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Overexpression of the Xenopus Tight-Junction Protein Claudin Causes Randomization of the Left–Right Body Axis

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Overexpression of the Xenopus Tight-Junction Protein Claudin Causes Randomization of the Left–Right Body Axis Developmental Biology 230, 217–229 (2001) doi:10.1006/dbio.2000.0116, available online at http://www.idealibrary.com on Overexpression of the Xenopus Tight-Junction Protein Claudin Causes Randomization of the Left–Right Body Axis Brenda J. Brizuela,1 Oliver Wessely, and E. M. De Robertis2 Howard Hughes Medical Institute and Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California 90095-1662 This study presents Xenopus claudin (Xcla), a tight-junction protein that is abundantly expressed in eggs and neuroecto- dermal precursors during early development. It was isolated via a differential screen for mRNAs enriched in microsomes in the Xenopus blastula. The Xcla protein contains four transmembrane domains and a carboxy-terminal cytoplasmic region with a putative PDZ-binding site. We show that this PDZ-binding site of Xcla is critical for its correct localization on the cell membrane and that a truncated form leads to delocalization of the tight-junction protein ZO-1. Overexpression of Xcla causes changes in the cell adhesion properties of blastomeres and leads to visceral situs randomization. The results suggest that left–right axial patterning is very sensitive to changes in regulation of cell–cell interactions and implicate a tight-junction protein in the determination of left–right asymmetry. © 2001 Academic Press Key Words: Xenopus; left–right asymmetry; Claudin; ZO-1; tight junctions; cell adhesion; cell–cell interactions. INTRODUCTION tight-junction proteins (Kubota et al., 1999; Fanning et al., 1999). Tight junctions provide an apical seal, also known as the Tight junctions contain integral membrane proteins that zonula occludens, in polarized epithelial and endothelial bridge adjacent cells and peripheral cytoplasmic proteins cells. Tight junctions are found associated with adherens that interact with the cytoskeleton (reviewed in Citi and junctions and gap junctions in most epithelial cell types and Cordenosi, 1998). In avian and mammalian epithelia the are linked intracellularly to the actin cytoskeleton (Kojima membrane-spanning component of the tight-junction com- et al., 1999; Fanning et al., 1998). These junctions play a plex includes the four-transmembrane proteins occludin major role in specific cell–cell recognition, sorting, and and family members of the more recently identified clau- communication and in the maintenance of the cohesive dins (Furuse et al., 1993, 1998a). The cytoplasmic compo- sheet of cells required for epithelial integrity and morpho- nent of the tight-junction complex contains several periph- genesis. Two important functions previously described for eral proteins, in particular zonula occludens-1 (ZO-1) tight junctions are a barrier function that allows epithelia to (Stevenson et al., 1986), ZO-2 (Gumbiner et al., 1991; Itoh separate fluid compartments (e.g., blood/brain) by control- et al., 1999a), and ZO-3 (Haskins et al., 1998). ZO-1 is ling the permeability of the paracellular pathway to ions thought to be responsible for clustering transmembrane and larger molecules and a fence function that restricts the proteins or receptors in their correct positions by binding to mobility of membrane proteins and lipids across the bound- the COOH-terminus of these proteins via 90-amino-acid ary between apical and basolateral membrane domains, establishing the polarity of epithelial cells (Fanning et al., repeats called PDZ domains, so designated because of their 1998; Citi, 1993; Mu¨ller and Hausen, 1995). More recently conservation in the PSD-95 (postsynaptic density-95), a third function in cell adhesion has been proposed for Dlg-A (discs-large), and ZO-1 proteins (Fanning and Ander- son, 1999). Tight junctions play an important role during 1 early embryonic development. For example, functional in- Present address: Zoology Department, North Carolina State University, Raleigh, NC 27695. tercellular tight junctions maintain epithelial integrity dur- 2 To whom correspondence should be addressed. E-mail: ing the formation of the blastocoele cavity in Xenopus [email protected]. (Cardellini et al., 1996; Merzdorf et al., 1998). The regula- 0012-1606/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 217 218 Brizuela, Wessely, and De Robertis membrane-bound or secreted proteins translated at these earliest stages of embryogenesis, we purified mRNAs associ- ated with the endoplasmic reticulum at the blastula stage and carried out a differential screen of microsomal versus nonmi- crosomal mRNAs. In the present study we characterize one of the clones isolated, Xcla. Although the function of this pro- tein was unknown at the time the studies were initiated, rapid progress in this active area of research revealed that Xcla was a member of the Claudin gene family of tight-junction pro- teins. Xcla mRNA is expressed maternally in the oocyte and blastula stages and has a dynamic zygotic expression pattern in epithelial cells at the gastrula, neurula, and tail-bud stages. We show that, like members of the claudin gene family in other systems, Xcla is an indispensable component of the tight-junction complex in Xenopus embryos. Overexpressed Xcla localizes at cell–cell contact points and increases cell adhesion, while a mutant construct lacking the C-terminal PDZ-binding domain changes ZO-1 localization. Perturbation of cell adhesion and cell migration have previously been correlated with randomization of left– right asymmetries in Xenopus embryos. Elimination of fibronectin fibrils from the blastocoele roof over which cardiac and visceral primordia move during development results in global randomization of left–right asymmetries (Yost, 1992). In addition, communication via gap junc- tions has been shown to play an essential role in left– right asymmetry in Xenopus (Levin and Mercola, 1998) and has been implicated in heterotaxia in humans (Britz- Cunningham et al., 1995). Furthermore, N-cadherin, a component of adherens junctions, is required for left– right asymmetry in the chick (Garcia-Castro et al., 2000). Interestingly, injection of synthetic mRNA encoding Xcla into Xenopus embryos causes bilateral expression of Xnr-1 in lateral plate mesoderm and left–right random- ization of the visceral organs (heart, gut, and gall bladder). This phenotype is not accompanied by a concomitant FIG. 1. (A) Alignment of the Xcla amino acid sequence with decrease in gap-junction communication. The results human Claudins 4, 3, and 5. (B) The putative Xenopus Claudin suggest that left–right axial patterning is very sensitive to protein has four transmembrane domains, two extracellular loops, changes in cell junction function. one intracellular loop, and a cytoplasmic carboxyl-terminus. (C, D) Immunostaining of Xcla-HA-injected animal caps in (C) nonper- meabilized and (D) permeabilized cell membranes; this demon- MATERIALS AND METHODS strates experimentally that the predicted structure shown in B is the correct topology and confirms that the protein is located in the cell membrane and is concentrated at regions of cell contact (inset Isolation of Microsomal RNA Fractions from in D). Xenopus Embryos To screen for membrane or secreted proteins that might function in early development, a method for microsome isola- tion was developed for Xenopus embryos from the methods of tion of tight-junction complex assembly is dependent on Adelman et al. (1973) and of Kopiczynki et al. (1998). Xenopus the localization of tight-junction transmembrane proteins embryos were allowed to develop to blastula stage 9 (8.5 h after within the cell (Fleming et al., 1993) and has been impli- fertilization), and 500 healthy embryos were selected, homoge- nized in a glass homogenizer with a loose pestle in 6 ml STKMH cated in the timing of blastocoele formation (Sheth et al., homogenization buffer (0.5 M sucrose, 50 mM Tris–HCl, pH 7.5, 2000). 25 mM KCl, 5 mM MgCl, 0.5 mg/ml heparin), and centrifuged In Xenopus, early development takes place in the absence of (45 s, 5220g) in an Eppendorf 5402 microcentrifuge to remove the RNA synthesis until the midblastula transition and com- yolk. The postyolk supernatant (PYS) was decanted from the pletely relies on maternally inherited proteins and mRNAs yolk pellet and adjusted to a final concentration of 150 mM KCl, (Newport and Kirschner, 1982). In an effort to identify 2 M sucrose, 0.5 mg/ml heparin. High Kϩ concentration is used Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Xenopus Claudin and Left–Right Asymmetry 219 FIG. 2. Xenopus claudin expression pattern studied by in situ hybridization. (A) Maternal expression of Xcla in oocyte stages I–IV. (B) RT-PCR of RNA isolated from 16-cell stage embryos; Xcla expression is enriched in the animal hemisphere. Vg1 was used as a vegetal control and histone H4 as a loading control. (C–F) Xcla mRNA in the animal pole of 2-, 4-, 8-, and 128-cell embryos. (G–J) Zygotic expression in the late gastrula, neurula, and neural tube stages. Note that the expression is repressed in the neural plate midline and in a transient band that appears to coincide with the midbrain/hindbrain boundary (arrowhead in I). (K and L) Tail-bud stage embryos. Xcla neural expression can be detected in the developing neuroepithelium at the tip of the tail, in the otic vesicles, and in the branchial arch endoderm (arrowheads in K), as well as in nasal placode and pronephros (arrowheads in L). to destabilize weak protein–protein interactions and help disso- (Tel-Test, Inc.) and tested by RT-PCR for the presence of chordin ciate free polysomes from membrane-bound polysomes (rough and Frzb-1 mRNA, which encode known secreted molecules. microsomes). The high sucrose density allows the PYS to form the bottom of a step gradient, producing a better separation of Isolation of Xcla cDNA the floating microsome fraction. The addition of heparin inhibits ribonuclease activity. The PYS was then placed in the bottom of The fractions that tested positive for microsomes marked by a sucrose density step gradient overlaid by 1.8 and 1.3 M chordin and Frzb-1 mRNA were transcribed into cDNA by the Cap STKMH.
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