Short cilia reduce fluid flow inside Kupffer′s vesicle and compromise charon asymmetric expression. (A-B′) Still images from wild-type siblings (A) and aei-/- mutant embryos (B) taken from Movies 1 and 2, respectively, in the supplementary material. Inserts A′ and B′ show vectors representing the fluid flow velocity and direction in each experimental situation. (C) Quantification of the average fluid flow velocity in wild type (n=5, 16.46±1.69 μm/s) and in aei–/- mutants (n=5, 6.74±2.41 μm/s). (D) Percentage of cilia in different regions of Kupffer′s vesicle (anterior, middle, posterior, dorsal and ventral) in wild-type (n=19) and aei-/- mutant embryos (n=14). (E) A 10-somite stage embryo showing nuclei stained with DAPI in blue and charon mRNA cytoplasmic localization in the epithelial cells that surround the lumen of Kupffer′s vesicle. (F,G) Expression pattern of charon in Kupffer′s vesicle of embryos overexpressing deltaD (F) and NICD (G). (H-J) charon expression at Kupffer′s vesicle is symmetric at the six-somite stage (H) and is asymmetric at the eight- and ten-somite stages (I,J). (K) Percentage of symmetric versus asymmetric charon expression in Kupffer′s vesicle in wild-type embryos from five- to 12-somite stages (n=18 for each stage). (L-O) Range of expression patterns of charon in Kupffer′s vesicle of aei-/- mutant embryos; charon expression may be symmetric (L), asymmetric with stronger expression on the right (M), asymmetric with stronger expression on the left (N) or reduced (O). (P) Percentages of the different charon expression patterns in Kupffer′s vesicle in aei-/- mutant embryos from the eight- to 12-somite stages (n=34 on average for each stage). (Q) charon expression pattern in Kupffer′s vesicle of one representative lrd1 morphant. (R) Percentage of symmetric versus asymmetric charon expression in Kupffer′s vesicle of lrd1 morphants from the eight- to 12-somite stages (n=27 on average for each stage).