(A) Confocal images of tie2:EGFP embryos treated with odanacatib show alterations in both myocardia (red) and valves (green). In particular, the atria of Ctsk-inhibited hearts are smaller, and the valve cells are less organized. The white line denotes the defined layers present in the WT valve. Percent values represent the number of animals exhibiting these phenotypes. n = 20 embryos from 3 independent experiments. Scale bar: 30 μm (left panels) and 10 μm (right panels). (B) Quantification of the number of control and odanacatib-treated embryos whose hearts “loop” and valves open and close (“clap”) at regular intervals. Each dot represents an experiment with 25 embryos, with 100 total embryos scored. (C) The cross-sectional area of the ventricles and atria is shown from n = 15–20 embryos. Data are presented as mean ± SEM; **P < 0.01,***P < 0.001 by 2-tailed Student’s t test or Dunnett’s corrected test (denoted by red box). V, ventricle; A, atrium. (D) In situ analyses of notch1b expression show reduced expression in AV valves of odanacatib-treated embryos (red arrow heads). Percent values represent the number of animals exhibiting these phenotypes. n = 30 embryos from 3 independent experiments. Scale bar: 50 μm. (E) Schematic depicts TALEN targeted sequences in ctsk, resulting INDELS, and genotyping strategy. Black arrowheads denote position of genotyping primers. (F) Restriction enzyme–based genotyping of PCR amplified samples that were left “uncut” (UC) or “cut” with the HaeIII restriction enzyme. (G) Confocal images of WT and ctsk mutants stained immunohistochemically for myosin (using MF20, red) show reduced size of atria in embryos homozygote for the 2 bp–deleted allele. Percent values represent the number of animals exhibiting these phenotypes. n = 30 embryos imaged from 3 independent experiments. Scale bar: 30 μm. (H) quantification of cross-sectional area of ventricles and atria in WT and ctsk-compromised embryos. Data are presented as mean ± SEM. ***P < 0.001, ****P < 0.0001 using 2-tailed Student’s t test or Dunnett’s corrected test (denoted by red box). (I) In situ analyses of notch1b expression show its boundary is expanded in AV and OFT valves (white arrowheads) of ctsk^2bp/2bp embryos (red arrow heads). Percent values represent the number of animals exhibiting these phenotypes. n = 25–30 embryos from 3 independent experiments. Scale bar: 50 μm.