Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.     

Dorsal Blastomeres in the Equatorial Region of the 32-Cell Xenopus Embryo Autonomously Produce Progeny Committed to the Organizer

For testing the autonomic differentiation abilities of dorsal equatorial blastomeres of 32-cell Xenopus embryos, their roles in head formation in normal development and the organizer-inducing capabilities of the dorsal-most vegetal cells, interspecific transplantations were made using Xenopus borealis and X. laevis . When transplanted into the ventral region, the dorsal blastomeres produced descendants that differentiated into prechordal mesoderm, notochord and somites in the recipient according to their fates. They induced formation of the secondary embryo with the head and tail. The prechordal mesoderm and notochord in the secondary structure consisted of progeny of the graft, whereas somites and the CNS were chimeric and the pronephros was composed of host cells. Replacement of the dorsal blastomeres by ventral equatorial cells caused complete arrest of head formation in the recipient. Without exception, the notochord was completely absent or very thin. These results confirm the assumption that the presumptive head organizer in the Xenopus embryo is localized in the dorsal equatorial region at the 32-cell stage and comes into existence not under the inductive influence of the dorsal-most vegetal cells, but owing to allocation of morphogenetic determinants residing in the fertilized egg to the dorsal equatorial blastomeres of the 32-cell embryo.     

Formation of the Neural Plate and the Mesoderm in Normally Developing Embryos of Xenopus laevis

Normally developing embryos of Xenopus were fixed at various stages between the blastula and early tail bud stage, and their serial sections were examined. The marginal belt of the blastula was characterized by abundance of cells with RNA-rich peripheral cytoplasm called mesoplasm. At the early gastrula stage, the marginal belt was folded into two layers giving rise to mesodermal material and marginal ectoderm. During gastrulation, the mesodermal material, which consisted of RNA-rich cells, spread to enclose the blastocoel and the endoderm, and a large part of it was shifted to the dorsal side of the embryo. It gradually established the mesodermal layer. The notochord was formed on the dorsal lip of the blastopore by involution, separately from preformed mesodermal material. The RNA-rich cells in the marginal ectoderm became columnar, forming a broad belt in the marginal zone. This belt was deformed and shifted to the dorsal side during gastrulation, eventually establishing the neural plate showing quantitative differentiation along the head-tail axis. Possible mechanisms involved in the formation of the neural plate and mesoderm were discussed with reference to the organizer and the mesoplasm.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

The Location of Dorsal Information in Frog Early Development

Dorsal information is necessary for the development of the dorsal axial structures which characterize the vertebrates. The nature of dorsal information in early embryos is not known, but its presence is required for the formation of dorsal mesoderm with Spermann organizer activity. In frogs, the dorsoventral axis is specified by a cortical/cytoplasmic rotation in the egg shortly after fertilization, and this dorsal information is limited to a few cells in the equatorial and vegetal region of early cleavage embryos. At the 8-cell stage, 2–4 cells can promote dorsal development, and at the 32-cell stage, 4–6 cells have dorsal information. Recent experiments have shown that growth factors can induce cells to form dorsal mesoderm and that lithium can act in those cells to enhance the induction. It will be important to determine the relationship between the location of dorsal information defined embryologically and factors involved in the development of dorsal mesoderm.     

Acquisition of developmental autonomy in the equatorial region of the Xenopus embryo

This paper describes a continuing effort to define the location and mode of action of morphogenetic determinants which direct the development of dorsal body axis structures in embryos of the frog Xenopus laevis. Earlier results demonstrated that presumptive endodermal cells in one vegetal quadrant of the 64-cell embryo can, under certain experimental conditions, induce partial or complete body axis formation by progeny of adjacent equatorial cells. (R.L. Gimlich and J.C. Gerhart, 1984, Dev. Biol. 104, 117-130). I have now assessed the importance of other blastomeres for embryonic axis formation in a series of transplantation experiments using cells from the equatorial level of the 32-cell embryo. The transplant recipients were embryos which had been irradiated with ultraviolet light before first cleavage. Without transplantation, embryos failed to develop the dorsal structures of the embryonic body axis. However, cells of these recipients were competent to respond to inductive signals from transplanted tissue and to participate in normal embryogenesis. Dorsal equatorial cells, but not their lateral or ventral counterparts, often caused partial or complete body axis development in irradiated recipients, and themselves formed much of the notochord and some prechordal and somitic mesoderm. These are the same structures that they would have formed in the normal donor. Thus, the dorsal equatorial blastomeres were often at least partially autonomous in developing according to their prospective fates. In addition, they induced progeny of neighboring host cells to contribute to the axial mesoderm and to form most of the central nervous system. The frequency with which such transplants caused complete axis formation in irradiated hosts increased when they were made at later and later cleavage stages. In contrast, the inductive activity of vegetal cells remained the same or declined during the cleavage period. These and other results suggest that the egg cytoplasmic region containing "axial determinants" is distributed to both endodermal and mesodermal precursors in the dorsal-most quadrant of the early blastula.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.     

The inducing capacity of the presumptive endoderm of Xenopus laevis studied by transfilter experiments

Summary The inducing capacity of the vegetal hemisphere of early amphibian blastulae was studied by placing a Nucleopore filter (pore size 0.4 m) between isolated presumptive endoderm and animal (ectodermal) caps. The inducing effect was shown to traverse the Nucleopore membrane. The reacting ectoderm differentiated into mainly ventral mesodermal derivatives. Expiants consisting of five animal caps also formed dorsal mesodermal and neural structures. Those results together with data published elsewhere suggest that, in addition to a vegetalizing factor, different mesodermal factors must be taken into consideration for the induction of either the ventral or the dorsal mesodermal derivatives. The neural structures are thought to be induced by the primarily induced dorsal mesodermal tissue. Electron microscopic (TEM) examination did not reveal any cell processes in the pores of the filter. The results indicate that transmissible factors rather than signals via cytoplasmic contacts or gap junctions are responsible for the mesodermal induction of ectodermal cells. The data support the view that in normogenesis the mesoderm is determined by the transfer of inducing factors from vegetal blastomeres to cells of the marginal zone (presumptive mesodermal cells).     

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.     

Early cellular interactions promote embryonic axis formation in Xenopus laevis

We have attempted to define the location and mode of action of axial determinants in the egg of Xenopus laevis. To this end, we transplanted small numbers of blastomeres from normal 64-cell stage embryos into synchronous recipient embryos which had been irradiated with ultraviolet light prior to first cleavage. Without transplantation, such embryos fail to develop dorsal structures of the embryonic body axis. We found that one to three blastomeres transplanted from the vegetal-most octet of cells can effect complete or partial rescue of of axis development in a recipient, provided that the donor cells derive from the quadrant just under the prospective dorsal marginal region. These same cells, when transplanted into the ventral vegetal quadrant of a normal 64-cell embryo, cause the formation of a complete second body axis. In contrast, other cells from the vegetal octet of normal donors fail to cause axis formation. When the rescuing donor cells are labeled with a lineage-restricted fluorescent marker, we find that their progeny do not contribute to the axial structures of the recipient. Progeny of the transplanted cells are found below the level of the blastopore in the early gastrula and eventually give rise to portions of the gut, as is their fate in normal development. These results, in agreement with those of Nieuwkoop (P.D. Nieuwkoop, 1977, Curr. Top. Dev. Biol. 11, 115-132), imply that the dorsal-most vegetal cells of the 64-cell embryo receive from the egg cytoplasm a set of determinants enabling them to induce neighboring cells to undertake axis formation. We discuss the relationship between axis induction in rescued irradiated embryos and axis determining processes in normal embryogenesis.     

Destruction of components of the neural induction system of the amphibian egg with ultraviolet irradiation

Ultraviolet irradiation of the vegetal hemisphere of the fertilized amphibian (Xenopus laevis) egg prior to first cleavage results in the embryo developing an incomplete set of neural structures. The effects of irradiation on various morphogenetic processes, including cell division, formation of the dorsal lip, invagination at gastrulation, and neural induction by the primary organizer, were examined. A decrease in the capacity for invagination during gastrulation and a diminution in the neural inducing capacity of the primary organizer were found to account for defective neurulation in irradiated embryos. Consequently, irradiation of the uncleaved egg leads to interference with the events of both gastrulation and neurulation.     

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction.

The development of the vertebrate nervous system is initiated in amphibia by inductive interactions between ectoderm and a region of the embryo called the organizer. The organizer tissue in the dorsal lip of the blastopore of Xenopus and Hensen's node in chick embryos have similar neural inducing properties when transplanted into ectopic sites in their respective embryos. To begin to determine the nature of the inducing signals of the organizer and whether they are conserved across species we have examined the ability of Hensen's node to induce neural tissue in Xenopus ectoderm. We show that Hensen's node induces large amounts of neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may reflect notoplate induction. The competence of the ectoderm to respond to Hensen's node extends much later in development than that to activin-A or to induction by vegetal cells, and parallels the extended competence to neural induction by axial mesoderm. The actions of activin-A and Hensen's node are further distinguished by their effects on lithium-treated ectoderm. These results suggest that neural induction can occur efficiently in response to inducing signals from organizer tissue arrested at a stage prior to gastrulation, and that such early interactions in the blastula may be an important component of neural induction in vertebrate embryos.     

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.     

Fates and Roles of the Presumptive Organizer Region in the 32-cell Embryo in Normal Development of Xenopus laevis

Using 32-cell Xenopus embryos series of extirpation experiments were performed in order to clarify whether or not the dorsal equatorial blastomeres were committed to differentiate to the axial mesodermal structures. First, these blastomeres designated as B1, B1', C1 and C1' and C1' were labeled using the technique of HRP injection or vital staining. They produce descendants which become localized in the organizer region of the early gastrula. These cells form the prechordal plate, notochord, somites, pharyngeal endoderm and neural tube at early neurula stage. The results of extirpation of the medial two or four of these blastomeres show that the entire head lacks or the tissues and organs of the head greatly reduce. This indicates that already at the 32-cell stage they have been committed to autonomously differentiate to form the axial mesodermal tissues of the head and that their roles in the head formation can neither be replaced nor complemented by any other blastomeres surrounding them. It is also shown that the vegetal yolk cells do not seem to play essential roles for development of the axial organs of the head. On the basis of the present results a view of establishment of the organizer of Xenopus eggs is proposed.     

Designation of the anterior/posterior axis in pregastrula Xenopus laevis

A new fate map for mesodermal tissues in Xenopus laevis predicted that the prime meridian, which runs from the animal pole to the vegetal pole through the center of Spemann's organizer, is the embryo's anterior midline, not its dorsal midline (M. C. Lane and W. C. Smith, 1999, Development 126, 423-434). In this report, we demonstrate by lineage labeling that the column 1 blastomeres at st. 6, which populate the prime meridian, give rise to the anterior end of the embryo. In addition, we surgically isolate and culture tissue centered on this meridian from early gastrulae. This tissue forms a patterned head with morphologically distinct ventral and dorsal structures. In situ hybridization and immunostaining reveal that the cultured heads contain the anterior tissues of all three germ layers, correctly patterned. Regardless of how we dissect early gastrulae along meridians running from the animal to the vegetal pole, both the formation of head structures and the expression of anterior marker genes always segregate with the prime meridian passing through Spemann's organizer. The prime meridian also gives rise to dorsal, axial mesoderm, but not uniquely, as specification tests show that dorsal mesoderm arises in fragments of the embryo which exclude the prime meridian. These results support the hypothesis that the midline that bisects Spemann's organizer is the embryo's anterior midline.     

Regional specification during embryogenesis in the inarticulate brachiopod Discinisca.

The process of embryogenesis is described for the inarticulate brachiopod Discinisca strigata of the family Discinidae. A fate map has been constructed for the early embryo. The animal half of the egg forms the dorsal ectoderm of the apical and mantle lobes. The vegetal half forms mesoderm and endoderm and is the site of gastrulation; it also forms the ectoderm of the ventral regions of the apical and mantle lobes of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg along the future plane of bilateral symmetry of the larva. The timing of regional specification in these embryos was examined by isolating animal, vegetal, or lateral regions at different times from the 2-cell stage through gastrulation. Animal halves isolated at the 8-cell and blastula stages formed an epithelial vesicle and did not gastrulate. When these halves were isolated from blastulae they formed the cell types typical of apical and mantle lobes. Vegetal halves isolated at all stages gastrulated and formed a more or less normal larva; the only defect these larvae had was the lack of an apical tuft, which normally forms from cells at the animal pole of the embryo. When lateral isolates were created at all developmental stages, these halves gastrulated. Cuts which separated presumptive anterior and posterior regions generated isolates at the 4-cell and blastula stages that formed essentially normal larvae; however, at the midgastrula stage these halves formed primarily anterior or posterior structures indicating that regional specification had taken place along the anterior-posterior axis. The plane of the first cleavage, which predicts the plane of bilateral symmetry, can be shifted by either changing the cleavage pattern that generates the bilateral 16-cell blastomere configuration or by isolating embryo halves prior to, or during, the 16-cell stage. These results indicate that while the plane of the first cleavage predicts the axis of bilateral symmetry, the axis is not established until the fourth cleavage. The development of Discinisca is compared to development in the inarticulate brachiopod Glottidia of the family Lingulidae and to Phoronis in the phylum Phoronida.     

Mesodermal and axial determinants contribute to mesoderm regionalization in <Emphasis Type="Italic">Bufo arenarum</Emphasis> embryos

The existence of mesodermal determinants in the equator of Bufo arenarum embryos has been previously demonstrated. In this work, their role in dorso-ventral regionalization of mesoderm was studied by transferring the determinants to animal blastomeres. The transfer was performed by cleavage reorientation and cytoplasmic microinjection. Forced inclination during early cleavage caused deviation of the third cleavage plane and annexation of equatorial cytoplasm into animal quartets. Animal blastomeres from embryos oriented with the dorsal side up, incorporated ventro-equatorial cytoplasm and formed blood cells, mesenchyme, and coelomic epithelium. In contrast, animal blastomeres from embryos oriented with the ventral side up, acquired dorso-equatorial cytoplasm and developed notochord, somites, mesenchyme, coelomic epithelium and nervous tissue. In order to investigate if this dorso-ventral differentiation pattern responds to an interaction of mesodermal and axial factors, isolated 8-cell-stage animal quartets were microinjected with subcortical cytoplasm from: (a) the ventro-equatorial region of synchronous embryos; (b) the vegetal pole of uncleaved eggs; (c) a combination of both cytoplasms. As expected, the implanted ventro-equatorial cytoplasm promoted ventral mesoderm differentiation. Conversely, the joint transfer of ventro-equatorial cytoplasm and vegetal pole cytoplasm behaved as the dorso-equatorial cytoplasm, promoting dorso-lateral mesoderm and neural formation. Thus, mesoderm regionalization in B. arenarum embryos seems to be caused by a concurrent action of both mesodermal and axial determinants.     

Differentiation behavior of the mesodermal germinal sone of differently old pregastrulation stages of Ambystoma mexicanum. Cultivation of untreated and lithium-treated isolates

Isolates from the dorsal, lateral and ventral area of the marginal zone are showing differentiation capacities already in the stage of early morula. The composition of the differentiation pattern alters: ecto- and mesodermal tissues are realized until the late morula. Beyond these, endodermal structures are visible with the stage of early blastula. After a mild Li treatment of the isolates the differentiation pattern of the marginal zone shows significant modifications. Ectodermal organs are registered to a lesser degree. Gut-structures are already visible within the stage of middle morula. The capacity for gut-differentiation is not restricted to only the lateral and ventral areas but is also observed in the dorsal region of the marginal zone. The results of experiments are discussed on the basis of Nieuwkoop's formation of the mesoderm in urodelean amphibians.     

Mesoderm formation in Eleutherodactylus coqui: body patterning in a frog with a large egg.

The direct developing frog, Eleutherodactylus coqui, develops from a large egg (diameter 3.5 mm). To investigate the effect of egg size on germ-layer formation, we studied mesoderm formation in E. coqui and compared it to that of Xenopus laevis (diameter 1.3 mm). First, we identified the position of prospective mesoderm in the 16-cell E. coqui embryo by cell-lineage tracing. Although the animal blastomeres are small, they form most of the blastocoel roof and make extensive contributions to some mesodermal tissues. Second, we performed recombinant analysis with X. laevis animal caps to define the distribution of mesoderm-inducing activity. Mesoderm-inducing activity in E. coqui was restricted around the marginal zone with strong activity in the superficial cells. Neither the vegetal pole nor the blastocoel floor had activity, although these same regions from X. laevis induced mesoderm. Third, we cloned Ecbra, a homologue of Xbra, an early mesoderm marker in X. laevis. Ecbra was expressed in the marginal ring close to the surface, similar to X. laevis, but E. coqui had weaker expression on the dorsal side. Our results suggest that mesoderm formation is shifted more animally and superficially in E. coqui compared to X. laevis.