Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor

The mesoderm of Xenopus laevis is formed through an inductive interaction in which a signal from the vegetal hemisphere of the blastula acts on overlying animal pole cells. We have recently reported that the Xenopus XTC cell line secretes a mesoderm-inducing factor (MIF) which may resemble the natural signal. In this paper, we describe the purification and biological effects of XTC-MIF. XTC-MIF is a hydrophobic protein with an isoelectric point of 7.8 and an apparent relative molecular mass (Mr) of 23,500. On reduction, XTC-MIF loses its biological activity and the protein dissociates into two inactive subunits with apparent Mr of about 15,000. These properties closely resemble those of transforming growth factor type beta (TGF-beta), and it is interesting that TGF-beta 2 has recently been shown to have mesoderm-inducing activity. The biological response to XTC-MIF is graded. After exposure to 0.2-1.0 ng ml-1 XTC-MIF, stage-8 animal pole explants form mesenchyme and mesothelium. At higher concentrations, up to about 5 ng ml-1, muscle is formed, occasionally with neural tissue. In response to concentrations of XTC-MIF greater than 5-10 ng ml-1, notochord and neural tissue are usually formed. The formation of notochord and neural tissue in response to XTC-MIF represents a qualitative difference between this inducing factor and the other known group of MIFs, the heparin-binding growth factors.     

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 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.     

Mesoderm Induction in Pleurodeles waltl: Distribution of Fibronectin and Chondroitin Sulfate in Induced Explants

In Pleurodeles , cell-matrix interactions play a major role in promoting active mesodermal cell migration during gastrulation. It was therefore important to determine whether the expression of define matrix molecules may be dependent on mesoderm induction. Results from induction experiments done with XTC cell line-conditioned medium show that mesoderm tissues induced in animal cap explants of Pleurodeles are identical to those from Xenopus . However, we also show that dorsally-induced explants in Pleurodeles elongate to a lesser degree than in Xenopus . This observation agrees well with the differences observed in the role of ECM in Pleurodeles and Xenopus gastrulation, respectively. Additional immunostaining studies demonstrate that the induction of mesodermal tissues is associated with the expression of chondroitin sulfate whereas fibronectin fibrils are already assembled in uninduced animal caps. These results suggest that mesoderm cell-matrix interactions in early amphibian embryo may be under the control of mesoderm induction.     

Mesendoderm extension and mantle closure in Xenopus laevis gastrulation: combined roles for integrin alpha(5)beta(1), fibronectin, and tissue geometry

We describe mesendoderm morphogenesis during gastrulation in the frog Xenopus laevis and investigate the mechanics of these movements with tissue explants. When a dorsal marginal zone explant is plated onto fibronectin, the mesendoderm moves away from the dorsal axial tissues as an intact sheet. Mesendodermal cells within these explants display monopolar protrusive activity and radially intercalate during explant extension. Live time-lapse confocal sequences of actin dynamics at the margin of these extending explants prompt us to propose that integrin-mediated traction drives these movements. We demonstrate that integrin alpha(5)beta(1) recognition of the synergy site located within the type III(9) repeat of fibronectin is required for mesendoderm extension. Normal mesendoderm morphogenesis occurs with a unique "cup-shaped" geometry of the extending mesendodermal mantle and coincides with a higher rate of tissue extension than that seen in the simpler dorsal marginal zone explant. These higher rates can be reconstituted with "in-the-round" configurations of several explants. We propose several mechanically based hypotheses to explain both the initial fibronectin-dependent extension of the mesendoderm and additional requirement of tissue geometry during the high-velocity closure of the mesendodermal mantle.     

Regulation of Xenopus embryonic cell adhesion by the small GTPase,rac

TGF-beta family signalling pathways are important for germ layer formation and gastrulation in vertebrate embryos and have been studied extensively using embryos of Xenopus laevis. Activin causes changes in cell movements and cell adhesion in Xenopus animal caps and dispersed animal cap cells. Rho family GTPases, including rac, mediate growth factor-induced changes in the actin cytoskeleton, and consequently, in cell adhesion and motility, in a number of different cell types. Ectopic expression of mutant rac isoforms in Xenopus embryos was combined with animal cap adhesion assays and a biochemical assay for rac activity to investigate the role of rac in activin-induced changes in cell adhesion. The results indicate that (1) the perturbation of rac signalling disrupts embryonic cell-cell adhesion, (2) that rac activity is required for activin-induced changes in cell adhesive behavior on fibronectin, and (3) that activin increases endogenous rac activity in animal cap explants.     

Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor

We have cultured explants of Xenopus blastular animal cap tissue from embryos that had received an earlier treatment with LiCl and from their untreated siblings, in various concentrations of XTC-cell-derived mesoderm-inducing factor (XTC-MIF, Smith, 1987; Smith et al. 1988). The pretreatment with lithium that we used transforms later morphogenesis in the whole embryo to give radialized body forms with anterior/dorsal levels of structure grossly over-represented. In addition, animal caps from 'Li+' embryos were allowed to develop without exposure to in vitro MIF (Li+ controls) and compared with normal uninduced control explants, and explants were made from normal early blastulae but given various initial treatments with LiCl in culture. The results confirm that the lithium ion itself will not induce mesoderm in competent, animal cap tissue of Xenopus. It does, however, enhance the responsiveness of this tissue to XTC-MIF, in a way that parallels its recently reported effect in the case of another mesoderm inducer of different character, bFGF (Slack et al. 1988). The effects observed are sufficient to imply that the altered body pattern that follows lithium treatment, in whole embryos, could be caused by modulation of the responses to an unaltered pattern of in situ inductive stimuli. We also observe evidence that appreciable inductive signals reach animal pole tissue beyond the limits of mesoderm formation in normal development. Relatively low concentrations of MIF prevent the development of an epidermis-specific marker in dissociated blastular animal cap cells (Symes et al. 1988). When such experiments are repeated in relation to the lithium pretreatment of embryos, such treatment is seen to have sensitized the cell population, so that the MIF concentration range that assures complete suppression of the marker is reduced. The results are discussed in relation to induction considered as pattern formation.     

Are beta 1 integrins involved in Xenopus gastrulation?

The molecular basis of vertebrate gastrulation is poorly understood. Work on urodele amphibians has implicated beta 1-containing integrins, but the limited information available for Xenopus indicates otherwise: peptides containing the RGD sequence do not inhibit gastrulation and induction of cell spreading in presumptive ectodermal cells by activin is not accompanied by an increase in synthesis of integrin beta 1. Here we report that beta 1-containing integrins are, nevertheless, the principal fibronectin receptors in the Xenopus gastrula, although their cell surface levels are low. Antibodies recognizing the external domain of the molecule can, unlike peptides containing the RGD site, block gastrulation when introduced into the blastocoel. These results allow us to propose a model to explain the role of integrin beta 1 in Xenopus gastrulation.     

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.     

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)     

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.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

Overexpression of a homeodomain protein confers axis-forming activity to uncommitted Xenopus embryonic cells.

The anteroposterior character of mesoderm induced by a peptide growth factor (XTC-MIF) was tested by transplantation into host Xenopus gastrulae. Both retinoic acid and a homeodomain protein were able to override the anteriorizing effect of the growth factor. Microinjection of a posteriorly expressed homeobox mRNA can respecify anteroposterior identity, transforming head mesoderm into tail-inducing mesoderm. Unexpectedly, overexpression of XIHbox 6 protein in the transplanted cells, without addition of growth factors, caused the formation of tail-like structures. The cells overexpressing XIHbox 6 were able to recruit cells from the host into the secondary axis. The results suggest that vertebrate homeodomain proteins are part of the biochemical pathway leading to the generation of the body axis.     

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.     

When does the anterior endomesderm meet the anterior-most neuroectoderm during Xenopus gastrulation?

During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements.     

SDF-1 alpha regulates mesendodermal cell migration during frog gastrulation

During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1alpha, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.     

Morphological changes of the surface of the egg of Xenopus laevis in the course of development: III. Scanning electron microscopy of gastrulation

Cell surface changes occurring before and during gastrulation in Xenopus laevis embryos have been examined by scanning electron microscopy (SEM). Our study covers the period of development from very young blastulae (stage 7) to late gastrulae (stage $\text{12}\text{1}\text{2}$. Before the onset of the epibolic movement there is evidence of locomotory activity of the cells lining the blastocoel at the animal pole. In the medim- (stage 8) and small-cell (stage 9) blastula, when pregastrulation movements are progressing rapidly, microvilli appear in the interstices between cells, both at the animal and at the vegetal pole. In the gastrula, most of the cells close to the blastopore have either their entire exposed surface or part of it covered with microvilli. On the other hand, the cells that have just reached the blastopore and have become clubshaped do not display microvilli on their surfaces; microvilli are also absent on the surface of the cells that have undergone invagination. The invaginated chorda-mesoderm is made up of single fibroblastlike cells with long thin filopodia which are interwoven with those of nearby cells. The observations are discussed in relation to changes in cell-to-cell connections and to the role of cell surface organization in the morphogenetic movements of gastrulation.     

Adhesive crosstalk in gastrulation

Recent studies show that signaling through integrin receptors is required for normal cell movements during Xenopus gastrulation. Integrins function in this process by modulating the activity of cadherin adhesion molecules within tissues undergoing convergence and extension movements.