Polycystin-2 Immunolocalization and Function in Zebrafish

Abstract

Polycystin-2 functions as a cation-permeable transient receptor potential ion channel in kidney epithelial cells and when mutated results in human autosomal dominant polycystic kidney disease. For further exploration of the in vivo functions of Polycystin-2, this study examined its expression and function during zebrafish embryogenesis. pkd2 mRNA is ubiquitously expressed, and its presence in the larval kidney could be confirmed by reverse transcription-PCR on isolated pronephroi. Immunostaining with anti-zebrafish Polycystin-2 antibody revealed protein expression in motile kidney epithelial cell cilia and intracellular cell membranes. Intracellular localization was segment specific; in the proximal nephron segment, Polycystin-2 was localized to basolateral cell membranes, whereas in the caudal pronephric segment, Polycystin-2 was concentrated in subapical cytoplasmic vesicles. Polycystin-2 also was expressed in muscle cells and in a variety of sensory cells that are associated with mechanotransduction, including cells of the ear, the lateral line organ, and the olfactory placodes. Disruption of Polycystin-2 mRNA expression resulted in pronephric kidney cysts, body axis curvature, organ laterality defects, and hydrocephalus-defects that could be rescued by expression of a human PKD2 mRNA. In-frame deletions in the first extracellular loop and C-terminal phosphofurin acidic cluster sorting protein-1 (PACS-1) binding sites in the cytoplasmic tail caused Polycystin-2 mislocalization to the apical cell surface. Unlike zebrafish intraflagellar transport protein (IFT) mutants, cyst formation was not associated with cilia defects and instead correlated with reduced kidney fluid output, expansion of caudal duct apical cell membranes, and occlusion of the caudal pronephric nephron segment.

TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment inXenopus

“…Here, we report for the first time that pkd2 loss of function affects early steps of Xenopus kidney development, that is, the emergence and/or the maintenance of the kidney field. This differs significantly from kidney pkd2 loss-of-function phenotypes described in mouse (McGrath et al, 2003;Wu et al, 2000) or zebrafish (Bisgrove et al, 2005;Obara et al, 2006;Schottenfeld et al, 2007;Sun et al, 2004), where early kidney development does not appear to be affected. However, during mouse pronephric development it is unclear whether Pkd2 does not have any function.…”

“…Although several features of pkd2 loss-of-function phenotypes are conserved among vertebrates, such as cystic kidneys and laterality defects, others already appear to be restricted to a species, as for example body curvature in zebrafish (Obara et al, 2006). Here, we report for the first time that pkd2 loss of function affects early steps of Xenopus kidney development, that is, the emergence and/or the maintenance of the kidney field.…”

“…Pkd2 mutants exhibit severe cardio-vascular defects, and form cysts in developing nephrons and pancreatic duct (Wu et al, 2000). Morpholino-based loss of function of pkd2 also results in the formation of pronephric cysts during zebrafish development (Obara et al, 2006;Sun et al, 2004) and in the formation of severe edema and of dilated pronephric tubules in Xenopus (Tran et al, 2010).…”

Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

“…Research into pkd2 function in zebrafish has further strengthened the idea that polycystin-2 functions in cilia. Knockdown of pkd2 by morpholino (MO) [3][4][5] or in mutants 5,6 produces phenotypes that are consistent with a role in cilia function, such as curved tails, pronephric cysts, and edema.…”

Ca2+ coding and decoding strategies for the specification of neural and renal precursor cells during development

“…TRPP2 is encoded by the PKD2 gene and in humans, mutations in PKD2 are responsible for autosomal dominant polycystic kidney disease (ADPKD) [67]. In addition, morpholino (MO)-based knock-down of pkd2 has been shown to result in the formation of pronephric cysts in zebrafish [68,69], and in the formation of severe oedema and dilated pronephric tubules in Xenopus [70].…”