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Fig. 5 Organ-wide activation of embryonic gene expression and cell proliferation in the epicardium in response to cryoinjury and ventricular resection.
(A) In situ hybridization for tbx18 shows an organ-wide expression (arrowheads) in the epicardium 3 days after cryoinjury compared to undetectable expression in uninjured hearts. Wound edges are indicated by black dashed lines. Scale bars are 100 μm. n = 3 heart control and 3 dpi. (B) Organ-wide GFP induction in the transgenic wt1b:GFP line shows activated epicardium after ventricular resection and after cryoinjury at 3 and 7 days after treatment. Induction of the transgene is visible in the thickened epicardium near the wound (white arrows) and in the epicardium lining the bulbus arteriosus and atrium as well (yellow arrows). Uninjured hearts show no GFP expression. Nuclei were stained with Dapi. Wound edges are indicated by white dashed lines. Scale bars are 100 μm. n = 5 hearts at all time points. (C) Organ-wide GFP induction in the transgenic wt1b:GFP line is only observed after ventricular resection but not in uninjured or sham amputated control hearts. Nuclei were stained with Dapi. Scale bars are 100 μm. (D) GFP expression in injured wt1b:GFP transgenic hearts is specific to the epicardium. Coexpression of tbx18 transcript detected via in situ hybridization and GFP detected via antibody staining at 3 days post amputation is shown. Scale bar is 100 μm. n = 3 hearts. Dashed line indicates wound edge. (E–F) Activation of epicardial proliferation in response to ventricular resection (E) and cryoinjury (F). Uninjured and amputated hearts at 3 dpa and 7 dpa (E) or cryoinjured hearts at 3 dpi and 7 dpi (F) of wt1b:GFP fish were stained for GFP and PCNA expression. Nuclei are stained with Dapi. Proliferating epicardial cells are indicated with white arrowheads. Note partial coverage of the wound with GFP-positive epicardium in E (arrow) and full coverage in F (arrow). Scale bars are 100 μm in the overview and 25 μm in the close ups. Wound plane is indicated by white dashed lines. n = 3 hearts (9 sections) for all conditions.