Shear stress promotes notch1 expression in a primary cilia-dependent manner. (A-B′) The hearts of Tg(actb2:Arl13b-GFP) embryos were examined by confocal microscopy to assess localization of primary cilia in the endocardium. (A) Tg(actb2:Arl13b-GFP) reporter expression in the heart. The schematic diagram indicates the orientation of the heart and confocal section relative to the whole embryo at 30hpf. (B) High-resolution view of a Tg(actb2:Arl13b-GFP) embryo merged with (B′) a bright field image demonstrating colocalization of the primary cilium base with an endocardial cell. (C,D) Whole-mount Tg(myl7:dsRed); Tg(Tp1:VenusPest) double transgenic (C) control and (D) ift88 morphants (MO) at 48hpf. The hearts are marked with dashed circles. (C′,D′) Confocal maximal projection of the heart from the individual embryos shown in C,D, overlaying cardiomyocytes (red) and Notch reporter (green). (E,F) Confocal optical section of the (E) control and (F) ift88 morphant embryo cardiomyocytes (red) at 80hpf. The insets marked by the dotted rectangle were magnified in E′,F′. (G) Quantification of ventricular Notch reporter (EGFP) MFI from whole-mount embryos at 48hpf. (H,I) Relative expression of (H) notch1b and (I) nrg1 in control and ift88 morphant hearts. (J-L) Expression of (J) Notch1, (K) Efnb2 and (L) Nrg1 in DMSO- or 50µM CBD-treated MLECs that were exposed to 2 dyn/cm2 shear stress for 4h compared with static DMSO- and CBD-treated controls. Red arrows highlight Arl13b-GFP in the endocardium. Blue arrows highlight Notch reporter signal in neural tissue. White arrows highlight trabeculae. *P≤0.05-0.01, **P≤0.01-0.001, ***P<0.001 compared with control morphants (one-sample t-test compared with 1.0-fold change or Student′s t-test). Error bars are s.e.m. Scale bars: 50µm in A; 10µm in B,B′,F&prime& 100µm in F. a, atrium; CBD, ciliobrevin D; lu, lumen; MFI, mean fluorescence intensity; v, ventricle.
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