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Figure 4 Confocal images of RNAScope section in situ hybridization to detect vegfc (white: A-D). Whole-mount confocal imaging of adult transgenic zebrafish hearts expressing the pan-endothelial marker fli1a:GFP (Green: E-O, Q-T) and lymphatic endothelial markers flt4:mCitrine (blue: E-O, Q-T), prox1:Gal4-UAS:RFP (red: H and N), lyve1:RFP (Red: O), mrc1a:eGFP (Green: P) and endothelial marker kdrl:mCherry (Red: P). (E-H) Response to amputation of flt4-expressing lymphatic vasculature during regeneration at 7 (E), 14 (F), 35 (G) and 69dpa (H). Lymphatic vasculature is observed to extent into the wound site at 35dpa to connect to the vessel on the BA (blue arrowhead) with some disconnections observed (white arrowhead). Response of flt4-expressing lymphatic vasculature to cryoinjury at 7 (I), 14 (J), 28 (K), 46 (L), 54 (M) and 60dpc (N). Expression prolife of regenerated lymphatic vasculature shows expression of prox1:Gal4-UAS:RFP (red: H and N), lyve1:RFP (Red: O) and mrc1a:eGFP (Green: P). (Q and S) Lymphatic vessels (blue arrowheads) extend to the apex in uninjured zebrafish. After amputation, there is little lymphangiogenesis and most of the regenerated vasculature lacks lymphatic marker expression with some minor extension into the wound site (R, blue arrowhead). Extensive lymphangiogenesis into and around the wound site (60dpc, T, blue arrowheads) is stimulated following damage to the myocardium with cryoinjury. (U) Quantification of the increase in area of flt4:mCitrine expression within a set 600 μm2 region centered on the wound site of sibling zebrafish subject to severe cryoinjury (n = 9) in comparison to that resulting from amputation (n = 7; *p=0.0132, unpaired t-test) or the uninjured apex (n = 4; *p=0.0407, unpaired t-test). (V) Quantification of increased branching of lymphatic vessels on the ventricle in response to cryoinjury (n = 9) in comparison to uninjured hearts uninjured hearts (n = 6; *p=0.0293, unpaired t-test) and those subjected to amputation (n = 7; *p=0.0140, unpaired t-test). Scale bars 200 μm (A-D) and 50 μm (insets).