Fig. 3.

flt1 deletion causes the upregulation of endothelial MAPK/ERK signaling and the downregulation of egr3 expression during cardiac regeneration. (A,B) Heat-map (A) and RT-qPCR validation (B) showing differentially regulated genes of interest in the border zone and injured area (BZI) of cryoinjured ventricles from flt1^+/+ and flt1^−/− zebrafish at 96 hpci. (C,C′) Immunostaining of cryoinjured ventricles from flt1^+/+ (n=4, C) and flt1^−/− (n=4, C′) zebrafish at 96 hpci for pERK (phosphorylated ERK, white). Red lines indicate the range of pERK⁺ injured area. Dotted orange line indicates the injured area. (D) Quantification of the percentage of pERK⁺ injured area in the indicated genotypes at 96 hpci. (E,E′) Immunostaining for pERK (green) and EGFP (cECs, magenta) with DAPI (blue) counterstaining on sections of cryoinjured ventricles from Tg(flt1:Mmu.Fos-EGFP);flt1^+/+ (n=4, E) and Tg(flt1:Mmu.Fos-EGFP);flt1^−/− (n=6, E′) zebrafish at 96 hpci. Arrowheads indicate pERK⁺ cECs in the BZI. Areas outlined are shown at higher magnification on the right. (F,G) Quantification showing the number (F) and percentage (G) of pERK⁺ cECs in the BZI of the indicated genotypes at 96 hpci. (H,I) Immunostaining for EGFP (green), MHC (myosin heavy chain, magenta) and Aldh1a2 (red) with DAPI (blue) counterstaining on representative sections from untouched (H) and cryoinjured (I) Pt(egr3:Gal4-VP16);Tg(5xUAS:EGFP) (abbreviated as egr3>EGFP) ventricles at 96 hpci. Arrowheads indicate EGFP⁺ cells. Areas outlined are shown at higher magnification on the right. White dotted lines indicate the injured area. epi, epicardium. Scale bars: 100 µm. Data are mean±s.e.m. (two-tailed, unpaired Student's t-test with P values shown in the graphs).