ABSTRACT
We have examined the role of cell-cell and cell-extracellular matrix (ECM) interactions during mesoderm differentiation and migration at the primitive streak of the mouse embryo with the use of function-perturbing antibodies. Explants of epiblast or mesoderm tissue dissected from the primitive streak of 7.5- to 7.8-day mouse embryos were cultured on a fibronectin substratum in serum-free, chemically defined medium. After 16-24 hours in culture, cells in explants of epiblast exhibited the typical close-packed morphology of epithelia, and the tissue remained as a coherent patch of cells that were shown to express transcripts of the cytokeratin Endo B by in situ analysis. In contrast, cells in explants of primitive streak mesoderm exhibited a greatly flattened, fibroblastic morphology, did not express Endo B transcripts, and migrated away from the center of the explant.
As epiblast cells in vivo undergo the epithelial-mes-enchymal transition at the primitive streak, they cease expressing the prominent calcium-sensitive cell adhesion molecule E-cadherin (uvomorulin, Cell-CAM 120/80). We asked whether the loss of E-cadherin expression was a passive result of differentiation or if it might play a more causative role in mesoderm differentiation and migration. Culture with function-perturbing antibodies against E-cadherin caused cells within epiblast explants to lose cell-cell contacts, to flatten, and to assume a mes-enchymal morphology; they were also induced to migrate. Anti-E-cadherin antibodies had no effect on explants of primitive streak mesoderm. In immunofluo-rescence studies, anti-E-cadherin-treated epiblast cells ceased to express SSEA-1, a carbohydrate moiety that is lost as mesoderm differentiates from the epiblast in vivo, and they also ceased to express E-cadherin itself. In contrast, these cells began to express the intermediate filament protein vimentin, a cytoskeletal protein characteristic of the primitive streak mesoderm at this stage of development.
As epiblast cells differentiate into mesoderm, their predominant adhesive interactions change from cell-cell to cell-substratum. Therefore, we also investigated the adhesive interactions between primitive streak tissues and extracellular matrix (ECM) components. Epiblast explants adhered well to fibronectin, more poorly to laminin and type IV collagen, and not at all to vitronectin. In contrast, mesoderm explants attached well to all these proteins. Furthermore, epiblast, but not mesoderm, displayed an anchorage-dependent viability in culture. After anti-E-cadherin treatment, epiblast cells that had assumed the mesenchymal morphology did attach to vitronectin, another characteristic shared with primitive streak mesoderm. Adhesion of epiblast to fibronectin and of primitive streak mesoderm to fibronectin, vitronectin, laminin, and type IV collagen was completely blocked by incubation with a broad-spectrum polyclonal serum, anti-ECM receptor (anti-ECMR) antiserum, which recognizes β1 and β3 integrins. Anti-ECMR-treated mesodermal explants recovered and attached, spread, and migrated normally after antibodies were removed. In addition, an antibody specific for α6β1 integrin, which mediates adhesion to laminin, selectively blocked attachment of mesoderm to laminin but not to fibronectin, indicating that α6β1 is a major laminin receptor for these cells.
We conclude that disruption of E-cadherin function in mammalian epiblast cells at the primitive streak in vitro causes them to acquire a phenotype characteristic of mesoderm, and we propose that similar mechanisms act during mesoderm differentiation in the intact embryo. Our results also show that the cell-substratum adhesion of primitive streak tissues is mediated by the integrin superfamily of receptors and that developmentally regulated changes in cell-ECM adhesion accompany the epithelial-mesenchymal transition at the mammalian primitive streak.
INTRODUCTION
Adhesive interactions among cells and between cells and their surrounding extracellular matrix (ECM) are important during morphogenetic processes, including the cell migrations occurring during embryonic development (Edelman, 1988; Takeichi, 1991; Hynes and Lander, 1992). Changes in cell-cell and cell-matrix adhesion accompany the ingression of primary mesenchyme cells during gastrulation in the sea urchin (Fink and McClay, 1985; Burdsal et al., 1991). In avian embryos, microinjection of antibodies against the fibronectin receptor, or of peptides mimicking the cellbinding domain of fibronectin, perturbs neural crest migration (Boucaut et al., 1984; Bronner-Fraser, 1985). In addition, cell interactions with an intact basal lamina containing crosslinked collagen are necessary for the completion of the cell movements of gastrulation in the sea urchin embryo (Wessel and McClay, 1987). In urodele amphibian embryos, a fibrillar ECM composed of fibronectin and laminin present on the basal surface of the blastocoel roof directs mesodermal cell migration during gastrulation (reviewed by Johnson et al., 1992). Also in amphibian embryos, microinjection of cell-binding domain peptides inhibits cellular movements during gastrulation (Boucaut et al., 1984; Darribere et al., 1988). These studies demonstrate the vital role that changes in cell-cell adhesion and cell-ECM interactions play in morphogenetic processes during development.
At 7 days of development in the mouse, cells present in the posterior epiblast (embryonic ectoderm) lose contacts with other cells in that region and form the primitive streak, the site of gastrulation. This loss of cell-cell contacts is accompanied by the loss of expression of cell-cell adhesion molecules. For example, in the mouse embryo, E-cadherin (uvomorulin, Cell-CAM 120/80) is expressed in the epiblast but is not expressed in primitive streak mesoderm; in the chick embryo, N-CAM and E-cadherin (L-CAM) are present in the epiblast but disappear from the primitive streak as mesoderm ingresses (Cunningham and Edelman, 1990; Damjanov et al., 1986).
As gastrulation continues, mesoderm cells actively migrate through and away from the primitive streak (Nakatsuji et al., 1986) and spread into embryonic regions anteriorly as two sheets, or mesodermal wings; mesodermal cells also migrate posteriorly into extraembryonic regions where they will contribute to extraembryonic membranes (Snell and Stevens, 1966; Lawson et al., 1991; reviewed by Cruz and Pedersen, 1991; and Lawson and Pedersen, 1992). Mesodermal migration takes place in the extracellular space between the epiblast and the visceral endoderm, where fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan have been detected by immunocytochemical studies (Wartiovaara et al., 1979; Adamson and Ayers; 1979; Leivo et al., 1980; Leivo, 1983). On the basis of these observations, in addition to functional studies in other embryonic systems, it appears that mesoderm cells use these ECM components as a migratory substratum during gastrulation. However, despite numerous descriptive studies (Spiegelman and Bennett, 1974; Solursh and Revel, 1978; Franke et al., 1983; Nakatsuji et al., 1986; Hashimoto et al., 1987; Hashimoto and Nakatsuji, 1989), the molecular mechanisms of gastrulation in the mouse embryo, including the factors that control ingression and migration, have yet to be identified.
To address these questions directly, we have developed a system for the culture of mouse primitive streak tissues and have used it to characterize the adhesive interactions of epiblast and primitive streak mesoderm. Making use of function-perturbing antibodies, we have studied the role of E-cadherin function during mesoderm differentiation. We have also examined the cell-substratum interactions of epiblast and mesoderm during gastrulation. The integrin superfamily of receptors (Hynes and Lander, 1992) are candidate molecules for mediating adhesion of mesoderm to ECM proteins; therefore, we have examined the attachment of explants of primitive streak tissues to purified ECM components in vitro and have made use of function-perturbing antibodies against integrins to determine whether these molecules are functional receptors in this system.
MATERIALS AND METHODS
Mice
Mice used in this study were Dub:ICR (originally obtained from Dominion Laboratories, Inc., Dublin, VA) and CD1:ICR (originally obtained from Charles River Laboratories, Wilmington MA). Both stocks were maintained as closed colonies in our animal facility. For all morphological results presented, tissues from Dub embryos were used, unless otherwise indicated. Animals were maintained on a cycle of 14 hours light/10 hours dark (light period, 6.00 a.m. to 8.00 p.m.). All embryos were obtained after natural mating. Noon of the day the vaginal plug was detected was designated 0.5 day of gestation.
Dissection and culture of explants
Primitive streak tissues were dissected from 7.5- to 7.8-day embryos. Mesoderm was dissected from embryos in which the primitive streak had advanced to approximately the distal tip of the egg cylinder (and mesoderm had migrated to the midline and partially into the anterior half of the egg cylinder in the mesodermal wings). Embryos that had developed to the late streak-stage (i.e., those in which head folds were present) were not used. The posterior halves of the embryonic region of each embryo were obtained by manual dissection with glass needles, as shown in Fig. 1. Separate epiblast and mesodermal tissues were isolated according to the protocol of Svajger and Levak-Svajger (1975). Briefly, the dissected posterior halves were placed in a solution of 0.5% trypsin (Calbiochem, San Diego, CA) and 2.5% pancreatin (Sigma Chemical Co., St. Louis, MO) in Ca2+- and Mg2+-free phosphate-buffered saline (PBS, 140 mM NaCl, 1.5 mM KH2PO4, 3 mM KCl, and 8 mM Na2HPO4) for 15 minutes at 4°C. For further dissection and stopping of enzymatic activity, the egg cylinders were transferred into medium containing serum (FM-II, Spindle 1980). The visceral endoderm was removed from the egg cylinders and discarded. Epiblast and mesoderm were teased apart with glass needles. Under a stereomicroscope the sheet of loosely connected mesodermal cells was easily distinguished from the thicker, more tightly coherent sheet of epiblast. Small tissue explants were transferred to prepared wells in 16-chamber slides (Nunc, Inc., Naperville, IL).
Dissection of egg cylinder. Dashed lines represent cuts made with glass needles under the dissecting microscope. The first horizontal cut separates extraembryonic regions, from embryonic regions and the second vertical cut separates the anterior embryonic region from the posterior region, which contains the primitive streak. EPC, ectoplacental cone; M, primitive streak mesoderm; VE, visceral endoderm. Modified from Tam (1990). Bar, 100 μm.
Dissection of egg cylinder. Dashed lines represent cuts made with glass needles under the dissecting microscope. The first horizontal cut separates extraembryonic regions, from embryonic regions and the second vertical cut separates the anterior embryonic region from the posterior region, which contains the primitive streak. EPC, ectoplacental cone; M, primitive streak mesoderm; VE, visceral endoderm. Modified from Tam (1990). Bar, 100 μm.
Separate wells in 16-chamber slides were incubated with solutions of fibronectin (20 μg/ml), laminin (35 μg/ml), vitronectin (20 μg/ml), or collagen type IV (35 μg/ml) in PBS for 2 hours. Human plasma fibronectin, mouse type IV collagen, and laminin were obtained from Collaborative Research, Inc. (Bedford, MA). Vitronectin was obtained from Telios Pharmaceuticals (San Diego, CA). In a control experiment, wells were coated with Cell-takTM (Collaborative Research, Inc.), a formulation of the repeating decapeptide in the adhesive protein of a marine mussel that is L- dopa- and hydroxyproline-rich and does not contain the tripeptide Arg-Gly-Asp (RGD; Waite and Tanzer, 1981; Waite, 1983). Wells were then washed three times with PBS and explants were transferred to the prepared wells and cultured in approximately 130 μl of a serum-free, chemically defined medium. The defined medium (T + 2× AA; Spindle, 1980) was supplemented with 0.8 mg/ml adenosine, 0.85 mg/ml guanosine, 0.73 mg/ml cytidine, 0.73 mg/ml uridine, 0.24 mg/ml thymidine, 0.2 mg/ml insulin, Mito+ serum extender (which contains defined factors including epidermal growth factor, transferrin, insulin, endothelial cell growth supplement, triiodothyronine, hydrocortisone, progesterone, testosterone, estradiol-17B, selenium, and O-phospho-rylethanolamine [Collaborative Research Inc.]), 100 μg/ml strep-tomycin, and 100 Units/ml penicillin G; 4 mg/ml of bovine serum albumin was added just before use. Explants were cultured at 37°C in an atmosphere of 5% CO2 and 95% air.
Antibodies
Anti-E-cadherin antibodies (anti-Cell-CAM 120/80 antibodies), which block the activity of E-cadherin (Damsky et al., 1983; Richa et al., 1985), were purified from a rabbit polyclonal serum. Preimmune and nonimmune antibodies were used at the same concentrations. Anti-ECMR antibodies are function-perturbing goat polyclonal antibodies that recognize a broad range of integrins, including the β1 and β3 families (Knudsen et al., 1981; Damsky et al., 1982; Sutherland et al., 1988). Anti-ECMR antibodies were purified on immobilized protein-G (Pierce, Rockford, IL). Antibody concentrations were determined at OD280, using an extinction coefficient of 1.4 for a 1 mg/ml solution. Nonimmune goat antibodies were purified in an identical fashion. GoH3, an antibody known to inhibit the function of α6β1 integrins, was obtained from the Central Blood Transfusion Laboratory, Amsterdam, The Netherlands (rat monoclonal antibody against CDW49F; Sonnenberg et al., 1987, 1988, 1990). Other antibodies used in immunofluorescence studies were anti-vimentin (Sigma) and anti-SSEA-1 monoclonal antibodies (gift of D. Solter, Wistar Institute, Philadelphia, PA).
Antibody perturbation studies
Two or three explants were transferred to each prepared well of the 16-chamber slides. The percentage of explants that attached and spread on the various ECM components was determined by counting explants viewed at 16× magnification on an inverted microscope (Zeiss Inc., Germany), and explants were photographed under differential interference contrast (DIC) optics. To determine the function of E-cadherin in maintaining epiblast integrity, we cultured epiblast explants for 16 hours on a fibronectin substratum and then added anti-E-cadherin antibodies to a final concentration of 90 μg/ml in culture medium. Explants were cultured with anti-E-cadherin anitbodies for 4-6 hours and were then washed 3 times with PBS. Explants were cultured for an additional 12-30 hours (depending on the experiment) to ensure that anti-E-cadherin antibodies were no longer present on the cell surface. In control experiments, mesoderm explants were cultured with anti-E-cadherin antibodies, and epiblast explants were cultured with pre-immune or non-immune antibodies.
To determine the function of integrins in epiblast and mesoderm attachment, we added function-perturbing anti-ECMR antibodies at the beginning of the culture period. Mesoderm explants were examined after 4 hours with 75 μg/ml anti-ECMR antibodies. At this time explants were washed 3 times with PBS and further cultured for 12-16 hours in the absence of antibodies. The effect of the anti-ECMR antibodies on epiblast attachment was examined after 12-16 hours, because of the slower kinetics of epiblast attachment. In control experiments, epiblast and mesoderm explants were cultured in an equal concentration of nonimmune goat antibodies.
To test the ability of cells from anti-E-cadherin-treated epiblast explants to reattach to vitronectin and fibronectin, we cultured epiblast explants on fibronectin with anti-E-cadherin antibodies for 4-6 hours and then for an additional 12 hours without antibodies. To remove cells from the fibronectin substratum, we cultured anti-E-cadherin-treated epiblast explants with 100 μg/ml anti-ECMR antibodies, which caused more than 80% of the cells to round up in 2 hours and allowed the gentle detachment of cells. Cells were dislodged with a gentle stream of medium from a pipette tip and were transferred to fibronectin- or vitronectin-coated wells. Cells were cultured for 4 hours without antibodies and the fraction of cells that had attached and spread on fibronectin and vitronectin was determined by cell counts. In control experiments, mesoderm cells that had attached and spread on fibronectin were also treated with the anti-ECMR antibodies, as described for epiblast, and were transferred to fibronectin- or vitronectin-coated wells.
Labelling of probes and in situ hybridization
Sense and antisense constructs specific for Endo B were the kind gift of R. Oshima (La Jolla Cancer Research Center, La Jolla, CA). Probes were labeled with digoxigenin via in vitro transcription with SP6 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) and the (SP6/T7) Dig-RNA Labeling kit (Boehringer Mannheim). The labeled mRNAs were quantified on gels by comparison with known standards, and the sensitivity of antibody detection of mRNAs was determined and normalized on blots.
In situ hybridization was performed on explanted tissues according to a protocol modified from that of Conlon and Rossant (1992). Explants were fixed for 1 hour in 4% paraformaldehyde and dehydrated by sequential incubation with 25%, 50%, and 75% methanol diluted in Ca2+-Mg2+-free-PBS containing 0.1% Tween- 20 (PBT). Final dehydration was in 2 changes of 100% methanol. Explants were stored overnight in 100% methanol at −20°C. Samples were rehydrated through the methanol:PBT series to PBT, treated with 6% hydrogen peroxide in PBT and then detergent extracted in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM EDTA, 50 mM Tris). After extraction, samples were refixed in 0.2% glu-taraldehyde and 4% paraformaldehyde in PBT for 15 minutes. Further RIPA buffer treatments were followed by washes in PBT. Explants were then incubated for 1 hour at 70°C in prehybridization buffer (50% formamide [Molecular Biology Grade; Fisher Sci-entific, Santa Clara, CA], 5× SSC [1× SSC=0.15 M sodium chloride, 0.015 M sodium citrate] pH 5, 50 μg/ml yeast RNA [Sigma], 1% SDS, and 50 μg/ml heparin [Sigma]). Digoxigeninlabeled probes were added to the hybridization buffer at a concentration of approximately 1 mg/ml. Explants were incubated with sense and antisense digoxigenin probes at a concentration of 1 μg/ml overnight at 70°C. Unbound probe was removed by a sequence of washes at 70°C in a solution of 50% formamide, 5× SSC (pH 5), and 1% SDS, followed by a 70°C wash in a 1:1 mixture of the 50% formamide, 5× SSC (pH 5), and 1% SDS solution and a solution of 0.5 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1% Tween-20. Further washes included a wash at room temperature in 0.5 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1% Tween-20; a wash at 37°C in the same solution plus RNase A (Sigma); and another wash at room temperature in 0.5 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1% Tween-20. Finally, explants were washed at room temperature in 50% formamide and 2× SSC (pH 5), followed by a wash at 65°C in the same solution. To block nonspecific binding of antibodies, we incubated explants at room temperature in 10% heat-inactivated sheep serum (Sigma) in TBST buffer (140 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl, pH 7.5, and 0.1% Tween-20). Alkaline phosphatase-conjugated anti-digoxigenin antibodies (Boehringer Mannheim) were preabsorbed with powder prepared from 12.5- to 13.5-day embryos before use. Explants were incubated with a 1:2000 dilution of antibodies for 4 hours at room temperature. To remove unbound antibodies we washed samples overnight at room temperature in TBST buffer with the addition of 2 mM levamisole, followed by incubation in NTMT buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, and 0.1% Tween-20) made fresh each day of use. Alkaline phosphatase activity was visualized by incubation with a chromogenic substrate consisting of 4.5 μl/ml NBT solution (75 mg/ml nitroblue tetrazolium salt [Boehringer Mannheim] in 70% dimethylformamide) and 3.5 μl/ml BCIP solution (50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate [Boehringer Mannheim] in dimethylformamide) in NTMT buffer. Color development was stopped by extensive washing with PBT.
Immunofluorescence and cytoskeletal staining
For immunocytochemistry, explants to be stained with antivimentin were simultaneously fixed and permeabilized in 1:1 methanol:acetone at −20°C for 5 minutes, rinsed 3 times with PBS, and incubated for 1 hour with a 1:30 dilution of the anti-vimentin antiserum (mouse IgM). Primary antibodies were removed by washing three times with PBS, and explants were then incubated for 45 minutes at room temperature with a 1:200 dilution of biotinylated anti-mouse IgM (Vector Laboratories, Inc., Burlingame, CA). Biotinylated antibodies were washed away, and samples were incubated in a 1:100 dilution of fluoresceinated streptavidin (Pierce) for 30-45 minutes at room temperature. Explants to be stained with anti-SSEA-1 (mouse IgM) or anti-α6 (GoH3) antibodies were fixed and permeabilized in 100% methanol at −20°C for 5 minutes. Samples were rinsed three times and incubated for 1 hour at room temperature with anti-SSEA-1 or were incubated overnight at 4°C with anti-α6 antibodies. For anti-SSEA-1 staining, biotinylated antibodies and fluoresceinated streptavidin were used as described for anti-vimentin staining. For anti- α6 integrin staining, biotinylated anti-rat secondary antibodies (Sigma) were used at a 1:100 dilution followed by streptavidin as described above. The actin cytoskeleton in mesoderm and epiblast explants was visualized in the following manner. Explants were fixed and permeabilized in 3.7% formaldehyde containing 100 μg/ml lysophosphatidyl choline, and actin stress fibers were stained with rhodamine-labeled phalloidin (Molecular Probes, Inc., Eugene, OR). Immunofluorescent staining was observed and photographed with a Zeiss microscope equipped with epifluorescence.
RESULTS
Characterization of in vitro explants of epiblast and mesoderm
Fragments of epiblast and mesoderm were dissected from the primitive streak region of 7.5- to 7.8-day embryos (the posterior half of the egg cylinder as shown in Fig. 1). Explants composed of 50-200 cells were then cultured on fibronectin-coated chamber slides in serum-free, chemically defined medium. After 16-24 hours in culture, epiblast and mesodermal tissues exhibited very different morphological features (Fig. 2). Cells in the epiblast explants had numerous cell-cell contacts and the close-packed morphology characteristic of epithelial cells in culture (Fig. 2A). In addition, almost all epiblast explants (95%±4% s.d., n=50 explants) maintained a coherent border as shown in the smaller explant in Fig. 2B. This is in contrast to explants of primitive streak mesoderm, in which cells had migrated away from the center of the explant (and each other) and had the characteristic appearance of fibroblastic cells in culture (Fig. 2C). Mesoderm cells were greatly flattened compared with epiblast, and mesoderm cells often exhibited an elongated shape, with a lamellipodium at the leading edge and retraction fibers at the rear of the cell.
Cultured explants of epiblast (A,B) and mesoderm (C) isolated from primitive streak-stage embryos. Explants were transferred to fibronectin-coated wells of 16-chamber glass slides, cultured for 16-24 hours in serum-free, chemically defined medium, and photographed under DIC optics. Bar, 40 μm.
Cultured explants of epiblast (A,B) and mesoderm (C) isolated from primitive streak-stage embryos. Explants were transferred to fibronectin-coated wells of 16-chamber glass slides, cultured for 16-24 hours in serum-free, chemically defined medium, and photographed under DIC optics. Bar, 40 μm.
The purity of the dissected tissues was assayed by in situ hybridization by using probes against the epiblast-specific cytokeratin Endo B (Jackson et al., 1981). Endo B mRNA was detected only when epiblast was probed with antisense RNAs (Fig. 3A, E). No transcripts were detected in mesoderm explants probed with antisense RNAs (Fig. 3B, F) and none were observed when either tissue was probed with sense RNAs (Fig. 3C, D, G, H). In three experiments, all epiblast explants (n=9) were positive for Endo B mRNAs while no mesoderm explants were positive (n=8). In both the in situ hybridization analysis and in the immunofluorescent studies described below, we found that 100% purity of tissues was routinely obtained.
In situ hybridization of cytokeratin (Endo B) transcripts in dissected primitive streak tissues. Digoxigenin-labeled sense and antisense probes for Endo B were generated for nonradioactive detection of Endo B mRNAs. (A-D) Bright-field microscopy. (E-H) Same field viewed under DIC optics. (A, E) Epiblast explants probed with antisense RNAs. (B, F) Mesoderm explants probed with anti-sense RNAs. (C, G) Epiblast explants probed with sense RNAs. (D, H) Mesoderm explants probed with sense RNAs. Bar, 40 μm.
In situ hybridization of cytokeratin (Endo B) transcripts in dissected primitive streak tissues. Digoxigenin-labeled sense and antisense probes for Endo B were generated for nonradioactive detection of Endo B mRNAs. (A-D) Bright-field microscopy. (E-H) Same field viewed under DIC optics. (A, E) Epiblast explants probed with antisense RNAs. (B, F) Mesoderm explants probed with anti-sense RNAs. (C, G) Epiblast explants probed with sense RNAs. (D, H) Mesoderm explants probed with sense RNAs. Bar, 40 μm.
Characteristic differences in epiblast and mesodermal explants were also observed when the organization of the actin cytoskeleton was examined. When explants were cultured on glass coverslips and stained with rhodaminelabeled phalloidin, cultured explants of both tissues exhibited prominently staining actin stress fibers (Fig. 4). However, patches of epiblast displayed strong bands of stress fibers that ran parallel to the coherent edge of the explant, indicating that the architecture of junctional complexes was maintained (Fig. 4A). In contrast, mesodermal cells migrating away from the edges of explants displayed stress fibers that ran parallel to the direction of migration, and that had no relation to other cells, thus reflecting the migratory status of the mesoderm cells (Fig. 4B).
Phalloidin staining of the actin-containing cytoskeleton in cultured explants of epiblast (A) and mesoderm (B) isolated from primitive streak-stage embryos. Explants were fixed, permeabilized, and stained with rhodamine-labeled phalloidin to visualize the organization of the cytoskeleton. Bar, 20 μm.
Phalloidin staining of the actin-containing cytoskeleton in cultured explants of epiblast (A) and mesoderm (B) isolated from primitive streak-stage embryos. Explants were fixed, permeabilized, and stained with rhodamine-labeled phalloidin to visualize the organization of the cytoskeleton. Bar, 20 μm.
Anti-E-cadherin antibodies disrupt cell-cell contacts in epiblast explants
E-cadherin is expressed from the onset of development in the mouse and antibodies against E-cadherin (anti-Cell CAM 120/80 antibodies) reversibly block compaction at the 8- to 16-cell stage of development (Hyafil et al., 1983; Damsky et al., 1983; Vestweber and Kemler, 1984). At later stages, E-cadherin is prominently expressed in the epiblast but not in the mesoderm of the primitive streak-stage embryo (Damjanov et al., 1986). To determine whether this loss of E-cadherin expression was a passive result of differentiation or if it might play a more causative role in mesoderm differentiation and migration at the primitive streak, we made use of function-perturbing antibodies specific for E-cadherin. Epiblast explants were cultured for 12-16 hours on fibronectin, and anti-E-cadherin antibodies were then added at a concentration of 90 μg/ml. After 4 hours in culture with anti-E-cadherin antibodies, cells throughout the epiblast explants had lost cell-cell contacts and begun to flatten to the substratum (Fig. 5A). After 16 hours in culture with anti-E-cadherin antibodies, cells displayed a more flattened and elongated morphology, and greater spaces separated cells within an explant (Fig. 5B). In any one experiment, 5-8% of cells in treated epiblast explants were completely rounded (i.e., did not spread on the substratum) after antibody treatment and died. While this may account for a small percentage of the spaces between previously contiguous cells in the center of an explant, after anti-E-cadherin treatment (and an additional 16 hours in culture) cells had migrated, as was evidenced by the fact that cells now occupied an area greater than that of the original explant (Fig. 5C). In the explant shown in Fig. 5C, the original diameter of the explant was approximately 200 μm and in this instance, some cells had migrated an additional 80-120 pm from the edge of the explant after anti-E-cadherin treatment and culture. Pre-immune or nonimmune serum, at the same concentration, had no effect on epiblast explants; they remained a coherent epithelial patch (Fig. 5D). In contrast, mesoderm explants were not affected by anti-E-cadherin treatment (Fig. 5E), although in 2 of 12 experiments the mesoderm cells had migrated as usual but were somewhat less flattened on the substratum compared with controls. The effects of anti-E-cadherin treatment on epiblast cells were apparent after 2-4 hours and were not reversible. After 4 hours of culture with anti-E-cadherin antibodies, explants were washed in PBS and cultured without antibodies for up to 30 hours (to ensure that antibodies were no longer present on the cell surface). In all cases, treated epiblast cells did not revert to an epithelial organization but continued to exhibit a fibroblastic morphology. The induced change in morphology was observed in every experiment with the anti-E-cadherin antibodies (consisting of 2 or 3 epiblast explants dissected from each of 1 to 3 littermates treated with anti-E-cadherin antibodies in each of 20 experiments).
Effect of anti-E-cadherin antibodies on epiblast and mesoderm explants. (A) Epiblast explant cultured for 4 hours with anti-E-cadherin antibodies. (B) Epiblast explant after 16 hours of culture with anti-E-cadherin antibodies. (C) Anti-E-cadherin-treated epiblast explant in which cells have significantly migrated from the edge of the explant after antibody treatment. The original center of the explant is indicated by the arrowhead. (D) Control epiblast cultured for 16 hours in pre-immune antibodies (D). Primitive streak mesoderm explants cultured for 16 hours in the presence of anti-E-cadherin antibodies. Bar, 40 μm.
Effect of anti-E-cadherin antibodies on epiblast and mesoderm explants. (A) Epiblast explant cultured for 4 hours with anti-E-cadherin antibodies. (B) Epiblast explant after 16 hours of culture with anti-E-cadherin antibodies. (C) Anti-E-cadherin-treated epiblast explant in which cells have significantly migrated from the edge of the explant after antibody treatment. The original center of the explant is indicated by the arrowhead. (D) Control epiblast cultured for 16 hours in pre-immune antibodies (D). Primitive streak mesoderm explants cultured for 16 hours in the presence of anti-E-cadherin antibodies. Bar, 40 μm.
Cells from epiblast explants treated with anti-E-cadherin antibodies cease expressing markers of epiblast and begin to express vimentin
Treatment of epiblast explants with anti-E-cadherin antibodies induced a change in treated cells that was typical of the epithelial-mesenchymal transition during gastrulation in vivo. We next asked whether this change in morphology was accompanied by changes in gene expression, which we assayed in a series of immunofluorescence experiments. Antibodies against SSEA-1 recognize a carbohydrate moiety that is present on epiblast cells but is lost as tissues, including the primitive streak mesoderm, differentiate in vivo (Solter and Knowles, 1978; Skreb et al., 1991). Except for a punctate spot of fluorescence on approximately 10% of cells, anti-E-cadherin-treated epiblast cells did not stain with anti-SSEA-1 monoclonal antibodies (Fig. 6A, n=6 explants). In contrast, all cells in control epiblast explants stained strongly for this antigen (Fig. 6B), and cells in explants of primitive streak mesoderm never stained positively for SSEA-1 (Fig. 6C).
Expression of SSEA-1 and vimentin in control and anti-E-cadherin-treated explants. (A) Anti-SSEA-1 staining of an anti-E-cadherin-treated epiblast explant. (B) Anti-SSEA-1 staining of a control epiblast explant. (C) Anti-SSEA-1 staining of a control mesoderm explant. (D) Anti-vimentin staining of an anti-E-cadherin-treated epiblast explant. (E) Anti-vimentin staining of a control epiblast explant. (F) Anti-vimentin staining of a control mesoderm explant. Bar, 20 μm.
Expression of SSEA-1 and vimentin in control and anti-E-cadherin-treated explants. (A) Anti-SSEA-1 staining of an anti-E-cadherin-treated epiblast explant. (B) Anti-SSEA-1 staining of a control epiblast explant. (C) Anti-SSEA-1 staining of a control mesoderm explant. (D) Anti-vimentin staining of an anti-E-cadherin-treated epiblast explant. (E) Anti-vimentin staining of a control epiblast explant. (F) Anti-vimentin staining of a control mesoderm explant. Bar, 20 μm.
We also examined the expression of vimentin, an intermediate filament protein that is expressed by mesenchymal cells and is newly expressed in primitive streak mesoderm as it traverses the streak (Jackson et al., 1981; Franke et al., 1983). Almost all cells (94%±3% s.d., n=6 explants) in anti-E-cadherin-treated epiblast explants stained strongly for intermediate filaments containing vimentin (Fig. 6D). In contrast, treated control epiblast explants never stained positively for vimentin (Fig. 6E). In control explants of primitive streak mesoderm 98% (±2% s.d., n=6 explants) of cells stained strongly with anti-vimentin antibodies (Fig. 6F).
When explants treated with anti-E-cadherin antibodies were cultured for 12-30 hours after antibodies were washed away, the cells within the treated explants maintained their mesenchymal morphology. Most cells (86%±3% s.d., n=4 explants) within anti-E-cadherin-treated epiblast explants that were cultured for an additional 24-30 hours without antibodies no longer stained with anti-E-cadherin antibod-ies (Fig. 7A). In contrast, cells within control epiblast explants continued to stain positively for E-cadherin (Fig. 7B).
Expression of E-cadherin after anti-E-cadherin treatment. An epiblast explant was cultured with anti-E-cadherin antibodies for 4-6 hours and antibodies were then washed away. The explant was cultured for an additional 24 hours without antibodies and then stained with anti-E cadherin antibodies (A). The small arrow indicates a cell which stained faintly for E-cadherin on its periphery. (B) Anti-E-cadherin staining of a control epiblast explant. Bar, 20 μm.
Expression of E-cadherin after anti-E-cadherin treatment. An epiblast explant was cultured with anti-E-cadherin antibodies for 4-6 hours and antibodies were then washed away. The explant was cultured for an additional 24 hours without antibodies and then stained with anti-E cadherin antibodies (A). The small arrow indicates a cell which stained faintly for E-cadherin on its periphery. (B) Anti-E-cadherin staining of a control epiblast explant. Bar, 20 μm.
Taken together, the morphological and immunocyto-chemical data suggest that disruption of E-cadherin function in epiblast is sufficient to trigger an irreversible transition to a mesodermal phenotype.
In vivo differentiation of epiblast to mesoderm and the acquisition of a migratory phenotype are accompanied by changes in cell-ECM interactions
In vivo, the epiblast is organized on a basal lamina containing fibronectin, laminin, and type IV collagen (Wartio-vaara et al., 1979; Adamson and Ayers; 1979; Leivo et al., 1980; Leivo, 1983). As mesoderm differentiates, it interacts with these ECM components as it migrates between the epiblast and the visceral endoderm. Because of these developmentally regulated changes in cell-ECM interactions, we examined cell-substratum interactions of primitive streak tissues in our culture system. Interestingly, there were quantitative differences between the attachment of epiblast to fibronectin and to the other ECM components, and there were also quantitative differences between epiblast and mesoderm in their attachment to ECM components (Fig. 8A). The percentage of epiblast explants that attached to fibronectin was far greater than the percentage that attached to laminin or type IV collagen (83%±13% s.d. versus 31%±18% s.d. and 24%±19% s.d., respectively). Epiblast explants spread on fibronectin (Fig. 8B) had the same mor-phological appearance as when they attached and spread on laminin or type IV collagen (data not shown). In striking contrast to their behavior on fibronectin (and to a lesser extent on laminin and type IV collagen), epiblast explants failed to attach to vitronectin (n=25 explants; Fig. 8C). Interestingly, epiblast, but not mesoderm, displayed anchorage-dependent viability upon culture; epiblast explants that had not attached and begun to spread by 4-6 hours failed to do so with overnight culture, and after 16 hours, these explants were composed of vesiculated, dead cells (Fig. 8C).
Attachment of epiblast and primitive streak mesoderm cells to various ECM proteins. Explants were isolated and transferred to wells of chamber slides coated with fibronectin, vitronectin, laminin, or type IV collagen. (A) Percentage of explants that attached and spread normally on each substratum. Error bars indicate s.d. (B) Appearance of attached and spread epiblast explant on fibronectin. (C) Unattached explant of epiblast cultured on vitronectin. Bar, 40 μm.
Attachment of epiblast and primitive streak mesoderm cells to various ECM proteins. Explants were isolated and transferred to wells of chamber slides coated with fibronectin, vitronectin, laminin, or type IV collagen. (A) Percentage of explants that attached and spread normally on each substratum. Error bars indicate s.d. (B) Appearance of attached and spread epiblast explant on fibronectin. (C) Unattached explant of epiblast cultured on vitronectin. Bar, 40 μm.
In contrast to epiblast, when primitive streak mesoderm was dissected from the embryo and its attachment assayed in vitro, the percentage of mesoderm explants that attached to fibronectin, vitronectin, laminin, and type IV collagen was routinely 97-100% (±1-3% s.d.; Fig. 8A; the morphol-ogy of adherent mesoderm explants is shown in Figs 9 and 10). Therefore, differentiation of mesoderm is accompanied by changes in the cell’s adhesive phenotype.
Inhibition of attachment of epiblast to fibronectin and of primitive streak mesoderm to fibronectin and vitronectin by anti-ECMR antibodies. (A) Unattached epiblast explant cultured on fibronectin with anti-ECMR antibodies. (B) Epiblast explant attached and spread on fibronectin when cultured with control antibodies. (C) Unattached primitive streak mesoderm explant cultured on fibronectin with anti-ECMR antibodies. (D) Primitive streak mesoderm explant attached and spread on fibronectin when cultured with control antibodies. Mesoderm explant attached and spread on fibronectin after anti-ECMR antibodies were washed away (E). (F) Unattached primitive streak mesoderm explant cultured on vitronectin with anti-ECMR antibodies. (G) Primitive streak mesoderm explant attached and spread on vitronectin when cultured with control antibodies. Mesoderm explant attached and spread on vitronectin after anti-ECMR antibodies were washed away (H). Bar, 40 μm.
Inhibition of attachment of epiblast to fibronectin and of primitive streak mesoderm to fibronectin and vitronectin by anti-ECMR antibodies. (A) Unattached epiblast explant cultured on fibronectin with anti-ECMR antibodies. (B) Epiblast explant attached and spread on fibronectin when cultured with control antibodies. (C) Unattached primitive streak mesoderm explant cultured on fibronectin with anti-ECMR antibodies. (D) Primitive streak mesoderm explant attached and spread on fibronectin when cultured with control antibodies. Mesoderm explant attached and spread on fibronectin after anti-ECMR antibodies were washed away (E). (F) Unattached primitive streak mesoderm explant cultured on vitronectin with anti-ECMR antibodies. (G) Primitive streak mesoderm explant attached and spread on vitronectin when cultured with control antibodies. Mesoderm explant attached and spread on vitronectin after anti-ECMR antibodies were washed away (H). Bar, 40 μm.
Inhibition of attachment of primitive streak mesoderm to laminin and type IV collagen. (A) Unattached primitive streak mesoderm explant cultured on laminin with anti-ECMR antibodies. (B) Primitive streak mesoderm explant attached and spread on laminin when cultured with control antibodies. Mesoderm explant attached and spread on laminin after anti-ECMR antibodies were washed away (C). (D) Unattached primitive streak mesoderm explant cultured on type IV collagen with anti-ECMR antibodies. (E) Primitive streak mesoderm explant attached and spread on type IV collagen when cultured with control antibodies. Mesoderm explant attached and spread on type IV collagen after anti-ECMR antibodies were washed away (F). (G) Primitive streak mesoderm explant attached and spread on a substratum of Cell-takTM when cultured with anti-ECMR antibodies. Bar, 40 μm.
Inhibition of attachment of primitive streak mesoderm to laminin and type IV collagen. (A) Unattached primitive streak mesoderm explant cultured on laminin with anti-ECMR antibodies. (B) Primitive streak mesoderm explant attached and spread on laminin when cultured with control antibodies. Mesoderm explant attached and spread on laminin after anti-ECMR antibodies were washed away (C). (D) Unattached primitive streak mesoderm explant cultured on type IV collagen with anti-ECMR antibodies. (E) Primitive streak mesoderm explant attached and spread on type IV collagen when cultured with control antibodies. Mesoderm explant attached and spread on type IV collagen after anti-ECMR antibodies were washed away (F). (G) Primitive streak mesoderm explant attached and spread on a substratum of Cell-takTM when cultured with anti-ECMR antibodies. Bar, 40 μm.
Adhesion to ECM glycoproteins is mediated primarily by the integrin superfamily of receptors (Hynes and Lander, 1992). Anti-ECMR antibodies, which recognize a broad range of integrins, have been shown to block the outgrowth of mouse trophectoderm cells from blastocysts in vitro (Richa et al., 1985; Sutherland et al., 1988). In the following experiments, these function-perturbing antibodies were used to determine whether integrins are active in epiblast and mesoderm adhesion. The effect of anti-ECMR antibodies on epiblast adhesion was tested only on attachment to fibronectin because this substratum was the only one that supported significant levels of adhesion. The effect of anti-ECMR antibodies on adhesion of primitive streak mesoderm to all four ECM glycoproteins was examined.
All epiblast cells cultured on fibronectin with 75 μg/ml anti-ECMR antibodies failed to attach (n=15; Fig. 9A), whereas 77% (±3% s.d.) of epiblast explants cultured in the same concentration of control antibodies attached and spread on the fibronectin substratum (Fig. 9B). Attachment of mesoderm to both fibronectin and vitronectin was completely blocked by anti-ECMR antibodies, and the explants remained as round, floating aggregates (Fig. 9C,F). Mesoderm explants attached quickly (within 1-4 hours) to fibronectin and vitronectin in the presence of nonimmune antibodies (Fig. 9D,G). The effect of the anti-ECMR anti-bodies on mesoderm attachment was reversible. After removal of the anti-ECMR antibodies (by washing the explants in PBS), the treated mesoderm explants attached, spread, and migrated normally after additional culture without antibodies (Fig. 9E,H). Not unexpectedly, the anti-ECMR-treated epiblast explants did not recover (data not shown), probably because of their anchorage dependence for viability.
Because mesoderm explants attached more slowly to laminin and type IV collagen (within 6-8 hours), the effect of the anti-ECMR antibodies was scored after 16 hours in culture. At this time, mesoderm explants remained as rounded clumps of cells which were unattached to the laminin or type IV collagen substratum (Fig. 10A,D). Nonimmune antibodies had no effect on attachment (Fig. 10B,E). After the removal of the anti-ECMR antibodies and an additional 24 hours in culture without antibodies, mesoderm explants attached, spread, and migrated normally on laminin and type IV collagen (Fig. 10C, F).
To determine whether the effect of the anti-ECMR antibodies was due to a specific interference between integrins and their ligands, we studied the ability of the antibodies to block adhesion to a substratum that is not mediated by integrins. Cell-takTM is a nonspecific attachment factor for tissue culture cells that does not contain the RGD tripeptide recognized by integrins (Waite and Tanzer, 1981; Waite, 1983). When mesoderm was cultured on Cell-takTM with anti-ECMR antibodies at the same concentrations as used in the previous experiments, the antibodies had no effect. Attachment occurred within 1-4 hours (Fig. 10G). Therefore, the anti-ECMR-mediated inhibition of attachment to the ECM glycoproteins was not attributable to steric hindrance caused by antibody binding to the cells but rather appeared to be due to specific interference with the interaction of integrin receptors and their ligands.
Primitive streak mesoderm cells use α6β1 as a laminin receptor
Several integrin receptors mediate adhesion to laminin (Hynes and Lander, 1992). To determine more specifically which integrins play a role in the attachment of primitive streak mesoderm to laminin, we examined the expression and function of α6β1 integrin, a well-characterized laminin receptor (Sonnenberg et al., 1988). GoH3 monoclonal antibodies, which recognize α6β1 integrin and block the function of this receptor (Sonnenberg et al., 1987, 1988, 1990) were used in immunofluorescence studies and in functional assays. Mesoderm cells stained prominently for α6β1 (Fig. 11A) and when explanted into wells coated with fibronectin or laminin and incubated with 75 μg/ml GoH3 antibodies, the cell’s attachment to laminin but not to fibronectin was specifically blocked (Fig. 11B,C). After the removal of the GoH3 antibodies, the mesoderm explants attached, spread, and migrated normally on laminin as well (Fig. 11D). These results confirm the role of the α6β1 integrin as a major receptor for laminin on primitive streak mesoderm cells.
Expression of α6β1 integrin in mesoderm and functional blockade by anti-α6β1 (GoH3) antibodies. GoH3 monoclonal antibodies stained a filamentous network of α6β1 integrins in the mesodermal cells (A). (B) Unattached primitive streak mesoderm explant cultured on laminin with GoH3 antibodies. (C) Primitive streak mesoderm explant attached and spread on fibronectin when cultured with GoH3 antibodies. Mesoderm explants attached and spread normally on laminin when antibodies were washed away (D). Bars: A, 20 pm; B-D, 40 μm.
Expression of α6β1 integrin in mesoderm and functional blockade by anti-α6β1 (GoH3) antibodies. GoH3 monoclonal antibodies stained a filamentous network of α6β1 integrins in the mesodermal cells (A). (B) Unattached primitive streak mesoderm explant cultured on laminin with GoH3 antibodies. (C) Primitive streak mesoderm explant attached and spread on fibronectin when cultured with GoH3 antibodies. Mesoderm explants attached and spread normally on laminin when antibodies were washed away (D). Bars: A, 20 pm; B-D, 40 μm.
Cells from epiblast explants treated with anti-E-cadherin antibodies acquire the ability to adhere to vitronectin
Because the anti-E-cadherin-treated epiblast cells had adopted a mesodermal phenotype with respect to morphology and protein expression, we asked whether they had also acquired adhesive behavior that was characteristic of mesoderm. Epiblast explants did not attach to vitronectin, whereas mesoderm explants attached and spread readily on it (Fig. 8A). We exploited this difference in the next set of experiments. Epiblast explants plated on fibronectin were treated with anti-E-cadherin antibodies which produced a field of elongated and flattened cells as described above (Fig. 12A). The cells were then incubated for 2 hours with anti-ECMR antibodies, which caused the cells to round up and lose contact with the fibronectin substratum and allowed their transfer to new wells (Fig. 12B). After transfer to fibronectin- or vitronectin-coated wells and 4 hours in culture (in the absence of antibodies), the percentage of cells that attached and spread to both substrata was determined by cell counts (Fig. 12C-E, appearance of treated epiblast cells attached to vitronectin). When control explants of epiblast were incubated with anti-ECMR antibodies, only a small percentage of cells (approximately 5%) on the edge of the explant rounded from the substratum (probably due to a lack of antibody accessibility in the interior of the explant). In addition, when control epiblast explants were trypsinized and the resulting small clumps of cells were transferred to new wells, no cells survived; therefore, the attachment of anti-E-cadherin-treated cells to vitronectin was compared to the attachment of primitive streak mesoderm cells which were removed from contact with fibronectin by anti-ECMR antibodies. The results of three experiments are presented in Fig. 12F. Primitive steak mesoderm cells reattached to fibronectin and vitronectin at high levels (73%±6% s.d. and 71%±8% s.d., respectively). Anti-E-cadherin-treated epiblast cells also readily reattached to fibronectin (71%±8% s.d.) and, when compared with untreated epiblast (Fig. 8A), anti-E-cadherin-treated cells now displayed a significant level of attachment to vitronectin (42%±10% s.d.).
Attachment of anti-E-cadherin-treated epiblast to vitronectin. (A) Epiblast cells treated with anti-E-cadherin antibodies and cultured for an additional 12-16 hours without antibodies. (B) Rounded appearance of treated epiblast cells after 2 hours of incubation with anti-ECMR antibodies. (C-E) Anti-E-cadherin-treated epiblast cells attached and spread on a vitronectin substratum. Bar, 40 μm. (F) Percentage of mesoderm and anti-E-cadherin-treated epiblast cells that attached and spread on vitronectin. Data are presented as the mean fraction of cells attached to fibronectin and vitronectin in three experiments (n =250 for mesoderm cells; n=225 for anti-E-cadherin-treated epiblast cells). Error bars indicate s.d.
Attachment of anti-E-cadherin-treated epiblast to vitronectin. (A) Epiblast cells treated with anti-E-cadherin antibodies and cultured for an additional 12-16 hours without antibodies. (B) Rounded appearance of treated epiblast cells after 2 hours of incubation with anti-ECMR antibodies. (C-E) Anti-E-cadherin-treated epiblast cells attached and spread on a vitronectin substratum. Bar, 40 μm. (F) Percentage of mesoderm and anti-E-cadherin-treated epiblast cells that attached and spread on vitronectin. Data are presented as the mean fraction of cells attached to fibronectin and vitronectin in three experiments (n =250 for mesoderm cells; n=225 for anti-E-cadherin-treated epiblast cells). Error bars indicate s.d.
DISCUSSION
In this study we used function-perturbing antibodies in a cell culture system to demonstrate the role of E-cadherin during the epithelial-mesenchymal transition that occurs at the primitive streak during mouse gastrulation. Perturbation of E-cadherin-mediated cell adhesion in explants of epiblast induced a more flattened, fibroblastic morphology and led to the expression of vimentin, an intermediate filament protein characteristic of mesenchymal cells and primitive streak mesoderm cells at this stage of development in vivo. In addition, when antibodies were washed away, treated epiblast cells did not resume their epithelial organization and no longer expressed E-cadherin or SSEA-1, two products that are lost as mesoderm differentiates in vivo (reviewed by Richa and Solter, 1986; Takeichi, 1991). We conclude that the loss of E-cadherin-mediated cell contacts in vitro triggered steps of differentiation in the treated cells that were characteristic of the differentiation pathway of primitive streak mesoderm.
E-cadherin-mediated cell adhesion is important for a number of morphogenetic events during development, including compaction and primitive endoderm segregation (Hyafil et al., 1983; Damsky et al., 1983; Vestweber and Kemler, 1984; Richa et al., 1985). Transfection and expression of recombinant cadherins lead to the sorting out of transfected cells in mixed cell populations, including populations of embryonic lung epithelia and mesenchyme (Nose et al., 1988; Friedlander et al., 1989). This implies that differential expression of cadherins and other cell adhesion molecules could play a critical role in tissue segregation during organogenesis. In another epithelial-mesenchymal transition, the formation of the secondary palate, cells destined to transform into mesenchyme turn off expression of E-cadherin (L-CAM) and syndecan. However, there is no evidence that the changes in E-cadherin (or syndecan) expression play a causative role in secondary palate formation or in the differentiation of tissues at the secondary palate (Hay, 1991), as we have demonstrated for the differentiation of tissues at the primitive streak.
What do the results obtained in vitro imply about the process of gastrulation in the intact embryo? One possibility is that the loss of E-cadherin-mediated cell contacts is one of the first steps in a synchronized pathway of differentiation at the primitive streak leading to the ingression of newly formed mesodermal cells. The antibody-induced perturbations of E-cadherin-mediated cell contacts in vitro may actuate a pathway that occurs naturally during the process of streak formation and gastrulation in vivo. Such a pathway might include the localized activity of proteinases, especially metalloproteinases, which are known to be prominently expressed during invasive processes such as the ingression of epiblast cells through the primitive streak (Sanders and Prasad, 1989; Alexander and Werb, 1991). Alternatively, the expression of a number of molecules that could initiate or transduce the signal for primitive streak formation has been described in the gastrulating mouse embryo. These include brachyury, goosecoid (which acts as an organizer during Xenopus gastrulation), and the transcripts for a number of genes such as FGF-4 and other fibroblast growth factor family members such as Wnt-2 (Wilkinson et al., 1988, 1990; Cho et al., 1991; Blum et al., 1992; and reviewed by Latimer and Pedersen 1993). The loss of E-cadherin expression may or may not be the catalyst for the organized cell movements of gastrulation in vivo; however, our data demonstrate that perturbation of E-cadherin function alone can induce a pathway of mesoderm-like differentiation. Furthermore, the effect of anti-E-cadherin treatment on epiblast differentiation was not reversible, as was the effect of anti-E-cadherin on the compacting 8- to 16-cell stage embryo (Damsky et al., 1983, Richa et al, 1985).
We also tested the ability of primitive streak tissues to attach to various ECM ligands and demonstrated that the integrin superfamily of receptors mediate adhesion in this embryonic system. The viability of epiblast, but not mesoderm, explants was anchorage dependent, and in vitro assays demonstrated that developmentally regulated changes in the adhesion of cells to vitronectin, laminin, and type IV collagen occur as epiblast differentiates into mesoderm at the primitive streak. In addition, epiblast cells acquired the ability to adhere to vitronectin, a characteristic of the mesoderm cells in vitro, after anti-E-cadherin-induced differentiation. The differences in the number of epiblast versus mesoderm explants that attached to vitronectin, laminin, and collagen type IV imply that quantitative or qualitative changes in integrin expression occur with mesoderm differentiation at the primitive streak. Changes in integrin expression may provide useful markers for the migratory mesoderm in mammals when more reagents that recognize mouse integrins become available to identify specific integrins functionally.
Because the events of gastrulation are staggering in their complexity, a simplified culture system, such as the one described in this report, provides a powerful tool for addressing questions concerning control mechanisms during vertebrate gastrulation. This culture system can be used to further characterize the differentiation of anti-E-cadherintreated epiblast tissues by in situ hybridization with additional primitive streak mesoderm-specific transcripts, such as PDGFα and Wnt-2 (Wilkinson et al., 1988; Mercola et al., 1990; Schatteman et al., 1992). Other mechanistic questions can also be addressed; for example, do peptide growth factors play a role in inducing the loss of E-cadherin expression in the posterior epiblast? Or, what role do peptide growth factors play in axial pattern formation and specification of mesodermal fate in the mouse, as has been previously investigated in the amphibian system (reviewed by Slack, 1990; Jessell and Melton, 1992)? The knowledge obtained by investigating these questions in vitro can then be used to improve experimental design when returning to the intact embryo.
ACKNOWLEDGEMENTS
The authors are grateful to Robert Oshima and Davor Solter for the kind gifts of Endo B constructs and anti-SSEA-1 antibodies, respectively. We also thank Ann Sutherland and Akiko Spindle for their generosity and for the development of the serum-free, chemically defined medium, Margaret Flannery for assistance, and Ann Sutherland, Zena Werb, Jean J. Latimer, Stephen G. Grant, and Mary McKenney for critical reading of this manuscript. This work was supported by a National Institutes of Health National Research Service Award 5 T32 ESO7106 from the National Institute of Envi-ronmental Health, by NIH Program Project Grant HD26732, and by the Office of Health and Environmental Research, US Department of Energy contract no. DE-AC03-76-SF01012.