Pkd2 Affects Cilia Length and Impacts LR Flow Dynamics and Dand5

Abstract

The left-right (LR) field recognizes the importance of the mechanism involving the calcium permeable channel Polycystin-2. However, whether the early LR symmetry breaking mechanism is exclusively via Polycystin-2 has not been tested. For that purpose, we need to be able to isolate the effects of decreasing the levels of Pkd2 protein from any eventual effects on flow dynamics. Here we demonstrate that curly-up (cup) homozygous mutants have abnormal flow dynamics. In addition, we performed one cell stage Pkd2 knockdowns and LR organizer specific Pkd2 knockdowns and observed that both techniques resulted in shorter cilia length and abnormal flow dynamics. We conclude that Pkd2 reduction leads to LR defects that cannot be assigned exclusively to its putative role in mediating mechanosensation because indirectly, by modifying cell shape or decreasing cilia length, Pkd2 deficit affects LR flow dynamics.

Keywords: Pkd2; cilia length; dand5; flow dynamics; left-right; zebrafish.

FIGURE 1

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.

FIGURE 2

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.

FIGURE 3

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.