Suramin changes the fate of Spemann's organizer and prevents neural induction in Xenopus laevis.

Suramin, a polyanionic compound, which has previously shown to dissociate platelet derived growth factor (PDGF) from its receptor, prevents the differentiation of neural (brain) structures of recombinants of dorsal blastopore lip (Spemann's organizer) and competent neuroectoderm. Furthermore, the suramin treatment changes the prospective differentiation pattern of isolated blastopore lip. While untreated dorsal blastopore lip will differentiate into dorsal mesodermal structures (notochord and somites), suramin treated dorsal blastopore lip will form ventral mesoderm structures, especially heart structures. The results are discussed in the context of the current opinion about the mode of action of different growth factor superfamilies.     

Amphibian embryos as a model system for organ engineering: in vitro induction and rescue of the heart anlage.

Beating hearts can be induced under in vitro conditions when the dorsal blastopore lip (including the zone of Spemann organizer) is treated with Suramin. In contrast, untreated organizer forms dorsal mesodermal derivatives as notochord and somites. When those in vitro produced heart precursor tissues are transplanted ectopically in the posterior trunk area of early larvae, secondary beating heart structures will be formed. Furthermore, the replacement of the heart primordium of the host embryo by heart tissue induced under in vitro conditions will result in the rescue of the heart anlage. This model could be a valuable tool for the study of the multi-step molecular mechanisms of heart structure induction under in vitro conditions and vasculogenesis after transplantation into the host embryo.     

In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.     

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.     

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.     

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.     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

Role of BMP-4 in the inducing ability of the head organizer in Xenopus laevis

BMP-4 has been implicated in the patterning of the Dorsal-Ventral axis of mesoderm and ectoderm. In this study, we describe the posteriorizing effect of BMP-4 on the neural inducing ability of dorsal mesoderm (dorsal lip region) in Xenopus gastrulae. Dorsal lip explants dissected from stage 10.25 embryos retained anterior inducing ability when precultured for 6 hrs until sibling embryos reach stage 12. When the dorsal lips from stage 10.25 embryos were treated with a range of BMP-4 concentrations, posterior tissues were induced in adjacent ectoderm in a dose-dependent manner. Thus activin-treated explants able to act as head inducers can also induce posterior structures in the presence of BMP-4. To investigate whether BMP-4 directly affects the inducing ability of dorsal mesoderm, we blocked the BMP-4 signaling pathway by injection of mRNA encoding a truncated form of the BMP-4 receptor (tBR) mRNA. Under these conditions, activin-treated explants induced anterior tissues following BMP-4 treatment. Taken together, these results indicate that BMP-4 may affect the head inducing ability of dorsal mesoderm and confer trunk-tail inducing ability during Xenopus gastrulation.     

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.     

Spemann's organizer--it's origin and derivatives (cellular-tissue and molecular-genetic aspects)

In 1924 H. Spemann and H. Mangold discovered that a piece of the dorsal lip of a blastopore from Triturus cristatus, after transplantation to the ventral side of another embryo, was able to cause the neighbouring tissues to change their fate and participate in the formation of a new embryo. The dorsal lip was termed "the organizer". Since then, for as long as 75 years, attempts have been made to establish the intimate mechanisms of the organizer activity. However, no real advance was achieved in their understanding. Within the last 15 years, genetic and molecular techniques have been vastly improved, to help in tracing the fate of many cell lineages, and in compiling more exactly the fate maps for different parts of the embryo. Using these data, I have attempted to trace the fate of Spemann's organizer after the early gastrula stage. Analysis of data on inductive abilities of the organizer cells, on the use of markers, and on the observation of expression of specific genes allowed to conclude that Spemann's organizer in amphibia and its homologues in other vertebrates too are heterogeneous: they are composed of distinct cell populations able to induce primarity the development of either the head or trunk parts of the embryo. These population, determined to become the head of the trunk organizers still at the blastula stage, may be located either in the single continuous cell layer (as in amphibia and birds) or separated among different tissue germs (as in mammals). When the dorsal-ventral orientation of the embryo is established and the organizer is switched on the very early invaginating cells of the dorsal blastopore lip (in the case of amphibia) move in advance of the entire invaginating mesoderm and by the end of gastrulation occupy the place just in front of the notochord. It is supposed that the early dorsal lip and the prechordal mesoderm (PCM) are one and the same cell population, i.e. during gastrulation Spemann's organizer transfers from the lip of blastopore to the prechordal zone. The PCM seems to play an exclusive role in the formation of a head in vertebrate, because some mutations in genes expressed in the PCM result in the entire head deletion. It is supposed that spreading of differentiating signals from the PCM occurs along the main body axis in both caudal and rostral directions. After the main body plan formation the PCM is replaced by adenohypophysis. This conclusion is drawn not only from the same topology of both these structures, but also from the similarities of a set of specific genetical markers expressed in these, that makes it possible to suppose the existence of deep connections and succession between them. The adenohypophysis seems to arise directly from the PCM, or cells of the ectoderm influenced by the PCM may be subsequently transformed into humoral cells of adenohypophysis. In this interpretation, adenohypophysis and the much earlier established PCM may be considered as derivatives of Spemann's organizer. This inference is supported by the fact that all the three above structures first originate in vertebrates only.     

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.     

Effects of Inducers on Inner and Outer Gastrula Ectoderm Layers of Xenopus laevis

Abstract. Gastrula ectoderm, isolated from Xenopus laevis , was cultured in Holtfreter solution or modified Leibovitz medium (L-15) by the sandwich-method with or without inducer. The ectoderm (SD cell layers) consists of two cell sheets, representing a superficial (S) and a deep (D) layer. In the L-15 medium rather than in Holtfreter solution, the two cell layers separate out into distinct cell masses. This difference in cell affinity under certain experimental conditions could indicate that the deep layer contains endodermal cells. However, an endodermal character of the deep layer can be ruled out by induction experiments with vegetalizing factor or dorsal blastopore lip as inducers. Under the influence of vegetalizing factor the outer as well as the inner ectoderm layer differentiated into mesodermal derivatives such as notochord and somites. The results of the experiments with dorsal blastopore lip as inducer indicate that both inner and outer ectoderm layers are responsive to the neural stimulus. The lower neural competence of the outer ectoderm layer observed by several authors in normogenesis is discussed with regard to the hypothesis about short distance diffusion of the neuralizing factor and/or close cell-to-cell contact between inducing tissue and ectodermal target cells.     

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.     

The role of the dorsal lip in the induction of heart mesoderm in Xenopus laevis

We have examined the tissue interactions responsible for the expression of heart-forming potency during gastrulation. By comparing the specification of different regions of the marginal zone, we show that heart-forming potency is expressed only in explants containing both the dorsal lip of the blastopore and deep mesoderm between 30 degrees and 45 degrees lateral to the dorsal midline. Embryos from which both of these 30 degrees-45 degrees dorsolateral regions have been removed undergo heart formation in two thirds of cases, as long as the dorsal lip is left intact. If the dorsal lip is removed along with the 30 degrees-45 degrees regions, heart formation does not occur. These results indicate that the dorsolateral deep mesoderm must interact with the dorsal lip in order to express heart-forming potency. Transplantation of the dorsal lip into the ventral marginal zone of host embryos results in the formation of a secondary axis; in over half of cases, this secondary axis includes a heart derived from the host mesoderm. These findings suggest that the establishment of heart mesoderm is initiated by a dorsalizing signal from the dorsal lip of the blastopore.     

Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus.

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.     

Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster

Three-dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.     

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.