Fig 1: Loss of ciliary genes disrupted ciliogenesis during cardiac OFT development.(A) Schematic of a cilium showing structural features and selected ciliary genes. Ciliary genes (red) were immunostained with axonemes (acetylated α-tubulin), basal bodies (γ-tubulin), and centrioles (NEK2). Nuclei were stained with DAPI (blue). Mlf1 and Pkhd1l1 localized to cilia axonemes; Sptbn5 localized along the entire cilia; Ppef2 and Tekt3 localized to basal bodies of cilia; and Agbl2 was observed both at the basal body and centriole of cilia. Boxed and color-coded numbers following each gene indicate the number of cardiac defects observed in knockout/mutant mouse hearts: red for OA + VSD, purple for OA + VSD + ASD, blue for MU-VSD, orange for ASD, and green for MU-VSD + ASD. Scale bars, 20 μm. (B) Immunofluorescence staining of cilia in the cardiac OFTs of ciliary gene knockout/mutant mice and control mice at E13.5. Axonemes (acetylated α-tubulin, green) and basal bodies (γ-tubulin, red) were shown. The right column showed higher magnification of the boxed area from the left. Scale bars, 20 and 10 μm. (C) Box plots of cilia prevalence (left) and cilia length (right) in the cardiac OFTs of ciliary gene knockout/mutant mice and control mice at E13.5. (Left): Error bars represent means ± SEM. (Right): The blue boxes showed the lower, median, and upper quartiles of measurements. Each black dot represented the cilia length from a single cell. Homozygous knockout/mutant mice exhibited shorter cilia and a lower cilia percentage compared to wild-type control mice, indicating disrupted ciliogenesis. Statistical significance was assessed by an unpaired two-tailed Student’s t test or Wilcoxon–Mann-Whitney test when appropriate. *P < 0.05, **P < 0.01, ***P < 0.001. n = 10 to 20 OFT sections from five hearts for each group were measured percentage of cilia and cilia length across all OFT sections (n > 400 cilia per group).