Endocardial primary cilia exist in the hearts at later stages. (A–F) Confocal stack projections of immunofluorescence showing cardiac cilia in wildtype and klf2 mutants at 3 dpf. Green, anti-GFP immunostaining; red, acetylated tubulin immunostaining. (D–F) greyscale of red channel only. (G) Quantification of cardiac cilia number in wildtype and klf2 mutants at 3 dpf (N = 16, 11, 6, 7 respectively). Mean + s.e.m. ANOVA analysis, ns, not significant. (H–M) Acetylated tubulin immunostaining showing primary cilia in endocardial cells (H, K), epicardial cells (I, L), and cardiomyocytes (J, M) at 3 dpf. (K–M) magnified white boxes in (H–J). (N–U) Confocal stack projections of immunofluorescence showing cardiac cilia in controlTricaine or BDM treatment or ablated Tg(vmhc:mCherry-NTR) hearts with or without Tricaine treatment at 1 dpt. (R–U) magnified white boxes in (N–Q). Red, mCherry fluorescence; green, acetylated tubulin immunostaining. (V) Quantification of cardiac cilia number in control or ablated hearts with or without Tricaine treatment at 1 dpt/4 dpf (N = 11, 11, 10, 13 respectively). Mean + s.e.m. ANOVA analysis, **P < 0.01, ***P < 0.001. Scale bars, (A–F, N–Q) 50 µm, (H–J) 10 µm, (K–M, R–U) 5 µm. dpf, days post fertilization, dpt, days post treatment. Dashed lines outline the hearts. Arrowheads point to cilia. A, atrium; AVC, atrioventricular canal; OFT, outflow tract; V, ventricle
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