Figure 3

(A) Schematic illustrates role for TGF-β, BMP, and Notch signals during AV valve development comparing mammals and fish. PG, proteoglycan; VPC, valve precursor cell; EMT, endocardial to mesenchymal transition. (B and C) In situ analyses of aggrecan (acana, red arrowheads) (B) and osteopontin (spp1, yellow arrowheads) (C) in 4 dpf embryos show defects in expression of the cardiac jelly are associated with loss of mesenchymal migration in gnptab/MLII morphants and mutants. Percent values indicate the number of embryos with the pictured phenotype. n = 50–65 embryos from 3 experiments. Scale bar: 50 μm. (D) Confocal images of WT and gnptab morphants stained immunohistochemically for myosin (red) and either pSmad1/5/8 or pSmad2 (green) illustrate reduced BMP and increased TGF-β signaling in MLII hearts. Boxed areas highlight region particularly affected, which are magnified in panels to the right. V, ventricle; A, atrium; OFT, outflow tract. n = 25 embryos from 3 independent matings. Scale bar: 20 μm. (E) Graphs show the percentage of cells in the ventricle and atrium with nuclear localized pSmad2 and pSmad1/5/8 staining. Data are presented as mean ± SEM. ****P < 0.0001 using 2-tailed Student’s t test. (F) Live confocal images of 4 dpf embryos expressing the BRE:dsEGFP (reports BMP signaling, denoted by white arrowheads) and myl7:RFP (labels cardiomyocytes red) confirm reduced BMP signaling (labeled green by BRE:dsEGPP) in the ventricle of MLII hearts, which is restored when TGF-β signaling is inhibited with SB505124. n = 10–12 embryos from 3 independent experiments. (G) Confocal images of tie2:EGFP⁺ (green) WT, gnptab-deficient morphants, and TGF-β–inhibited gnptab-deficient morphants treated with SB505124. Embryos stained immunohistochemically for myosin (red). n = 25–30 embryos from 3 independent experiments. Scale bar: 30 μm. (H) Graphs show percent of embryos whose hearts loop normally and that exhibit edema. For simplicity, each dot represents the average value obtained from an experiment containing 25–30 embryos. Total n > 100 embryo per condition. Data represent mean ± SEM. ****P < 0.0001 using Dunnett’s test with correction.