We found that E-cadherin and epidermal growth factor receptor (EGFR) are associated in mammary epithelial cells and that E-cadherin engagement in these cells induces transient activation of EGFR, as previously seen in keratinocytes (37). In contrast, EGFR does not associate with and is not activated by N-cadherin. Analysis of cells expressing chimeric cadherins revealed that the extracellular domain of E-cadherin is required for interaction with and activation of EGFR. This activation results in tyrosine phosphorylation of known EGFR substrates and reduction in focal adhesions. These interactions, however, are not necessary for suppression of cell motility by E-cadherin.

E-cadherin has been termed an "invasion suppressor," yet the mechanism of this suppression is not known. In contrast, several reports indicate N-cadherin does not suppress but, rather, promotes cell motility and invasion. Here, by characterizing a series of chimeric cadherins we defined a previously uncharacterized region consisting of the transmembrane domain and an adjacent portion of the cytoplasmic segment that is responsible for the difference in ability of E- and N-cadherin to suppress movement of mammary carcinoma cells, as quantified from time-lapse video recordings. A mutation in this region enabled N-cadherin to suppress motility, indicating that both E- and N-cadherin can suppress, but the activity of N-cadherin is latent, presumably repressed by binding of a specific inhibitor. To define regions common to E- and N-cadherin that are required for suppression, we analyzed a series of deletion mutants. We found that suppression of movement requires E-cadherin amino acids 699-710. Strikingly, beta-catenin binding is not sufficient for and p120ctn is not involved in suppression of these mammary carcinoma cells. Furthermore, the comparable region of N-cadherin can substitute for this required region in E-cadherin and is required for suppression by the mutant form of N-cadherin that is capable of suppressing. Variations in expression of factors that bind to the two regions we have identified may explain previously observed differences in response of tumor cells to cadherins.

Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G(12) subfamily comprised of Galpha(12) and Galpha(13) interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator beta-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 519-524). Because of the importance of beta-catenin in cadherin-mediated cell-cell adhesion, we examined whether G(12) subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G(12) proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G(12) proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G(12) binding ability was not affected by activated G(12) proteins, indicating a requirement for direct G(12)-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G(12) proteins reversed the inhibition via a mechanism that was independent of G(12)-mediated Rho activation. These results validate the G(12)-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G(12) proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

To the Editor: Plakoglobin, a member of the arm-repeat family of proteins, binds to classical cadherins and desmosomal cadherins (Zhurinsky et al., 2000Zhurinsky J. Shtutman M. Ben-Ze'ev A. Plakoglobin and beta–catenin. protein interactions, regulation and biological roles.J Cell Sci. 2000; 113: 3127-3139Crossref PubMed Google Scholar). Mutations in plakoglobin cause Naxos disease, which involves arrhythmogenic right ventricular cardiomyopathy, palmoplantar keratoderma, and woolly hair (McKoy et al., 2000McKoy G. Protonotarios N. Crosby A. et al.Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease).Lancet. 2000; 355: 2119-2124Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Plakoglobin also is required for the cell separation effect of pemphigus autoantibodies (Caldelari et al., 2001Caldelari R. de Bruin A. Baumann D. Suter M.M. Bierkamp C. Balmer V. Muller E. A central role for the armadillo protein plakoglobin in the autoimmune disease pemphigus vulgaris.J Cell Biol. 2001; 153: 823-834Crossref PubMed Scopus (152) Google Scholar). In recent studies, we found a discrepancy between the exon/intron structure of the human plakoglobin gene as described in Whittock et al., 2000Whittock N.V. Eady R.A. Mcgrath J.A. Genomic organization and amplification of the human plakoglobin gene (JUP).Exp Dermatol. 2000; 5: 323-326Crossref Scopus (16) Google Scholar and in the public (NCBI) and Celera human genome databases. Using sequence information from plakoglobin cDNA, Whittock et al amplified virtually the entire plakoglobin gene by polymerase chain reaction (PCR). They found the gene to contain 13 exons spanning approximately 17 kb of genomic DNA. In particular, they found that the 5′-most 328 bp of cDNA existed in the genome as a single exon, as shown in Figure 1 (“single exon”). In contrast, the plakoglobin gene sequence in the Celera database is annotated and indicates that this 328 bp sequence is split into two exons separated by a 14.7 kb intron (see Figure 1, “two exons”). The plakoglobin sequence in the public database is incomplete (exons 2–13 are not present) and has not yet been annotated, but exon 1 and ≈ 3.8 kb of intron 1 (as shown in Figure 1, “two exons”) are present and are virtually identical to the corresponding sequences in the Celera database. (The only exception is a ≈ 300 bp region within intron 1, about 2.7 kb downstream of exon 1, which differs in sequence between the Celera and public databases.) We verified the “two exons” structure by performing PCR on human genomic DNA (kindly provided by Richard Wenstrup, MD, Children's Hospital, Cincinnati, OH) with a series of primer pairs, as shown in Figure 1. All primer pairs generated fragments with sizes that were consistent with the “two exon” structure, and inconsistent with the “single exon” structure. Further, one PCR product (U2:D2) was sequenced on both strands and matched the database sequences at the intron–exon 2 junction. Based on this confirmation of the database sequence, we propose that the exons of the plakoglobin gene be renumbered 1–14. Whittock et al., 2000Whittock N.V. Eady R.A. Mcgrath J.A. Genomic organization and amplification of the human plakoglobin gene (JUP).Exp Dermatol. 2000; 5: 323-326Crossref Scopus (16) Google Scholar had confirmed the single exon structure by PCR amplification using upstream and downstream primers (A and B in Figure 1) within “exon 1.” Primers corresponding to the 5′- and 3′-ends of “exon 1” should not have yielded a PCR product if these sequences were separated by 14.7 kB; however, the Whittock et al upstream primer A (Figure 1) spanned the junction between the split portions of the exon and, in retrospect, it seems likely that the 3′ portion of this oligonucleotide primed synthesis from within the actual exon 2, misleadingly suggesting these sequences constituted a single exon. The presence of the 14.7 kb intron between exons 1 and 2 is clearly significant for analyses of the effects of plakoglobin mutations and for studies of plakoglobin gene regulation. For example, Potter et al., 2001Potter E. Braun S. Lehmann U. Brabant G. Molecular cloning of a functional promoter of the human plakoglobin gene.Eur J Endocrin. 2001; 145: 625-633Crossref PubMed Scopus (25) Google Scholar noted that the immediate 5′ flanking region of the plakoglobin gene (5′ in Figure 1) has a high CpG dinucleotide content and demonstrated that expression of plakoglobin is downregulated in some cell lines as a result of hypermethylation in this region. Analysis of the intron sequence shows that the 1 kb immediately downstream of the first exon has an exceptionally high CpG content, strongly supporting the suggestion of Potter et al., 2001Potter E. Braun S. Lehmann U. Brabant G. Molecular cloning of a functional promoter of the human plakoglobin gene.Eur J Endocrin. 2001; 145: 625-633Crossref PubMed Scopus (25) Google Scholar. Furthermore, regulogram analysis (Jegga et al.Jegga A.G. Sherwood S. Carman J. Pinski A. Phillips J. Pestian J. Aronow B.J. TRAFAC: A web accessible system for the detection of potential cis-regulatory regions in conserved gene orthologs.http://www.trafac.chmcc.org/Google Scholar) of the plakoglobin gene reveals a significant cis-acting element cluster within the 14.7 kb intron (Figure 2). In particular, there is a 300 bp block of sequence 1 kb into the first intron that contains a cluster of cis-regulatory elements, which is highly conserved in human and mouse plakoglobin genes. The many possible cis-acting elements and the high CpG content underscore the potential significance of this region for gene regulation, and the importance of a precise understanding of the gene in this region. This study was financially supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R.B.), the Howard Hughes Medical Institute (B.J.A.), and postdoctoral award DAMD17-01-1-0204 from the DOD Breast Cancer Research Program (M.A.W.).

Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G12 subfamily comprised of Galpha12 and Galpha13 interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator beta-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). Because of the importance of beta-catenin in cadherin-mediated cell-cell adhesion, we examined whether G12 subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G12 proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G12 proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G12 binding ability was not affected by activated G12 proteins, indicating a requirement for direct G12-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G12 proteins reversed the inhibition via a mechanism that was independent of G12-mediated Rho activation. These results validate the G12-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G12 proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

: Tumor invasion is a major obstacle to effective clinical management of breast cancer. To identify new targets for anti-invasive therapies, we have focused on the mechanisms by which the cell adhesion molecule E-cadherin suppresses tumor invasion. A related cadherin, N-cadherin, does not suppress cell movement, even though it is as effective as E-cadherin at mediating adhesion. We analyzed deletion mutants and exploited the difference between E- and N-cadherin to define regions of E-cadherin required for suppression of movement. We localized the key region that differs between E-cadherin and N-cadherin to a region consisting of the transmembrane segment and a small portion of the cytoplasmic domain, but demonstrated that E-cadherin does not regulate motility through sequestering pl 20, at physiological levels of expression. We also found that the catenin- binding domain is also required. Further, we identified two components that are tyrosine phosphorylated after E-cadherin contact, but determined they play no role in suppression of motility. We developed a new assay for analyzing the effect of cadherins on cell movement, which revealed that E-cadherin, but not N-cadherin, suppresses movement in intact monolayers of cells.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.

Expression of the calcium-dependent adhesion molecule E-cadherin suppresses the invasion of cells in vitro, but the mechanism of this effect is unknown. To investigate this mechanism, we analyzed the effects of expressing E-cadherin in mouse L-cells and rat astrocyte-like WC5 cells. Increased cellular adhesion mediated by E-cadherin reduced invasion in WC5 cells and in some L-cells, but not in others. In all cases, suppression of invasion was correlated with decreased cell movement as assessed in an in vitro wound-filling assay and a transwell motility assay. To define the relationship between adhesion mediated by E-cadherin and suppression of motility, we analyzed the effects of deleting different regions of the E-cadherin cytoplasmic domain. E-cadherin lacking the entire cytoplasmic domain did not mediate calcium-dependent adhesion and did not reduce cell motility when expressed in WC5 cells. E-cadherin lacking a portion of the catenin-binding domain did not associate with the cytoskeleton and did not promote adhesion, yet still suppressed the motility of WC5 cells. In addition, E-cadherin that retains an intact catenin-binding domain, but lacks a juxtamembrane portion of the cytoplasmic domain, mediated effective adhesion, but did not suppress motility. These results indicate E-cadherin mediates adhesion and suppresses cell motility via distinct of E-cadherin plays a key role in suppressing motility.

Cultured Schwann cells secreted low levels (30 pg/ml/1.5×106cells) of a 45-kDa neuregulin protein and showed constitutive activation of a neuregulin receptor, Erb-B3, suggesting the existence of an autocrine loop involving neuregulins in Schwann cells. RT-PCR analyses indicated that Schwann cells and fibroblasts in culture produced SMDF/n-ARIA and NDF but not GGF neuregulin messages. Schwann cell and fibroblast neuregulin messages encoded both β and α domains; Schwann cell transcripts encoded only transmembrane neuregulin forms while fibroblast messages encoded transmembrane and secreted forms. SMDF/n-ARIA and NDF messages were also expressed in early postnatal rat sciatic nerve, suggesting a role for neuregulins in peripheral nerve development. An anti-neuregulin antibody inhibited the mitogenic response of Schwann cells to cultured neurons and to extracts of cultured neurons or embryonic brain, consistent with the accepted paracrine role of neuregulins on Schwann cells. Surprisingly, the same antibody inhibited Schwann cell proliferation stimulated by several unrelated mitogens including bFGF, HGF, and TGF-β1. These data implicate both paracrine and autocrine pathways involving neuregulin form(s) in Schwann cell mitogenic responses.

Publisher Summary This chapter provides an overview of cell adhesion—from early experiments implicating differential cell–cell adhesion as an important developmental mechanism to the methods used to isolate and characterize cell adhesion molecules (CAMs). More than 50 CAMs have been identified and structural characterization has allowed these molecules to be classified into families. The initial definition of CAMs was operational, based on their ability to mediate cell–cell adhesion in assays in vitro . Many of the CAMs (the desmogleins and desmocollins of desmosomes) and the cadherins, found within adherens junctions, do appear to function as adhesive molecules in vivo . CAMs do not serve simply as intercellular glue—for example, contacts involving N-cadherin, Ll/Ng-CAM, or N-CAM trigger changes in intracellular second-messenger systems that affect neurite extension and contact mediated by E-cadherin affects cell motility. The structure of the membrane-associated protein tyrosine phosphatase μ that includes an immunoglobulin domain that mediates cell–cell adhesion and a cadherin-related segment in its cytoplasmic region is strongly suggestive of a role in cell-contact regulation of cellular differentiation. In the case of some integrin receptors, ligand binding is known to affect gene expression. The field of cell adhesion is moving beyond identifying and structurally characterizing CAMs and into the more exciting realm of precisely defining their roles in cellular physiology and differentiation.

We examined the effects of nerve growth factor (NGF) and cell-cell contact on expression of the neural cell adhesion molecule L1 in PC12 cells. After 7 d exposure to NGF, but not after exposure to EGF, FGF, TGF beta, or dibutyryl cAMP (dbcAMP), L1 mRNA levels increased fourfold. This increase was not blocked by K252a, an inhibitor of the high-affinity NGF receptor, although neurite extension was completely inhibited. L1 mRNA levels also increased in NGF-treated mutant PC12 cells (PC12nnr5) that lack the high-affinity NGF receptor. The effect of NGF on L1 mRNA was greatest in cells cultured at high density, but its effect on cells cultured at low density was augmented by antibody to L1 (to mimic L1 homophilic binding). Various extracellular matrix components had no differential effects on L1 mRNA levels in either the presence or absence of NGF. Together, these findings suggest that NGF regulates L1 expression by a mechanism that is independent of the high-affinity NGF receptor and that this regulation is modulated by cell-cell contact but not by cell-extracellular matrix interactions.

Although previous studies suggest that P 0 is expressed only in myelinating Schwann cells, monoclonal antibody 1E8 reacts with P 0 , yet also stains early Schwann cell precursors and non‐myelinating Schwann cells (Bhattacharyya et al.: Neuron 7:831–844, 1991). We therefore characterized the 1E8 epitope and analyzed P 0 mRNA expression during development. Immunoblot analyses of P 0 fusion proteins and of deglycosylated P 0 indicated that the 1E8 epitope is polypeptide. Northern blot and polymerase chain reaction (PCR) analyses revealed that P 0 is encoded by a single mRNA that is expressed in chicken embryos as early as E4 and in rat embryos as early as E14. These data indicate that the antigen recognized by 1E8 in early chicken embryos is P 0 and that, during development of both chickens and rats, P 0 mRNA is expressed long before myelination. © 1995 Wiley‐Liss, Inc.

Somatic cell hybrids between SNB-19 human glioblastoma cells and human D98OR HeLa cells were produced and analyzed for their ability to form tumors in nude mice and to invade reconstituted extracellular matrix (Matrigel). Whereas both the SNB-19 and D98OR HeLa parental cells form tumors, four of six hybrid lines did not form tumors, even after periods up to six months, suggesting that each cell type can complement the tumorigenicity of the other. SNB-19 cells showed high rates of Matrigel invasion at all cell densities examined, whereas D98OR HeLa cells showed lower rates of invasion that were further reduced at high cell density. All six hybrid cell lines displayed a combination of these properties: at low cell density, the hybrids showed high rates of invasion, similar to the SNB-19 cells, but the invasion rate diminished at higher cell densities, similar to the D98OR HeLa cells. Taken together, these results provide new experimental evidence that several distinct genetic changes are involved in generating the tumorigenic and invasive phenotype of glioblastoma cells.

We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas.

The neural crest gives rise to a variety of cell types including Schwann cells of the peripheral nervous system. Schwann cell precursors begin to differentiate early and migrate along specific pathways in the embryo before associating with nerve trunks. To determine whether motor axons direct the migration of Schwann cell precursors along specific pathways, we tested the effect of ablating the ventral half of the neural tube, which contains motor neuron cell bodies. The ventral neural tube was removed unilaterally from lumbar regions of chicken embryos at stage 17, when neural crest cells are just beginning to migrate and before motor axons have extended out of the neural tube. At several stages after ventral tube ablation, sections of the lumbar region of these embryos were stained with anti-acetylated tubulin to label developing axons, HNK-1 to label migrating neural crest cells and 1E8 to label Schwann cell precursors. In many embryos the ablation of motor neurons was incomplete. The staining patterns in these embryos support the idea that some Schwann cells are derived from the neural tube. In embryos with complete motor neuron ablation, at stage 18, HNK-1-positive neural crest cells had migrated to normal locations in both control and ablated sides of the embryo, suggesting that motor axons or the ventral neural tube are not required for proper migration of neural crest cells. However, by stage 19, cells that were positive for HNK-1 or 1E8 were no longer seen in the region of the ventral root, nor ventral to the ventral root region. Because Schwann cell precursors require neural-derived factors for their survival in vitro, we tested whether neural crest cells that migrate to the region of the ventral root in ventral neural tube-ablated embryos then die. Nile Blue staining for dead and dying cells in ventral neural tube-ablated embryos provided no evidence for cell death at stage 18. These results suggest that motor axons arrest the migration of Schwann cell precursors during neural crest migration.

A monoclonal antibody, 1E8, which recognizes the peripheral myelin protein, P0, specific for chicken Schwann cells and their precursors (Bhattacharyya et al., Neuron 7:831-844, 1991), was used to immunoselect Schwann cells from embryonic day 14 (E14) chicken sciatic nerve. When cultured, these immunoselected cells displayed properties characteristic of perinatal rodent Schwann cells, including S100-immunoreactivity and O4 antigen-immunoreactivity. In addition, the purified chicken Schwann cells divided slowly when cultured alone, but when co-cultured with chicken or rat sensory neurons, they bound to axons and proliferated. Proliferation was also stimulated by the addition of bovine brain membrane extracts or chicken brain membranes. The 1E8 monoclonal antibody was also used to test the effect of axonal contact on P0 expression. Chicken Schwann cells purified using the 1E8 monoclonal antibody gradually lost P0 when cultured alone. These cells remained 1E8-negative even after prolonged co-culture with embryonic rat dorsal root ganglion neurons or chicken sensory ganglia. These results demonstrate that chicken Schwann cells behave like rodent Schwann cells in their expression of specific antigens, interactions with axons, and regulation of P0 expression. In addition, chicken Schwann cells respond to neuronal signals from the rat and cow, illustrating the cross-species conservation of these signals.

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.

The WC5 rat cerebellar cell line, infected with a Rous sarcoma virus (RSV) that is temperature-sensitive for pp60v-src transformation, expresses high levels of the neural cell adhesion molecule, N-CAM, when grown at the non-permissive temperature for pp60v-src activity. At the permissive temperature, N-CAM expression is 4- to 10-fold reduced and the cells aggregate poorly. To evaluate the effects of variations in N-CAM expression, we compared the invasive ability of transformed WC5 cells that express low levels of N-CAM with transformed cells in which N-CAM-mediated adhesion was restored. WC5 cells were transfected with expression vectors containing cDNAs encoding the 120 or 180 kDa forms of chicken N-CAM linked to constitutive promoters. Several permanently transfected lines that expressed chicken N-CAM at the cell surface were isolated. These cell lines showed enhanced aggregation at the permissive temperature relative to untransfected WC5 cells or cells transfected with control constructs. By comparing the ability of control and transfected WC5 cells to invade reconstituted extracellular matrix, we tested the effect of variations in N-CAM-mediated adhesion on invasion. Clones that expressed high levels of N-CAM showed invasion rates that were similar to control cells, indicating that increasing N-CAM-mediated adhesion does not inhibit the invasiveness of RSV-transformed WC5 cells.