19–28 larvae per time point for each treatment. For clarity, only relevant comparisons (burn in red, or Lm-infected transection in blue, to the control transection) are shown on the graph; ***p<0.001, ****p<0.0001.

Figure 3—figure supplement 1. VimGFP-positive cells at the wound recover over time at the Lm-infected tail wound.

(A) an example of an image with vimentin expression showing a 50 × 100 micron box placed strategically to count vimGFP-positive cells is shown (see Materials and methods). (B) Merge of z-projections of vimGFP-positive cell images (green) with z-projections of corresponding SHG images (white) from Figure 3F. (C) sum projections of z series acquired by spinning disk confocal microscope using Tg(vim:GFP) larvae following uninfected or Lm-infected transection. Scale bar is 100 micron. (D) Graph showing quantitation of vimGFP-positive cells at the wound over time. Values are least square means and SE from three biological replicates, with associated p values. Total N = 27–48 larvae per time point for each treatment. ***<0.0001.

Figure 3—figure supplement 2. Tail wound infection in the presence of hly mutant of L. monocytogenes causes no delay in vimGFP-positive cells and is associated with limited inflammation.

(A) representative images of sum projections of z series acquired by spinning disk confocal microscope using Tg(vim:GFP) larvae following tail transection in the presence of mCherry-expressing wild-type (Wt) or hly Lm over time. Scale bar is 100 micron. N = 4–6 larvae per time point for Wt Lm and N = 11–12 larvae per time point for hly Lm; one experimental repeat. (B) representative images of sum projections of z series acquired by spinning disk confocal microscope using transgenic larvae Tg(mpeg1:GFP) larvae following tail transection in the presence of mCherry-expressing wild-type (Wt) or hly Lm over time. Scale bar is 100 micron. N = 5–7 larvae per time point for Wt Lm and N = 12 larvae per time point for hly Lm; one biological repeat.