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Claudins are tetratransmembrane tight junction proteins and play important roles in regulating paracellular permeability of different nephron segments of the kidney. However, the roles of claudins in kidney development remain largely unknown. Here we studied the expression and functions of claudin-6 in Xenopus pronephros development. Xenopus claudin-6 is expressed in the developing pronephric tubule and duct but not glomus. Knockdown of claudin-6 by specific morpholino led to severe defects in pronephros tubular morphogenesis and blocked the terminal differentiation of the tubule cells. The claudin-6 morpholino targeted tubule cells showed failure of apical accumulation of actin and reduced lateral expression of tight junction protein Na/K-ATPase, suggesting an incomplete epithelization likely due to defected cell adhesions and apical-lateral polarity. Our work uncovered a novel role for claudin-6 in embryonic kidney development.
Fig. 1.
Specific expression of XCldn6 in developing pronephros. (A and B) Expression of XCldn6 in the embryos at stage 25 (A) and 34 (B) revealed by whole-mount in situ hybridization. Pr, pronephros; PT, pronephric tubule; PD, pronephric duct. (C–F) Expression of XCldn6 protein in the pronephric tubule cells in embryos at indicated stages revealed by immunostaining. XCldn6 is no longer detected at the apical surface of the tubule cells at stage 39 (arrowheads in F).
Fig. 2.
The XCldn6 antibody and XCldn6-MO are specific. (A) Injection of the XCldn6 morpholino completely abolished the staining with the XCldn6 antibody in the pronephric tubule (lower panel), compared with the control (upper panel). (B) Endogenous XCldn6 level was dramatically reduced in embryos injected with the XCldn6-MO as revealed by western bolt using the XCldn6 antibody. (C) Embryos injected with the XCldn6-MO developed pericardial edema (arrowhead) at stage 43.
Fig. 3.
Xcldn6 is required for pronephros development. (A and B) Expression patterns of Pax2, Lim1 and Pax8 in control embryos and embryos injected with XCldn6-MO or together with rescuing XCldn6.1a mRNA at stage 36 revealed by whole-mount in situ hybridization. The percentages of embryos with deformed pronephric tubules are shown in (B). (C and D) Injection of XCldn6-MO severely reduced the expression of PDZK1 and CLC-K in stage 36 embryos which were rescued by co-expression of XCldn6.1a mRNA. The statistical data are shown in (D).
Fig. 4.
Effects of XCldn6 knockdown on the polarity, cytoskeleton and junction protein expression in pronephric tubule cells. (A and B) Immunostaining of PKCζ (red) in pronephric tubule cells of control embryos and embryos injected with XCldn6-MO. The targeted areas were labeled green by tracing GFP (B'). (C and D) Immunostaining of p-ERM (red) in pronephric tubule cells of control embryos and embryos injected with XCldn6-MO. The targeted areas were labeled green by tracing GFP (D'). (E) Immunostaining of p-ERM (red) in a pronephric tube in which part of the cells were targeted with XCldn6-MO. The targeted areas are outlined which were labeled green by tracing GFP (Eâ²). (F, G) Immunostaining of β-actin and Na/K-ATPase (red) in pronephric tubes in which part of the cells were targeted with XCldn6-MO. The targeted areas labeled by tracing GFP (green) are outlined. All embryos were at stage 36. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Supplemental Fig. 1.
The Cldn6 genes in Xenopus laevis. (A)Alignment of the Cldn6 proteins of Xenopus laevis. The protein sequences used were from: XCldn6.1a: NM_001093287; XCldn6.2a: NM_001088863; XCldn6.1b: NM_001088861; XCldn6.2b: NM_001088604. The sequences were aligned using ClustalW. The fragment used to develop the antibody against XCldn6 was outlined. (B) The genomic organization of the 4 Cldn6 genes in Xenopus laevis. The Cldn6.1 and 6.2 genes are clustered tandemly in reverse directions with intervals of 12.8 kb and 9.8 kb respectively.
