cup^–/– mutants have fluid flow defects in the LRO. (A,B) Fluid flow heatmap and quantification of cup siblings with straight tail (n = 8) and cup^–/– mutants with curly tail (n = 9), respectively. Asterisks represent statistical significance (Wilcoxon Test, p-value < 0.05). Cilia beat frequency (CBF) of cup siblings and cup^–/– mutants, respectively; cup siblings show an average CBF of 34.8 Hz and cup^–/– mutants an average of 34.4 Hz (paired t-test, p-value < 0.05). (C,D) Representative image of cell shapes from one KV from cup siblings and one KV from cup^–/– mutants, respectively. (E) Quantification of differences in length to width ratio and (F) differences in cellular length and width in cup siblings (n = 6) and cup mutants (n = 5); asterisks represent statistical significance (paired t-test, p-value < 0.05). (G) Number of cells present in cup mutants and sibling in the middle plane. (H) 3D cilia length measurements in live embryos injected with 50 pg of arl13b-mCherry mRNA (arl13b; n = 16) and injected with pkd2 MisMO (n = 6), pkd2 MO (n = 6), cup mutants (n = 8), and cup siblings (n = 13) with arl13b-mCherry mRNA; asterisks represent statistical significance (paired t-test, p-value < 0.05) (I) Motile/Immotile cilia ratio in the same cup mutants and cup siblings as in panel (H). Scale bars 10 μm. L, left; R, right; A, anterior; P, posterior.

pkd2 knockdown in the DFCs rescues KV fluid flow pattern. (A) Immunostaining for cortical actin and rhodamine showing the result of a successful injection of pkd2 MO into DFCs, anterior (A’) and posterior (A”) panels in the different channels allow for better contrast/brightness balance; (B) Immunostaining for Pkd2 in WT and pkd2MO^DFCs embryos; (C) Representative images of KV architecture from one WT embryo and one pkd2 MO^DFCs injected embryo. (D) Quantification of the differences in length to width ratio in the KV of WT (n = 6) and pkd2 MO^DFCs (n = 9). (E) Cilia distribution in the antero-posterior axis of the KV of WT embryos (n = 22) and embryos injected with pkd2 MisMO (n = 6), pkd2 MO (n = 48) and pkd2 MO^DFCs (n = 15). (F) Quantification of the differences in cellular length, width, and height, in the same WT and pkd2 MO^DFCs injected embryos from panel (D). (G–I) Fluid flow heatmap and quantification of WT (n = 8), pkd2 MO^DFCs (n = 6) and pkd2 MO 1-cell stage embryos (n = 7), respectively. Asterisks represent statistical significance (Wilcoxon Test, p-value < 0.05). (J) 3D cilia length measurement in WT (n = 23), pkd2 MisMO (n = 8), pkd2 MO 1-cell stage (n = 25), rescue (n = 19), and pkd2 MO^DFCs (n = 10). (K) Motile/Immotile cilia ratio in live embryos injected with arl13b-mCherry mRNA (n = 8) and injected with pkd2 MisMO (n = 10), pkd2 MO (n = 6) and pkd2 MO rescued with Xenopus pkd2 mRNA (n = 5). Asterisks represent statistical significance (p < 0.05) with paired t-test. Scale bars 10 μm. L, left; R, right; A, anterior; P, posterior.

Comparative readouts for lack of fluid flow and knockdown of Pkd2 in the DFCs (A–D)dand5 expression pattern quantification by in situ hybridization of WT embryos, pkd2 MO, pkd2 MO^DFCs, and dnah7 MO. (E)dand5 expression level in fold change quantified by qRT-PCR; asterisks represent statistical significance (paired t-test, p-value < 0.05). (F–J) Organ situs quantification by scoring heart and liver laterality in the same larvae in WT, pkd2 MO^DFCs, pkd2 MO 1-cell stage, pkd2 Mismatch MO and dnah7 MO.