The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that 13-catenin is the linker protein between E-cadherin and c-catenin and that E-cadherin does not bind directly to a-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of a-catenin are independent binding sites for 8-catenin. In addition to 13-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike 13-catenin, p120does not bind a-catenin in vitro, although a complex of p120 and endogenous oi-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and x-catenin revealed two catenin pools in cell lysates: an -1000-to '2000-kDa complex bound to E-cadherin and an -220-kDa pool that did not contain E-cadherin. Only 18-catenin in the -220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.The cadherin superfamily comprises glycoproteins responsible for calcium-dependent, homotypic cell interactions (1). Ecadherin is generally expressed in epithelial tissues and has been shown to regulate cell-cell adhesion (2), cell migration (3), morphogenesis (4), and the establishment of membrane polarity (5).Homotypic interactions between extracellular domains of cadherins are necessary but not sufficient for cell-cell adhesion (2). Linkage of the cadherin cytoplasmic domain to three cytosolic proteins, named a-catenin, ,B-catenin, and plakoglobin (y-catenin), is required (6-8). Although a-catenin, 3-catenin, and plakoglobin can be coimmunoprecipitated in a complex with E-cadherin (1, 7), the binding order of proteinprotein interactions has not been resolved. Insight into this problem is important for understanding functions of the cadherin-catenin complex and the regulation of cadherincatenin complex assembly. Here, we define the binding order of protein-protein interactions in the cadherin-catenin complex using genetic and biochemical approaches. MATERIALS AND METHODSStrain and Microbiological Techniques. All cloning procedures and bacterial transformation were performed by standard procedures outlined by Sambrook et at (9). Yeast strain Y190 (MATa gal4 gal80 his3-200 trpl-901 ade2-101 ura3-52

E-cadherin is maternally expressed in most vertebrate species, but its function during early development of the vertebrate embryo proper is unknown. To directly examine E-cadherin gene (cdh1) function in zebrafish, morpholino oligonucleotides (MOs) that inhibit E-cadherin protein (Cdh1) expression were injected into embryos. Cdh1 knockdown reduced embryo survival. In early cdh1 MO-injected embryos, the cleavage plane orientation between blastomeres was irregular and adhesion defects prevented normal compaction. Cdh1 knockdown inhibited epiboly cell movements. Epiboly delay caused yolk cell lysis and produced embryos with a bifurcated embryonic axis. Cdh1 knockdown inhibited gastrulation cell movements, causing defects in convergence and extension. Additionally, prechordal plate derivatives were absent in Cdh1 knockdown embryos even though presumptive prechordal plate markers were induced normally. E-cadherin mRNA coinjection demonstrated the specificity of cdh1 MO-induced defects. Our experiments illustrate the importance of cdh1 in regulating morphogenetic cell movements and tissue formation in the early embryo.

Summary Initiation of motile cell behavior in embryonic development occurs during late blastula stages when gastrulation begins. At this stage, the strong adhesion of blastomeres has to be modulated to enable dynamic behavior, similar to epithelial-to-mesenchymal transitions. We show that in zebrafish MZspg embryos mutant for the stem cell transcription factor Pou5f1/Oct4, which are severely delayed in the epiboly gastrulation movement, all blastomeres are defective in E-cad endosomal trafficking and E-cad accumulates at the plasma membrane. We find that Pou5f1-dependent control of EGF expression regulates endosomal E-cad trafficking. EGFR may act via modulation of p120 activity. Loss of E-cad dynamics reduces cohesion of cells in reaggregation assays. Quantitative analysis of cell behavior indicates that dynamic E-cad endosomal trafficking is required for epiboly cell movements. We hypothesize that dynamic control of E-cad trafficking is essential to effectively generate new adhesion sites when cells move relative to each other.