Department of Cell Biology, University of Virginia Health System, School of Medicine, PO Box 800732, Charlottesville, VA, 22908-0732, USA
*Author for correspondence (e-mail: dwd3m{at}virginia.edu)
Accepted June 18, 2001
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SUMMARY |
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Key words: Integrin, Fibronectin, Gastrulation, Epiboly, Cell polarity, Xenopus
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INTRODUCTION |
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In contrast to salamanders and other vertebrate embryos, the FN matrix of the BCR in Xenopus laevis has been suggested by some investigators to play a minor, if any, role in gastrulation. While FN has been shown to support Xenopus mesoderm migration in vitro (Winklbauer, 1990), the disruption of integrin/FN interactions in vivo does not block gastrulation, as defined by eventual blastopore closure and mesoderm involution (Howard et al., 1992; Ramos and DeSimone, 1996; Ramos et al., 1996; Winklbauer, 1989; Winklbauer and Keller, 1996; Winklbauer and Stoltz, 1995). Keller and Jansa have further shown that involution and dorsal mesoderm extension occur even in the absence of a BCR (Keller and Jansa, 1992), suggesting that the primary mechanism regulating dorsal mesoderm movement in Xenopus is convergent extension. However, others have reported that inhibition of integrin/FN interactions in Xenopus results in delayed blastopore closure, axial defects and ectodermal thickening (Johnson et al., 1993; Ramos and DeSimone, 1996; Ramos et al., 1996). This indicates that FN may play more subtle but important roles in multiple morphogenetic processes at gastrulation.
Recent evidence obtained from Xenopus (Djiane et al., 2000; Medina et al., 2000; Medina and Steinbeisser, 2000; Tada and Smith, 2000) and zebrafish (Heisenberg et al., 2000) suggests that cell-cell adhesion is regulated through the Wnt/Fz signaling pathway at gastrulation. Convergent extension is inhibited in Xenopus explants overexpressing the Fz7 receptor, but this defect can be rescued by co-expressing a dominant-negative construct of CDC42, a member of the Rho family of small GTPases (Djiane et al., 2000). Gastrulation defects resulting from disruptions in the Wnt signaling pathway may arise from a loss of planar cell polarity (PCP) in marginal zone cells (Djiane et al., 2000; Wallingford et al., 2000). The establishment of PCP in Xenopus is presumed to involve interactions of members of the wingless/Wnt family with frizzled receptors (Djiane et al., 2000; Tada and Smith, 2000). Thus, it appears that cell adhesion and cell polarity pathways converge in these embryos and interact to regulate cell rearrangements at gastrulation. Integrin/ligand interactions have also been tied to the establishment of cell polarity, and the differentiation of MDCK cells and keratinocytes (Ojakian and Schwimmer, 1994; Watt et al., 1993). It has been proposed that integrin/ECM engagement provides initial spatial cues that help set up subsequent epithelial cell polarity (Yeaman et al., 1999). Interestingly, in mouse (George et al., 1993), chick (Harrisson et al., 1993) and amphibians (Johnson et al., 1993), disruption of integrin/FN interactions results in ectodermal epithelial defects that could be interpreted as stemming from a loss of cell polarity.
We have re-examined the role that FN plays in Xenopus gastrulation using monoclonal antibodies (mAbs) that inhibit FN/integrin interactions and matrix assembly in vivo. We show that FN is required for the cellular rearrangements that drive epiboly in the marginal zone at gastrulation. Integrin-based interactions with FN are necessary for the establishment of cell polarity in the deep layers of the dorsal marginal zone (DMZ) and BCR. These polarized cells either participate actively in intercalative behaviors in the DMZ or are required to maintain epithelial integrity in the BCR.
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MATERIALS AND METHODS |
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Dextran-labeled embryos were surface biotinylated at stage 9 using 1 mg/ml sulfo-NHS biotin (Pierce) in 0.1xMBS (modified Barths saline) for 5 minutes. Embryos were washed extensively in 0.1xMBS with 100 mM Tris-HCl (pH 7.4) to quench exogenous biotin. Embryos were injected with antibody as described above and fixed at stage 17. Biotin labeling was detected with strepavidin-HRP using Fluorescein Tyramide as a substrate (Davidson and Keller, 1999). Embryos were bisected and processed for confocal microscopy as described below.
Confocal microscopy
Embryos stored in methanol were rehydrated in PBS/0.05% Tween-20 (PBST), bisected with a scalpel and processed for immunostaining. Embryo halves were blocked for 2 hours in PBST/DMSO (PBS, 0.1% Triton X-100, 1% DMSO, 5% calf serum) and incubated in primary antibody diluted in PBST/DMSO (rabbit anti-FN polyclonal 1:1000, mouse anti-tubulin 1:1000, mouse or rabbit anti-hemagglutinin (HA) 1:500) overnight mat 4°C. Embryos were washed and incubated overnight in appropriate secondary antibodies in PBST/DMSO. Embryos were washed extensively in PBST, dehydrated in methanol, cleared in BBA (benzyl alcohol/benzyl benzoate) and mounted for confocal microscopy as described (Davidson and Keller, 1999). Images were collected on a Zeiss CSLM 400.
Embryos were stained with anti-tubulin antibody, as described above, in order to determine spindle orientation. Sagittal and oblique confocal sections of bisected embryos were used to determine spindle orientation in deep and superficial layers of the BCR. For a spindle to be counted out of the BCR epithelial plane, the axis of the spindle had to be 45° or more away from the plane of the epithelium.
Digital image capture and timelapse
Antibody-injected embryos were mounted in clay wells in 0.1x MBS and digital images captured with Isee software (Innovision, Raleigh NC) using a Hamamatsu Orca camera mounted on a Zeiss Axiophot microscope. A motorized stage (Ludl) allowed for serial capture of images at 1 minute intervals from nine sibling embryos in each experiment. Morphometric measurements were made using the Isee software.
HAß1 constructs
The integrin HAß1 construct was produced by subcloning the Xenopus ß1 cytoplasmic tail (starting at amino acid 752) onto the extracellular and transmembrane domains of viral hemagluttinin (Doms, 1986). The entire construct was subcloned into the pSP64T vector (D. G. Ransom, PhD thesis, University of Virginia). This construct will be described in detail elsewhere (B. J. Dzamba et al., unpublished). RNA transcripts were produced from the pSP64T vector by standard methods and injected into the animal poles of embryos at the four-cell stage. Expression of the HA constructs was detected with a mouse monoclonal anti-HA antibody (site A; a gift from Judith White).
Deep cell layer explants
Fertilized eggs were injected with dextran as described above. At stage 10, DMZs were cut from labeled embryos and the deep cell layers lining the blastocoel were teased free from the superficial and underlying intermediate layers. The labeled deep cell layers were typically 2-3 cell layers thick. A labeled deep cell fragment was then placed on either FN (20ug/ml) or BSA-coated coverslips. The DMZ from an unlabeled embryo was excised and, after removal of the deep cell layers lining the blastocoel, the region consisting of the superficial and intermediate deep cells (2-3 layers) was used to overlay the labeled fragment on the substrate. Explants were kept flat with a small fragment of coverslip anchored with silicone grease. Explants were cultured in Danilchiks solution for up to 3 hours and cell intercalation recorded by digital image capture. Explants were fixed in 3.7% formaldehyde in MEMFA (0.1 M Mops, pH 7.4, 2 mM EGTA, 1 mM MgSO4), dehydrated, cleared in BBA, mounted and examined by confocal microscopy as described above.
Dsh-GFP localization assays
Differential recruitment of the Dishevelled (Dsh) protein to the cell membrane was assayed using DMZ explants. 50 pg of synthetic XDsh-GFP RNA (a gift from S. Sokol) was injected into fertilized eggs prior to first cleavage. This amount of XDsh-GFP had no discernable effect on normal development. At stage 10, dorsal marginal zones were cut from embryos and trimmed to remove involuting marginal zone tissue, placed on bovine serum albumin (BSA)-coated coverslips, or on coverslips that had been previously coated with various adhesive substrates (0.23 µM plasma FN, GST fusion proteins, or 10 µM poly-lysine) and blocked with mAb 4B12 and/or BSA (1% solution). Preparation of the 9.11-GST and Hep II-GST fusion proteins is described elsewhere (Ramos and DeSimone, 1996). XDsh-GFP localization was monitored by confocal microscopy over a 2 hour period.
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RESULTS |
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Epiboly is correlated with the spreading of the superficial layer of the BCR. The relative area index (RAI)(Keller, 1978) is a measure of the area occupied by the clonal derivatives of a population of superficial cells (Fig. 5A). In embryos injected with the control mAb 4H2, there was a steady increase in the DMZ RAI during gastrulation (Fig. 5B). In embryos injected with either function-blocking mAb, the DMZ RAI showed an initial increase, before decreasing later in gastrulation (Fig. 5B). There was no loss of daughter cells in any of the clonal lineages examined, so the decrease in RAI reflects a decrease in surface area of superficial cells in the mAb 4B12- and mAb 1F7-injected embryos. The lack of superficial layer spreading during mid-gastrulation correlated temporally and spatially with lack of radial intercalation in the marginal zone (Fig. 4). The decrease in RAI seen in these embryos probably reflects a certain amount of compression observed in the DMZ before buckling at later stages (see below).
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DISCUSSION |
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Disrupting FN matrix assembly delays, but does not block, blastopore closure. The cellular rearrangements that drive blastopore closure are not clear. However embryos that lack a BCR roof can still close the blastopore (Keller and Jansa, 1992). This indicates that the majority of the morphogenetic processes behind blastopore closure are independent of the BCR, and supports our observation that closure is independent of FN matrix. Embryos treated with LiCl display a similar phenotype at gastrulation, with delayed blastopore closure, symmetrical dorsal and ventral mesoderm involution and a failure of epiboly (Cooke and Smith, 1988; Kao and Elinson, 1988; Klein and Moody, 1989; Regen and Steinhardt, 1988). Despite these defects, blastopore closure still occurs in these embryos. These data suggest that blastopore closure proceeds through a cascade of steps and that more than one mechanism is involved. Disrupting FN matrix assembly probably inhibits cell rearrangements early in the process, but mechanisms acting later can compensate to close the blastopore.
It is likely that that the short axis observed in mAb-injected embryos reflects a lack of radial intercalation in the DMZ. Radial intercalation initially occurs in the animal region of the BCR, as epiboly extends the BCR to encompass the embryo during gastrulation. The early stages of epiboly occur before gastrulation initiates and are, therefore, independent of FN matrix assembly. A later phase of radial intercalation begins in the marginal zone at gastrulation (Keller, 1978) and contributes to the lengthening of the dorsal axis (Wilson and Keller, 1991). This later stage of epiboly in the marginal zone is coincident with the initiation of FN matrix assembly and is a prelude to convergent extension movements in the same tissue (Wilson and Keller, 1991). In embryos without a FN matrix, there is no evidence for spreading of the superficial layers, or radial intercalation in the deep layers of the DMZ, suggesting that there is a failure in epiboly and perhaps a subsequent failure of convergent extension.
The situation in the animal pole of the BCR is apparently different because the roof thins before the assembly of a FN matrix. In mAb-injected embryos, the roof thins and then undergoes a subsequent thickening after stage 10.5. Although the layer index of the BCR increases, this thickening is not due to increased proliferation of deep cells, because no increase in the number of mitotic indices in this cell population was observed in the absence of a FN matrix (data not shown). The randomization of spindle orientation in the absence of FN suggests that the increase in the BCR layer index is due to a loss of normal BCR cell orientation. Thus, it would appear that in the marginal zone, FN supports intercalation, while in the animal pole of the BCR it acts to stabilize the existing architecture. There is precedent for such an interpretation because the epiboly observed during gastrulation is biphasic (Keller, 1978) and the behaviors of deep cells in the animal cap and marginal zone are not equivalent (Keller, 1980a). Observations in Pleurodeles also support a model in which there is a mechanistic difference in the epibolic movements of the BCR and the marginal zone. In Pleurodeles, superficial ectoderm involution occurs to a greater extent than in Xenopus and is dependent upon epiboly in the animal cap early in gastrulation. Disruption of FN/integrin interactions in this amphibian results in a thickened convoluted BCR (Johnson et al., 1993). This is what one would expect if FN-independent epiboly were to occur in the BCR while involution was blocked in the marginal zone.
One of the more intriguing observations to arise from analyses of Xenopus embryos that lack a FN matrix is the apparent dissociation of morphogenetic movements within the three primary germ layers. It is unlikely that this is the direct mechanical result of a loss of integrin-FN ligation, and suggests that interruption of integrin/FN binding affects intracellular pathways that can alter the activity of other morphogenetic machines during gastrulation. Bottle cells form the dorsal lip, yet involution of superficial ectoderm does not occur, despite involution and extension of the deeper mesoderm. Several mechanisms have been proposed to drive the dorsal extension of deep mesoderm and endoderm cells during gastrulation (Keller and Tibbetts, 1989; Winklbauer and Schurfeld, 1999). Dissociation of these coordinated cellular rearrangements could result in increased tension at the dorsal lip and cause the characteristic buckling we observed in embryos that lack a FN matrix (Fig. 3, Fig. 6J). It is significant that in both mouse (George et al., 1993) and chick (Harrisson et al., 1993), the absence of integrin/FN interactions in these embryos is associated with a shortened axis, ectodermal thickening and multiple mesodermal defects, some of the same characteristics that we observe in Xenopus. This suggests that integrin/FN ligation provides a signal that is essential in gastrulation and highly conserved across species.
FN is permissive for radial intercalation
Our evidence suggests that FN is required for BCR deep cell radial intercalation. Similar to what is observed in vivo, cells in the deep layer explant on a FN substrate undergo intercalation, while those on a non-adherent (BSA) substrate do not. There are several possible explanations to account for cell dependence on FN for intercalation. The simplest is that the FN substrate acts as a boundary and that as cells approach that boundary, they are captured. In our explant assay, many cells were observed to undergo intercalation distant from the substrate, suggesting that boundary capture is not the mechanism driving intercalation in this explant. A second possibility is that our results reflect simple mixing. This would suggest that adherence to FN signals a subsequent reduction in cell adhesion to FN (e.g. decreased integrin affinity and/or avidity) and increased adhesion to neighboring cells to promote cell mixing. This is again unlikely because in our assay, labeled cells initially adherent to FN are never observed to leave the substrate but more superficial cells can intercalate between them. In addition, cells distant from the FN substrate undergo intercalation activity without having contacted FN. This observation would suggest that the positional relationships between cells are altered upon integrin/FN ligation in the absence of any change in the ability of a cell to bind FN, and that this behavior is passed on to neighboring cells.
An interesting observation in these experiments is that FN is not required for patches of BCR deep cells to re-incorporate into the BCR (Fig. 8B-H). It is thought that the ability of BCR patches to re-incorporate is mediated through cadherins (Wacker et al., 2000). Our data suggest that FN can alter the way cells of the BCR interact with each other, possibly through crosstalk with cadherins. It is unlikely that integrin binding to FN abolishes cadherin function, because the BCR remains intact. However, if integrin ligation modulated cadherin function and reduced cell-cell adhesion, this could promote cell rearrangements in the BCR without blocking incorporation (Zhong et al., 1999). We are presently examining the possibility that integrin ligation modulates cadherin function in the BCR during gastrulation.
Radial intercalation requires ß1-containing integrins
Our use of antibodies that block the 5ß1 integrin-binding sites on FN demonstrates that the cell behaviors we observe are integrin mediated. If FN conveys a signal via integrins across the plasma membrane, then it should be able to interfere with the intracellular transmission of this signal. The HAß1 construct presumably acts as a dominant negative receptor by clustering ß1 tails and depleting cytoplasmic components needed for the normal activity of endogenous ß1-containing integrins, as reported previously for similar integrin chimeric constructs (Chen et al., 1994; LaFlamme et al., 1994). In embryos, these constructs give the same phenotypes with respect to BCR morphology as the mAb-dependent blocking of FN fibril assembly. This evidence supports the hypothesis that the effects we see are based upon integrin-mediated signals and that these signals are at least partially responsible for local radial intercalation behaviors in the DMZ.
FN matrix and cell polarity
In FN matrix-deficient embryos, the BCR thickens in the animal pole region and does not thin in the marginal zones during gastrulation. If thinning is driven through radial intercalation of multiple layers of BCR deep cells, then these cells must somehow interpret the vertical plane of the epithelium. Cell polarity is evident in the BCR in the uniform orientation of mitotic spindles within the horizontal plane of the epithelium in both the superficial and deep cell layers. One could imagine that tension across the BCR could influence spindle orientation. However, in embryos lacking a matrix, spindle orientation remains unchanged in the superficial layer, while it is randomized in deep layers. It is unlikely, therefore, that tension alone could account for spindle orientation in the BCR. Such an interpretation is further supported by the observation that small patches of dominant negative HAß1 integrin expression disrupt morphogenesis locally (data not shown). Another possibility is that these deep cells have intrinsic polarity and that this directs mitotic spindle orientation in a manner similar to that reported for Drosophila (Eaton et al., 1995; Gho and Schweisguth, 1998).
Recent studies in Xenopus indicate that a pathway acting through Dishevelled, similar to the Drosophila planar cell polarity (PCP) cascade (Adler, 1992), may regulate cell polarity in the DMZ (Axelrod et al., 1998; Djiane et al., 2000; Rothbacher et al., 2000; Tada and Smith, 2000; Wallingford et al., 2000). Our results indicate that integrin ligation to FN can initiate the rapid relocalization of Dsh to the cell membrane. This occurs in stage 10 marginal zone tissue that undergoes convergent extension, as well as in animal caps that do not. Because we do not manipulate either Wnt or Fz expression in these experiments, these results suggest that the deep cells of the BCR have the information needed to initiate Dsh relocalization but that FN can trigger the pathway prematurely. These effects are also specific to integrins, because we can block Dsh relocalization with an antibody (mAb 4B12; Ramos et al., 1996) that interferes with integrin binding to the CCBD of FN. Furthermore, Dsh localization to the plasma membrane is not supported by the non-integrin-dependent adhesive substrates poly-L-lysine and the HepII domain of FN. The Dsh mobilization that we report is not as robust as that seen when members of the Wnt signaling pathway are overexpressed (Boutros et al., 2000; Medina et al., 2000; Medina and Steinbeisser, 2000; Rothbacher et al., 2000; Umbhauer et al., 2000), but is comparable with that observed previously in the absence of overexpression of Wnts and/or their receptors (Wallingford et al., 2000). Although it has been proposed that membrane localization of Dsh may be an indication of a non-canonical Wnt signaling pathway (Axelrod et al., 1998; Wallingford et al., 2000), the physiological consequences of Dsh membrane localization remain unclear (Boutros et al., 2000; Medina et al., 2000; Umbhauer et al., 2000). Our experiments indicate that integrin/FN ligation is permissive for the initiation of polarity and is required for cell intercalation, and that this is correlated with the intracellular mobilization of Dsh to the cell membrane.
A role for FN induced polarity in the DMZ
In Xenopus embryos, cells of the DMZ undergo radial intercalation prior to mediolateral cell intercalation (Wilson and Keller, 1991), and can transmit the ability to intercalate to non-involuted tissue through physical contact (Keller et al., 1992b). We provide evidence that FN acting through integrins can confer polarized behavior to deep cells of the DMZ. Cell polarity is transmitted through cell-cell contact to cells distant from the substrate and is required for cells to undergo radial intercalation. It is likely that FN provides an integrin-dependent signal that allows for polarity to be established in the marginal zone. Interestingly, this putative signaling event permits the mobilization of Dsh, a molecule more frequently associated with the Wnt/Fz signaling pathways (Boutros and Mlodzik, 1999). Previous reports, however, have suggested a link between integrins and Wnt signaling (DAmico et al., 2000; Novak et al., 1998; Tu et al., 1999; Yoganathan et al., 2000), as well as the mobilization of Dsh following integrin ligation (Torres and Nelson, 2000). Significantly, interference with XFz7 (Djiane et al., 2000), XWnt11 (Tada and Smith, 2000) or Dsh (Wallingford et al., 2000) in the DMZ blocks convergent extension movements and alters cell-cell adhesion (Medina et al., 2000) without altering the ability of cells to adhere to FN (M. M., G. N. Wheeler and D. W. D., unpublished) (Tada and Smith, 2000). Our model predicts that integrin signaling is in a pathway parallel to that described for Wnt/Fz, and that interplay between these pathways, possibly through Dsh, establishes cell polarity in the DMZ of the embryo within a correct temporal framework. While the molecules that mediate cell polarity continue to be characterized, the signals that provide the initial asymmetry required for the development of polarity remain unknown. Our evidence suggests that FN matrix may provide a cue important in the establishment of cellular asymmetry that is crucial to subsequent morphogenetic rearrangements in Xenopus.
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ACKNOWLEDGMENTS |
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