Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo

The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix.     

Mechanisms of mesendoderm internalization in the Xenopus gastrula: lessons from the ventral side.

Two main processes are involved in driving ventral mesendoderm internalization in the Xenopus gastrula. First, vegetal rotation, an active movement of the vegetal cell mass, initiates gastrulation by rolling the peripheral blastocoel floor against the blastocoel roof. In this way, the leading edge of the internalized mesendoderm is established, that remains separated from the blastocoel roof by Brachet's cleft. Second, in a process of active involution, blastopore lip cells translocate on arc-like trails around the tip of Brachet's cleft. Hereby the lower, Xbra-negative part of the lip moves toward the interior, to contribute mainly to endoderm. In contrast, the upper, Xbra-expressing part moves toward the blastocoel roof-apposed surface of the involuted mesoderm, and eventually becomes inserted into this surface. Vegetal rotation and active mesoderm surface insertion persist over much of gastrulation ventrally. Both processes are also active dorsally. In fact, internalization processes generally spread from dorsal to ventral, though at different rates, which suggests that they are independently controlled. Ventrally and laterally, mesoderm occurs not only in the marginal zone, but also in the adjacent blastocoel roof. Such blastocoel roof mesoderm shares properties with the remaining, ectodermal roof, that are related to its function as substratum for mesendoderm migration. It repels involuted mesoderm, thus contributing to separation of cell layers, and it assembles a fibronectin matrix. These properties change as the blastocoel roof mesoderm moves into the blastopore lip during gastrulation.     

Xenopus embryonic cell adhesion to fibronectin: position-specific activation of RGD/synergy site-dependent migratory behavior at gastrulation

During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg- Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.     

Fibronectin visualized by scanning electron microscopy immunocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae

In amphibian gastrulae, scanning electron microscopy (SEM) has shown the presence of a network of extracellular fibrils on the inner aspect of the ectoderm layer, which serves as the substratum for migration by the presumptive mesoderm cells. In vitro experiments have shown that the fibril network promotes attachment and migration by mesoderm cells, and probably guides the migration by contact guidance. Filopodia of the migrating cells showed preferential attachment to the fibrils. Use of a colloidal gold probe for SEM immunocytochemistry has shown that fibrils observed by SEM contain fibronectin, probably as a major component. This provides direct evidence that the extracellular matrix containing fibronectin provides the substratum and guides cell migration in morphogenetic movement.     

Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling

In vertebrates, PDGFA and its receptor, PDGFRalpha, are expressed in the early embryo. Impairing their function causes an array of developmental defects, but the underlying target processes that are directly controlled by these factors are not well known. We show that in the Xenopus gastrula, PDGFA/PDGFRalpha signaling is required for the directional migration of mesodermal cells on the extracellular matrix of the blastocoel roof. Blocking PDGFRalpha function in the mesoderm does not inhibit migration per se, but results in movement that is randomized and no longer directed towards the animal pole. Likewise, compromising PDGFA function in the blastocoel roof substratum abolishes directionality of movement. Overexpression of wild-type PDGFA, or inhibition of PDGFA both lead to randomized migration, disorientation of polarized mesodermal cells, decreased movement towards the animal pole, and reduced head formation and axis elongation. This is consistent with an instructive role for PDGFA in the guidance of mesoderm migration.     

Mesodermal cell migration during Xenopus gastrulation

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula.

During Xenopus gastrulation, the mesoderm migrates across a fibronectin (FN)-containing substrate, the inner surface of the blastocoel roof (BCR). A possible role for FN is to promote the extension of cytoplasmic processes which serve as locomotory organelles for mesoderm cells. To test this idea, the interaction of prospective head mesoderm (HM) cells with FN was examined in vitro. Nonattached HM cells extend filiform processes from an active region of the cell surface. This spontaneous activity is modulated by cell attachment to FN. Additional active regions appear, and cytoplasmic lamellae extend from these sites, leading to cell spreading and translocation. Thus, although FN seems not to induce processes de novo, it modulates a spontaneous protrusive activity to yield the extension of lamellae along the substrate surface. As putative locomotory organelles, HM cell protrusions were characterized functionally. They adhere rapidly and selectively to in situ substrates, preferentially to FN, and retract upon attachment. During translocation, the passive cell body is moved by the activity of the protrusions. Lamellae continuously extend, retract, or split into parts. This leads to an intermittent, nonpersistent mode of translocation. The polarity of HM cells, as expressed in the arrangement of protrusions, bears no constant relationship to the orientation of the cell body, and a cell can change its direction of movement without a corresponding rotation of the cell body. This may be relevant with respect to the mechanism by which mesoderm cells translate guidance cues of the BCR into a polarized, oriented cell structure during directional migration in situ.     

Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel roles for integrin and fibronectin

Fibronectin (FN) is reported to be important for early morphogenetic movements in a variety of vertebrate embryos, but the cellular basis for this requirement is unclear. We have used confocal and digital time-lapse microscopy to analyze cell behaviors in Xenopus gastrulae injected with monoclonal antibodies directed against the central cell-binding domain of fibronectin. Among the defects observed is a disruption of fibronectin matrix assembly, resulting in a failure of radial intercalation movements, which are required for blastocoel roof thinning and epiboly. We identified two phases of FN-dependent cellular rearrangements in the blastocoel roof. The first involves maintenance of early roof thinning in the animal cap, and the second is required for the initiation of radial intercalation movements in the marginal zone. A novel explant system was used to establish that radial intercalation in the blastocoel roof requires integrin-dependent contact of deep cells with fibronectin. Deep cell adhesion to fibronectin is sufficient to initiate intercalation behavior in cell layers some distance from the substrate. Expression of a dominant-negative beta1 integrin construct in embryos results in localized depletion of the fibronectin matrix and thickening of the blastocoel roof. Lack of fibronectin fibrils in vivo is correlated with blastocoel roof thickening and a loss of deep cell polarity. The integrin-dependent binding of deep cells to fibronectin is sufficient to drive membrane localization of Dishevelled-GFP, suggesting that a convergence of integrin and Wnt signaling pathways acts to regulate radial intercalation in Xenopus embryos.     

Mesoderm cell migration in the vertebrate gastrula

Mesoderm migration across the inner surface of the outer embryonic layer is an essential morphogenetic mechanism in vertebrate gastrulation. Conserved traits of this process are (1) cadherin-dependent cohesion of the mesoderm, and (2) a predominant role for fibronectin in mediating mesoderm cell-substrate interactions. Compared to lower vertebrates, differentiation of the outer substrate-forming cell layer is accelerated in amniotes, providing mesoderm cells with a basement membrane substrate instead of a loose network of extracellular matrix fibrils. Guidance cues which determine the direction of mesoderm migration have been demonstrated in the fibrillar matrix of the amphibian gastrula.     

Precardiac cell migration: fibronectin localization at mesoderm-endoderm interface during directional movement

The pathway of directional movement of chick precardiac mesoderm cells was studied by indirect immunofluorescence and by scanning electron microscopy. Directional movement of the precardiac cells begins at stage 6 from the lateral sides of the embryo at the level of Hensen's node. The cells move anteriorly in an arc to the embryo's midline. By stage 8 the cells arrive at the lateral sides of the anterior intestinal portal and movement ceases. The interval of this directional movement is approximately 10 hr. During migration the precardiac cells are in close association with the underlying endoderm. As migration proceeds, the cells encounter increasing amounts of fibrils in the substratum at the mesoderm-endoderm interface. Concomitant with increasing fibril formation there is an increase in fibronectin (FN) in the heart-forming region. During stage 5 FN first appears in the lateral heart-forming regions and increases in amount during the period of cell migration. By stage 7 a concentration difference of FN is apparent in the lateral regions with more FN cephalad and decreasing amounts caudad. At stages 7 and 8 large amounts of extracellular FN-associated fibrils are observed at the lateral sides of the anterior intestinal portal where the cells stop moving. The precardiac cells moving into this region are oriented perpendicular to the anterior intestinal portal and in close association with these fibrils. There is no evidence that the fibrillar meshwork forming the substratum of the precardiac mesoderm cells is physically oriented as a guide for directional movement. The correlations between FN distribution at the mesoderm-endoderm interface and directional cell movement suggest that the precardiac cells may migrate by haptotaxis, i.e., by moving along the substratum toward areas of greater adhesiveness.     

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens female X Rana esculenta male

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.     

The 140-kDa fibronectin receptor complex is required for mesodermal cell adhesion during gastrulation in the amphibian Pleurodeles waltlii

We have studied the localization and function of a 140-kDa glycoprotein complex implicated in cell adhesion to fibronectin- and laminin-rich extracellular matrices in Pleurodeles waltlii gastrulae. In particular, we have shown that antibodies directed against highly purified avian fibronectin (FN) receptor complex cross-react with two major polypeptides of apparent molecular weights of 140,000 and 100,000 and a third minor component of 90,000. Using sections of embryos or whole mounts, we have also discovered that the putative FN receptor is widely distributed on the early embryonic cell surface. We have also found that the basal surface of the roof of the blastocoel, a region particularly enriched in an extracellular matrix consisting of fibronectin- and laminin-rich fibrils, is rich in receptor complex. We have prepared monovalent Fab' fragments of this antibody and have found that they cause detachment of cells previously attached to substrata coated with fibronectin, and they also arrest gastrulation when injected into the blastocoel of early gastrulae. Thus, it appears that the fibronectin receptor complex plays a significant functional role in cell attachment of gastrula-stage cells in vitro and in cell migration in vivo during gastrulation.     

Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.     

A PTP-PEST-like protein affects alpha5beta1-integrin-dependent matrix assembly, cell adhesion, and migration in Xenopus gastrula

During amphibian gastrulation, mesodermal cell movements depend on both cell-cell and cell-matrix interactions. Ectodermal cells from the blastocoel roof use alpha5beta1 integrins to assemble a fibronectin-rich extracellular matrix on which mesodermal cells migrate using the same alpha5beta1 integrin. In this report, we show that the tyrosine phosphatase xPTP-PESTr can prevent fibronectin fibril formation when overexpressed in ectodermal cells resulting in delayed gastrulation. In addition, isolated ectodermal cells overexpressing xPTP-PESTr are able to spread on fibronectin using the alpha5beta1 integrin in the absence of activin-A induction and before the onset of gastrulation. We further show that while the inhibition of fibrillogenesis depends on the phosphatase activity of xPTP-PESTr, induction of cell spreading does not. Finally, while cell spreading is usually associated with cell migration, xPTP-PESTr promotes ectodermal cell spreading on fibronectin but also reduces cell migration in response to activin-A, suggesting an adverse effect on cell translocation. We propose that xPTP-PESTr overexpression adversely affect cell migration by preventing de-adhesion of cells from the substrate.     

Roles for Xenopus aquaporin-3b (aqp3.L) during gastrulation: Fibrillar fibronectin and tissue boundary establishment in the dorsal margin

Aquaporins and aquaglyceroporins are a large family of membrane channel proteins that allow rapid movement of water and small, uncharged solutes into and out of cells along concentration gradients. Recently, aquaporins have been gaining recognition for more complex biological roles than the regulation of cellular osmotic homeostasis. We have identified a specific expression pattern for Xenopus aqp3b (also called aqp3.L) during gastrulation, where it is localized to the sensorial (deep) layer of the blastocoel roof and dorsal margin. Interference with aqp3b expression resulted in loss of fibrillar fibronectin matrix in Brachet's cleft at the dorsal marginal zone, but not on the free surface of the blastocoel. Detailed observation showed that the absence of fibronectin matrix correlated with compromised border integrities between involuted mesendoderm and noninvoluted ectoderm in the marginal zone. Knockdown of aqp3b also led to delayed closure of the blastopore, suggesting defects in gastrulation movements. Radial intercalation was not affected in aqp3b morphants, while the data presented are consistent with impeded convergent extension movements of the dorsal mesoderm in response to loss of aqp3b. Our emerging model suggests that aqp3b is part of a mechanism that promotes proper interaction between cells and the extracellular matrix, thereby playing a critical role in gastrulation.     

The Spemann organizer meets the anterior‐most neuroectoderm at the equator of early gastrulae in amphibian species

The dorsal blastopore lip (known as the Spemann organizer) is important for making the body plan in amphibian gastrulation. The organizer is believed to involute inward and migrate animally to make physical contact with the prospective head neuroectoderm at the blastocoel roof of mid‐ to late‐gastrula. However, we found that this physical contact was already established at the equatorial region of very early gastrula in a wide variety of amphibian species. Here we propose a unified model of amphibian gastrulation movement. In the model, the organizer is present at the blastocoel roof of blastulae, moves vegetally to locate at the region that lies from the blastocoel floor to the dorsal lip at the onset of gastrulation. The organizer located at the blastocoel floor contributes to the anterior axial mesoderm including the prechordal plate, and the organizer at the dorsal lip ends up as the posterior axial mesoderm. During the early step of gastrulation, the anterior organizer moves to establish the physical contact with the prospective neuroectoderm through the “subduction and zippering” movements. Subduction makes a trench between the anterior organizer and the prospective neuroectoderm, and the tissues face each other via the trench. Zippering movement, with forming Brachet's cleft, gradually closes the gap to establish the contact between them. The contact is completed at the equator of early gastrulae and it continues throughout the gastrulation. After the contact is established, the dorsal axis is formed posteriorly, but not anteriorly. The model also implies the possibility of constructing a common model of gastrulation among chordate species.     

Planar Polarity in the Ciliated Epidermis of Xenopus Embryos

The coordinated orientation of ciliary beat in the larval epidermis of amphibians, evident in an organized streamline pattern, suggests a planar polarity of the epithelium, i.e., a polarity within the plane of the cell sheet. It has been proposed that the direction of ciliary beat is determined at mid gastrula by a gradient of a diffusible factor produced by the mesoderm. To analyze whether ectoderm in isolation can establish a uniform direction of ciliary beat, and at what stage its polarity is specified in the embryo, ectoderm of Xenopus laevis embryos of different stages was cultured in vitro on substrates. On concanavalin A, ectoderm isolated at early gastrula stages, i.e., prior to any contact with mesoderm, can autonomously coordinate the direction of ciliary beat, at least in small regions. A uniform planar polarity is expressed by ectoderm explanted from the early mid gastrula onward. On fibronectin, which promotes migration, the direction of movement correlates well with the direction of ciliary beat, and directional migration can even override the inherent polarity specified prior to explantation. Embryos which lack dorsal mesoderm nevertheless develop a highly organized streamline pattern, excluding a strict requirement for dorsal mesoderm for the determination of planar polarity. However, in spite of the early specification of planar polarity found for isolated tissue, rotated ectodermal transplants in situ can readjust their polarity in accordance with that of the host.     

Regulation of Xenopus gastrulation by ErbB signaling

During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Different signals are implicated in these processes. Our previous studies reveal that signals through ErbB receptor tyrosine kinases modulate Xenopus gastrulation, but the mechanisms employed are not understood. Here we report that ErbB signals control both C&E and head mesoderm migration. Inhibition of ErbB pathway blocks elongation of dorsal marginal zone explants and activin-treated animal caps without removing mesodermal gene expression. Bipolar cell shape and cell mixing in the dorsal region are impaired. Inhibition of ErbB signaling also interferes with migration of prechordal mesoderm on fibronectin. Cell-cell and cell-matrix interaction and cell spreading are reduced when ErbB signaling is blocked. Using antisense morpholino oligonucleotides, we show that ErbB4 is involved in Xenopus gastrulation morphogenesis, and it partially regulates cell movements through modulation of cell adhesion and membrane protrusions. Our results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to non-canonical Wnt and FGF signals, that controls gastrulation. We further demonstrate that regulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation.     

A maternal-effect mutation disturbs extracellular matrix organization in the early Pleurodeles waltl embryo

Summary In the early development of the urodele amphibian Pleurodeles waltl, a fibronectin-containing extracellular matrix underlies the inner face of the blastocoel roof. When gastrulation occurs, the fibronectin fibrils provide a suitable substrate for mesodermal-cell migration. Delay in morphogenetic movements of gastrulation has been described in embryos from mutant females (ac/ac) of Pleurodeles waltl. Studies of abnormal mutant gastrulae with fluorescent lectins and immunostaining for fibronectin reveal that they lack a normal matrix. The fibronectin-containing extracellular material always gives rise to a granular pattern without fibronectin-fibril formation. Fibronectin and ₅₁ syntheses occur normally in maternal-effect embryos. In vitro, mesodermal cells from early mutant gastrulae adhere and migrate on fibronectin-conditioned substrata.