Hyls1 D226G leads to perinatal lethality. (A) Survival of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG animals. N ≥ 8 mice per genotype. (B) Quantification of the number of digits per limb (left) and representative images (right) in Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG P0 animals. Arrows show extra digits. N ≥ 7 mice per genotype. (C) Percentage of pups with normal or open palate in Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG P0 animals. N ≥ 5 mice per genotype. (D) Analysis of kidney area in E18.5 (left) and P0 (middle) animals and representative P0 images (right) in Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG animals. Circled points indicate the data used in the representative images. N ≥ 4 mice per genotype. (E) Representative images of immunofluorescence analysis of cilia (ARL13B) and centrosomes (γ-TUBULIN) in the kidneys, trachea, lungs, heart, brain, and thymus in control (Hyls1^+/+ or Hyls1^+/DG) and Hyls1^DG/DG P0 animals. LV: lateral ventricle. (F) Quantification of cilia (ARL13B) number per centrosome (γ-TUBULIN) in the kidneys, trachea, lungs, heart, brain, and thymus from immunofluorescence images in E. N = 3 mice per genotype. Data are represented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (D) and an unpaired two-tailed Student’s t test with Welch’s correction (F). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Only significant results are indicated. Number of digits per limb was assessed using two-way ANOVA with post-hoc analysis and results are summarized in supplementary material. Scale bar is 5 μm in E.