Brizuela BJ et al. (2001), Overexpression of the Xenopus tight-junction pr...

XB-ART-9661

Dev Biol 2001 Feb 15;2302:217-29. doi: 10.1006/dbio.2000.0116.

Show Gene links Show Anatomy links

Overexpression of the Xenopus tight-junction protein claudin causes randomization of the left-right body axis.

Brizuela BJ , Wessely O , De Robertis EM .

???displayArticle.abstract???
This study presents Xenopus claudin (Xcla), a tight-junction protein that is abundantly expressed in eggs and neuroectodermal 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.

???displayArticle.pubmedLink??? 11161574
???displayArticle.link??? Dev Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: actl6a cldn6.2 gdf1 nodal1 ocln prl.1 tjp1 tjp2 tjp3

???attribute.lit??? ???displayArticles.show???

	FIG. 1. (A) Alignment of the Xcla amino acid sequence with human Claudins 4, 3, and 5. (B) The putative Xenopus Claudin protein has four transmembrane domains, two extracellular loops, one intracellular loop, and a cytoplasmic carboxyl-terminus. (C, D) Immunostaining of Xcla-HA-injected animal caps in (C) nonpermeabilized and (D) permeabilized cell membranes; this demonstrates 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 in D).
	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).
	FIG. 3. The PDZ binding domain is required for Xcla localization at cell contact points in L-cells. (A) Schematic representation of the epitope-tagged constructs used; see text for details. Representative examples from two independent experiments, in which many microscope fields involving hundreds of cells were examined. (C, D) Xcla-HA and (E) Xcla-DC-HA transfected into L-cells and analyzed by immunofluorescence using an anti-HA antibody. Note that Xcla-HA is localized at contact points between cells, whereas Xcla-DC was never found in intracellular locations (compare arrows in C–E). (B) Phase contrast; (C) fluorescence image of cells shown in (B). (D and E) Confocal microscopy.
	FIG. 4. Wild-type Xcla, but not Xcla-DC, colocalizes with ZO-1 in the cell surface membrane. Confocal microscopy of Xenopus animal cap explants injected with synthetic mRNA encoding Xcla-HA (A–C) or Xcla-DC-HA (D and E) and uninjected control (F). The explants were stained with anti-HA monoclonal antibody to visualize the exogenous Xcla protein (red) and with anti-ZO-1 polyclonal antibody to show the endogenous ZO-1 protein (green). Xcla-HA and ZO-1 are colocalized in spots along the membrane in Xcla-HA-injected caps (C), but located mostly in intracellular organelles from the membrane in Xcla-DC-HA-injected caps (E). Endogenous ZO-1 protein is localized in the membrane of uninjected caps (F).
	FIG. 5. Xcla overexpression affects cell adhesion. (A–D) Embryos co-injected once into the A4 blastomere at the 32-cell stage with lacZ and prolactin, Xcla, Xcla-DC, or Xcla plus Xcla-DC mRNAs, followed by staining for b-galactosidase activity at stage 11. Overexpression of wild-type Xcla (B) causes cells to be more adherent than in prolactin mRNA-injected control embryos (A), whereas overexpression of mutated Xcla-DC (C) also causes cells to adhere, but they disperse as clumps of cells. This altered cell adhesion can be rescued to normal by co-injection of Xcla plus Xcla-DC (D). (E–H) Dissociated animal caps injected with Xcla/GFP (F), Xcla-DC/GFP (G), or control prolactin/GFP (H) were mixed with dissociated animal caps injected with rhodamine-dextran (red cells in F–H) and allowed to reaggregate by the addition of Ca21. Both Xcla and Xcla-DC mRNA lead to cell sorting from rhodamine-dextran injected cells, while prolactin-injected control cells disperse evenly throughout the aggregate.
	FIG. 6. Overexpression of Xcla mRNA causes bilateral Xnr-1 expression and left–right randomization. (A–H) Embryos were injected at the four-cell stage with Xcla or Xcla-DC mRNA and scored for inversions at stage 45 (B–E) or analyzed by Xnr-1 in situ hybridization at stage 22 (F–H). (B, D, and F) Uninjected control embryos. (C) Inverted heart and gall bladder (compare arrowheads in B and C) and (E) inverted gut coiling (compare with D) in Xcla-DC-injected embryos. (F) Normal left-sided Xnr-1 expression in control embryo from dorsal view. Bilateral Xnr-1 expression from dorsal view in (G) Xcla and (H) Xcla-DC mRNA-injected embryos.
	FIG. 7. Gap junction communication (GJC) is not inhibited by overexpression of Xcla or Xcla-DC. Albino embryos injected with either Xcla (A and B) or Xcla-DC (C and D) prior to the first cleavage, followed by one injection of a mixture of Lucifer yellow (LY) and rhodamine-dextran (RLD) at the 16-cell stage, are shown under fluorescence. The presence of a cell with LY only (arrows) adjacent to a cell which was injected with both LY 1 RLD (arrowheads) indicates dye transfer through gap junctions. Heptanol is an inhibitor of GJC; the heptanol-treated embryo shown in E and F is an example of inhibition of GJC. The heptanol treatment was less effective in our case (11% versus 5% for Levin and Mercola, 1998), perhaps due to subtle technical differences. (G and H) Percentage of embryos showing LY transfer after heptanol treatment (n 5 27), no treatment (n 5 49 albino; n 5 32 pigmented), Xcla injection (n 5 24) or Xcla-DC injection (n 5 37 albino; n 5 69 pigmented). Ectopic expression of Xcla somewhat increased the percentage of embryos with GJC (G). This observation might be explained if the tighter adhesion of cells injected with Xcla mRNA led to an increase in gap-junction communication. (H) Dorsal injections into pigmented embryos showing a higher frequency of GJC that is, nevertheless, not affected by Xcla-DC mRNA microinjection.
	FIG. 8. A model for tight-junction assembly. Oligomers of occludin may form a complex with ZO-1. Claudin binding to ZO-1 may then facilitate the assembly of the mature apical tight junction. Transmembrane proteins located at the tight junction associate with cytoplasmic proteins ZO-2 and ZO-3, which also bind to ZO-1 and actin.