Stahlman Cardiovascular Laboratories, Program for Developmental Biology and The Division of Cardiovascular Medicine, Vanderbilt University, Nashville, TN, USA
* These authors contributed equally to this study
Author for correspondence (e-mail: david.bader{at}mcmail.vanderbilt.edu)
Accepted March 19, 2001
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SUMMARY |
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Key words: Heart, Blood vessels, Cell adhesion, Chick, Bves
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INTRODUCTION |
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Genetic mutations in mice interrupt specific events in the development of coronary vessels, such as cell migration and adhesion. For example, migration of epicardial epithelium is blocked in Vcam1-null mice, while early events in heart tube formation appear relatively normal (Kwee et al., 1995). Adhesion between epicardium and myocardium is severely disrupted in 4 integrin-null mice (Igta4-null Mouse Genome Informatics) resulting in the lack of proper coronary circulation and the eventual failure of cardiac morphogenesis (Yang et al., 1995). More recently, Tevosian et al. identified a FOG2-dependent signaling pathway in cardiac myocytes that regulates epithelial-mesenchymal transition in epicardium generating vasculogenic mesenchyme (Tevosian et al., 2000). These Fog2-null mice (Zfpm-null Mouse Genome Informatics) have an intact epicardium but do not undergo epithelial-mesenchymal transition and thus lack coronary vessels. Additionally, many mutations that affect myocardial differentiation result in detects of vascular patterning and remodeling illustrating the interplay between the myocardial and vasculogenic components of the developing heart (reviewed by Fishman and Chien, 1997). Thus, it is clear that regulation of cell adhesion and migration is essential in the development of coronary vessels.
Using a screen for gene products that regulate heart morphogenesis, we have identified a novel gene product coding for a protein expressed in cells of the developing coronary vascular system. Bves (blood vessel/epicardial substance) is present in the PEO, migrating epicardium, delaminated mesenchyme and coronary smooth muscle of the chicken and mouse (Reese et al., 1999; Reese and Bader, 1999). Recently, Andree et al. identified three highly related murine genes, popeye 1, popeye 2 and popeye 3, that encoded multiple messages by differential splicing (Andree et al., 2000). Sequence analysis has determined that mouse Bves is popeye 1a. Bves protein is broadly distributed in epicardial/epithelial cells but appears as a punctate perinuclear spot in mesenchymal cells after epithelial-mesenchymal transition. Bves-positive cells migrate throughout the heart to regions of forming blood vessels. As Bves-positive cells arrive at forming vascular channels, Bves protein is only detected in differentiating smooth muscle. While changes in Bves distribution are correlated with specific changes in cellular adhesion and motility, the subcellular localization and function of the molecule in this developmental process are unknown.
In this study, we show that Bves undergoes dynamic changes in its subcellular distribution during epithelial, mesenchymal and differentiative phases of coronary vessel development, suggesting that Bves may play a role in cell adhesion and/or migration. We also demonstrate that Bves is membrane protein with a short intracellular N terminus, three transmembrane helices and a long extracellular C-terminal domain. This structure is highly conserved within all members of the Bves/popeye gene family identified in vertebrates and invertebrates (A. M. W. and D. M. B., unpublished). Confocal microscopy shows that Bves is confined to the lateral domain of the cell membrane in epithelial cells and colocalizes with E-cadherin. When epicardial cells are dissociated and exist as single cells, Bves is not detected at the cell membrane but accumulates in the Golgi until cell contact is made. Bves is present in filopodia and at cell borders prior to the appearance of E-cadherin, suggesting that it plays a role in the newly proposed adhesion zipper model of cell adhesion (Vasioukhin et al., 2000). Importantly, transfection of Bves into L-cells, a normally non-adhesive cell line, readily confers adhesion. Finally, epicardial epithelial migration from the PEO explants is blocked by a Bves antibody. Taken together, these data provide direct evidence that Bves is a member of a new class of cell adhesion molecules and suggest a role for this protein in coronary vessel development.
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MATERIALS AND METHODS |
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Tissues and cell culture
Methods for obtaining cryosections of embryonic chick tissues were standard. PEOs were isolated from stage 18 embryos as previously described (Dettman et al., 1998) and grown in Dulbeccos modified Eagles medium (DMEM) with 5% fetal bovine serum. Rat epicardial cells (REC, Eid et al., 1994) and CLL 1.3 (L-cell, ATCC) were cultured in DME with 4 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose and 1.0 mM sodium pyruvate supplemented with 10% FBS and 10 µg/ml Penn/Strep. To generate stable L-cell lines that expressed Bves, L-cells were transfected with pCIneo containing full-length mouse Bves cDNA (Reese et al., 1999) with a FLAG tag at the 3' end of the coding sequence. Cell line L-F was obtained by transfection with pCIneoBves with the endogenous Kozak and initiation sequence, while cell line L-K was transfected with pCIneoBves with an engineered Kozak and initiation sequence 5' to the protein-coding sequences. 1 µg of each construct was used for transfection with the FuGENE 6 Transfection reagent (Roche). Cells were transfected for 48 hours, selected using growth medium containing 400 µg/ml G418 and resistant cells were maintained in medium containing 200 µg/ml G418.
L-cell aggregation assay
L-F and L-K cell lines and non-transfected cells were tested for cellular adhesion activity in standard rotation and hanging drop suspension cultures (Kubota et al., 1989; Kubota et al., 1999; Thoreson et al., 2000). Production of Bves protein in transfected cells was confirmed by immunochemical analysis with Bves and FLAG antibodies. The ratio of number of cells in aggregates to total cell number was used as the measure of aggregation (Nakada et al., 2000). In addition, the same aggregation assays were used to determine the ability of Bves-transfected cells to sort form non-transfected cells. In this case, non-transfected L-cells were labeled with DiI (Molecular Probes) using the manufacturers instructions, mixed with non-labeled L-K cells and allowed to aggregate as described above. Samples were stained with DAPI in order to visualize the total number of cells in aggregates and the contribution of non-transfected L-cells was visualized by DiI. In addition, the same assay was conducted in the presence of blocking antibody to determine whether aggregation is directly related to the function of Bves in L-cells. Two different concentrations of antibody were used (1:100 or 1:50) in the cell suspension and were analyzed as described above.
Wound healing assay
RECs were grown to confluence in chamber slides. The resulting epithelium was wounded by tearing the epithelial sheet with a pipette tip across the slide (Nobes and Hall, 1999). The culture was gently washed with PBS and incubated in normal growth medium for 8 hours. Bves and E-cadherin protein localization were detected by epifluorescence or confocal microscopy after processing for immunocytochemistry.
Golgi disruption
Subconfluent cultures of REC cells were treated with Brefedin A (BFA)(Sigma) at 50 µg/ml in growth medium for 6 hours. This treatment is known to disrupt Golgi structure (Godi et al., 1998; Yamaji et al., 2000). Cells were then processed for immunofluorescence microscopy using anti-Bves and anti-ß-COP, a standard marker of the Golgi. The purpose of this experiment was to determine disruption of the Golgi apparatus altered the intracellular localization of Bves. Replicate, non-treated cultures served as controls.
Antibody blocking studies
Anti-Bves D033 and B846 and were affinity purified and exhaustedly dialyzed against DMEM. Anti-LEK1 (Goodwin et al., 1999), a rabbit antiserum against a nucleoprotein, was also tested and had no effect in any assay when compared to controls. Antisera were added to culture medium in concentrations from 50 ng to 5 µg/ml and assayed for their ability to alter epithelial migration and epithelial-mesenchymal transition from chick PEO cultures and formation of epithelial sheets from individual RECs. Stage 18 chick embryos were removed using sterile technique and placed in a 35 mm dish with PBS. Under the microscope, the embryo was placed on its side, left side up. Fine forceps were used to remove the PEO from the arterial pole of the looping heart tube (Dettman et al., 1998) and cultured for 3-5 days with PEO culture medium in the presence or absence of antisera. Cultures were photographed and scored for migration of epithelial sheets and subsequent production of mesenchyme at the culture periphery (Mikawa and Gourdie, 1996; Dettman et al., 1998). For analysis of epithelial sheet formation, REC cultures were plated at subconfluent density in the presence of antisera or allowed to attach for 8 hours before antisera addition. Formation of epithelial sheets was monitored by phase microscopy.
In vitro transcription/translation
To determine whether Bves inserts into membranes, in vitro transcription/translation (Promega) in the presence or absence of canine pancreatic microsomes (Promega) was performed with full-length Bves cDNA and transmembrane domain-truncated (tm) Bves cDNA. Soluble and membrane-bound fractions were separated by centrifugation at 14,000 g. Radiolabeled proteins were solubilized in SDS sample buffer and analyzed by standard autoradiographic methods after electrophoresis on 7.5% PAGE with known molecular weight standards.
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RESULTS |
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Bves is an integral membrane protein
The previous data suggest that Bves has a dynamic subcellular distribution during vasculogenesis. To understand the molecular basis of Bves function in these events, computer modeling of the predicted amino acid sequence was conducted. These analyses (Erik et al., 1998) predict that Bves contains a short intracellular N terminus (amino acids 1-40), three transmembrane helices with short intervening loops (amino acids 41-139) and a long C-terminal extracellular domain (amino acids 140-387; Fig. 3A). Epitope mapping studies have mapped the N and C termini to the intra- and extracellular domains (D. E. R., unpublished). While our analyses and those of Andree et al. (Andree et al., 2000) identify the three transmembrane domains, no other protein motifs with any predicted function were found in the extracellular or intracellular domains. Still, there is a high degree of sequence homology among members of the Bves/popeye family in vertebrates.
The predicted transmembrane domains are a common feature of this family and potentially crucial to the function of Bves. To determine whether these domains regulate Bves insertion into cell membranes, in vitro transcription/translation analysis of Bves cDNA was performed in the presence or absence of canine pancreatic microsomes. When full-length Bves cDNAs were used as a template for transcription/translation in the absence of microsomes, a band of approximately 45 kDa was produced. When this reaction was carried out in the presence of microsomes, radioactive products were readily detected in the membrane fractions, confirming the work of Andree et al. (Andree et al., 2000). In addition, a slight upward shift in mobility of these proteins on SDS/PAGE was seen, suggesting a modification of the translated products (Schmidt-Rose and Jentsch, 1997). In constructs where the transmembrane domains were deleted, truncated protein was produced but not detected in the microsomal fraction (Fig. 3B) demonstrating that the region containing the predicted transmembrane helices is functional and plays a role in membrane insertion.
Having determined that Bves is a membrane protein and that it is localized to the cell periphery of epicardial epithelial cells (Fig. 1), we sought to determine whether Bves was confined to a specific membrane compartment in established epithelia. For these analyses, confocal microscopy was performed on immunostained epicardial cell (REC) cultures. Analysis of images obtained in the x-y axis revealed that Bves was present around the entire cell in confluent cultures (Fig. 3C). When the same images were viewed in the x-z axis, Bves staining was confined to the lateral portion of the cell membrane (Fig. 3C). Colocalization studies with E-cadherin (Fig. 3D), which accumulates in the lateral membrane compartment (Grindstaff et al., 1998), showed extensive but not complete overlap with Bves. Similar analyses with anti-connexin 43 showed little colocalization with Bves (data not shown).
In addition to the cell membrane staining observed in vivo and in vitro, an immunoreactive perinuclear spot was seen in RECs and delaminated vasculogenic mesenchyme. This staining was observed with both anti-Bves antisera and was competed for by immune peptide. Colocalization studies with Bves antibodies and immunochemical markers of cell organelles were conducted using REC cell lines. As seen in Fig. 3, markers of the Golgi such as anti-ß-COP showed extensive overlap with Bves antibodies. To corroborate these data and confirm that Bves protein was present in the Golgi region, REC cells were treated with Brefeldin A, a compound known to disrupt Golgi structure (Wan et al., 1992; Yamaji et al., 2000). Anti-ß-COP staining of Brefeldin A-treated cells confirmed Golgi disruption (Fig. 4). When the same cells were viewed with Bves antibodies, perinuclear staining was eliminated, indicating the intracellular deposition of Bves. Interestingly, membrane deposition of Bves in these cells was unaffected by this treatment and epithelial sheets remained intact. In addition, in vivo immunolocalization of ß-COP and Bves co-exist in the perinuclear region of delaminated mesenchymal cells in developing chick heart (Fig. 1F-H). These results suggest that the accumulation of immunoreactive Bves in an intracellular compartment.
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A newly proposed adhesion zipper model for initiation of cell adhesion (Vasioukhin et al., 2000) predicts that molecules involved in this process should localize to points of cell contact, and that E-cadherin is one of the first molecules to participate in this event. To determine whether Bves accumulates at points of cell/cell contact, RECs were grown to confluence and an in vitro model of wound healing was employed (Nobes and Hall, 1999). In addition, the accumulation of Bves was compared with that of E-cadherin during this process. As seen in Fig. 5J, Bves appeared at the cell surface in a punctate pattern and accumulated at points of contact and cell borders prior to the arrival of E-cadherin. As the wound area healed, overlap of Bves and E-cadherin staining was again apparent around the entire cell. The presence of Bves in structures that mediate cell adhesion prior to the accumulation of E-cadherin suggests a role for Bves in the initial phases of intercellular adhesion during development.
Bves is an adhesion molecule
The results described above lead us to the hypothesis that Bves is a cell adhesion molecule. To test this hypothesis, the L-cell aggregation assay for cell adhesion was employed (Kubota et al., 1989; Nakada et al., 2000; Thoreson et al., 2000). Bves-transfected and non-transfected cells were used to perform aggregation assays under Ca2+-free conditions. As seen in Fig. 5A, control L-cells do not aggregate and are seen as individual cells in these assays. Stable transfection of Bves constructs into L-cells resulted in the conspicuous formation of cell aggregates and promotion of cell adhesion significantly over control non-transfected cells (Fig. 6). We next tested whether the presence of Bves on interacting cells was required for cell adhesion by mixing equal numbers of transfected and non-transfected cells. Cell sorting was monitored by labeling non-transfected cells with DiI and visualizing all cells with DAPI staining. Three examples are given and demonstrate that Bves-transfected cells were readily detected in aggregates while non-transfected DiI-labeled cells were excluded from cell clusters (Fig. 6D-F). Fig. 6E shows an aggregate with over 50 cells, one of which is DiI labeled. These data demonstrate that only Bves-producing cells aggregate in this assay and suggest that while Bves has no homology to any known adhesion molecule, it acts as a Ca2+-independent, homophilic cell adhesion molecule. To confirm these results, the aggregation assay was conducted in the presence of blocking antibody to disrupt Bves function. In this case, we have detected a large reduction in the size and number of aggregates (Fig. 6G,H and graph) in a manner dependent on antibody concentration, supporting the hypothesis that Bves expression results in cell aggregation.
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DISCUSSION |
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Bves is a novel cell adhesion molecule
Computer, biochemical and morphological analyses show that Bves is a membrane protein and that it mediates cell adhesion in a Ca2+-independent manner. Still, Bves does not fall into any known family of cell adhesion molecule. Computer predictions and epitope mapping data from our laboratory (D. E. R., unpublished) demonstrate that the long C-terminal domain is extracellular and separated from the short intracellular N terminus by three transmembrane helices that are necessary for membrane insertion. Transmembrane sequences are found in all Bves/popeye transcripts, except popeye 3b, and suggest that they are a conserved feature of this family. To our knowledge, the present study is the first report of an adhesion molecule with three transmembrane domains. In addition, the extracellular C terminus of Bves does not contain any of the domains or motifs characteristics of other established adhesion molecules such as immunoglobulins, fibronectins, epidermal growth factor or cadherin repeats (Kreis and Vale, 1993; Suzuki, 1996; Gumbiner, 1996). While no obvious similarities to other adhesion molecules exist, there is remarkable sequence conservation of this extracellular domain in all Bves/Popeye molecules (Reese et al., 1999; Reese and Bader, 1999; Andree et al., 2000). Thus, while it is clear that Bves can mediate cell adhesion, the molecular basis of this function has yet to be determined. Future analyses using modifications of the Bves molecule should identify the domain/domains required for its adhesive activity and provide evidence for novel molecular structures that regulate the basic property of cell adhesion.
Bves and its potential role in cell adhesion
Our data provide direct evidence that Bves is a Ca2+-independent cell adhesion molecule. In addition, Bves accumulates at points of cell contact prior to the appearance of E-cadherin which is thought to be one of the earliest markers of cell adhesion (Yonemura et al., 1995; Adams et al., 1998). Thus, we postulate that it plays an early role in cell adhesion. Vasioukhin et al. have proposed an adhesion zipper model to describe the role of filopodia in the mechanism of intercellular adhesion in developing epithelia (Vasioukhin et al., 2000). In this model, filopodia slide along each other upon contact and project into the opposing cell membrane. Embedded filopodia are stabilized by clusters of adherent junction molecules, such as E-cadherin and their binding proteins, to initiate the cell adhesion. With our current data showing that Bves mediates cell adhesion, its presence at points of cell contact and adjoining cell boundaries prior to E-cadherin suggests an early role in the adhesion process. In other studies, we show that recombinant Bves is produced and accumulates in an intracellular positions of fibroblasts but is not trafficked to the membrane even at confluence (D. E. R., unpublished). In contrast, when the same molecule is transfected into epithelial cells, Bves is readily detected at the membrane. These studies indicate that contact alone is not a sufficient signal to initiate membrane trafficking of Bves. Thus, we posit that a signaling system that regulates adhesion and specific to certain cell lineages and developmental stages is required for trafficking of Bves to the membrane and its subsequent role in adhesion. The signals that regulate Bves trafficking to the membrane during the process of epithelialization during development and its removal during epithelial/mesenchymal transition are currently under investigation.
The potential role of Bves in coronary vessel development
Our data show that Bves can act as an adhesion molecule. In addition, treatment of the PEO with antibody B846 clearly disrupts epicardial migration, as well as the epithelial sheet formation in the REC cell culture, suggesting that Bves has a function in cell adhesion and/or migration in coronary vasculogenesis. Although we do not currently know the biochemical mechanisms that underlie antibody disruption of Bves function, it is possible that B846 binding to Bves may stabilize or immobilize the protein in the membrane, leading to inhibition of cell movement and mesenchyme formation from the PEO.
Our current hypothesis is that Bves plays a role in the regulation of cell adhesion during coronary vessel development. As the epicardial epithelium migrates over the heart, Bves, along with other cell adhesion molecules, is present at the cell surface, maintaining epithelial integrity while permitting migration. Specific cells in the epicardium respond to signals from cardiac muscle, quite possibly mediated through a downstream target of FOG2 (Tevosian et al., 2000), that remove Bves from the cell surface. As mesenchymal cells undergo their directed migration to developing vascular beds, Bves expression is still maintained but accumulates in a perinuclear region. Once vasculogenic mesenchyme reaches forming channels within the myocardium, signals from the endothelium cause smooth muscle progenitors to traffic Bves to the membrane again where it once more participates in adhesion between differentiating smooth muscle cells. Although many questions remain concerning Bves trafficking and function, its potential for regulation of cell adhesion and migration during the generation of coronary vessels suggest a critical role in this vasculogenic process.
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ACKNOWLEDGMENTS |
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