Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain

Cell, Vol. 69, 225-236,

April 17, 1992, Copyright

0 1992 by Cell Press

Regulation of Embryonic Cell Adhesion by the Cadherin Cytoplasmic Domain Chris Kintner Molecular Neurobiology Laboratory Salk Institute for Biological Studies P.O. Box 85800 San Diego, California 92188

Summary Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of ceil adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytopiasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytopiasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types. Introduction The cadherins are cell surface glycoproteins that mediate calcium-dependent cell adhesion (reviewed in Kemier et al., 1989; Takeichi, 1991). In differentiated tissues, cadherins can be found at sites of focal ceil contact where they are localized to intermediate, or adherens, junctions (Boller et al., 1985; Volk and Geiger, 1988). The molecular cloning of cadherins from different tissues and species, including E-cadherin (also known as uvomorulin), L-CAM, N-cadherin (also known as A-CAM), and P-cadherin, has demonstrated that these molecules span the plasma membrane once and share extensive sequence similarity in regions on both sides of the membrane. Conserved regions in the cadherin extracellular domain bind homotypically and specifically, thereby linking together adjacent cells expressing the same cadherin type. Conserved regions in the cadherin intracellular domain apparently bind to cytoplasmic components, thereby linking the cadherins to the cytoskeletal network. The intracellular domain of the cadherins has been analyzed primarily by introducing E-cadherin into nonadherent fibroblasts by DNA transfection. Fibroblasts expressing an intact E-cadherin show dramatic increases in the rate of cell aggregation (Mege et al., 1988; Nagafuchi et al., 1987; Ozawa et al., 1989). In addition, these cells are more epithelial-like; they form focal contact sites where

cadherin protein is concentrated, partition the (Na+,K+)ATPase to lateral surfaces, and have an increased frequency of dye coupling (Matsuzaki et al., 1990; McNeil1 et al., 1990). None of thesechangesoccurs in fibroblasts that express a form of E-cadherin with 70 aa deleted from the carboxyl terminus, even though this deleted form is still expressed on the cell surface (Nagafuchi and Takeichi, 1988; Ozawa et al., 1989). This mutant protein also fails to interact with three intracellular polypeptides called a-, p-, and y-catenin, which are known to bind intact E-cadherin (McCreaand Gumbiner, 1991; Nagafuchi and Takeichi, 1989; Ozawa et al., 1989,199O). Recent studies show that a-catenin shares sequence similarity with vinculin (Herrenknecht et al., 1991; Nagafuchi et al., 1991) and that f3-catenin is very similar to the product of the Drosophila segment polarity gene called armadillo and to a component of desmosomes called plakoglobin (McCrea et al., 1991). Thus, cadherin-mediated adhesion appears to require interactions with cytoplasmic components whose structural features may be shared by proteins that comprise multiple types of cell-cell and cell-matrix contacts. Cytoplasmic interactions underlying cadherin-mediated adhesion could be the basis for regulating cadherin function, particularly during embryogenesis, when the expression of different cadherin types has been proposed to direct the morphogenesis of different epithelial tissues (Edeiman, 1988; Takeichi, 1991). In Xenopusembryos, for instance, a maternal cadherin related to mouse E- and P-cadherin is expressed in the unspecialized occluding epithelium that forms on the surface of the embryo during early cleavage stages (Angres et al., 1991; Choi et al., 1990; Ginsberg et al., 1991). During gastruiation, the portion of this epithelium covering the animal pole, called ectoderm, diverges morphologically along two very different pathways of epithelial differentiation. The ectoderm either gives rise to a specialized epitheiium-comprising epidermis, in which case it expresses E-cadherin (Choi and Gumbiner, 1989; Levi et al., 1991). Or alternatively, the ectoderm gives rise to the neuroepithelium of the neural tube, in which case it expresses N-cadherin (Detrick et al., 1990; Hatta and Takeichi, 1986). Studies in which N-cadherin was ectopically expressed in ectoderm by RNA injection support the notion that the morphogenesis of these ectodermal derivatives is caused in part by the expression of different cadherin types. Injecting N-cadherin RNA into the animal pole of fertilized eggs results in increased ceil adhesion in the ectodermal cell layer, as measured by the formation of boundaries over which cell mixing does not occur (Detrick et al., 1990; Fujimora et al., 1990). Moreover, in embryos injected with large amounts of N-cadherin RNA, the ectoderm prematurely loses its occluding epithelial morphology at the beginning of gastrulation (Detrick et al., 1990; see Figure 1). Thus, the expression of N-cadherin could explain in part the morphological changes that occur in ectoderm during early neural development, including the formation of a boundary between

Cell 226

Figure

1. Histology

of Embryos

Expressing

Various

Forms

of N-Cadherin

Embryos were injected with RNA encoding various forms of N-cadherin and then processed for histology by embedding in plastic and staining with methylene blue. (A) Shown is a section through a control embryo at late gastrulation to illustrate the normal morphology of the outer occluding epithelium in the ectodermal cell layer. The outer surface is oriented toward the top of the photograph. (6) Shown is a section from an embryo at a similar age as in (A) but injected with 1 .O ng of N-cadherin RNA (Detrick et al., 1990). The outer occluding epithelium is oriented as in the photograph shown in (A). Note that the morphology of the ectodermal cells in both layers is altered relative to the control and that contacts between cells in the outer layer are perturbed. (C) Shown is a section from an embryo injected with N-cadAE RNA and processed for histology at the same stage as the embryo in (A). The outer occluding epithelium is oriented toward the right side of the photograph. Note that the integrity of the entire ectodermal layer is completely lost in these embryos.

Cadherin 227

Regulation

in Embryos

neural and nonneural ectoderm, and the transformation of ectoderm from an occluding epithelium to the neuroepithelium of the neural tube. The ability of N-cadherin to alter the morphology of ectoderm both in ectopic expression experiments and in normal development may reflect its ability to inhibit the function of other cadherins expressed in ectoderm. One potential inhibitory mechanism is based on the fact that the cytoplasmic domains of different cadherin types share a high degree of sequence similarity, suggesting that all cadherins interact with cytoplasmic components such as the catenins. Thus, cadherins coexpressed in the same cell could compete for binding to cytoplasmic components needed for function. One test of this model is to determine whether a nonfunctional cadherin, still capable of interacting with cytoplasmic components, acts as a dominantnegative mutant of cell adhesion (Herskowitz, 1987). In other words, a cadherin lacking an extracellular-binding domain but containing a cytoplasmic domain should inhibit cadherin-mediated adhesion by competing for the binding of intracellular components. Here I report the results obtained with a mutant form of Xenopus N-cadherin in which nearly all the extracellular amino acids have been deleted. Introduction of mRNA encoding this mutant N-cadherin into the ectoderm of Xenopus embryos dramatically inhibits cell adhesion. Inhibition of adhesion occurs in the ectodermal cell layer at a time when the embryo expresses other cadherins besides N-cadherin, indicating that inhibition occurs via cytoplasmic sequences shared by these different cadherins. These results highlight the importance of the cytoplasmic interactions in cadherin-mediated cell adhesion and suggest that these interactions may regulate cadherin function in cases in which cells express more that one cadherin type.

Results N-Cadherin with an Extracellular Deletion Blocks Cell Adhesion The effects of ectopic N-cadherin expression on ectoderm were examined by injecting synthetic N-cadherin RNA into the animal pole of fertilized eggs. In embryos injected with a relatively large amount of N-cadherin RNA (1.0 ng), a lesion appeared in the ectoderm during gastrulation (Detrick et al., 1990). Plastic sections through these embryos indicated that the morphology of ectoderm in regions of high ectopic N-cadherin expression differed from that in normal embryos (Figures 1A and 1 B). Notably, the cells in the outer layer of ectoderm lacked the close apposition normally found in an occluding epithelium and appeared more spindle shaped in morphology(Figure 1B). One interpretation of this result is that N-cadherin alters the morphology of ectoderm by inhibiting the function of other

(D) Shown is a section through an embryo at the beginning of gastrulation epithelium is oriented toward the left side of the photograph. Note that cells in cavity. (E) Shown is a transverse section of an embryo at a similar stage as in (A) that morphology of the outer occluding epithelium changes dramatically at

cadherins expressed in ectoderm. To determine whether inhibition occurs via the cytoplasmic interactions required for cadherin function, a deleted form of N-cadherin was constructed, called N-cadAE, in which most of the sequences encoding the extracellular portion were removed from an N-cadherin cDNA (see Experimental Procedures; Figure 2). The protein encoded by N-cadAE lacks almost all of the “pre” region and the extracellular amino acids in the mature polypeptide, but contains a signal peptide, transmembrane domain, and the complete intracellular domain. The effects of N-cadAE on cell adhesion were tested by injecting N-cadAE RNA into one animal pole blastomere of embryos at the 4-8 cell stage, thereby introducing RNA into approximately one-quarter of the ectodermal cell layer. The resulting embryos appeared to develop normally until mid gastrulation. At this point, the embryos stop gastrulating, as monitored bytheclosureof the blastopore, and within 1 hr the integrity of the ectodermal cell layer was completely lost. Plastic sections through the injected embryos showed that the ectodermal cells in the region of the lesion had lost all normal sites of cell contact (Figure 1C). All embryos (Table 1) injected with 1 .O ng of N-cadhE RNA showed this extreme phenotype. In addition, mosaic embryos were generated by coinjecting N-cadAE RNA along with 8-galactosidase RNA, to mark the injected cells, into one animal blastomere of an embryo at the 16 cell stage. The ectodermal cells derived from the injected blastomere were altered in morphology, while neighboring cells were normal in appearance, indicating that the N-cadAE phenotype is cell autonomous (Figure 1 E). The effects of N-cadAE on cell adhesion were analyzed by examining the ability of ectodermal cells injected with N-cadAE RNA to aggregate in vitro(see Experimental Procedures). When animal caps from control embryos were placed intocalcium-free medium, they dissociated into single cells that rapidly reaggregated into tissue masses once calcium was added back to the medium. In contrast, when animal caps were isolated from N-cadAE RNA-injected embryos, they dissociated completely into single cells even in the presence of calcium. No sign of cell aggregation was evident among ectodermal cells injected with N-cadAE, which instead appeared the same when cultured in calcium as control cells appeared when cultured without calcium. After 24 hr in culture, however, the ectodermal cells injected with N-cadAE showed signs of reaggregating, presumably because the injected N-cadAE RNA and protein eventually turned over. Thus, these results indicate that N-cadhE is as effective as calcium depletion at disrupting embryonic cell adhesion in vitro. Characteristics of the N-cadAE Phenotype Several features of the N-cadAE phenotype were apparent from examining embryos that had been injected with differ-

that was injected with 1 .O ng of N-cadAE RNA. The outer occluding the inner cell layer have already begun to dissociate into the blastocoel that was injected once with NcadAE the boundary marked by the arrow.

RNA at the 16 cell stage.

Note

Cell 226

EXTRACELLULAR SIG,

PRE

INTRACELLULAR

Figure 2. Schematic Diagram of the nant-Negative Mutants of N-Cadherin

EC1 -3

===-v

’

N-CADAE

I161 INTRACELLULAR

PEP.,

N-CADAC Terminal Deletions

1161 F&m+’

-

I T127 I T87

Ema”

A-

IT70 I T36

P

T27

I

T6 9

79 I

Internal Deletions

lTAQ-79

A

TAQ-144

ent concentrations of N-cadAE RNA, or with P-galactosidase RNA as a control. First, in embryos injected with 0.5 ng or more of N-cadAE RNA, the onset of the N-cadAE phenotype appeared to be fixed at a particular developmental stage. The outer ectodermal cell layer, for example, never dissociated before mid gastrulation. Since RNA is likely to be translated within a few hours after injection, this lag period indicates that N-cadAE is more effective at inhibiting adhesion in late stage embryos. In addition, the N-cadAE phenotype always occurred in the inner ectodermal cell layer before

Table 1. Inhibition N-Cadherin

of Cell Adhesion

with Different

Mutant

RNA Injected”

No. of Experiment9

Phenotype

N-cadAE N-cadAC T127 T07 T70 T36 T27 T6 TAQ-79 TAG-i 44

6 4 2 2 2 2 3 3 3 3

+ + + + + + +

Forms

of

(Stager

(stage (stage (stage (stage (stage (stage (stage

11) 11) 11) 12) 12) 12) 12)

+ (stage

12)

’ One nanogram of each RNA was injected into the animal pole of fertilized eggs as described in Experimental Procedures. b At least 40 embryos injected with each RNA were analyzed in each independent experiment. ’ Injected embryos were scored as positive (+) when obvious signs of cell dissociation were observed in the outer ectodermal cell layer. Without exception, all embryos injected in agiven RNA gave the same phenotype. A small variation in the onset of cell dissociation was consistently observed with the different RNAs, and this is noted as the earliest stage in which the phenotype was observed for each case.

Domi-

The mutant Ntadherin called N-cadAE (extracellular) was generated by removing the sequences between two BamHl sites in an N-cadherin cDNA (Detrick et al., 1990). This in-frame deletion removes most of the sequences encoding the extracellular domain downstream of the signal peptide and just upstream of the transmembrane domain (top diagram). The second diagram from the top also shows the location of the peptide sequence used to generate the PEP.1 antibody (Choi et al., 1990) and the portion of the intracellular domain that contains the catenin-binding region (Ozawa et al., 1990). The extracellular cysteines in N-cadAE (Cys) were converted to serines to yield a second mutant of N-cadherin called N-cadAC (third diagram from the top). Finally, increasingly larger portions of the cytoplasmic domain were deleted from N-cadAC. Each deletion is designated by the number of amino acids remaining in the cytoplasmic tail, which is 161 aa long in the intact N-cadherin polypeptide. Internal deletions are designated by the stretch of amino acids in the cytoplasmic domain that were removed.

it occurred in the outer occluding epithelium, suggesting that the adhesion of different cell types is affected differently by N-cadAE. This difference between the two layers was evident by examining plastic sections of embryos at different developmental stages. The cells in the inner ectodermal layer showed signs of losing cell contacts and passing freely into the blastocoel before the start of gastrulation (Figure 1 D), while cells in the outer occulding layer remained normal in appearance until mid gastrulation. Second, the severity of the N-cadAE phenotype decreased as embryos were injected with less N-cadAE RNA. The first change in the severity of the N-cadAE phenotype was a delay, in that embryos would complete gastrulation before the outer ectodermal cells began to dissociate. In embryos injected with even less N-cadhE RNA (10 pg), the outer ectodermal cell layer remained intact but the inner cell layer showed signs of dissociation (Figures 3A and 38). If left to develop, most of these embryos appeared externally to be normal. However, tissue sections of these embryos revealed avariety of histological defects, all of which occurred in the ectodermal derivatives formed from areas injected with RNA. An example is shown in Figure 3C of the morphological abnormalities observed in embryos with a weak N-cadAE phenotype. In this example, the neural tube on one side of this tadpole embryo was injected with N-cadAE RNA as marked by the coinjection of j3-galactosidase RNA. While neural tissue on the control side of this embryo has formed columnar epithelium, the cells on the injected side have lost the columnar morphology and show a chaotic organization. Thus the alterations observed in embryos injected with limiting amounts of N-cadAE RNA were also consistent with defects in cell adhesion and morphogenesis. Third, the N-cadAE phenotype did not correlate with the

Eitherin

Regulation

Figure

3. Micrographs

in Embryos

of Sections

Prepared

from Embryos

Injected

with Limiting

Amounts

of NcadAE

RNA

Embryos were injected with varying amounts of N-cadAE along with a small amount of b-galactosidase RNA (see Experimental Procedures). Embryos were fixed at different stages, stained with X-Gal, sectioned in paraplast, and counterstained with hematoxylin eosin. (A) A transverse section of a control embryo at stage 14 showing a region near the neural plate. (6) A transverse section of an embryo from the same region shown in (A) but injected with approximately 10 pg of RNA and processed at stage 14 for histology. Note that the inner cells show signs of dissociation, while the outer cells have remained relatively intact. X-Gal staining (blue) shows that both layers have received the injected RNA. (C)Shown is an example of a mild NtadAE phenotype in an embryo processed at early tadpole stages. This section gives a cross-sectional view through anterior regions of the embryo at the level of the forming eye cup. Note that the cytoarchitecture of the nervous system in the area of RNA injection as indicated by X-Gal staining is disorganized relative to the control side.

normal expression pattern of N-cadherin. Areas of the embryos where N-cadherin expression does not occur, such as the epidermis, dissociated in response to NcadAE. Moreover, the N-cadAE phenotype appeared in embryos before gastrulation (Figure 3B), when Ncadherin RNA is not yet

detectably expressed (Detrick et al., 1999). These observations suggest that NcadAE affects cell adhesion molecules other than N-cadherin. Together these observations indicate that N-cadAE is a potent inhibitor of cell adhesion. Since the N-cadAE phe-

Cell 230

Control r,

+l

N-cadAE RNA N-cadAE + ’ AE/T70 RNA I1w ‘mo +

t_

+’

r -

+’

c

Maternal Cadherin

+

Homodimer

+

Heterodimer

*

N-cadAE

116-

12

3

Figure 4. Western of N-Cadherin

Analysis

45

67

of Embryos

Injected

with Mutant

Forms

Extracts were prepared under reducing (+) and nonreducing (-) conditions at mid gastrulation from embryos injected with different amounts of RNA encoding different forms of N-cadherin. Extracts from approximately 5 embryos were electrophoresed in a 10% polyacrylamide gel containing SDS, electrophoretically transferred to a nylon membrane, and reacted with the anti-PEP.1 antibody. Sound antibody was detected using chemoluminescence and autoradiography. The left side of the figure shows the position of molecular weight markers. The right side designates the position of various cadherin species. Extracts from control embryos contain one band corresponding to the maternal cadherin (lanes 1 and 2). Extracts prepared under reducing conditions from embryos injected with N-cadAE RNA contain one additional band (lanes 3 and 5). The levels of the N-cadAE protein are approximately the same as the level of the maternal cadherin when embryos are injected with IO pg of N-cadAE RNA (lane 5) and are approximately 50-fold higher when injected with 1 ng of N-cadAE RNA (lane 3). Extracts prepared from embryos injected with N-cadAE RNA under nonreducing conditions contain additional bands (lane 4). The most prominent of these bands is a putative homodimer of the N-cadAE protein. When embryos are injected with RNAs encoding both a shortened N-cadAE (AgE/T70) and the intact N-cadhE (in a ratio of lO:l, respectively), a heterodimer smaller in size than the homodimer is observed (lane 6). The intensity of the bands corresponding to the heterodimer and the homodimer varies appropriately in extracts of embryos injected with different ratios of RNAs encoding the intact and shortened version of the N-cadAE (data not shown). The identities of the minor bands observed in lanes 4 and 6 are unknown, but they could represent breakdown products of N-cadAE.

notype can occur when N-cadherin is not yet expressed in embryos, N-cadAE must block the action of other adhesion molecules such as the maternal cadherin. Expression of N-cadAE in Embryos The expression of N-cadAE RNA in early embryos was examined by Western analysis using a rabbit antibody, anti-PEP.l, which recognizes a peptide sequence conserved in the intracellular domain of most cadherins (Choi et al., 1990). PEP.1 detects two prominent protein species in extracts prepared under reducing conditions from embryos injected with N-cadAE RNA (Figure 4, lanes 3 and 5). The larger of these two proteins (120 kd) is also present in extracts of control embryos (Figure 4, lane 2) and is likely to correspond to the maternal Xenopus cadherin first described by Gumbiner and colleagues using the same rabbit antibody (Choi et al., 1990). The smaller protein

detected in the N-cadAE extract is not present in control extracts and has the molecular weight predicted for the protein product of the N-cadAE RNA. The levels of the N-cadAE product appeared to about 50-fold higher than those of the maternal cadherin in extracts of embryos that developed the full N-cadAE phenotype following injection with 1 .Ong of N-cadhE RNA (Figure 4, lane 3). This result suggests that the N-cadAE product needs to be in excess in order to inhibit embryonic adhesion completely. In contrast, similar levels of the N-cadAE product and the maternal cadherin were detected in embryos following injection with enough N-cadAE RNA(10 pg) to generate morphological abnormalities such as those shown in Figure 3C (Figure 4, lane 5). This result suggests that N-cadAE can regulate embryonic cell adhesion when expressed at levels about the same as those observed for the endogenous cadherins. Extracts prepared from N-cadAE-injected embryos under nonreducing conditions gave a more complicated protein pattern upon Western analysis. Several new bands were present, all of which migrate between the N-cadAE peptide and the maternal Xenopus cadherin. The most abundant of these new bands indicates that N-cadAE disulfide bonds to itself, because it has an apparent molecular weight predicted for a dimer of the N-cadAE protein (Figure 4, lane 4). This possibility was tested by coinjecting embryos with RNA encoding N-cadhE and RNA encoding a form of N-cadAE that lacks sequences at the carboxyl terminus. This terminal deletion of N-cadAE produces a smaller protein and removes the region of N-cadAE recognized by the anti-PEP.1 antibody. Extracts from these embryos prepared under nonreducing conditions contained a predominant band at a molecular weight predicted for a heterodimer between the intact and the shortened N-cadAE protein (Figure 4, lane 6), demonstrating that the N-cadhE protein is dimerizing via disulfide bonds. Sequences in N-cadAgE That Inhibit Cell Adhesion The regions of N-cadAE that inhibit cell adhesion were localized in order to determine how N-cadAE acts as a dominant-negative mutant. Since NcadAE can inhibit adhesion when the maternal cadherin is the only known cell adhesion molecule expressed in embryos, the regions in N-cadAE that convey the dominant-negative phenotype are likely to share significant sequence similarity with this cadherin. Most of the sequences shared by these two polypeptides are found within the cytoplasmic domain (Detrick et al., 1990; Ginsberg et al., 1991). In contrast, the region of the exodomain still present in N-cadAE corresponds to the portion least conserved among mature cadherin polypeptides, with the exception of 4 cysteine residues whose position near the transmembrane domain is invariant in all known cadherins. The extracellular domain of N-cadhE still contains 3 of these cysteines, while the 4th, which lies closer to the amino terminus, was removed in the process of generating the extracellular deletion (see Figure 2). These cysteines are the most likely source of the disulfide bonds that form between N-cadhE as described above, and they could potentially disulfide bond with other cell surface molecules and thereby interfere with their ex-

;$herin

Regulation in Embryos

pression or function. To test this possibility, a form of N-cadAE was generated, called N-cadAC, in which the sequence encoding these cysteines was changed to codons encoding serines. Upon Western analysis with the PEP.1 antibody, the N-cadAC product appeared to be the same size as that of N-cadAE, but did not form any detectable oligomeric proteins when analyzed under nonreducing conditions (data not shown). Thus, removing the extracellular cysteines in N-cadAE prevented the formation of intermolecular disulfide bonds. More importantly, the embryos injected with N-cadAC displayed the same phenotype observed originally in the embryos injected with N-cadAE (Table 1). Thus, these results rule out the possibility that the formation of disulfide bonds is necessary for N-cadAE to generate a dominant-negative phenotype. Intracellular Sequences That Inhibit Cell Adhesion The extracellular domain in N-cadAC lacks any significant sequence similarity with other cadherins, indicating that intracellular sequences in N-cadAC are responsible for the dominant-negative phenotype. The region in the cytoplasmic domain of N-cadAC that inhibits cell adhesion was localized by first generating a set of terminal deletions. These deletions remove progressively more sequences from the carboxyl terminus, proceeding toward the transmembrane domain (see Figure 2). One expectation was that the sequences in the terminal 70 aaof the cytoplasmic domain should produce a dominant-negative phenotype because these sequences have been previously shown to be necessary for the function of E-cadherin and for binding to intracellular components, the catenins (Nagafuchi and Takeichi, 1989; Ozawa et al., 1990). Surprisingly, the terminal deletion series did not give the expected result. Forms of N-cadAC lacking the catenin-binding region were still capable of dissociating ectodermal cells when expressed in early embryos. For example, T70 lacks the carboxyl half of the cytoplasmic domain in N-cadAC (Figure 2) and yet completely dissociates ectodermal cells at mid gastrulation when expressed in embryos (Table 1). These results show that sequences in the amino half of the cadherin cytoplasmic domain inhibit cell adhesion, indicating that a portion of the cytoplasmic domain outside the catenin-binding region mediates protein interactions required for cadherin function. To localize this region more precisely, further deletions were generated that removed even more of the cytoplasmic domain from N-cadAC (Figure 2). From this series, only one form, called T6, did not inhibit cell adhesion (Table 1). This protein lacks all but 6 aa of the cadherin intracellular domain. At the same time, T27 inhibited cell adhesion, indicating that the region of the cytoplasmic domain between aa 6 and aa 27 is capable of generating a dominant-negative phenotype. Comparing the sequence of Xenopus N-cadherin, E-IP-cadherin, and E-cadherin in this region shows that 40% of the amino acids are identical. If conservative amino acid changes are taken into account, then 66% of the amino acids are similar in this region. Finally, in a contiguous stretch of 12 aa in the center of this region, 10 aa are conserved in the sequence of these three Xenopus cadherins. Thus, this conserved region of the cytoplasmic domain presumably

contains a site that interacts with cytoplasmic essary for cadherin function.

proteins nec-

The Catenin-Binding Region Inhibits Cell Adhesion The results described above indicate that the cytoplasmic sequence between aa 6 and aa 27 of N-cadAC inhibits cell adhesion when expressed in embryos. To determine whether other regions of the cytoplasmic domain can also inhibit cell adhesion, internal deletions were generated in the cytoplasmic domain of N-cadAC. As the conserved region between aa 6 and aa 27 clearly inhibits cell adhesion on its own, these deletions also included this region. The first of the internal deletions, called TA9-79, removed the portion of the cytoplasmic domain between aa 9 and aa 79, leaving the carboxy-terminal 82 aa, where catenin binding is thought to occur. Expression of this form in embryos produced a phenotype very similar to that of N-cadAC (Table 1). The ectoderm in the injected embryo showed signs of dissociation at mid gastrulation, followed by a complete dissociation of ectodermal cells by late gastrulation. This result shows that the terminal 82 aa in the cytoplasmic domain of N-cadherin are capable of producing a dominant-negative phenotype. Finally, a second internal deletion was generated that removed more of the intracellular domain, resulting in a form called TA9-144, which contained the carboxy-terminal 17 aa. Expression of this form in embryos had no apparent effect on cell adhesion. N-cadAC inhibits the Binding of acatenin to E-Cadherin The results described above indicate that N-cadAC can inhibit embryonic cell adhesion through sequences in which catenin binding is thought to occur. Kemler and colleagues have shown that forms of uvomorulin (mouse E-cadherin) with similar extracellular deletions as N-cadAC are still capable of interacting with the catenins, as measured by immunoprecipitation (Ozawa et al., 1990). Together these results suggest that N-cadAC inhibits the function of other cadherins by binding up the supply of catenins in embryos. To demonstrate this competition more directly, the binding of a-catenin to Xenopus E-cadherin was measured in the presence and absence of N-cadAC. Because E-cadherin is not normally expressed in embryos at significant levels when the N-cadAC phenotypes arise (see Figure 4) asynthetic E-cadherin RNA was used to generate embryos that express E-cadherin during early cleavage stages. Xenopus E-cadherin RNA was injected into fertilized eggs along with a lo-fold excess of N-cadAC RNA, or of T6 RNA as a control. Detergent extracts were prepared from injected embryos at mid gastrulation from which E-cadherin and associated proteins were isolated by immunoprecipitation using an anti-Xenopus E-cadherin monoclonal antibody coupled to Sepharose beads (McCrea and Gumbiner, 1991). The a-catenin recovered in these immunoprecipitates by association with E-cadherin was detected on Western analysis using a rabbit antibody that recognizes Xenopus a-catenin (Herrenknecht et al., 1991). The results from this analysis show that a-catenin (102 kd) can be isolated in association with E-cadherin from

Cell 232

A

B

200 -

zoo-

E-cadherin 11697 -

a-catenin

66 -

11697 66 -

12

Figure

5. Binding

12

of a-Catenin

tion of cadherin function was examined here by expressing a dominant-negative form of N-cadherin, called N-cadAE, in the ectodermal cell layer of frog embryos. Complete dissociation of the ectodermal cell layer occurred when the N-cadAE product was expressed in embryos at levels lo- to 50-fold higher than the levels of the endogenous cadherins. Moreover, effects on the morphogenesis of the ectodermal cell layer and its derivatives (Figure 3C) were observed when N-cadAE was expressed in embryos at approximately the same level as the endogenous cadherins. These results suggest that different cadherins can compete for function via their cytoplasmic domain and that this regulation of cadherin function may occur during morphogenesis when emerging tissues transiently coexpress several different cadherin types.

to E-Cadherin

E-cadherin RNA (100 pg) was injected into embryos along with either N-cadAC RNA (1 ng) or 16 RNA (1 ng). Ecadherin and associated proteins were immunoprecipitated from extracts prepared from RNAinjected embryos at mid gastrulation, separated by electrophoresis in 15% acrylamide gels containing SDS, and electrophoretically transferred to nylon membranes. (A) Membrane probed with an a-catenin antibody (Herrenknecht et al., 1991) using chemoluminescence and autoradiography. The autoradiogram presented here was intentionally overexposed in order to show all a-catenin reactivity above background. The high background in this Western is likely to represent the fact that the immunoprecipitates were washed under mild conditions, and the a-catenin antibody, like most peptide antibodies, gives high background staining when used across species. The left side of the figure shows the position of molecular weight markers while the right side designates the position of a-catenin (102 kd). Note that a-catenin is detected in immunoprecipitates of E-cadherin from extracts of embryos expressing the T6 but not the N-cadAC proteins. (B) Membrane probed with aXenopus E-cadherin monoclonal antibody (McCrea and Gumbiner, 1991) using chemoluminescence and autoradiography. The left side of the figure shows the position of molecular weight markers while the right side designates the position of the E-cadherin (130 kd). Note that immunoprecipitates contain similar levels of E-cadherin.

embryos that also express T6 by RNA injection (Figure 5A, lane 1). In contrast, a-catenin was not isolated in detectable amounts in association with E-cadherin from embryos that also express N-cadAC (Figure 5A, lane 2). Equal amounts of E-cadherin were immunoprecipitated from both extracts, indicating that the coinjection of N-cadAC RNA is not simply interfering with the expression of Ecadherin RNA (Figure 5B, lanes 1 and 2). Thus, the simplest interpretation of these results is that N-cadAC inhibits the association of a-catenin to other cadherins.

Intracellular interactions between the cadherin cytoplasmic tail and intracellular proteins have been proposed to link the cadherins to the cytoskeletal network as well as regulate cadherin function (Nagafuchi and Takeichi, 1989; Ozawa et al., 1990). The regulation of cadherin function is likely to be particularly important during embryogenesis, when tissues undergo dramatic changes in morphology due in part to changes in the expression of different cadherin types. The role of cytoplasmic domain in the regula-

Expression of the N-cadAE Phenotype Removing most of the extracellular domain of N-cadherin results in a molecule, N-cadAE, that produces an obvious phenotype when expressed in ectoderm of early embryos. The first signs of the N-cadAE phenotype occur at very late blastula stages when cells in the inner ectodermal cell layer dissociate into the blastocoel. The N-cadAE phenotype then appears in the outer occluding epithelium, which dissociates at mid gastrulation, or approximately 10-l 1 hr after RNA injection. The lag period between the injection of the RNA and the appearance of the N-cadAE phenotype indicates that the N-cadAE protein is not simply killing the embryo, since nonspecific toxic effects from RNA injection are usually apparent before late cleavage stages. However, since RNA is likely to be expressed as protein within a few hours after injection into fertilized eggs (Kintner, 1988) the lag period raises the question of why occluding epithelium formed at early stages is resistant to N-cadAE while that formed at later stages is not. One explanation is based on the observation that the N-cadAE phenotype appears at a point when the outer occluding epithelium switches from expressing the maternal cadherin to Ecadherin and N-cadherin (Choi and Gumbiner, 1989; Detrick et al., 1990; Levi et al., 1991). Thus, the lag period may reflect the fact that N-cadAE is more effective at inhibiting the function of these other cadherins than it is at inhibiting the maternal cadherin. An alternative explanation is that the lag period reflects a difference in the way occluding epithelium is generated at early and late stages of development. At early stages, the cells in the outer occluding epithelium are generated by cell division using maternal components stored in the egg. The maternal cadherin, for example, is synthesized during oocyte maturation and stored near the egg cortex where it is presumably poised for insertion into the outer occluding epithelium as it forms during rapid cleavage stages (Angres et al., 1991; Choi et al., 1990; Ginsberg et al., 1991). Thus, these stored components may preclude N-cadAE protein that is newly synthesized during early cleavage stages from acting until gastrulation stages. This possibility can be tested by introducing N-cadAE RNA in oocytes so that the N-cadAE can act as a maternal protein. During gastrulation, the outer occluding epithelium is also generated by an expansion that does not rely on cell division, as in early

zitherin

Regulation

in Embryos

stages, but rather on the intercalation of cells from the adjacent inner layer of ectoderm (Keller, 1980). By blocking adhesion of the cells in the inner layer of ectoderm before gastrulation begins, N-cadhE may alsoeffectively prevent these cells from contributing to the outer occluding epithelium at the stage when their contribution is essential for maintaining the surface epithelium. Dimerization of N-cadAE The N-cadherin mutant N-cadAE contains 3 of the 4 CySteine residues that are present near the transmembrane domain of all known cadherins. These cysteines apparently allow the N-cadAE protein to disulfide bond readily with itself, but several observations indicate that the formation of these bonds is due to the fact that N-cadAE contains 3 rather than the normal 4 cysteines. First, the Western analysis shown in Figure 4 indicates that disulfide bonds do not form readily between N-cadhE and the maternal cadherin. Second, the predicted dimers were also not observed in embryos that had been also coinjected with N-cadhE RNA and RNA encoding either Xenopus N-cadherin, or Ecadherin (unpublished data). Finally, deleting these extracellular cysteines from N-cadAE prevented the formation of intermolecular disulfide bonds but had no obvious effect on the dominant-negative phenotype. Thus, N-cadAE, with its 3 rather than 4 cysteines, can be considered a neomorphic mutation that is probably not relevant to the normal function of intact cadherin proteins. Nonetheless, the fact that N-cadAE efficiently disulfide bonds to itself suggests that there is a mechanism for bringing together cadherin proteins so that lateral interactions can occur. Such lateral interactions have been proposed to cluster the cadherin into adhesion sites and thereby increase the avidity of cadherin binding on the cell surface. Cytoplasmic Interactions Required for Cell Adhesion The N-cadAE phenotype does not correlate with N-cadherin expression, indicating that N-cadAE has a dominant-negative effect on adhesion molecules in early embryos other than N-cadherin. This observation simplifies the analysis of the dominant-negative phenotype because the target of the NcadAE mutation must be proteins that share significant sequence similarity with N-cadAE. The molecules most similar to N-cadherin in early embryos are the maternal cadherin and E-cadherin (Angres et al., 1991; Choi et al., 1990; Choi and Gumbiner, 1989; Ginsberg et al., 1991; Levi et al., 1991). Most of sequences that are shared between N-cadAE and these other cadherins are contained within the cytoplasmic domain, indicating that intracellular sequences are the targets of the dominantnegative phenotype (Detrick et al., 1990; Ginsberg et al., 1991). Several observations support this conclusion. First, the extracellular cysteines in N-cadAE could be changed to serines with no apparent effect on the dominantnegative phenotype. This change removed the last sequence similarity that can be found in the extracellular domain of N-cadAE and other cadherins. Second, the dominant-negative phenotype was lost when the intracellular domain of N-cadAC was deleted (TS in Figure 2).

Previous analysis of fibroblasts transfected with deleted forms of Ecadherin concluded that cadherin-mediated adhesion requires an intact cytoplasmic tail (Nagafuchi and Takeichi, 1989; Ozawa et al., 1990). The simplest interpretation of this result is that the cytoplasmic domain is required for essential interactions with cytoplasmic components. An alternative interpretation, however, is that the intracellular deletion causes a conformational change that propagates across the plasma membrane to the extracellular domain, resulting in a decrease in the affinity of extracellular cadherin binding. The results with the dominant-negative mutants favor the idea that the cadherin cytoplasmic tail is required for protein interactions rather than for correct protein conformation, because the functional activity of the intracellular domain can be separated from an active extracellular domain. Moreover, these results indicate that the cytoplasmic interactions proposed from studies with E-cadherin are also shared to some extent by other members of the cadherin family. Targets of the Dominant-Negative Phenotype The role of the maternal cadherin in early Xenopus embryos has been previously analyzed by blocking antibody studies. Early embryos dissociated in calcium-free medium and then reaggregated in the presence of blocking antibodies directed against the maternal cadherin formed loose cell aggregates (Angres et al., 1991). One interpretation of this result is that early embryonic cells can adhere by means other than the maternal cadherin. In contrast, the experiments reported here show that cell adhesion in ectoderm and its derivatives can be completely inhibited after gastrulation by expressing N-cadAC. The difference between these results might be explained by a difference in the efficacy of the two methods in blocking adhesion. Alternatively, the results presented here could be an indication that N-cadAC inhibits adhesion molecules other than the cadherins. Onegroupof proteins potentially inhibited by N-cadAC are the cadherin-like desmosomal proteins, desmoglein and the desmocollins (Collins et al., 1991; Goodwin et al., 1990; Koch et al., 1991, 1990; Mechanic et al., 1991; Nilles et al., 1991; Parker et al., 1991; Wheeler et al., 1991). In particular, the cytoplasmic domain of these proteins shows some similarity with the cadherin cytoplasmic domain, including within regions where catenin binding might occur. Moreover, the cadherin-like desmosomal proteins interact with a protein, called plakoglobin, which may be the same or very similar to j3-catenin (Franke et al., 1989; McCrea et al., 1991). Thus, N-cadAC could potentially act as a dominant-negative mutant for other types of specialized cell contacts, including desmosomes. This possibility can be examined in more detail when more is known about the expression of cadherin-like desmosomal proteins in Xenopus embryos and by analyzing the dominant-negative phenotype at an ultrastructural level. Deletion mapping indicates that at least two regions in the cytoplasmic domain of N-cadAC produce a dominantnegative phenotype. One of these regions unexpectedly maps in a portion of the cytoplasmic domain very close to the transmembrane domain. The sequence in this region

is conserved amongst the cytoplasmic domains of the cadherins but not the cadherin-like desmosomal proteins. Previous studies indicated that deleting this region of the intracellular domain from E-cadherin had no effect on adhesion in transfected fibroblasts, as measured by cell aggregation (Ozawa et al., 1990). The discrepancy between these two results may reflect the different assays used in these studies. Thus, the amino half of the E-cadherin cytoplasmic domain may not be required for cell aggregation in transfected fibroblasts, but it may be required in cells that form a stable occluding epithelium in vivo. The fact that this region of the intracellular domain is conserved among different cadherins also suggests that the function of these sequences may have been overlooked in transfected fibroblasts. Catenin Binding The second region that produces a dominant-negative phenotype is contained roughly within the portion of the cytoplasmic domain where catenin binding is thought to occur (Nagafuchi and Takeichi, 1989; Ozawa et al., 1989). Several lines of evidence support the notion that the dominant-negative phenotype can result from the competitive binding of catenins to this region of the cadherin cytoplasmic domain. First, previous studies have shown that catenins bind to the terminal 70 aa of E-cadherin transfected into fibroblasts (Ozawa et al., 1990). Second, catenin binding to this region still occurs even when the extracellular domain of E-cadherin is deleted, indicating that catenin binding does not depend on extracellular cadherin binding (Ozawa et al., 1990). Finally, the results in Figure 5 show that a-catenin binding to E-cadherin expressed in embryos by RNA injection can be inhibited by the coexpression of N-cadAC. In sum, the dominant-negative mutant of N-cadherin supports the view that interactions with cytoplasmic components are critical for cadherin function. The dominantnegative phenotype suggests that the cytoplasmic domain may be a site for cadherin regulation, such as in cases when cells express more than one cadherin type. Finally, the dominant-negative mutants may be relevant to oncogenesis, where any mechanism that inhibits cell adhesion could play a role in tumor invasion during metastasis (Vleminckx et al., 1991). Experimental

Procedures

Xenopus Embryos and RNA Injections Xenopus embryos were obtained from adult frogs (Nasco) by inducing egg laying with hormones and artificial fertilization as previously described (Detrick et al., 1990). Embryos were staged according to Nieuwkoop and Faber (1967). In most experiments, two animal blastomeres were injected in each embryo at the 2-4 cell stage, with 10-20 nl of water containing RNA at a concentration of 1 O-200 ng/pl (Kintner, 1988). Embryos (40-50) were injected with each RNA, of which 5%10% were usually lost to injection damage, while the remaining embryos were scored for a phenotype. To generate mosaic embryos, only one blastomere was injected in embryos at the 8-16 cell stage. To mark the injected regions, the test RNA was coinjected with a small amount (0.5 nglpl) of fi-galactosidase RNA (Detrick et al., 1990). For cell dissociation experiments, embryos were injected in all four animal quadrants at the 4 cell stage in order to distribute the injected RNA homogeneously in all ectodermal cells. In all experiments, embryos

were also injected with just P-galactosidase RNA to control for nonspecific effects of RNA injection. Cell dissociation experiments involved isolating animal caps from embryos at stages 8-10. Control animal caps were dissociated by incubation in medium lacking calcium and gently sucking the caps through a narrowed opening of a Pasteur pipette (Angres et al., 1991). Histology Injected embryos were fixed in 2.0% glutaraldehyde, 2.0% formaldehyde in 0.1 M phosphate buffer (pH 6.8) for 1 hr on ice. Fixed embryos were washed in phosphate buffer plus 0.1% Triton X-100 and then stained in whole mount with X-Gal as described previously (Detrick et al., 1990). Stained embryos were dehydrated and embedded either in paraplast as described previously(Kintner, 1988)or in plastic following the recommendations of the manufacturer (Polysciences). Paraplast sections were cut on a rotary microtome at a thickness of IO Km, mounted on glass slides, and stained with hematoxylin eosin. Plastic sections were cut on an ultramicrotome at a thickness of 1 vrn, mounted on gelatin-coated glass slides, and stained in 1 .O% methylene blue in 1 .O% Borax. Generation of the N-cadAE and N-cadAC Mutants The starting N-cadherin cDNA was an EcoRl fragment inserted into SP72 (Promega) in which most of the 5’ untranslated sequences were already removed by exonuclease Ill digestion (called AXNcadl in Detrick et al., 1990). This deletion also removed the portion of the SP72 polylinker between Xhol and EcoRI. This plasmid could be linearized with EcoRl and transcribed with SP6 RNA polymerase to generate an N-cadherin transcript that is expressed efficiently in early embryos (Detrick et al., 1990). N-cadAE (Figure 1) was generated from AXNcadl by removing the restriction fragments between the BamHl sites at nt 90 and 2004 (see Detrick et al., 1990). This in-frame deletion removes most of the sequences encoding the extracellular domain in N-cadherin. N-cadAC was generated by converting the codons encoding extracellular cysteines in N-cadAE to serines. NtadAC was constructed from a polymerase chain reaction (PCR) fragment that was primed at the 5’end using the nucleotide sequences overlapping a unique Dralll site near the the initiation codon (5’-CACAGCATCACCATGTGCCGG-37 (the “Dralll primer”). The d’end was primed by sequences that overlap the region containing the cysteines but changed the cysteine codons TGC or TGT to serine codons TCC or TCT, respectively (BI-TGGAGTATTCATGCTCAGAAGAGGAGACC-3’). PCR reactions used Taql polymerase with the instructions and buffers provided by the manufacturer (Cetus). The PCR product was blunted with mung bean nuclease, digested with Dralll, and purified by polyacrylamide gel electrophoresis. After gel elution, the purified fragment was cloned into N-cadAE, which was first digested with Pstl, digested with mung bean nuclease to blunt the 3’ Pstl overhang, and then digested with Dralll. Recombinants were analyzed by nucleotide sequencing. The sequence of the recombinants revealed that cysteine codons had been converted to serine codons but that an extra adenosine residue had been inserted into the sequence where the PCR product was blunt ligated into the N-cadAE vector. The extra adenosine was presumably added on by the terminal transferase activity associated with Taq polymerase. A second PCR fragment was therefore generated in order to remove this extra adenosine from N-cadAC(A). This PCR fragment was primed at the 5’ end with sequences that overlapped a unique Earl restriction site and removed the extra adenosine residue in the N-cadAC(A) sequence (5%TCTCCTClTCTGAGCATGAATACTCCAGCACTAGAGC-39. The 3’end was primed by sequences that overlap a unique Ncol site present in the 3’end of N-cadAC(A) (V-TGGTTCCATGGTGTCAGGCTGC33. A PCR fragment was generated from these two primers using N-cadAC(A) DNA as a template, digested with Ncol and Earl, purified as described above, and ligated into the N-cadAC(A) vector DNA digested with Earl and Ncol. Recombinants were sequenced, yielding a form of N-cadAC lacking the inserted adenosine residue. Terminal Deletions of N-cadAC Terminal deletions in the sequences encoding the were generated by exonuclease Ill digestion of Briefly, the N-cadAC DNA was digested at Hindlll ent in the 3’ untranslated region of the N-cadAC

cytoplasmic domain the N-cadAC DNA. and Sac1 sites presDNA (Detrick et al.,

Cadherin 235

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1990). After different periods of exonuclease Ill digestion, the resulting DNA was blunted by mung bean nuclease digestion, treated briefly with Klenow polymerase, and ligated to “nonsense” Xbal linkers (New England Biolabs, #1062). The sequence of these linkers encodes a stop codon in all three reading frames. After digestion with Xbal, ligation, and transformation into bacteria, the deleted clones were analyzed first by restriction analysis and then by nucleotide sequence. For one of these deletions, T70, the region encoding the shortened cytoplasmic domain was combined with sequences encoding the extracellular domain of N-cadAE in order to generate the shortened form of N-cadAE (AE/T70) used in the experiment shown in Figure 4. The exonuclease Ill digestions generated the terminal deletions, called T127, T87, T70, and T36, shown in Figure 2. To make even smaller deletions in the cytoplasmic domain of N-cadAC, the appropriate PCR fragments were generated. T27 shown in Figure 2 was generated by first producing a PCR fragment that was primed at the 5’ end by the “Dralll primer” (see above), while the 3’ end was primed by sequences in NcadAC that ended at aa 27 in the cytoplasmic domain, followed by an artificial nonsense codon and an artificial Xbal site (5’-ACGCTCTAGACATACTTCAGAATG-3’). The PCR product was synthesized with these primers using N-cadAC as a template, digested with BamHl and Xbal, and purified as described above. The purified product was ligated into the T127 vector DNA that had been first digested with BamHl and Xbal. Recombinants were analyzed by nucleotide sequence. The T6 deletion shown in Figure 2 was generated in a similar manner except the S’end of the PCR fragment was primed by sequences in N-cadAC that ended at aa 6 in the cytoplasmic domain, followed by an artificial stop codon and Xbal restriction site (5’ACGCTCTAGAGCTCCT-TGTCCCGGGCG-37. Internal Deletions In the N-cadAC Cytoplasmic Domain Internal deletions were introduced into N-cadAC by using a PCR product that fused aa 9 in the cytoplasmic domain either to a unique Pvull site present at aa 79, or to a unique Apal site at aa 144 in the cytoplasmic domain. The TA9-79 deletion (Figure 2) was generated with a PCR product that was primed at the 5’ end by the “Dralll primer,” while the 3’end was primed by a sequence in N-cadAC that ended at aa 9 in the cytoplasmic tail, followed by an in-frame Pvull site (S-CTCCGCAGCTGTTGCTTGCCGCTCCTTGTC-3’). The PCR product generated with these two primers was digested with BamHl and Pvull and ligated into N-cadAC digested with the same enzymes. Recombinants were characterized by nucleotide sequencing. The TA9-144 deletion shown in Figure 2 was generated in a similar manner except that the PCR product was primed at the 3’end by a sequence in N-cadAC that ended at aa 9 in the cytoplasmic tail, followed by an in-frame Apal site (5’-CTCGGGGCCCTTTGCTTGCCCTCCTTGTC-3r). The PCR product was digested with BamHl and Apal and ligated into N-cadAC digested with the same enzymes.

RNA Synthesis Capped RNA was synthesized in vitro from DNA templates RNA polymerase as described previously (Kintner, 1988). DNA was removed by DNAase I digestion, and RNA was chromatography on Sephadex G-50. Eluted RNA was tracted, ethanol precipitated twice, and assayed for integrity dehyde gel electrophoresis (Sambrook et al., 1989).

using SP6 Template purified by phenol exby formal-

Interaction of E-Cadherln with acatenin A cDNA was isolated from an st17 cDNA library (Kintner and Melton, 1987) which was shown by several criteria to encode the Xenopus cadherin first expressed embryonically in epidermis during gastrulation (Choi and Gumbiner, 1989; Levi et al., 1991). The E-cadherin cDNA was subcloned into the sp64T vector (Krieg and Melton, 1984) and used to generate synthetic RNA as described above. To measure the binding of E-cadherin to a-catenin, embryos at the 2 cell stage were injected twice with 10 pg of E-cadherin RNA along with 100 pg of either N-cadAC or T6 RNA. At mid gastrulation, extracts were prepared from 40 injected embryos by homogenization in 0.4 ml of 0.1 M NaPO, (pH 7.2), 1 .O% Nonidet P-40,1 .O% Triton X-100,1 mM CaChplusprotease inhibitors and centrifugation at 10,006 rpm in a microcentrifuge for 10 min to clear the extract of yolk and lipids. E-cadherin and associated proteins were isolated from the extracts by adding an E-cadherin

monoclonal antibody coupled to Sepharose beads (McCrea and Gumbiner, 1991) and rocking the extracts for 8 hr. The beads were collected by low speed centrifugation and washed five times with 100 bead volumes of the same buffer used for homogenization. The washed beads were boiled for 10 min in 0.1 ml of Laemmli’s sample buffer and then removed by centrifugation. lmmunoprecipitated proteins were separated on 15% acrylamide gels containing SDS, transferred to a solid support, and probed with antibodies as described below. Western Analysis Extracts were prepared by homogenizing 15 embryos in 10 mM TrisHCI (pH 7.5) 0.1 M NaCI, 1 .O% Nonidet P-40,2 mM CaC12 plus protease inhibitors as described previously (Detrick et al., 1990). Homogenates were cleared of yolk by centrifugation at 10,000 rpm in a microcentrifuge, then added directly to Laemmli loading buffer, with or without 0.1 M j&mercaptoethanol, and boiled. Samples were electrophoresed in a 10% polyacrylamide gel containing SDS and transferred to an Immobilon-P membrane (Millipore). The portion of each membrane containing the molecular weight markers was stained separately with amido black. The remaining membrane was blocked with Blotto and reacted with antibodies as described previously (Kintner, 1988). Antibody binding was detected using a horseradish peroxidase secondary antibody, chemoluminescence, and autoradiography (ECL, Amersham). Acknowledgments I would like to thank Anna Newman for technical assistance. I am particularly grateful to Dr. Barry Gumbiner for insightful discussions about cadherins and for providing the anti-PEP.1 and E-cadherin antibodies. Dr. Rolf Kemler kindly provided antibodies against atatenin. Drs. Greg Lemke, Ajay Chitnis, Christine Holt, and Rebecca Riehl provided helpful comments on the manuscript. The support of the National Institutes of Health is gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenf” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

30, 1991; revised

February

3, 1992.

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