Bves: prototype of a new class of cell adhesion molecules expressed during coronary artery development

Aya M. Wada*, David E. Reese* and David M. Bader{ddagger}

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

{ddagger}Author for correspondence (e-mail: david.bader{at}mcmail.vanderbilt.edu)

Accepted March 19, 2001


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bves is a protein expressed in cells of the developing coronary vascular system, specifically in the proepicardium, migrating epithelial epicardium, delaminated vasculogenic mesenchyme and vascular smooth muscle cells. Here, we show that Bves protein undergoes a dynamic subcellular redistribution during coronary vessel development. Bves is a membrane protein with three predicted transmembrane helices, an extracellular C terminus and an intracellular N terminus, and is confined to the lateral membrane compartment of epithelial cells. When epicardial cells are dissociated into single cells in vitro, Bves accumulates in a perinuclear region until cells make contact, at which time Bves is trafficked to the cell membrane. Bves accumulates at points of cell/cell contact, such as filopodia and cell borders, before the appearance of E-cadherin, suggesting an early role in cell adhesion. While Bves shares no homology with any known adhesion molecule, transfection of Bves into L-cells readily confers adhesive behavior to these cells. Finally, Bves antibodies inhibit epithelial migration of vasculogenic cells from the proepicardium. This study provides direct evidence that Bves is a novel cell adhesion molecule and suggests a role for Bves in coronary vasculogenesis.

Key words: Heart, Blood vessels, Cell adhesion, Chick, Bves


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation of coronary vessels is one of the most complex and intricate processes yet described in cardiac development. It begins with the recruitment of mesothelial cells from the liver diverticulum to form the proepicardial organ (PEO) that migrates as an epithelium to and over the surface of the heart to form the epicardium (Manasek, 1968; Manner, 1993; Mikawa and Gourdie, 1996; Gittenberger-DeGroot, 1998). Next, a subpopulation of epicardial cells undergoes an epithelial-mesenchymal transition and migrates away from the epicardium through a connective tissue space and then into the myocardium (Viragh and Challice, 1981; Mikawa and Gourdie, 1996; Dettman et al., 1998). Cell lineage studies show that a subset of these mesenchymal cells differentiate into endothelial cells that line developing vascular channels, while other mesenchymally derived cells differentiate into either vascular smooth muscle cells or fibroblasts (Mikawa and Gourdie, 1996; Dettman et al., 1998). The vascular channels link and remodel to form arteries and veins that join with the proximal aorta and coronary sinus to complete formation of the coronary system that provides the blood supply to the heart muscle itself.

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 {alpha}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.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunofluorescence with tissue sections and cells
Immunofluorescent localization procedures have been described in detail (Reese et al., 1999). Polyclonal antisera DO33 (Reese et al., 1999) and newly developed B846 (DPTLNDKKVKKLEPQMS; amino acids 266-283 of the extracellular domain of mouse Bves) were generated in rabbits (Biosynthesis) followed by affinity purification with peptide-conjugated column (Reese et al., 1999). These sera have been shown to reactive with Bves protein produced from the cloned mRNA. In addition, both sera react in immunofluorescence analysis in a manner competed by immunopeptide (Reese et al., 1999; data not shown). Throughout the text, we refer to this immunoreactive Bves as Bves. Dilution of primary antibodies: affinity-purified DO33 and B846 anti-Bves, 1:100 for cell culture and 1:300 for tissue sections; monoclonal antibody (mAb) anti-smooth muscle {alpha}-actin (Sigma), 1:200; mAb MF20, 1:1; mAb anti-E-cadherin (Transduction Laboratory), 1:200; anti-connexin 43 (Sigma), 1:200; anti-ß-COP (Sigma), 1:25; anti-PAN-cadherin (Sigma) 1:200; anti-Golgi 58K (Sigma), 1:50. Secondary antibodies against rabbit or mouse IgG were conjugated to either Cy2 or Cy3 (Jackson Immunoresearch), used at suggested dilutions, and visualized by epifluorescent and confocal microscopes.

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 Dulbecco’s modified Eagle’s 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 manufacturer’s 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 ({delta}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.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bves has a dynamic subcellular distribution during coronary vessel formation
We have developed a series of new antisera to conserved extracellular regions of human, mouse and chicken Bves to analyze protein trafficking during development and cell differentiation. (The location of epitopes for the two Bves antibodies used in this study is given in Fig. 3.) Although our previous studies identified Bves in the epicardial epithelium, delaminated mesenchyme and derived coronary vascular smooth muscle, our new antisera clearly defined subcellular distribution during development. A section through the atrioventricular junction of the chick heart shows the production of mesenchyme from the epicardium (Fig. 1A). Using antiserum B846, Bves is detected in the lateral boundaries of epicardial epithelium (Fig. 1B,C, arrows). In the epicardial epithelium, PAN-cadherin staining is detected at cell-cell boundaries where Bves localization is also detected (Fig. 1D). The same colocalization was detected with E-cadherin and Bves (data not shown), suggesting that Bves is present at cell boundaries in epicaridal epithelium. As cells undergo epithelial-mesenchymal transition, Bves distribution abruptly changes so that it is confined to a perinuclear spot and is not present in detectable levels at the cell surface (Fig. 1B,E,F, arrowheads). Bves localization was excluded from DAPI-stained nuclei, but is co-localized with ß-COP (data not shown) and Golgi 58K antibodies. This suggests that Bves subcellular distribution is changed to the perinuclear region in cells undergoing epithelial-mesenchymal transition (Fig. 1C-H). More extensive EM localization of Bves to cisternae of the Golgi will be necessary to prove that this perinuclear staining is conclusively present in the Golgi. As coronary vasculogenesis proceeds, Bves-positive cells accumulate in areas of developing vessels (Fig. 2A,B). The higher density of Bves-positive cells and their eventual differentiation into smooth muscle illustrates the recruitment of vasculogenic mesenchyme to developing arteries. The recruitment of Bves-positive mesenchyme to developing vessels is more obvious in 3D composed confocal immunofluorescence with Bves (Fig. 2B) where Bves-positive cells (red) accumulate around developing arteries (shown in green, {alpha}-smooth muscle actin).



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Fig. 3. Bves is an integral membrane protein. (A) Schematic representation of Bves protein structure shows the predicted structure of Bves and the relative positions of antibody epitopes. (B) TNT reactions were conducted in the presence and absence of dog pancreatic microsomes. Both full-length and truncated Bves ({delta}tm), which lacks the putative transmembrane domain, were used. The first lane shows that truncated Bves is produced and present in the lysate, but it is not captured in the microsomal fraction (lane 2). In contrast, full-length Bves, produced in the absence of microsomes (lane 3), is captured by microsomes (lane 4) and shows a slight increase in mobility. (C) Confocal analysis of Bves distribution in REC epithelium is presented. Analysis of the x-y axis shows the peripheral distribution of Bves. x-z axis analysis demonstrates that Bves is located in the lateral cell compartment. (D) Bves (red) and E-cadherin (green) staining show the colocalization (yellow) of the two proteins in the membrane.

 


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Fig. 1. Subcellular distribution of Bves in the epicardium and mesenchyme. (A) A section through the developing atrioventricular junction stained with D033 shows the localization of Bves (red, Cy3) in the developing epicardium (epi) and delaminated vasculogenic mesenchyme. MF20 (green, Cy2) shows the position of the myocardium. (B) A higher power view shows Bves localization in the epicardium (B846) in the periphery of epithelial cells (arrows) while Bves appears as a spot in mesenchyme (arrowhead, B,E). (C-E) Bves (red) localization in epicardial epithelium is seen co-labeled with PAN-cadherin antibody (green). DAPI (blue) marks nuclei. Localization of Bves at the cell-cell boundaries coincided with cadherin (C,D, arrows). Nuclear co-localization shows the ‘spot’ is in a perinuclear region of the delaminated mesenchyme (E, F-H, arrowhead). (F-H) Bves-positive mesenchymes in the subepicardial space. Colocalization of Golgi 58K protein antibody (green) with Bves antibody (red) reveals the Bves-positive ‘spot’ is confined the perinuclear region (F,G are merged in H).

 


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Fig. 2. Localization of Bves during coronary vessel development. Bves-positive mesenchymal cells accumulate in the tunica media of developing arteries. Anti-smooth muscle actin (green) shows differentiated smooth muscle cells. (A) Delaminated mesenchyme from the epicardium accumulates around a developing vessel (v). (B) Bves-positive cells differentiated into vascular smooth muscle (Reese et al., 1999) and additional Bves-positive cells continue to be recruited to the vessel shown in the 3D composed image. (C-E; C, Bves; D, merge; E, smooth muscle actin) Bves has two staining patterns in cells of the developing artery. Outer, Bves-positive cells have the characteristic punctate staining of mesenchymal cells, while Bves is broadly distributed in differentiated smooth muscle cells of the inner lamellae. Arrows distinguish the boundary between the two staining patterns. (F) Later, all smooth muscle cells of the tunica media of coronary vessels have a peripheral staining pattern.

 
Careful examination of developing vessels reveals two pattern of Bves staining in the arterial wall (Fig. 2C-E). Bves remains as a punctate spot in cells of newly forming outer lamellae, while it has a more widely distributed pattern in smooth muscle cells of older, more differentiated inner lamellae and perinuclear staining is lost or greatly diminished. Later in coronary vessel development, Bves-positive cells are found exclusively in the tunica media of arteries (Reese et al., 1999). Bves staining is broadly distributed in these smooth muscle cells, outlining their characteristic ‘corkscrew’ appearance (Fig. 2F). These data show that the subcellular distribution of Bves changes with the phase of coronary vessel development.

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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Fig. 4. Bves localization in the Golgi. Immunofluorescent microscopy for Bves (Cy3, red) and ß-COP (Cy2, green) localization in REC cells reveals extensive overlap in the Golgi and additional Bves staining at the membrane top. Treatment of cell lines with BFA diminishes Golgi staining but Bves at the membrane is not disturbed bottom.

 
Bves is an early marker of cell adhesion
Changes in Bves subcellular distribution correlate with changes in cell adhesion and motility during coronary vasculogenesis. We characterized Bves trafficking by plating RECs at low density and allowing cells to reach confluence. In single cells, Bves was not present at the cell membrane in detectable amounts but was present in the perinuclear region. (Fig. 5A, arrowheads). As cells contacted each other, Bves was again seen at the cell membrane and was present along cell borders (Fig. 5A). Co-labeling with anti-E-cadherin showed that Bves accumulated at the cell membrane before E-cadherin (see below). Bves staining was absent from the free surfaces of cells during sheet formation but as cells became highly confluent, Bves, as well as E-cadherin, were detected in the plasma membrane around the entirety of the cell (Figs 4, 5). Cytoplasmic accumulation of Bves was greatly diminished in mature epithelial sheets (Fig. 4A). These results show that Bves has a dynamic subcellular distribution during formation of epithelial sheets.



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Fig. 5. Bves localization during epithelium formation. (A) REC culture during epithelial formation (A) and at confluence (B). Prominent staining at the periphery of single cells and at the free surfaces of cells is absent (A,C, arrowhead). As the cells become confluent and form the epithelial sheet, uniform cell membrane localization is observed (B). Higher power images (C-F) show the deposition of Bves during sheet formation. Note that Bves localization is detected at the point of cell contact or newly made cell-cell boundaries (D,E, arrows). As the epithelial sheet forms, Bves accumulates around the entire cell surface. In mature epithelial sheets, cytoplasmic staining is greatly diminished (B,F). (G-I) Blocking antibody alters the generation of epithelium in REC. REC form epithelial sheets in Ca2+-free conditions (G). When Bves blocking antibody was added at 1:100 dilution in the medium, semi-confluent REC cells were unable to form the epithelial sheet (H). Higher magnification of treated culture is given in I. (J-K) Bves accumulates at points of cell contact and cell borders prior to E-cadherin. The leading edges of REC epithelial sheets during wound healing in vitro are shown 8 hours after injury. (J) Anti-Bves (B846) is detected in cell processes, punctuate structures along the cell/cell boundaries and as continuous borders between cells (arrows, J,L). (K) E-cadherin in the same cells is broadly distributed in the cytoplasm and at low levels in processes and at cell edges. (L) Merged image of J and K.

 
We next tested the ability of anti-Bves antisera to alter the generation of epicardial sheets in this system. REC cells were grown in the Ca2+-free condition to avoid the effect of Ca2+-dependent adhesion molecules such as E-cadherin. Cells at semi-confluence density were then treated with blocking antibody B846. Control cultures produced confluent epithelial sheet during this time. In contrast, after overnight incubation with this antibody, generation of epithelial sheets dramatically inhibited (Fig. 5G-I). If confluent epicardial sheets were allowed to form prior to antibody treatment, anti-Bves B846 did not disrupt the epicardial sheet. These results show that the blocking antibody can alter the generation of epithelial sheet.

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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Fig. 6. Transfection of full-length Bves promotes aggregation in L-cells. L-cells were transfected with full-length Bves (B, L-F cells) or full-length Bves with an engineered Kozak sequence (C, L-K cells). Aggregation assays were performed on transfected and non-transfected cells as described in Materials and Methods and the degree of adhesion was calculated. Non-transfected cells did not aggregate (A). Graphs show that both L-F cells and L-K cells gained adhesive activity significantly over controls. (D-E) Bves-expressing cells are distinct from the non-transfected cells. Equal numbers of non-transfected L-cells were labeled with DiI, and Bves-transfected L-K cells were mixed and allowed to aggregate. Cells were stained with DAPI to visualize all cells. In the three examples given, note that aggregates are composed almost exclusively of transfected cells. (G,H) Promotion of aggregation in L-K cells was reduced in the presence of blocking antibody. When blocking antibody was added at 1:100 dilution to the cell suspension, reduction of aggregates were observed (see graph). Increase of antibody concentration (1:50 dilution) results in decrease in aggregates (H, graph) compared with aggregates without blocking antibody (G), suggesting that aggregation of L-cells results in expression of Bves. The ratio of aggregate to total cell is given with the s.e.m.

 
Anti-Bves (B846) inhibits epicardial sheet movement and subsequent epithelial-mesenchymal transition
To examine the possible role of Bves during coronary vasculogenesis, we sought to disrupt Bves function using blocking antibodies with PEO explant cultures. PEO was dissected out from chick embryos at HH stage 18 and placed in a culture dish containing M199 medium. Explants were allowed to attach to the culture dish by overnight incubation, and at this point, PEO cells begin to spread out as an epithelium. In some cultures, blocking antibody was added to the explants and incubated for the next 48 hours. Others were maintained without antibody. As previously shown (Dettman et al., 1998), cells in control cultures first migrate as an epithelial sheet from the PEO then undergo mesenchyme formation at the edge of the advancing sheet (seen as a light fringe in Fig. 7). When antibody B846 was added to the culture medium, epicardial sheet migration was completely blocked and the PEO remained as a coherent structure at this concentration of antibody. Individual cells were counted at the periphery of each PEO in control and treated groups and showed a drastic reduction in mesenchyme production with addition of B846 (Fig. 7, graph). Anti-Bves D033 had no effect on epicardial sheet migration or epithelial-mesenchymal transition during the 72 hours of culture. Thus, while the molecular mechanism underlying antibody inhibition of Bves function is currently unknown, these data suggest a role for Bves in coronary vasculogenesis.



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Fig. 7. Bves antibody B846 blocks epithelial migration of the PEO. Control untreated PEOs show epithelial migration and mesenchyme formation from the explant (top left). Epithelial-mesenchymal transition is seen as the light fringe at the edge of the culture. Treatment with B846 blocks epithelial migration (top right). The number of mesenchymal cells that migrate out from the PEO was counted after DAPI staining (graph). Forty individual PEO samples were counted for control and treated groups.

 

    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bves or Bves-like molecules are expressed in multiple cell types
Our studies detect Bves protein in cells of the developing coronary vascular system. We have recently developed new antisera that more precisely localize Bves subcellular distribution in vivo. These data reveal that Bves or Bves-like molecules are more widely expressed, being detected in cardiac, skeletal and specific populations of smooth muscle myocytes, brain ependima, intestinal epithelium, tracheal epithelium, specific pancreatic ducts, developing ectoderm and various epithelial cell lines (this study; D. E. R., A. M. W., V. Soukoulis and D. M. B., unpublished). Interestingly, Bves has the same membrane labeling pattern in all of these cell types suggesting a common function in a variety of cell types. The recent study of Andree et al. (Andree et al., 2000) has demonstrated that at least three murine genes encode multiple RNAs and that mouse bves is popeye1a. While in situ hybridization analyses show enrichment of specific popeye1-3 transcripts in cardiac and skeletal muscle, their data and our own show that expression of these gene products, specifically bves/popeye1a, is widespread in the developing embryo. Thus, while the assignment of each mRNA with each protein product is not yet complete, it appears that bves/popeye is an emerging gene family and that Bves or Bves-like molecules will have a role in cell adhesion in a broad range of cell types.

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.


    ACKNOWLEDGMENTS
 
We thank Drs Brigid Hogan, Steve Hanks, Al Reynolds and Maureen Gannon, and all the members of our laboratory for their helpful comments. This work was supported by an NIH grant (HL 63325).


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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