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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.
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 CâE). (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
(AâC) 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. (AâD) 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). (EâH) 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 FâH) 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 leftâright randomization. (AâH) Embryos were
injected at the four-cell stage with Xcla or Xcla-DC mRNA and
scored for inversions at stage 45 (BâE) or analyzed by Xnr-1 in
situ hybridization at stage 22 (FâH). (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.
