A conditionally replicative adenovirus, CRAd-S-pK7, can target endometriosis with a cell-killing effect.

BACKGROUND: Novel therapeutic approaches for endometriosis based on molecular strategies may prove to be useful. Conditionally replicative adenoviruses (CRAds) are designed to exploit key differences between target and normal cells. The wild-type adenovirus (Adwt) promoter can be replaced by tissue-specific promoters, allowing viral replication only in target cells. Viral infectivity can be enhanced by altering Ad tropism via fiber modification. We investigated whether CRAds can be used to target endometriosis and determined the most efficient transcriptional- and transductional-targeting strategy. METHODS: An in vitro study was carried out using human endometriotic cell lines, 11Z (epithelial) and 22B (stromal), normal human ovarian surface epithelial cell line (NOSE006) and primary human endometriosis cells. A total of 9 promoters and 12 Ad tropism modifications were screened by means of a luciferase reporter assay. From this screening data, three CRAds (CRAd-S-pK7, CRAd-S-RGD, CRAd-S-F5/3sigma1, all incorporating the survivin promoter but with different fiber modifications) were selected to perform experiments using Adwt and a replication-deficient virus as controls. CRAds were constructed using a plasmid recombination system. Viral-binding capacity, rates of entry and DNA replication were evaluated by quantitative real-time PCR of viral genome copy. Cell-killing effects were determined by crystal violet staining and a cell viability assay for different concentrations of viral particles per cell. RESULTS: Comparison of promoters demonstrated that the survivin promoter exhibited the highest induction in both endometriotic cell lines. Among the fiber-modified viruses, the polylysine modification (pK7) showed the best infection enhancement. CRAd-S-pK7 was validated as the optimal CRAd to target endometriosis in terms of binding ability, entry kinetics, DNA replication and cell-killing effect. CRAd-S-pK7 also exhibited a high level of DNA replication in primary endometriosis cells. CONCLUSIONS: CRAd-S-pK7 has the best infection and cell-killing effect in the context of endometriosis. It could prove to be a useful novel method to target refractory cases of endometriosis.

Cyclooxygenase-2 regulates survival, migration, and invasion of human endometriotic cells through multiple mechanisms.

Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity that affects up to 20% of women of child-bearing age. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)), is highly expressed in endometriotic tissues and results in increased concentrations of peritoneal PGE(2) in women. In this study, we determined the expression of COX-2 protein in ectopic and eutopic endometria in humans and the role of COX-2 in endometriotic cell survival, migration, and invasion in humans. Our results indicate that COX-2 protein is abundantly expressed in ectopic endometria compared with eutopic endometria. Comparatively, expression of COX-2 protein is higher in eutopic endometria from women with endometriosis compared with women without endometriosis. Inhibition of COX-2 decreases survival, migration, and invasion of endometriotic cells that are associated with decreased production of PGE(2). Cell growth inhibitory effects of COX-2 inhibition/silencing are mediated through nuclear poly (ADP-ribose) polymerase-mediated apoptosis. Cell motility and invasion inhibitory effects of COX-2 inhibition/silencing are mediated through matrix metalloproteinase-2 and -9 activities. Interestingly, effects of COX-2 inhibition is more profound in endometriotic epithelial than in stromal cells. Furthermore, inhibition of COX-2 affects invasion rather than migration of endometriotic epithelial and stromal cells. It is the first evidence showing that inhibition of COX-2 decreases endometriotic epithelial and stromal cell survival, migration, and invasion in humans. Our results support the emerging concept that COX-2/PGE(2) promotes the pathophysiology and pathogenesis of endometriosis in humans.

[Endometriosis--a stem cell disease?]. / Endometriose--eine Stammzellerkrankung?

Endometriosis is an estrogen-dependent and chronic disease with an unknown etiology and pathogenesis. It is however likely and well accepted that retrograde menstruation of endometrial cells into the pelvic cavity is the origin of this disease in many cases. Here we discuss a model in which retrogradely menstruated endometrial cells have different inherent developmental properties because they represent in fact a mixture of different developmental cell stages. These stages can be distinguished in part by the expression of marker proteins such as cytokeratin (intermediate filament protein of epithelial cells) or E-cadherin (intercellular adhesion protein of epithelial cells and metastasis suppressor molecule). Cytokeratin-positive E-cadherin negative cells, for example, would be less differentiated epithelial cells than cytokeratin-positive E-cadherin positive cells. In analogy to findings in other cell systems we assume that the cells which are undifferentiated or not fully differentiated still have the potential to give rise to differentiated daughter cells and, on the other hand, could be maintained as a pool of rather undifferentiated cells and capable of self renewal. This feature would be similar to stem cells (SC) and cells with plasticity. Interestingly we find epithelial cells of different developmental stages in deep infiltrating (e. g. of colon) or peritoneal endometriotic lesions. Therefore we conclude that less differentiated cells in retrogradely menstruated endometrial cell populations possibly representing SC features or plasticity might be the cellular source of primary endometriotic lesions and those present in lesions may contribute to the persistence of the disease by detaching and forming secondary lesions.

Identification of an invasive, N-cadherin-expressing epithelial cell type in endometriosis using a new cell culture model.

Studies of molecular, cellular, and pathophysiological parameters in endometriosis are primarily hampered by a lack of in vitro model systems, such as endometriotic cell lines. To overcome this we successfully established cell lines from peritoneal endometriotic biopsies and characterized them at the molecular and cellular level. Two types of cells could be transformed: one exhibiting stromal cell features (cytokeratin/E-cadherin-negative), the other epithelial-like (cytokeratin-positive/E-cadherin-negative, invasive in vitro). Using a Matrigel assay the epithelial-like cell lines proved as invasive as metastatic carcinoma cells, possibly through the influence of N-cadherin implicated as a path-finding cadherin allowing cellular invasion and migration in both normal and pathophysiological processes. Our results support the idea that endometriosis, although not neoplastic, shares features with malignant cells and that metastasis in endometriosis may include mechanisms proposed for micrometastasis in cancer. Thus our cell lines will not only be useful tools for analyzing molecular and cellular events relating to endometriosis, but may also represent a paradigm for invasion and metastasis in general.

mARVCF cellular localisation and binding to cadherins is influenced by the cellular context but not by alternative splicing.

ARVCF, a member of the catenin family, is thought to contribute to the morphoregulatory function of the cadherin-catenin complex. Recently, we reported the isolation and characterisation of murine ARVCF (mARVCF), particularly its interaction with M-cadherin. Here, we describe the identification of novel mARVCF isoforms that arise by alternative splicing. At the N-terminus, alternative splicing results in the inclusion or omission of a coiled-coil region probably important for protein-protein interactions. At the C-terminus, four isoforms also differ by domains potentially important for selective protein-protein interaction. The eight putative mARVCF isoforms were expressed as EGFP-fusion proteins in six different cell lines that exhibit a distinct pattern of cadherins. Apparently, binding of the mARVCF isoforms to M-, N-, or E-cadherin is generally unaffected by their altered N- and C-termini, as revealed by the MOM recruitment assay. However, mARVCF isoforms reproducibly exhibit differential localisation in distinct cellular environments. For example, mARVCF isoforms are unable to colocalise with N-cadherin in EJ28 carcinoma cells but do so in HeLa cells. Our results suggest that the subcellular localisation of mARVCF may be determined not only by the presence or absence of an appropriate interaction partner, in this case cadherins, but also by the cellular context.

A model system for studying postnatal myogenesis with tetracycline-responsive, genetically engineered clonal myoblasts in vitro and in vivo.

The aim of this work was to introduce a tetracycline-responsive (Tet-off) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clones expressing tetracycline-responsive transactivator protein tTA. Like parental i28 myoblasts, these clones differentiated well in vitro. The second step introduced the firefly (Photinus pyralis) luciferase gene into one of the stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 microg ml(-1)) resulted in at least 100-fold decreases in luciferase activity within 8 h in both growing and differentiating myoblast cultures. Enzyme activity was rapidly restored after tetracycline was removed (8 h). After successful implantation of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycycline for at least 1 month. The tetracycline-responsive master clones are potentially powerful tools for studying the function of various genes in postnatal myogenesis.

In search of pathogenic mechanisms in endometriosis: the challenge for molecular cell biology.

Endometriosis, defined histologically as the presence of endometrium-like glands and stroma outside the uterus, is a chronic, invasive and metastasising disease. It shares features with malignant tumours (invasion and metastasis) but is not neoplastic. Despite the fact that endometriosis is one of the most frequent gynaecological diseases, it is under researched, puzzling and highly debated. The aetiology and pathogenesis is little understood although it is agreed that implantation, at least in many cases, is responsible for endometriosis. This theory advocates retrograde menstruation as the underlying phenomenon, where cells of the menstrual efflux provide the cellular source for endometriotic lesion formation. Causative therapy and non-invasive diagnostics of endometriosis do not exist. Thus, there is a substantial but unmet need for molecular and cellular research to unravel the pathogenic mechanisms of endometriosis as a basis for developing novel diagnostic and therapeutic concepts. In this review, we specifically focus on the cellular basis of lesion formation, the possible modulation of this by cytokines and other factors and the characteristics of endometriotic cells in terms of invasion and metastasis. Considering available experimental information, we concentrate on arguments and ideas in favour of an endometriotic founder cell population exhibiting substantial plasticity for differentiation and self-renewal. Perhaps present in the menstrual efflux or arising by metaplasia (a complementary theory to implantation), this cell type might respond to stimuli present in the ectopic host environment and establish the endometriotic phenotype.

The armadillo repeat region targets ARVCF to cadherin-based cellular junctions.

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

The putative role of cell adhesion molecules in endometriosis: can we learn from tumour metastasis?

Endometriosis, one of the most frequent diseases in gynaecology, is a considerable threat to the physical, psychological and social integrity of women. The etiology and pathogenesis of this important disease, defined as the ectopic location of endometrium-like glandular epithelium and stroma outside the uterine cavity, is poorly understood. Clinical observations and in vitro experiments imply that endometriotic cells are invasive and able to metastasize. Analogous to tumour metastasis, it is likely that cell adhesion molecules are central for the invasion and metastasis of endometriotic cells. Investigation of these molecules in endometriosis should increase our understanding of the molecular mechanisms involved in the pathogenesis of this disease.

M-cadherin and its sisters in development of striated muscle.

Cadherins are calcium-dependent, transmembrane intercellular adhesion proteins with morphoregulatory functions in the development and maintenance of tissues. In the development of striated muscle, the expression and function of mainly M-, N-, and R-cadherin has been studied so far. While these three cadherins are expressed in skeletal muscle cells, of these only N-cadherin is expressed in cardiac muscle. In this review, M-, N-, and R-cadherin are discussed as important players in the terminal differentiation and possibly also in the commitment of skeletal muscle cells. Furthermore, reports are described which evaluate the essential role of N-cadherin in the formation of heart tissue.

The M-cadherin catenin complex interacts with microtubules in skeletal muscle cells: implications for the fusion of myoblasts.

M-cadherin, a calcium-dependent intercellular adhesion molecule, is expressed in skeletal muscle cells. Its pattern of expression, both in vivo and in cell culture as well as functional studies, have implied that M-cadherin is important for skeletal muscle development, in particular the fusion of myoblasts into myotubes. M-cadherin formed complexes with the catenins in skeletal muscle cells similar to E-cadherin in epithelial cells. This suggested that the muscle-specific function of the M-cadherin catenin complex might be mediated by additional interactions with yet unidentified cellular components, especially cytoskeletal elements. These include the microtubules which also have been implicated in the fusion process of myoblasts. Here we present evidence that the M-cadherin catenin complex interacts with microtubules in myogenic cells by using three independent experimental approaches. (1) Analysis by laser scan microscopy revealed that the destruction of microtubules by nocodazole leads to an altered cell surface distribution of M-cadherin in differentiating myogenic cells. In contrast, disruption of actin filaments had little effect on the surface distribution of M-cadherin. (2) M-cadherin antibodies coimmunoprecipitated tubulin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated epithelial cells ectopically expressing M-cadherin. Vice versa, tubulin antibodies coimmunoprecipitated M-cadherin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated M-cadherin-expressing epithelial cells. (3) M-cadherin and the catenins, but not a panel of control proteins, were copolymerized with tubulin from myogenic cell extracts even after repeated cycles of assembly and disassemly of tubulin. Moreover, neither M-cadherin nor E-cadherin could be found in a complex with microtubules in epithelial cells ectopically expressing M-cadherin. Our data are consistent with the idea that the interaction of M-cadherin with microtubules might be essential to keep the myoblasts aligned during fusion, a process in which both M-cadherin and microtubules have been implicated.

Tracing cellular and molecular mechanisms involved in endometriosis.

The aetiology and pathogenesis of endometriosis, defined as the presence of endometrium-like tissue outside the uterine cavity, is largely unknown. In this paper we present and discuss possibilities to study the putative pathogenic properties of endometriotic cells in vitro. The current focus of our investigations is on the invasive phenotype of the disease, assuming that this might contribute to the pathogenesis of endometriosis. So far, we have shown that: (i) cytokeratin-positive and E-cadherin-negative endometriotic cells have an invasive phenotype in a collagen invasion assay in vitro similar to metastatic carcinoma cells; (ii) the invasiveness of endometriotic but not of eutopic endometrial cells can be stimulated by a heat-stable protein present in peritoneal fluid; and (iii) the endometriotic cell line EEC145T, which we established, may be a useful tool for the identification of gene products which are, positively or negatively, invasion-related. Finally, our studies suggest that the invasive phenotype in endometriosis shares aspects with tumour metastasis, but might also have unique mechanisms.

M-cadherin-mediated cell adhesion and complex formation with the catenins in myogenic mouse cells.

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK- cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with alpha-catenin/beta-catenin or with alpha-catenin/plakoglobin, as E-and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.

Nonmalignant epithelial cells, potentially invasive in human endometriosis, lack the tumor suppressor molecule E-cadherin.

Endometriosis is one of the most frequent diseases in gynecology. It is a histologically defined nonmalignant disease in which endometrium-like tissue is found outside the uterus (for example, peritoneum, gut, or lung). The pathogenesis of endometriosis is unknown, but invasive mechanisms have been implicated in the development of the disease. Indeed, primary cells from human endometriotic biopsies but not from human endometrial biopsies are invasive in an in vitro collagen invasion assay. In this study, these in vitro invasive endometriotic cells were found to be nonmalignant epithelial cells lacking E-cadherin, which acts as an invasion suppressor molecule in carcinomas. Immunocytochemistry showed that the E-cadherin-negative epithelial cell type was increased in sections of endometriosis tissue as compared with sections of eutopic endometrium. On the basis of these data we propose that the E-cadherin-negative invasive endometriotic cells seen in vitro represent the cell population that migrates to ectopic (extrauterine) locations and thus causes endometriosis in vivo. Accordingly, the loss of E-cadherin expression is postulated to constitute a crucial mechanism in the pathogenesis of endometriosis.

Effects of divalent cations on M-cadherin expression and distribution during primary rat myogenesis in vitro.

In the process of myogenesis, cadherins are thought to be involved in the initial cell-cell recognition and possible initiation of myoblast fusion to form multinucleated myotubes. Of the cadherins, M-cadherin, but not N-cadherin, is down-regulated upon inhibition of myogenesis, suggesting that M-cadherin may be a key receptor involved in myogenesis. M-cadherin binds in a calcium-dependent manner, and depletion of divalent cations inhibits myoblast fusion. We analyzed the regulation of M-cadherin protein and mRNA levels in primary rat myogenic cultures in the presence and absence of divalent cations. In untreated cultures M-cadherin was localized to various myogenic cell-cell contacts. M-cadherin protein and mRNA levels showed a peak at day 2 after the initiation of growth. When divalent cations were removed from the cell culture medium, myoblast fusion was inhibited and immunocytochemical analysis revealed a failure of M-cadherin to localize to cell-cell contacts. Analysis of M-cadherin protein and mRNA in fusion-inhibited cultures still revealed a peak at day 2. However, by day 3, M-cadherin protein levels in the fusion-inhibited cultures were reduced in both the detergent-soluble and -insoluble fractions in comparison with the untreated cultures. Interestingly, beta-catenin, a protein associated with cadherins, was frequently observed at intercellular contacts in the fusion-inhibited cultures. We could also show that the intracellular levels of beta-catenin protein remained constant regardless of the presence or absence of divalent cations. In summary, the dynamic regulation of M-cadherin in muscle-fusion-related events is an indication of the importance of M-cadherin for myoblast fusion and myogenic differentiation.

Invasiveness of endometriotic cells in vitro.

The pathogenesis of endometriosis is not known. The currently favoured theory is that viable endometrial cells, shed from the endometrium into the pelvic cavity by retrograde menstruation, reattach and invade other tissues. We used a collagen gel invasion assay to assess invasive potential of endometriotic cells. The invasion indices of cells from peritoneal endometriotic lesions and a metastatic bladder carcinoma cell line (EJ28) were similar (2.2-15.6 vs 8.4-11.6) whereas cells from normal endometrium and non-metastatic carcinoma cells (RT112) were non-invasive (indices < 1). Invasiveness of endometriotic cells might contribute to the pathogenesis of endometriosis.

Molecular cloning, characterization, and mapping of a full-length cDNA encoding human UDP-galactose 4'-epimerase.

Galactose metabolism in all organisms is catalyzed by three enzymatic steps: the galactokinase, galactose-1-phosphate uridyltransferase, and UDP galactose 4'-epimerase reactions. We report here the molecular cloning, characterization, and mapping of a full-length cDNA encoding human UDP-galactose 4'-epimerase (GALE). Our cDNA is 1488 bp long and matches the mRNA size of 1.5 kg detected in fibroblasts and lymphoblasts. The human GALE cDNA encodes a predicted protein of 348 amino acids with a molecular mass of 38,266. The human GALE enzyme is 87% identical to the rat protein, 53% identical to the homologous GAL10 protein from the yeast Kluyveromyces lactis, and 51% identical to the galE protein from the prokaryote Escherichia coli. This extraordinary degree of sequence identity has allowed us to build a homology model of the human protein based on the bacterial crystal structure. This predicted human structure is very similar to the E. coli galE enzyme, suggesting that both enzymes use similar mechanisms. The human gene encoding GALE maps, as expected, to a single locus on chromosome 1 and appears to be compact. The human GALE gene is structurally intact in 19 patients with epimerase-deficiency galactosemia, an inborn error of metabolism secondary to GALE deficiency. Therefore, we propose that this disorder is due to small mutations within the gene.

Involvement of M-cadherin in terminal differentiation of skeletal muscle cells.

Cadherins are a gene family encoding calcium-dependent cell adhesion proteins which are thought to act in the establishment and maintenance of tissue organization. M-cadherin, one member of the family, has been found in myogenic cells of somitic origin during embryogenesis and in the adult. These findings have suggested that M-cadherin is involved in the regulation of morphogenesis of skeletal muscle cells. Therefore, we investigated the function of M-cadherin in the fusion of myoblasts into myotubes (terminal differentiation) in cell culture. Furthermore, we tested whether M-cadherin might influence (a) the expression of troponin T, a typical marker of biochemical differentiation of skeletal muscle cells, and (b) withdrawal of myoblasts from the cell cycle (called terminal commitment). The studies were performed by using antagonistic peptides which correspond to sequences of the putative M-cadherin binding domain. Analogous peptides of N-cadherin have previously been shown to interfere functionally with the N-cadherin-mediated cell adhesion. In the presence of antagonistic M-cadherin peptides, the fusion of myoblasts into myotubes was inhibited. Analysis of troponin T revealed that it was downregulated at the protein level although its mRNA was still detectable. In addition, withdrawal from the cell cycle typical for terminal commitment of muscle cells was not complete in fusion-blocked myogenic cells. Finally, expression of M-cadherin antisense RNA reducing the expression of the endogenous M-cadherin protein interfered with the fusion process of myoblasts. Our data imply that M-cadherin-mediated myoblast interaction plays an important role in terminal differentiation of skeletal muscle cells.

Contactus adherens, a special type of plaque-bearing adhering junction containing M-cadherin, in the granule cell layer of the cerebellar glomerulus.

In the glomeruli of the granule cell layer of mammalian cerebellum, neuronal extensions are interconnected by numerous small, nearly isodiametric (diameters up to 0.1 micron), junctions previously classified as puncta adherentia related to the vinculin-containing, actin microfilament-anchoring junctions of the zonula adherens of epithelial and certain other cells. Using immunofluorescence and immunoelectron microscopy, we have found, however, that these junctions are negative for E- and VE-cadherin, for desmosomal cadherins, and also for vinculin, alpha-actinin, and desmoplakin, but they do contain, in addition to the protein plakoglobin common to all forms of adhering junctions, the plaque proteins alpha- and beta-catenin and the transmembrane glycoprotein M-cadherin previously found as a spread--i.e., not junction bound--plasma membrane protein in certain fetal and regenerating muscle cells and in satellite cells of adult skeletal muscle. We conclude that these M-cadherin-containing junctions of the granule cell layer represent a special type of adhering junction, for which we propose the term contactus adherens (from the Latin contactus, for touch, site of bordering upon, also influence), and we discuss the differences between the various adhering junctions on the basis of their molecular constituents.

Expression of M-cadherin protein in myogenic cells during prenatal mouse development and differentiation of embryonic stem cells in culture.

Molecules regulating morphogenesis by cell-cell interactions are the cadherins, a class of calcium-dependent adhesion molecules. One of its members, M-cadherin, has been isolated from a myoblast cell line (Donalies et al. [1991] Proc. Natl. Acad. Sci. U.S.A. 88:8024-8028). In mouse development, expression of M-cadherin mRNA first appears at day 8.5 of gestation (E8.5) in somites and has been postulated to be down-regulated in developing muscle masses (Moore and Walsh [1993] Development 117:1409-1420). Affinity-purified polyclonal M-cadherin antibodies, detecting a protein of approximately 120 kDa, were used to study the cell expression pattern of M-cadherin protein. It was first visualized in somites at E10 1/3 and could be confined to desmin positive, myotomal cells. At all subsequent prenatal stages, M-cadherin was only found in myogenic cells of somitic origin. The detection of the protein at E10 1/3 suggests a translational delay of M-cadherin mRNA of 1 to 2 days (E8.5 vs. E10 1/3). This was further supported by the finding that during differentiation of ES cell line BLC6 into skeletal muscle cells in culture, expression of M-cadherin mRNA can be detected 2 days prior to M-cadherin protein. During prenatal development, the pattern of M-cadherin expression changes: In E10 1/3 embryos and also in myotomal cells of later stages, M-cadherin is evenly distributed on the cell surface. In developing muscle masses (tested at E16 to E18), however, M-cadherin protein becomes clustered most likely at sites of cell-cell contact as indicated by double-labelling experiments: M-cadherin-staining is the positive image of laminin negative areas excluding the presence of a basal lamina at M-cadherin positive sites. Furthermore, M-cadherin is coexpressed with the neuronal cell adhesion molecule N-CAM which has been shown to mediate cell-cell contact in myogenic cells. In summary, our results are in line with the idea that M-cadherin might play a central role in myogenic morphogenesis.