Figure 6—figure supplement 4.

Figure 6—figure supplement 4. Post-injury induction of sFlt4 limits myocardial wall expansion and scar internalization. (A–C) Imaged sections of a 56dpc hearts immune-labeled for macrophage (IB4, green) and neutrophil (Mpx, Red). Hearts were heart shocked just prior to injury and during regeneration to induce sFlt4 (B) or control for this induction (A). (C) Separate quantification of Mpx and IB4-positive cells in and within 100 μm of the woundsite (average of at least three images through woundsite, n = 6 per group). There is no difference in macrophage, but elevation of neutrophils is observed in some sFlt4-induced hearts despite the late stage (4/6), however this elevation across the group is not significantly different from the control. (D–G) AFOG-histology images showing fibrin in red and collagen in blue in post-injury heat shocked (non-transgenic (nonTg; D and F) and transgenic (hsp70l:sflt4; sFlt4; E and G) zebrafish 14 (D and E) and 56 days following cryoinjury (F and G). There is persistence of scar tissue even in nonTg zebrafish, but regeneration of cardiac myocardial wall in significantly reduced with induction of sFlt4. (F, insert) Quantified regions of the wound area. (H) Quantification of scar area (red, F insert; largest sectioned scar area, threshold to select tissue) shows no significant reduction in both nonTg and sFlt4 zebafish. (I) Quantification of the regenerated myocardial area (blue, F insert; thickened myocardial wall proximal to the scar) shows significant increase nonTg zebrafish between 14dpc and 56dpc, but no such increase is observed with sFlt4 induction (n = 6; **p=0.0022) (J) Quantification of the minimum distance of scar tissue from the epicardium (green, F insert; scar internalization with regeneration of the myocardial wall) is significantly larger nonTg zebrafish compared to sFlt4 induced siblings (n = 6, *p=0.0152; one outlier removed). (K and L) Immunostaining of PCNA and Mef2 showing cardiomyoctye proliferation 3 day post cryoinjury. (M) Quantification of PCNA/Mef2c-positive cells shows no effect on the induction of cardiomyocyte proliferation with expression of sFlt4 (average of at least three images through wounded region, double positive and Mef2c-only cells counted within 500 μm2 window proximal to woundsite, n = 6 per group). (N and O) Immuno-staining of PCNA and Raldh2 showing epicardial proliferation 3 day post cryoinjury. (P) Quantification of PCNA/Raldh2-positive cells shows no inhibition of epicardium activation with induction of sFlt4 (average of at least three images through wounded region all double positive and Raldh2-only cells within the entire 708 μm2 imaging window were counted, n = 6 per group). Scale bars 200 μm.