α-catenin Function in the Blastula
The function of αEC at the late blastula stage was investigated first by comparing the effects of replacing C-cadherin, the major cadherin at this early stage, with mutant forms either lacking the CBD domain (C-cad ΔCBD), and thus unable to bind αEC, or with the CBD domain replaced by the C-terminus of αEC. This was achieved by depleting the endogenous C-cadherin mRNA using an antisense oligo as described previously 
followed by injection of the appropriate mRNA into cultured oocytes before fertilization. show sections of late blastulae (St.9) stained for C-cadherin (red, upper panels) and αEC (green, lower panels) after no treatment (), depletion of C-cadherin (), depletion of C-cadherin + 300 pg wild type C-cadherin mRNA (), and depletion of C-cadherin + 300 pg C-cad ΔCBD (). The degree of cell dissociation can be seen in the lower panels, where spaces between the cells can be more easily seen (dark areas) due to the background staining of the cell contents. Depletion of C-cadherin mRNA caused the loss of C-cadherin protein from the cell surface and cell dissociation. It’s replacement by wild-type C-cadherin replaced cell surface C-cadherin protein and rescued cell adhesion. However, replacement by C-cad ΔCBD did not rescue cell adhesion. Although some of the mutant protein was expressed on the surface (upper panel in ),
it was not associated with αEC (as expected, since this lacks the CBD domain), and was non-functional in cell adhesion. The result is shown more clearly in , in which sections stained for C-cadherin have been observed. The high magnification 3D projections show that C-cadherin is concentrated in plaques on the cell surfaces in untreated embryos (), which are lost after C-cadherin depletion (), and regained at higher density after replacement by wild-type C-cadherin (). When replaced by C-cad ΔCBD, however, less of the mutant protein was expressed on the surface, and more remained trapped in the cytoplasm ().
The C-cadhering catenin binding domain (CBD) is required for cell adhesion . (A–D
We then carried out a similar experiment in which C-cadherin was replaced by a construct in which the CBD domain of mouse E-cadherin was replaced by the C-terminal half of αEC (nEαC).In contrast to the C-cad ΔCBD protein, nEαC was efficiently expressed on the cell surface and rescued cell adhesion (). In these rescued embryos, there was no cell surface staining with anti-β−catenin antibody, indicating that the rescue was by the nEαC protein and not by residual wild-type C-cadherin protein ().These data suggest that the primary function of the CBD is to bind αEC, and that this binding to C-cadherin is essential for its normal expression and/or stability on the cell surface, and for its adhesive function.
C-terminus of α-E-catenin is sufficient to drive cadherin membrane expression.
To test this further, we depleted early embryonic αEC directly, using the antisense oligo described previously 
, or a novel translation-blocking morpholino oligo (αEC-MO) complementary to the translation start site of Xenopus laevis
αEC. shows the blastocoelic surfaces of whole animal caps stained for C-cadherin from embryos that were untreated, αEC-depleted, or αEC-depleted then rescued with full-length human αEC mRNA. Pixel intensity measurements using the Zeiss LSM software were used to quantitate the amount of C-cadherin antibody staining (). Embryos depleted of αEC were also injected with full-length C-cadherin mRNA. In these embryos, some of the full-length protein was expressed on the cell membrane, but this was reduced compared to controls, and it did not rescue the dissociation effect of αEC depletion ().
α-E-catenin is required for C-cadherin expression on the cell surface in the blastula.
Thus the two approaches; replacement of C-cadherin with a mutant that lacked the αEC binding domain, or reducing directly the αEC protein levels, both gave the same result; that αEC is required for cell adhesion because it controls the expression levels of C-cadherin in the adherens junctions of the blastula cells.
Alpha Catenin Function in the Post-gastrula Ectoderm
The ectoderm was used as a model in this study, since its cells can be directly visualized on the outside of the embryo, and it differentiates into two components, the neural and non-neural ectoderm, each with a distinct expression pattern of cadherins and α-catenins. The non-neural ectoderm expresses C- and E-cadherins, whilst the neural ectoderm expresses C- and N-cadherins, each with characteristic sub-cellular distributions 
. The neural ectoderm expresses αNC 
and αEC 
, whilst the non-neural (epidermal) ectoderm expresses only αEC 
. In-situ hybridization of Xenopus
whole mounts confirmed the previously reported patterns of expression of these two alpha catenins in other species ( A, B, C, D, E, F
The distribution of α-N-catenin and α-E-catenin mRNAs in the neural and non-neural ectoderm.
To test the roles of the two α-catenins at post-gastula stages, we injected morpholino oligos complementary to the translation start site of each mRNA into individual blastomeres at the 8-cell stage whose descendants would either enter the neural (single dorsal animal blastomere) or non-neural (single ventral animal blastomere) ectoderm respectively. The efficiency of the αEC-MO is shown in . The antibody used to detect αNC did not cross react on western blots, so its efficiency was assayed by immunocytochemistry (see below). shows an en face
view of the non-neural ectoderm stained for C-cadherin (red) and αEC (green). In clones of cells depleted of αEC (), C-cadherin expression on the cell surface was lost. When human alpha-E-catenin mRNA, which is not recognized by the αEC-MO, was injected into the same blastomere immediately after the MO, the effect was rescued (). Thus, αEC has the same function in controlling cell surface expression of C-cadherin as seen in the blastula. However, the effect of αEC depletion on E-cadherin expression was completely different. En face
views of αEC-depleted clones showed the expression of E-cadherin on junctions to be unaffected (). Staining of E-cadherin was indistinguishable in αEC-depleted cells (green) and untreated cells. However, examination of transverse sections revealed that the non-neural ectoderm was multi-layered in αEC-depleted regions () compared to the untreated non-neural ectoderm from the same embryo (), which shows the usual two cell layers. This multi-layered arrangement resembled the cyst-like structures described in mice lacking αEC in the epidermis 
. When αEC was depleted globally by injection of the αEC-MO into both cells at the 2-cell stage, there was a loss of αEC throughout the embryo, but high levels of E-cadherin remained present in the non-neural ectoderm, and there was some up-regulation of expression of E-cadherin in deeper cells in the embryo (). Interestingly, western blotting data showed that the overall levels of C-cadherin protein were only modestly reduced after αEC depletion (). To visualize the formation of cyst-like structures in αEC-depleted non-neural ectoderm, we depleted αEC in one side of the embryo and made time lapse movies. In these embryos, large cyst-like structures started to appear immediately after the neural folds closed, when the non-neural ectoderm cells normally start to intercalate and spread by epiboly (Movie S1)
Antisense morpholino mediated depletion of α-E-catenin in the non-Neural Ectoderm.
Next we depleted the α-catenins in the neural ectoderm. Clones of cells depleted of αEC, αNC, or both, were generated on one side of the neural ectoderm by injection of MO, together with a lineage tracer, into one dorsal animal cell at the 8-cell stage. As it did in the non-neural ectoderm, depletion of αEC caused a dramatic decrease in junctional localization of C-cadherin (, the αEC-depleted cells are green). shows a clone of αNC-depleted cells. The tissue was stained with an antibody against αNC, and shows the degree of depletion in injected, compared to uninjected cells in the same neural plate. The neighboring panel shows the same cells stained for C-cadherin. The staining was reduced, but not completely absent compared to αEC-depletion (compare with ). The surface areas of the apices of the neural plate cells were increased in αNC depleted cells. This effect was also seen after depletion of N-cadherin, and is caused by the loss of the F-actin in the apical regions of the neural plate cells 
. It is hard to know whether the difference is due to loss of the concentration effect on C-cadherin caused by the lack of apical constriction of the neural plate cells, or by an actual decrease in overall C-cadherin levels on the cell surface. However, unlike the effect of αEC depletion, a significant amount of C-cadherin remained in the cell junctions after αNC depletion. The opposite was true for N-cadherin. Depletion of αNC, but not αEC, caused complete loss of N-cadherin from the cell surface in the neural ectoderm (). shows a clone of αEC-depleted cells (green) in the neural plate, stained for N-cadherin (red). There was no reduction of N-cadherin expression. shows a clone of αNC-depleted cells (green) in the neural plate. N-cadherin was completely lost from the cell surface. In chick neural epithelium, the depletion of α-N-catenin has also been shown to cause a loss of N-cadherin expression from the cell surface 
Depletion of α-E-catenin and α-N-catenin in the neural ectoderm.
In summary, αEC is required for C-cadherin expression on the cell surface in both the neural and non-neural ectoderm, but is not required for E-cadherin expression in the non-neural ectoderm, or N-cadherin expression in the neural ectoderm. αNC is essential for N-cadherin cell membrane expression in the neural ectoderm, but not required for C-cadherin expression. These data indicate that there is considerable specificity of function of the α-catenins in the post-gastrula embryo.
Previous work showed that loss of E-cadherin in the non-neural ectoderm, or N-cadherin in the neural ectoderm, caused the loss of F-actin in the cortical cytoplasm, and abrogation of the morphogenetic movements of these tissues 
. To assay the functions of the α-catenins in these effects, we first assayed the result of depleting αEC and αNC in the non-neural and neural ectoderms respectively, and depletion of both in the neural ectoderm. The effect of αEC depletion in the non-neural ectoderm, the formation of large cysts, has already been noted (Movie S1
). Depletion of αNC in both sides of the neural plate, by injection of both dorsal animal cells at the 8-cell stage, caused an effect identical to that caused by the depletion of N-cadherin, the failure of the neural plate to invaginate (). This was rescued by the subsequent injection of morpholino-resistant form of αNC mRNA into each morpholino-injected cell (). For the time-lapse movie showing the rescue of the morphogenetic movements, see Movie S2
. αEC depletion in the neural plate caused a similar, but less severe phenotype (), and these embryos did eventually close their neural tubes, but were significantly delayed. Depletion of both α-catenins simultaneously in the neural plate caused complete cell dissociation of the neural plate during neurulation ().
α-N-catenin is critical for the normal morphogenesis of the neural tube.
In light of these phenotypic effects, we compared the amount of F-actin after αEC and αNC depletion in the neural and non-neural ectoderms, using Alexa488-phalloidin to stain for F-actin. Depleted clones of cells were identified using the lineage tracer RLDX, co-injected with the morpholinos into single cells at the 8-cell stage. Pixel intensity measurements were used to quantitate F-actin levels. αEC depletion in the non-neural ectoderm caused a dramatic loss of F-actin throughout the cells (), even though E-cadherin junctional expression was unaltered. Depletion of either αEC or αNC in the neural plate caused a decrease in F-actin (). Depletion of both α-catenins in the neural plate caused complete loss of F-actin and cell dissociation.
Cortical F-actin staining in alpha catenin-depleted neural and non-neural ectodermal cells.
These data suggest that α-catenins have specificity in their binding to cadherin complexes containing different cadherins. To assay the degree to which each cadherin interacts with the other cadherins, and with the two α-catenins, we carried out co-immunoprecipation experiments. First, HA-tagged C-cadherin or E-cadherin was expressed in the non-neural ectoderm by injection at the 8-cell stage. Immunoprecipitation with anti-HA of lysates from the two batches of embryos showed that αEC was co-immunoprecipated by both C- and E- cadherin, indicating that αEC binds to both cadherins in the non-neural ectoderm (). This result was also found in the complementary experiment of expressing myc-tagged αEC into the non-neural ectoderm and immunoprecipitating with anti-myc. Although this antibody did not immunoprecipate efficiently, both cadherins were present in the precipitate at low levels (). Interestingly in this experiment, neither E- or C-cadherin interacted strongly, if at all, with the other, suggesting the possibility that they may be in different junctional complexes in the non-neural ectoderm (). These data also suggest that the continued presence of E-cadherin in cell junctions in αEC-depleted embryos is not due simply to the fact that it doesn’t bind αEC. To eliminate the possibility that the membrane-localized E-cadherin seen in αEC-depleted embryos was due to binding of residual αEC present, we immunoprecipated E-cadherin from lysates of embryos injected with αEC-MO (). No αEC was found in immunoprecipates, although β-catenin was present, indicating that E-cadherin is not expressed on the surface through binding to residual αEC in these experiments.
Co-Immunoprecipitations of cadherin-catenin complexes.
Next, we assayed for interactions in the neural ectoderm. Either Myc-tagged N-cadherin and HA-tagged αNC together, or HA-tagged αNC alone, were expressed in the neural ectoderm by injection of mRNAs into both dorsal animal cells at the 8-cell stage. N-cadherin and αNC each co-immunoprecipated the other, and αNC co-immunoprecipated C-cadherin (). However, N-cadherin did not co-immunoprecipate any endogenous αEC, even though it was abundant in the input lysate (). To eliminate the possibility that overexpression of tagged αNC in this experiment may have swamped out any possibility of interaction between N-cadherin and endogenous αEC, we repeated the experiment by expressing Myc-tagged N-cadherin only in the neural ectoderm. Once again, no endogenous αEC was co-immunoprecipated by anti-myc, although an abundant amount of β-catenin was co-immunprecipated (). These data show that N-cadherin interacts specifically with αNC, and not αEC, in the neural ectoderm. This explains the fact that N-cadherin on the cell surface is abrogated by depletion of αNC, but not αEC (). They also show that C-cadherin reacts with both alpha catenins, which may explain the partial reduction in C-cadherin levels on the cell surface when αNC is depleted (). In order to test whether N-cadherin can interact with αEC in the absence of αNC, we ectopically expressed Myc-tagged N-cadherin specifically in the non neural ectoderm and immunoprecipitated using an antibody against myc. In contrast to the neural ectoderm, only a sparse amount of beta catenin and virtually no αEC was co-immunoprecipitated by N-cadherin-Myc (). These data suggest that N-cadherin may be utilizing a different set of catenins and binding partners when expressed ectopically in the presumptive epidermis.
The most parsimonious explanation of the immunoprecipitation and depletion data in the non-neural ectoderm would be that there are different cadherin-containing junctions present, some containing C-cadherin but not E-cadherin (because they do not co-immunoprecipate), and that either E-cadherin-containing junctions do not bind αEC, or do not require binding (because αEC depletion did not cause a loss of E-cadherin from cell junctions). To test these possibilities, high resolution confocal imaging was carried out on cleared ventral ectoderm tissues co-stained for E-cadherin and αEC (), or C-cadherin and αEC (). The results supported this hypothesis. First, E-cadherin (green in )
was found to be distributed throughout the baso-lateral cell surfaces, but concentrated in a sub-apical belt of junctions. αEC in the same fields of view (red in ) was concentrated apically, and merged images showed that E-cadherin and αEC only partially overlapped. Around the apical surface of the cells, areas of red but no green could be seen, in addition to yellow areas where the staining overlapped. Baso-laterally, most of the E-cadherin-positive areas did not overlap with αEC staining as shown by the 3D side-view of a single cell surface (). In contrast, there was almost complete overlap of the C-cadherin and αEC staining, which was confined to the more apical regions of the cells (). Although these images are at the limits of resolution of the confocal microscope, there is a clear difference between the distributions of the cadherins themselves, and in their interactions with αEC. To view the 3D projections see movies S3
Three-dimensional projections of high resolution confocal images of cleared non-neural ectodermal cells.
These data suggest that the dependence on αEC binding for E-cadherin junctional expression may be context-dependent in the non-neural ectoderm. In some junctions the two proteins overlapped. In others they did not. Depletion of αEC showed that E-cadherin expression at the cell surface is not αEC–dependent, although we cannot rule out the possibility that some of it has gone after αEC depletion. Ectopic expression of E-cadherin in the blastula offered the opportunity to test this context dependence. We showed previously that E-cadherin can rescue the depletion of C-cadherin in the blastula 
. However, we did not test whether this was αEC-dependent. We therefore depleted αEC in cultured oocytes, and subsequently injected them with E-cadherin-HA mRNA before fertilization. Animal caps were removed at the late blastula stage, and viewed en face
under the confocal microscope. The results are shown in . E-cadherin did not rescue the loss of cell adhesion or the F-actin cytoskelton in the absence of maternal αEC in the blastocelic roof cells and these remained dissociated ( A, B, C
), although the E-cadherin protein was translated and was present at high levels in these embryos ( F
). However, when the superficial layer of cells in the animal caps was examined, in samples where all the deeper (blastocelic roof) cells had fallen off, E-cadherin was seen to be expressed at cell-cell contact sites and rescued the dissociation of αEC- depleted cells ( D,E
). Imaging the animal caps from the outside also showed the same result (data not shown). These results suggest that E-cadherin expression and function in cell adhesion is context-dependent. In the deep cells of the animal cap, E-cadherin required αEC, whereas in the superficial cells it did not.
E-cadherin requires α-E-catenin in the non-polarized blastocelic roof cells, but not in the polarized superficial animal cap cells for junctional localization and cell adhesion.