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The homophilic nature of cadherin-mediated cell-cell adhesion provides an organism with the opportunity of altering the adhesive capabilities of its cells by selectively modulating the expression of different cadherin types. Differential cadherin expression is of major importance in regulating the cell rearrangements involved in the processes which shape tissues and organs during embryogenesis. The pregastrula embryo of Xenopus laevis expresses two maternally supplied cadherins: XB/U-cadherin and EP-cadherin. Since these two proteins are almost 92% identical at the amino acid level, it was unclear whether heterophilic interactions between them were possible. Different functional roles can only be ascribed to the two cadherins if the possibility of heterophilic binding between them can be excluded. We describe a simple and straightforward assay which can be used to assess interactions between adhesion molecules. A combination of antisense oligonucleotide and enzyme treatments eliminates endogenous cadherins in Xenopus oocytes and subsequent injection of a specific mRNA yields oocytes carrying only one or the other cadherin. After removal of the vitelline membranes, two oocytes expressing the appropriate cadherins will adhere to one another when they are placed in close contact. By scoring for adhesion in homotypic and heterotypic pairings, we demonstrate that XB/U-cadherin and EP-cadherin do not interact with one another.
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9232599
???displayArticle.link???Mech Dev
Fig. l. Amino acid sequence identities between XB/U-cadherin and EP-,
P-, and B-cadherin. The shaded region spans amino acid positions 45-87
and indicates the domain responsible for homophilic binding between
cadherins. Not unexpectedly, this region is more conserved than the rest
of the molecule in the P- and B-cadherin isoforms but, while the overall
identity to XB/U-cadherin is high, EP-cadherin displays a considerable
sequence divergence within this region.
Fig. 2. Adhesion between paired oocytes. To score for adhesion the oocyte pairs were either gently pried apart or one of the oocytes was lifted up as depicted
in (A). These particular oocytes were not microinjected and therefore express both maternal cadherins but oocyte pairs expressing only a single cadherin
appeared superficially similar. (B,C) A phase contrast and an immunofluorescent image, respectively, of the contact site between two oocytes. The cadherins
in (C) were labeled with the aXcad antibody which does not discriminate between the two maternal isoforms. A repeat of the labeling procedure with an inert
primary antibody did not yield any significant staining. Bar in (A), 250 #m; bar in (B,C), 200 #m.
Fig. 3. Antisense treatment gives rise to a stable breakdown product of the
endogenous mRNA, but injected synthetic mRNA is not affected. (A) The
endogenous messages for both maternal cadberins can be seen in this
Northern blot as a single band of 3.8 kb in both uninjected and sense
(S) injected oocytes (arrow). A breakdown product of 2.7 kb appears
upon injection of the antisense oligonncleotide (AS) and this fragment is
maintained for up to 6 h after injection (arrowhead). Ten ng of the antisense
oligonucleotide yields a somewhat more efficient degradation than
injection of 5 ng. Injection of 1 ng of XB/U-cadherin mRNA subsequent to
the antisense treatment gives rise to a band of 2.9 kb (double arrowhead)
above the breakdown product. (B) A Xenopus actin probe served as a
loading control after decay of the cadherin probes.
Fig. 4. Antisense treatment prevents de novo synthesis of cadherin protein.
Western blotting demonstrates that injection of the sense oligonucleotide
(S) has no effect on the regeneration of the cadherin protein complement
after collagenase treatment. The antisense oligonucleotide (AS), on the
other hand, results in a gradual disappearance of endogenous protein. If
mRNA for either XB/U- or EP-cadherin is injected into antisense-treated
oocytes, expression of that cadherin is maintained, even up to 20 h after the
antisense treatment.
Fig. 5. XB/U-cadherin and EP-cadherin do not interact heterophilically.
The bars indicate the percentage of oocyte pairs displaying adhesion after
various treatments. Sense-injected oocytes (S + S) adhere in virtually
100% of the cases while antisense treatment (AS + AS) reduces adhesion
to about 10%. Injection of either XB/U- or EP-cadherin subsequent to
anfisense treatment reconstitutes adhesion when oocytes are placed in
homotypic pairings (XB + XB or EP + EP) but heterotypic pairings
(XB + EP) fail to show a corresponding 'rescue' effect. The error bars
represent the standard deviations derived from five independent assays.
