Fig. 5.

Fig. 5. egr3 expression in the heart is regulated by biomechanical forces

(A) In situ hybridization for egr3 expression in 48 hpf tnnt2a^+/+ and tnnt2a^−/− sibling embryos. (B) Illustration of bead insertion into a 48 hpf heart. (C) Pt(egr3:Gal4-VP16); Tg(UAS:eGFP) expression (gray) at 72 hpf in bead-inserted larva versus sham; myocardial cells are marked by Tg(myl7:BFP-CAAX) expression (magenta) and EdCs by Tg(kdrl:Hsa.HRAS-mCherry) expression (cyan). Red dotted circle highlights inserted bead; yellow arrowhead points to egr3⁺ cells near the bead. (D) Quantification of total volume of egr3 reporter⁺ cells in bead-inserted larvae and sham; n = 3 sham and 5 experimental larvae; one experiment. (E) Confocal images of representative hearts from control [Tg(fli1a:Gal4);Tg(UAS:Kaede)] and endothelial-specific egr3-overexpressing [Tg(fli1a:Gal4);Tg(UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)] tnnt2a MO–injected embryos immunostained for Alcama at 54 hpf; magnified regions show the AV canal of tnnt2a MO–injected embryos with rescued Alcama expression in the Tg(UAS:egr3-p2a-dTomato) embryo. (F) Quantification of Alcama⁺ EdCs per cardiac region of tnnt2a MO–injected embryos at 54 hpf; n = 6 control and 6 egr3 OE embryos, n = 0.00 and 8.00 EdCs (ventricle), n = 0.00 and 13.00 EdCs (AV canal), n = 0.00 and 4.83 EdCs (Atrium); two independent experiments. (G) Oscillatory shear stress induces the expression of the mechanosensitive transcription factor gene egr3 in valve EdCs, where it orchestrates valvulogenesis by promoting Alcama expression and their migration via Nr4a2b. (H) Schematic of aortic valve collection from LVAD patients and control donors. (I) Relative mRNA levels of EGR3, KLF2, NR4A2, and SPP1 from aortic valves of LVAD patients and control donors; n = 4 and 4 to 6; one experiment. Ct values can be found in data S1. (D), (F), and (I) Student’s t test, mean ± SEM. (B) and (H) Illustrations were generated with Biorender.com. Scale bars, 20 μm.