Injured area is outlined in all images (a,b) lyve1b+ lymphatic capillaries (blue arrows) are detected in the injured area of 36 dpci Tg(fli1:EGFP); Tg(lyve1b:dsRed2) wt sibling (a) but not in flt4-/- hearts (b). (c) Quantification of lyve1b+ lymphatic vessel coverage in the injured area of 35–38 dpi flt4-/- hearts (nwt = 3, nflt4-/-=8, *p<0.001). (d–i) lyve1b+ lymphatic capillaries (blue arrows) are detected in the injured area of 28 dpci (d) and 65 dpci (g) Tg(fli1:EGFP); Tg(lyve1b:dsRed2) wt siblings, but not in vegfc+/- heterozygous fish (e,h). (f,i) Quantification of lyve1b+ lymphatic vessel coverage in the injured area of 21–28 dpi vegfc+/- hearts (nwt = 3 hearts, nvegfc +/-=4, *p<0.005) and 65–66 dpi vegfc +/- hearts (nwt = 10 hearts, nvegfc+/-=10, *p<0.005). (j,k) AFOG-stained sections at 36 dpci showing lack of regeneration in flt4 -/- hearts (k) as compared to wt siblings (j). Collagenous scar is stained in blue, fibrin in red, and cardiac muscle in orange. Black arrow points to scar. (l) Increased scar area (calculated as percent of ventricle) in flt4-/- hearts (nwt = 8 hearts, nflt4-/-=11, *p<0.05). (m,n) AFOG-stained sections at 65 dpci showing lack of regeneration in vegfc+/- hearts (n) as compared to wt siblings (m). Black arrow points to scar. (o) Increased scar area (calculated as percent of ventricle) in vegfc+/- hearts (nwt = 6 hearts, nvegfc+/-=3, *p<0.001). (p,q) lyve1b+ isolated LEC clusters (blue arrows) are normally detected in the injured area of 29 dpci Tg(flt1_9a_cFos:GFP);Tg(lyve1b:dsRed);cxcr4a-/- (q) and wt sibling (p) hearts. (r) Quantification of lyve1b+ lymphatic vessel coverage (relative to wt sibling) in 29 dpi cxcr4a -/- hearts (nwt = 4 hearts, ncxcr4a-/-=5). (s,t) AFOG-stained sections at 77 dpci showing impaired regeneration in cxcr4a-/- mutant (t) as compared to wt siblings (s). Black arrow points to scar. (u) Increased scar area (calculated as percent of ventricle) in cxcr4a-/- mutant hearts (nwt = 5 hearts, ncxcr4a-/-=5, *p<0.005). Scale bars are 200 µm. All fish size are 25–30 mm.
|
