Gastrulation and larval pattern in Xenopus after blastocoelic injection of a Xenopus-derived inducing factor: experiments testing models for the normal organization of mesoderm

When a Xenopus XTC cell-derived mesoderm-inducing factor (MIF) is injected into the blastocoel of Xenopus embryos before gastrulation, they develop almost normally until just after the onset of mesoderm involution at the internal blastoporal lip. Cells from the entire lining of the blastocoel roof and inner marginal zone then undergo a synchronous, sudden change of contact and arrangement which resembles the transformation undergone by normal mesoderm at its time of involution at the vegetal edge of the marginal zone. We describe a dose-dependent spectrum of subsequent abnormalities in gastrulation and, in cases where gastrulation partially recovers, in the resulting larval pattern. Because of such recovery, embryos injected with widely different doses may appear equally abnormal at the early gastrula stage but very different by control larval stages. Extra spinocaudal axial patterns, in the area of ectopic mesoderm, are seen after MIF doses that just permit recovery of gastrulation. The sudden cellular transformation corresponding to involution, in the ectopically specified mesoderm, spreads throughout the animal cap within 15 min in individuals, at a time significantly later than the earliest normal transformation in the marginal zone. No systematic alteration could, however, be detected in its timing, in relation to a 250-fold range of injected MIF concentration or a 3.5-hr difference in time of injection. The severity of the effects on final embryonic pattern is largely independent of the blastular stage of injections. Splitting of the total injected dose into two, separated by 2 to 3 hr of blastular development, reveals that the degree of effect on gastrulation and patterning depends only upon the highest experienced concentration at any time before response. When fibroblast growth factor (bFGF), a different effective mesoderm inducer, is similarly injected, a similar abnormal cell behavior and ectopic mesoderm formation are seen, but beginning only at midgastrular stages some 1.5 hr beyond that characteristic of XTC-MIF. The findings are introduced and discussed in terms of models for the natural organization of the time course of gastrulation and mesodermal pattern.     

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.     

Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells

When Xenopus embryos are cultured in calcium- and magnesium-free medium (CMFM), the blastomeres lose adhesion but continue dividing to form a loose heap of cells. If divalent cations are restored at the early gastrula stage the cells re-adhere and eventually form muscle (a mesodermal cell type) as well as epidermis. If, however, the cells are dispersed during culture in CMFM, muscle does not form following reaggregation although epidermis does. This suggests that culturing blastomeres in a heap allows the transmission of mesoderm-induction signals from cell to cell while dispersion effectively dilutes the signal. In this paper, we have attempted to substitute for cell proximity by culturing dispersed blastomeres in XTC mesoderm-inducing factor (MIF). We find that dispersed cells do not respond to XTC-MIF by forming mesodermal cell types after reaggregation, but the factor does inhibit epidermal differentiation. One interpretation of this observation is that an early stage in mesoderm induction is the suppression of epidermal differentiation and that formation of mesoderm may require contact-mediated signals that are produced in response to XTC-MIF. We have gone on to study the suppression of epidermal differentiation in more detail. We find that this is a dose-dependent phenomenon that can occur in single cells in the absence of cell division. Animal pole blastomeres become more difficult to divert from epidermal differentiation at later stages of development and by stage 12 they are 'determined' to this fate. Fibroblast growth factor (FGF) also suppresses epidermal differentiation in isolated animal pole blastomeres and transforming growth factor-beta 1 acts synergistically with FGF in doing so.     

A mesoderm-inducing factor is produced by Xenopus cell line

Inductive interactions play a major role in the diversification of cell types during vertebrate development. These interactions have been extensively studied in amphibian embryos (usually Xenopus laevis) where the earliest is mesoderm induction, in which an equatorial mesodermal rudiment is induced from the animal hemisphere under the influence of signal from the vegetal hemisphere. The molecular basis of mesoderm induction is unknown, although Tiedemann has isolated a protein form 9- to 13-day chick embryos that has the properties one would expect of a mesoderm-inducing factor. However, the relevance of this molecule to the events of early amphibian development is unclear, and it is a matter of some importance to discover a Xenopus mesoderm-inducing factor. In this paper I show that the Xenopus XTC cell line secretes mesoderm-inducing activity into the culture medium. Isolated animal pole regions cultured in XTC-conditioned medium differentiate into muscle and notochord, while controls form 'atypical epidermis'. Three different cell lines -XL, XL177 and KR- secrete no such activity indicates that the active principle is heat stable, trypsin sensitive, nondialysable, and has an apparent relative molecular mass of about 16,000. Work is in progress to characterize the activity further and to discover whether the mesoderm-inducing factor is also present in normal embryos.     

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.     

The mitochondrial-apoptotic pathway is triggered in Xenopus mesoderm cells deprived of PDGF receptor signaling during gastrulation

Platelet-derived growth factor receptor (PDGFR) signaling is required for normal gastrulation in Xenopus laevis. Embryos deprived of PDGFR signaling develop with a range of gastrulation-specific defects including spina bifida, shortened anteroposterior axis, and reduced anterior structures. These defects arise because the involuting mesoderm fails to move appropriately. In this study, we determine that inhibition of PDGFR signaling causes prospective head mesoderm cells to appear in the blastocoel cavity at the onset of gastrulation, stage 10. These aberrant cells undergo apoptosis via the caspase 3 pathway at an embryonic checkpoint called the early gastrula transition (EGT). They are TUNEL-positive and have increased levels of caspase 3 activity compared to control embryos. Apoptotic death of these mesoderm cells can be prevented by co-injection of mRNA encoding Bcl-2 or by injection of either a general caspase inhibitor or a caspase 3-specific inhibitor. Prevention of cell death, however, is not sufficient to rescue gastrulation defects in these embryos. Based on these data, we propose that PDGFR signaling is necessary for survival of prospective head mesoderm cells, and also plays an essential role in the control of their cell movement during gastrulation.     

Generation of the germ layers along the animal-vegetal axis in Xenopus laevis

After completion of gastrulation, typical vertebrate embryos consist of three cell sheets, called germ layers. The outer layer, the ectoderm, which produces the cells of the epidermis and the nervous system; the inner layer, the endoderm, producing the lining of the digestive tube and its associated organs (pancreas, liver, lungs etc.) and the middle layer, the mesoderm, which gives rise to several organs (heart, kidney, gonads), connective tissues (bone, muscles, tendons, blood vessels), and blood cells. The formation of the germ layers is one of the earliest embryonic events to subdivide multicellular embryos into a few compartments. In Xenopus laevis, the spatial domains of three germ layers are largely separated along the animal-vegetal axis even before gastrulation; ectoderm in the animal pole region; mesoderm in the equatorial region and endoderm in the vegetal pole region. In this review, we summarise the recent advances in our understanding of the formation of the germ layers in Xenopus laevis.     

Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.

A main achievement of gastrulation is the movement of the endoderm and mesoderm from the surface of the embryo to the interior. Despite its fundamental importance, this internalization process is not well understood in amphibians. We show that in Xenopus, an active distortion of the vegetal cell mass, vegetal rotation, leads to a dramatic expansion of the blastocoel floor and a concomitant turning around of the marginal zone which constitutes the first and major step of mesoderm involution. This vigorous inward surging of the vegetal region into the blastocoel can be analyzed in explanted slices of the gastrula, and is apparently driven by cell rearrangement. Thus, the prospective endoderm, previously thought to be moved passively, provides the main driving force for the internalization of the mesendoderm during the first half of gastrulation. For further involution, and for normal positioning of the involuted mesoderm and its rapid advance toward the animal pole, fibronectin-independent interaction with the blastocoel roof is required.     

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.     

eFGF regulates Xbra expression during Xenopus gastrulation.

We show that, in addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm-inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero-posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. We show that during blastula stages eFGF and Xbra are able to activate the expression of each other, suggesting that they are components of an autocatalytic regulatory loop. Moreover, we show that Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid-blastula transition results in the up-regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF-Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages.     

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.     

Expression of nicotinic acetylcholine receptors in aneural Xenopus embryos

During gastrulation in vertebrate embryos, the mesoderm moves inward and under the ectoderm and these two cell layers subsequently differentiate in close proximity to each other, providing an opportunity for the exchange of inductive signals. This study examines whether the activation of muscle nicotinic acetylcholine receptor (AChR) genes and the subsequent expression of receptors in Xenopus myotomal muscle are dependent on interaction between the ectoderm and the mesoderm, or their derivatives, after the onset of gastrulation. We eliminated such interaction by inducing total exogastrulation of Xenopus embryos. During exogastrulation, the mesoderm moves away from the ectoderm, and the nervous system fails to develop. Single channel recordings from the myotomal muscle of exogastrulated embryos revealed the presence of two major classes of AChRs, which could be distinguished on the basis of channel conductance. The current amplitudes, conductances, reversal potentials, and open times of these channels closely resembled those reported for the two major classes of AChR channels normally expressed in vivo. We conclude that interaction between ectoderm and mesoderm following the onset of gastrulation is not required for the future expression of the major classes of AChRs in myotomal muscle.     

Mediolateral cell intercalation in the dorsal, axial mesoderm of Xenopus laevis

The pattern of mediolateral cell intercalation in mesodermal tissues during gastrulation and neurulation of Xenopus laevis was determined by tracing cells labeled with fluorescein dextran amine (FDA). Patches of the involuting marginal zone (IMZ) of early gastrula stage embryos, labeled by injection of FDA at the one-cell stage, were grafted to the corresponding regions of unlabeled host embryos. The host embryos were fixed at several stages, serially sectioned, and examined with fluorescence microscopy and three-dimensional reconstruction. Patterns of mixing of labeled and unlabeled cells show that mediolateral cell intercalation occurs in the posterior, dorsal mesoderm as this region undergoes convergent extension and differentiates into somites and notochord. In contrast, it does not occur in any dorsoventral sector of the anterior, leading edge of the mesodermal mantle. These results, taken with other evidence, suggest that the mesoderm of Xenopus consists of two subpopulations, each with a characteristic morphogenetic movement, cell behavior, and tissue fate. The migrating mesoderm (1) does not show convergent extension; (2) migrates and spreads on the blastocoel roof; (3) is dependent on this substratum for its morphogenesis; (4) shows little mediolateral intercalation; (5) consists of the anterior, early-involuting region of the mesodermal mantle; and (6) differentiates into head, heart, blood island, and lateral body wall mesoderm. The extending mesoderm (1) shows convergent extension; (2) is independent of the blastocoel roof in its morphogenesis; (3) shows extensive mediolateral intercalation; (4) consists of the posterior, late-involuting parts of the mesodermal mantle; and (5) differentiates into somite and notochord.