The zebrafish Kupffer's vesicle as a model system for the molecular mechanisms by which the lack of Polycystin-2 leads to stimulation of CFTR

Abstract

In autosomal dominant polycystic kidney disease (ADPKD), cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR). Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV) as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca(2+)-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition.

Keywords: (ADPKD); (CFTR); (KV); Autosomal dominant polycystic kidney disease; Cystic fibrosis transmembrane conductance regulator; Kupffer's vesicle; Polycystin-2; Zebrafish.

Fig. 1.

Polycystin-2 and CFTR expression. (A) Localization of KV (squared region) in the body of a 10–11 s.s. zebrafish embryo. (B,C) RNA in situ hybridizations for pkd2 (B) and cftr (C) in 10–11 s.s. WT embryos. Both pkd2 and cftr transcripts are detected in the KV region (right squares), neural floorplate (arrow heads), brain and pronephric ducts primordia (arrows). (D-S) Confocal images for the immunolocalization of Polycystin-2 in KV cells at the 10–11 s.s. In WT embryos (D-K), Polycystin-2 is detected clustered near the nuclei (white arrow in F), along cilia (white arrow heads in F) and at the basal body (dashed arrows in H,J and K). In pkd2-morphants (L-S), the Polycystin-2 signal is markedly reduced and, although still detected at the basal body (dashed arrow in P,R and S), it is no longer detected along cilia. (D,L) maximal intensity z-stack projection; (E-K;M-S) z-section. Polycystin-2 (green), acetylated α-tubulin (red), γ-tubulin, (purple), nuclei (blue). Scale bars: 10 µm. (T,U) Lateral view of pkd2-morphant (T) and WT (U) larvae at 72 hpf. (V) Heart position defects: pkd2-morphants – 33% right-sided, 21% central; cftr-morphants – 21% right-sided, 17% central; and WT siblings – 0.7% right-sided, 1.0% central. Left-sided (light grey), central (dark grey) and right-sided hearts (black). n, number of scored embryos.

Fig. 2.

KV volume. (A-J) Confocal live-microscopy scans of the whole KV of 10–11 s.s. ras:GFP transgenic embryos. The middle focal plane along the xy axis and the respective orthogonal views (along xz and yz axes) are shown for the most representative WT (A), pkd2-morphant (B), pkd2-mismatch MO (C), 0.14% (v/v) DMSO-treated WT (D), cftr-morphant (E), double-morphant (F), 30 µM CFTRinh-172-treated WT (G) and pkd2-morphant (H), and 10 µM forskolin+40 µM IBMX-treated WT (I) and pkd2-morphant (J) embryos. C is a control for B. D is a control for G,H,I and J. KV^volume is indicated in µm³ and in picol. Scale bars: 10 µm. (K) Estimated KV volumes (µm³) for WT (n=16), pkd2-mismatch MO (n=12), pkd2-morphant (n=11), cftr-morphant (n=8), double-morphant (n=6), 0.14% (v/v) DMSO-treated WT (n=10), 30 µM CFTRinh-172-treated WT (n=10) and pkd2-morphant (n=10), 10 µM forskolin+40 µM IBMX-treated WT (n=11) and pkd2-morphant (n=12) embryos. Mean±s.d.; ^ψP≤0.05 and ^ψψP<0.0001, significantly different from WT; *P<0.05 and **P<0.0001, significantly different from pkd2-morphants.

Fig. 3.

KV-lining cells. (A) Number of cells counted in the whole KV live-microscopy scans of 10–11 s.s. WT (n=14) and pkd2-morphant (n=10) embryos. (B) Number of cilia counted in WT (n=6) and pkd2-morphant (n=6) embryos immunodetected for acetylated α-tubulin. (C) Cellular length, width and height of WT (n=6) and pkd2-morphant (n=11) embryos immunodetected for actin cytoskeleton. The box plot and the respective max and min values are indicated. *P<0.01 and **P<0.0001, significantly different from WT. (D) Estimated apical surface area of KV-lining cells. (E) Schematic representation of cell shape of WT and pkd2-morphant KVs.

Fig. 4.

KV inflation live-dynamics. (A) KV luminal area of pkd2-morphants and WT embryos measured along development from 1 to 4 s.s. For each time point mean±s.d. are indicated. Number of tested embryos: WT, n=11; pkd2-morphant, n=8. ^ψP<0.05, significantly different from the previous time point in pkd2-morphants; *P<0.05, significantly different from WT at the corresponding time point. (B-I) Bright field images captured for the same embryo along development are shown for the most representative WT (B-E) and pkd2-morphant (F-I). Scale bars: 10 µm.

Fig. 5.

The zebrafish KV as a model organ for CFTR stimulation in ADPKD cysts. (A,B) In WT embryos, the KV inflation is ensured by the CFTR-mediated transport of Cl⁻ and by the subsequent movement of water towards the organ lumen (A). In KV epithelial cells (B), Polycystin-2 (PC2) located at the cilia membrane allows the entrance of Ca²⁺ when stimulated by the luminal fluid-flow. This ciliary wave activates the Ca²⁺ release from the ER pools, in a Polycystin-2-dependent manner, initiating a Ca²⁺-signaling of unknown effectors. Through inhibition of adenylyl cyclases 5 and 6 (AC5/AC6) and activation of phosphodiesterase 1A (PDE1A), the Ca²⁺ transients maintain the basal intracellular levels of cAMP required for the normal rate of CFTR activity. (C,D) Mimicking ADPKD cysts, the pkd2-knockdown enhances CFTR-mediated ion and fluid secretion into the KV, resulting in its significant enlargement (C). The reduced Ca²⁺ oscillations are expected to activate AC5/AC6 and inhibit PDE1A, raising the intracellular levels of cAMP and, thus, driving the overstimulation of CFTR (D). Ca²⁺ and Polycystin-2 (red); Cl⁻ and CFTR (green); cAMP (yellow); H₂O (blue); AC5, AC6, and PDE1A (grey). Full black arrows – known activations; dashed lines and arrows – expected inhibitions/activations; line and arrow widths are proportional to the expected level of activation.