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Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordial germ cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo. Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmental potency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. The dorsal mesoderm of the marginal zone is determined before the mid-blastula transition by factors located after cortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGF and activins), maternal receptors and maternal nuclear factors (β-catenin, Smad and Fast proteins), which form multiprotein transcriptional complexes, act together to initiate pattern formation. Following mid-blastula transition in Xenopus laevis (Daudin) embryos, secreted nodal-related (Xnr) factors become important for endoderm and mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced by high concentrations of activin (vegetalizing factor) or nodal-related factors, especially Xnr5 and Xnr6, which depend on Wnt/β-catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these and other factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuning of gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF-β superfamily. The homeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation. Medical and ethical problems of future stem cell therapy are briefly discussed. [ABSTRACT FROM AUTHOR]

Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordial germ cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo. Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmental potency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. The dorsal mesoderm of the marginal zone is determined before the mid-blastula transition by factors located after cortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGF and activins), maternal receptors and maternal nuclear factors (β-catenin, Smad and Fast proteins), which form multiprotein transcriptional complexes, act together to initiate pattern formation. Following mid-blastula transition in Xenopus laevis (Daudin) embryos, secreted nodal-related (Xnr) factors become important for endoderm and mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced by high concentrations of activin (vegetalizing factor) or nodal-related factors, especially Xnr5 and Xnr6, which depend on Wnt/β-catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these and other factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuning of gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF-β superfamily. The homeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation. Medical and ethical problems of future stem cell therapy are briefly discussed. [ABSTRACT FROM AUTHOR]

The synuclein family consists of three small intracellular proteins mainly expressed in neural tissues, and has been associated with human neurodegenerative diseases. We have examined the spatial and temporal expression patterns of three synuclein genes during embryogenesis of Xenopus laevis. The Xenopus synucleins were firstly expressed in the developing nervous system at the tail bud stages. At tadpole stages, Xenopus snca was expressed in the brain, branchial arch and somite, and sncbb signals were detected in entire brain and spinal cord. However, sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion. RT-PCR indicated that expression of synucleins was up-regulated at the end of neurulation, and then maintained at later examined stages. Our study provides the spatiotemporal expression patterns of the synuclein family genes in Xenopus embryos, and forms a basis for further functional analysis of synucleins., (Copyright © 2011 Wiley-Liss, Inc.)

We describe a novel basic leucine zipper transcription factor, XXBP-1, which interacts with BMP-4 in a positive feedback loop. It is a maternal factor and is zygotically expressed in the dorsal blastopore lip and ventral ectoderm with the exception of the prospective neural plate during gastrulation. Overexpression of XXBP-1 leads to ventralization of early embryos as described for BMP-4, and inhibits neuralization of dissociated ectoderm. Consistent with mediating BMP signaling, we show that the ectopic expression of XXBP-1 recovers the expression of epidermal keratin and reverses the dorsalization imposed by truncated BMP receptor type I, indicating that it may act downstream of the BMP receptor. Its effects can be partially mimicked by a fusion construct containing the VP16 activator domain and the XXBP-1 DNA-binding domain. In contrast, fusing the DNA-binding domain to the even-skipped repressor domain leads to upregulation of the neural markers NCAM and nrp-1 in animal cap assay. Taken together, the results suggest a role for XXBP-1 in the control of neural differentiation, possibly as an activator.

I have taught developmental biology in Essen for 30 years. Since my department is named Zoophysiologie (Zoophysiology), besides Developmental Biology, I also have to teach General Animal Physiology. This explains why the time for teaching developmental biology is restricted to a lecture course, a laboratory course and several seminar courses. However, I also try to demonstrate in the lecture courses on General Physiology the close relationship between developmental biology, physiology, morphology, anatomy, teratology, carcinogenesis, evolution and ecology (importance of environmental factors on embryogenesis). Students are informed that developmental biology is a core discipline of biology. In the last decade, knowledge about molecular mechanisms in different organisms has exponentially increased. The students are trained to understand the close relationship between conserved gene structure, gene function and signaling pathways, in addition to or as an extension of, classical concepts. Public reports about the human genome project and stem cell research (especially therapeutic and reproductive cloning) have shown that developmental biology, both in traditional view and at the molecular level, is essential for the understanding of these complex topics and for serious and non-emotional debate.

The interaction between early proneural genes and lateral inhibition determines the number of primary neurons. The mechanism for regulating the size of the proneural domain, however, has not been clarified. We show here that inhibition of the function of XETOR in Xenopus, a homolog of human oncoprotein ETO/MTG8, leads to a neurogenic phenotype of expanded proneural domain without alteration in the density of primary neurons. This result suggests that XETOR is a prerequisite for regulating the size of the proneural domain. We further show that such a regulation is accomplished by establishing a negative feedback loop between XETOR and proneural genes except Xngnr-1, as well as by antagonism between XETOR and lateral inhibition., (Copyright 2002 Elsevier Science Ireland Ltd.)

We identified a novel cDNA, XCL-2, encoding an m-type calpain, a calcium-dependent intracellular protease. This protein has all characteristic structures and active sites of canonical calpains. Zygotic transcription of the gene was first detected at stage 10. It is expressed exclusively in the ventral circumblastoporal collar and the mesoderm-free zone at the most anterior tip of neural fold in late gastrulae and neurulae. In later stages, expression is only found in cement gland and proctodeum. It is also expressed in a tissue-specific manner. In adult tissues, various levels of expression were detected in brain, eye, heart, intestine, kidney, lung, stomach and testis, but not in liver, muscle, nerve, ovary, skin and spleen. Overexpression of wild-type XCL-2 suggests that this gene is involved in gastrulation movement and convergent extension during gastrulation and neurulation. Overexpression of a dominant-negative mutant caused a phenotype morphologically similar to, but histologically different from, that caused by overexpression of wild-type XCL-2. The mutant phenotype can be rescued by injection of wild-type XCL-2. These data suggest that XCL-2 plays an important role in convergent extension movements during embryogenesis in Xenopus laevis.

We have identified a cDNA coding for a Xenopus MARCKS-like protein (XMLP) from a cDNA library prepared from activin-treated ectoderm. Using whole-mount in situ hybridization and RT-PCR, we found XMLP maternal transcripts during the cleavage stages. After MBT, the signals were restricted to the neural plate. Subsequently XMLP was expressed predominantly in the brain, somites and pronephros. Ectopic expression of XMLP resulted in eye and axis defects and in a change of the expression pattern of Krox 20, a neural marker for rhombomeres 3 and 5. Injected XMLP caused apoptosis. It was characterized by loss of intercellular adhesion contacts, transient plasma membrane ruffling at gastrula, and epithelial disruption attailbud stage. Overexpression of mutant XMLPs showed that this phenotype was correlated with its putative PSD domain and glycine at position 2. The embryos injected with a morpholino oligo complementaryto XMLPmRNA showed malformations of the anterior axis and eye defects. Extirpation experiments indicated that the phenotypes might be correlated with disturbed morphorgenetic movements rather than an inhibition of induction process. Overexpression of XCYP26 resulted in a shift of the expression pattern of XMLP. In the early tailbud stage (stage 20) the signal stripe in the XCYP26 injected half of the embryo got diffuse or even disappeared. This observation suggests that retinoic acid plays an important role in the regulation of XMLP. Our results suggest that XMLP might participate in pattern formation of the embryonic axis and the central nervous system.

We have isolated a full-length cDNA encoding Xenopus L-arginine:glycine amidinotransferase (XAT), which shares a highly conserved sequence with human, chick and rat amidinotransferase. Although there are some studies about its structure and function in energy metabolism of adult tissues in some other species, little is known about its roles during early embryonic development. Characterization of embryonic expression indicates that XAT is differentially expressed around the yolk plug including the dorsal blastopore area at early gastrula stages and is extensively expressed in the midline of the neural plate of early neurula stages. Sections reveal that its transcripts are located in the notochord. In the tailbud stage signals are found both in the notochord and trunk area, whereas only faint signals can be found in the cephalic part.

Ornithine decarboxylase (ODC) is involved in the biosynthesis of polyamines and hence has been found in almost all types of cells studied. Therefore it is frequently used as internal standard. We isolated a cDNA, XODC2, which is a paralogue to ubiquitous ODC and expressed in a spatial and temporal manner during the early embryogenesis of Xenopus laevis. Expression of XODC2was first detected at the animal pole at stage 9. During neurula stages the signals were found both in the extreme anterior and posterior part of the dorsal body axis. In tailbud stages the expression is further shifted to both the tail and head areas and gradually restricted to distinct tissues: forebrain, inner layer of epidermis of the head area, stomodeal-hypophyseal anlage, frontal gland, ear vesicle, branchial arches, the front tip of neural tube and proctodeum. In addition, signals were also found in the inner layer of epidermis underneath the cement gland during early tailbud stages while in later tailbud stages signals were detected at the apical zone of the cement gland. Comparative studies indeed could confirm that XODC1 in contrast to XODC2 is expressed ubiquitously throughout the whole embryos during early development of Xenopus laevis.

After the Hans Spemann and Hilde Mangold discovery of the importance of the dorsal blastopore lip for axis formation in the early embryo (Nobelprize for Spemann, 1935), the scientific community tried in a goldrush-like manner to find the inducing factors responsible for the programming of early embyronic determination and differentiation. The slow progress towards a solution of this problem caused a fading of interest on behalf of most laboratories. This article describes the activities of a few laboratories in Finland, Japan and Germany, which continued their studies despite tremendous experimental difficulties. Finally only Heinz Tiedemann's group in Berlin was the first which could isolate a mesoderm/endoderm inducing factor in highly purified form, the so-called vegetalizing factor, now known as activin. Furthermore this article describes the identification of neuralizing factors like Chordin, Cerberus and Dickkopf in the zone of the Spemann-Mangold organizer. The finding that BMP-4 acts as an antagonist to these factors located on the dorsal side led to a new understanding of the mechanisms of action of inducing (neuralizing) factors and early embryonic pattern formation. Moreover, the observations that closely related genes and their products were also found in Drosophila, Zebrafish, Mice and Human were the basis for new concepts of evolutionary mechanisms (dorsal/ventral and anterior/posterior polarity or conserved processes in eye-development of all 7 animal phyla).

Early observations on the morphology of total exogastrulae from urodeles (Axolotl) had provided evidence for essential vertical signalling mechanisms in the process of neural induction. Conversely, more recent studies with anurans (Xenopus laevis) making use of molecular markers for neural-specific gene expression appear to support the idea of planar signalling as providing sufficient information for neural differentiation along the anterior-posterior axis. In an attempt to resolve this apparent contradiction, we report on the comparative analysis of morphology and gene expression characteristics with explants prepared from both urodeles (Triturus alpestris) and anurans (Xenopus laevis). For this purpose, we have made use of a refined experimental protocol for the preparation of exogastrulae that is intended to combine the advantages of the Holtfreter type exogastrula and the Keller sandwich techniques, and which we refer to as pseudoexogastrula explants. Analysis of histology and expression of several neural and ectodermal marker genes in such explants suggests that neural differentiation is induced in both species, but only within the intermediate zone between ectoderm and endomesoderm. Therefore, experiments with Xenopus and Triturus explants described in this communication argue against planar signalling events as being sufficient to generate a specific anterior/posterior neural pattern.

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.

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.

The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26-mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non-responsive territories.

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105000 g), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.

Using the powerful RDA-PCR-technique we could identify a novel Xenopus specific Sox-gene (xSox3) a transcription factor closely related to the sox sub-group B, which contains a HMG box. In normogenesis the xSox3 gene is expressed in the presumptive central nervous system. Furthermore a maternal component is also found in oocytes and in early cleavage stages in the animal hemisphere only. By whole-mount in situ hybridization the first zygotic transcription activities can be detected in the late blastula in the dorsal ectoderm and the dorsal and lateral part of the marginal zone. The expression reaches the highest level atthe late gastrula till the late neurula and fades after stage 30. The expression is restricted from gastrulation onwards to the presumptive brain area and the lens epithelium. Furthermore we could show that the gene is expressed in isolated Spemann organizer with adjacent neuroectoderm. The signal can be suppressed by suramin treatment, which inhibits neural development and causes a shift of dorsal to ventral mesoderm. The treatment of whole embryos with LiCl and UV results in an overexpression or an inhibition of the expression, respectively. In exogastrulae (pseudo-exogastrulae) the gene is expressed in the close vicinity to the endomesoderm only, but not in the distal most part of the ectoderm. This result indicates that it is unlikely that the gene can be activated by planar signals. The gene can also be activated in dissociated gastrula ectoderm without mesodermal or neural inducers. That means that the gene can be expressed in ectodermal cells in a cell autonomous manner.

Traditionally the whole animal cap (ventral plus dorsal ectoderm) of amphibian blastula and gastrula stages was considered as a homogeneous cell mass, because both the isolated dorsal and ventral ectoderm without induction differentiated into ciliated (atypical) epidermis. Recent results suggest a predisposition of the dorsal and ventral ectoderm. We used a special experimental approach, i.e. injection of activin as inducer into the blastocoel of intact Xenopus blastulae before the isolation of animal caps and fluorescein-dextran-amine (FDA) as a lineage tracer. In recombinants of FDA-labeled and unlabeled ectoderm we showed that the cells of the dorsal ectoderm mainly differentiate into neural tissue and notochord when they remain at their original dorsal position. In contrast, when small pieces of dorsal ectoderm are transplanted to the ventral part of animal caps, most of the descendants form epidermis. However, when small pieces of the ventral ectoderm are transplanted to the dorsal side, they significantly contribute to neural tissue and notochord. These results suggest that the prepattern in Xenopus animal caps of the late blastula and early gastrula stages is labile and reversible. Still more important is the fact that the fate of individual cells depends on the site of their localization within the animal cap. This means that cells in the dorsal most or ventral most part of the animal cap, respectively, will not randomly differentiate into all cell types, but predominantly into dorsal or ventral derivatives, respectively.

A central topic of embryology is the establishment of the body plan during embryogenesis. Starting with maternal factors distributed in the early cleavage stages in distinct patterns and gradients cell-to-cell interactions including early embryonic induction result in the formation of mesoderm and the organizer area. While many facts are known about the role of growth factors like activin (closely related to the vegetalizing factor), processed Vg1, BMPs and FGF for mesoderm formation, the establishment of the central nervous system is not yet well understood. However, there is growing evidence that neural induction is a multistep process at the level of the dorsal mesoderm (organizer) and the reacting neuroectoderm. Therefore the existence of only one neuralizing factor is unlikely. We report about data that follistatin protein is not a direct neural inducer. Furthermore our comparative studies of Xenopus and Triturus exogastrulae indicate that planar signals are unlikely in the Triturus embryo (urodeles) during the early steps of neural induction. Vertical signals emanating from the chordamesoderm are essential for the terminal neuralization and regionalization of the central nervous system during gastrulation for both Xenopus and Triturus. The putative role of neuralizing factors and BMP/activin-like molecules for the stabilization or shift of neuroectoderm into different pathways of differentiation (epidermis or neural default state) is discussed.

The classical Einsteck-test (Spemann and Mangold, Roux Arch. Dev. Biol. 100: 599-638, 1924) and data from total exogastrulae (Holtfreter, 1933) suggest that vertical signals are transmitted between the chordamesoderm (organizer) and reacting ectoderm in the early phase of neural induction. In contrast to these results with Axoloti (urodeles), several authors observed the expression of neural specific genes in Xenopus exogastrulae, isolated dorsal blastopore lip with adjacent ectoderm (open-face explants) and Keller-sandwiches. Our data with Xenopus (anurans) also show that the expression of neural specific genes takes place in exogastrulae. However, when we prepared open face explants and exogastrula-like structures by microdissection at very early gastrula stage, the signal of a class II beta-tubulin, characteristic of terminal neural differentiation, is not found in the ectoderm. These results suggest that planar signals transmitted from the chordamesoderm into the ectodermal part can fairly be excluded under these experimental conditions. In similar experiments with Triturus alpestris we could not observe either the differentiation of neural structures in the ectodermal part of exogastrulae. These results confirm earlier experiments of Holtfreter performed with Ambystoma mexicanum (Axoltl) embryos. On the basis of the published data of different authors and our results, we cannot exclude the existence of planar signals for early and/or transient expressed genes before the onset of gastrulation in Xenopus, which make the neuroectoderm susceptible for the response to vertical signals during gastrulation. On the other hand our experiments with Triturus alpestris suggest that planar neural signals are unlikely in this species. These differences between Triturus and Xenopus embryos are discussed in the context of the peculiarities in morphological structure, competence and speed of development of the two species.

A high neuralizing activity has been determined in forebrain of 7.5-day old chick embryos using Rana temporaria early gastrula ectoderm as reacting tissue (Mikhailov and Gorgolyuk, Soviet Scientific Reviews, Section of Physiology and General Biology, Vol. 1: 267-306, 1987). The corresponding protease-sensitive agent was extracted, partially purified by chromatography on DEAE-Toyopearl and Heparin-Ultragel columns, and its neuralizing activity was tested in vitro on ectoderm isolated from early gastrulae of R. temporaria, Triturus alpestris, and Xenopus laevis at different concentrations and for different periods of time (animal cap assay). Induction of neural structures was found in R. temporaria and T. alpestris explants (up to 100 and 60%, respectively), but not in cultures of X. laevis ectoderm. Under our experimental conditions, so-called "autoneuralization" of the ectoderm explants can safely be excluded. The results are discussed in relation to the neural competence of amphibian ectoderm and the mechanisms of neuralizing actions of different factors which might be involved in neural induction and patterning.

Factors of the TGF-beta superfamily (activin, vegetalizing factor) and the FGF family determine endoderm and mesoderm. The dorsoventral polarity of the mesoderm depends on additional factors (BMP-4, Wnt-8, noggin). Activin can directly activate gene transcription by signal transduction. Mesoderm is determined by factors prelocalized in the marginal zone. Its differentiation depends also on the animal ectoderm. Neural inducing factors have been partially purified. A masked neuralizing factor in the ectoderm is activated by induction of the ectoderm to the nervous system. Phorbolester can evoke neuralization signaling.

Ectoderm was isolated from early gastrulae of Triturus alpestris and induced with recombinant basic fibroblast growth factor (b-FGF). Neural tissue differentiated in about 38% of the explants which were induced by 2,5 μg/ml FGF. These explants do not contain other tissues, or contain only small amounts of mesenchyme and melanophores which are probably derived from induced neural crest. It is therefore unlikely that these neural tissues are secondarily induced. The other explants contain predominantly blastema tissue, endothelium/ mesothelium, small amounts of skeletal muscle and, rarely, notochord besides neural tissues. The mitotic rate was enhanced in about 20% of the induced explants. Possible mechanisms for the unexpected neural-inducing activity of b-FGF in Triturus ectoderm are discussed.

Mesoderm formation in the amphibian embryo is thought to be induced in the ectoderm of the animal region by signals emanating from the endoderm of the vegetal region after cleavage up to the mid-blastula. During this process the dorsal vegetal zone is thought to stimulate the dorsal animal zone to establish the Spemann organizer, which will in turn trigger the overlaying neuroectoderm during gastrulation resulting in the development of the central nervous system. In this concept it is assumed that the animal hemisphere is an uncommitted area, which receives its instructions from the vegetal region of the embryo. However, the experiments of this paper show that the 4 animal blastomeres of the eight-cell stage will form dorsal mesodermal structures in over 50% of the cases. The results support the view that developmental determinants are distributed in distinct gradients already in the early cleavage stages and that in the embryo the mesoderm is determined by factors prelocalized in the marginal zone. The spatial and temporal activation of certain genes in a distinct pattern is not simply emanating from certain areas or centers, but is a result of complex interactions between the vegetal and animal hemisphere and vice versa.

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus.

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.

Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGFβ superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11 + ). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration.

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.

A neural-specific beta-tubulin mRNA is expressed in the developing central nervous system shown by whole-mount in situ hybridization experiments. Of special interest is the fact that from the late blastula (stage 9; Nieuwkoop and Faber, 1967; Hausen and Ribesell, 1991) until the early neurula (stage 13) the signal can be found not only in the presumptive neural plate but also in the presumptive epidermis. Later in development (from stage 13) the specific mRNA becomes restricted to the presumptive brain and spinal cord area. The results are discussed in the context of predisposition and (pre)determination.

Heteroplastic combinations were made between Xenopus laevis presumptive neural plate and competent ectoderm of Xenopus borealis. Primarily induced presumptive neural plate cells (Xenopus laevis) can easily be distinguished from Xenopus borealis cells by specific quinacrine fluorescence of the nuclei. It was clearly shown that presumptive neural plate, which has primarily been induced by the underlying chordamesoderm exerts homoiogenetic inducing activity on competent ectoderm. The inducing activity is increased in pieces of presumptive neural plates, when the superficial layer has been removed from the adjacent deep layers. The enhancement can be explained by the fact that the removal of the superficial layer acting as barrier allows the inducing stimulus to be easily propagated from the apical (distal) side of the deep layers of the presumptive neural plate.

Neuralization (archencephalic brain formation) takes place after dissociation and delayed reaggregation of animal caps of early gastrula without inducer (Grunz, H. and L. Tacke: Cell Differ. Dev. 28, 211-218 (1989)). This autoneuralization can be prevented by the cell supernatant from dissociated ectoderm of Xenopus laevis, which contains extracellular matrix components. After phenol extraction of the supernatant, the aqueous phase does no longer show inhibitory activity. It can be concluded from these results that glycoconjugates responsible for the prevention of neuralization represent glycoproteins or proteoglycans which are loosely attached to integral plasma membrane components. Single early gastrula ectoderm cells mixed with non-competent late gastrula ectoderm or endoderm, which primarily form common aggregates, do not differentiate into neural derivatives. In these reaggregates the ectoderm cells remain separated from each other by heterologous cells (non-competent ectoderm or endodermal cells) during the period of competence. These data indicate that the quick recovery of extracellular matrix components together with the restoration of the former organization of the plasma membrane is responsible for the prevention of neuralization.

In the field of early embryonic induction and differentiation we can observe an exponential increase of research activities over the last three years. The reasons for this rapid exploration are the application of powerful techniques of molecular biology and molecular genetics and the recent accumulation of knowledge about the close functional correlation between growth factors, embryonic induction factors, the products of oncogenes (or proto-oncogenes), and transcription factors. The highly probable role of the cellular and viral oncogenes in regulation of the differentiation and function of normal and malignant cells has stimulated the interest of scientists working on the molecular basis of malignant cell transformation. An excellent model to study mesoderm and neural induction and differentiation on the cellular and molecular level is the embryo of the south African clawed frog (Xenopus laevis), which is now a favored vertebrate system in many laboratories.