Gene expression and pattern formation during early embryonic development in amphibians

Horst Grunz

Universität GH Essen, FB 9 Abteilung Zoophysiologie, Universitätsstr. 5, 45117 Essen, Germany

(Fax, 49-201 183-4197; Email, h.grunz@uni-essen.de)

Temporal and spatial gene expression and inductive interactions control the establishment of the body plan during embryogenesis in invertebrates and vertebrates. The best-studied vertebrate model system is the amphibian embryo. Seventy-five years after the famous organizer experiment of Hans Spemann and Hilde Mangold in 1924 our knowledge of the molecular mechanisms of the multi-step formation of embryonic axis has substantially improved. Although in the 30s and 40s the interest of many laboratories was focussed on neural induction (determination of the central nervous system), only crude factors from so-called heterogeneous inducers (liver, bone marrow, etc.,) could be isolated by the traditional biochemical techniques available at this time. An important breakthrough was the characterization and purification of a mesoderm inducing factor, the so-called vegetalizing factor (homologous to Activin) in highly purified from chicken embryos. Much later after the introduction of molecular techniques Vg1 and Activin (both belonging to the TGF-b family) and FGFs could be identified as important factors for mesoderm formation. It was in the 90s that secreted neuralizing factors (chordin, noggin, follistatin and cerberus) could be detected, which are expressed at the dorsal side of the early embryo including the Spemann organizer. In contrast to the classical view, these proteins act as antagonists to factors like BMP-4 localized on the ventral side. Of special interest was the fact that in Drosophila sog, homologous to chordin, determines the ventral side, while dpp, homologous to BMP-4, participates in the formation of the dorsal side. These data of evolutionary conserved genes in both invertebrates and vertebrates support the view that they are descendents of common ancestors, the urbilateralia, living around 300 million years ago. The expression of those genes coding for secreted proteins is closely related to inductive interactions between cells and germ layers. Recently it was shown that planar signals are not sufficient to generate a specific anterior/posterior pattern during the primary steps of neural induction, i.e., formation of the central nervous system in amphibians.

1. Introduction

A central topic in biology and developmental biology is the formation of the animal/vegetal, dorsal/ventral and anterior/posterior polarities during early embryogenesis (ontogenesis). Comparing the expression of certain genes in invertebrates and vertebrates it was shown that many common mechanisms leading to spatial polarities in the early embryo are evolutionarily (phylogenetically) conserved. Moreover, homologous genes are responsible for the presumptive dorsal side in vertebrates, while they programme the ventral side in invertebrates (and vice versa; see below). These data support the view that invertebrates and vertebrates (both bilateralia) had a common ancestor (urbilateralia) about 300–600 million years ago. The new molecular methods in developmental biology and molecular genetics dramatically changed our knowledge about segmentation of the invertebrate and vertebrate body during evolution. So-called homeotic genes (segment identity genes) are supposed to play a central role in the evolution of monotonous segmented organisms, for example myriapodes, to insects with highly specialized body segments including appendices like wings and legs. Recent results have changed the traditional view about analogous and homologous organs. The finding that pax6 and homologous genes in human, mice, amphibians, squid and fly (Drosophila) play a central role as a master control gene in eye development was a spectacular one (Halder et al 1995a, b; Mansouri et al 1999). Traditionally, it has been assumed that the approximately 40 eye types in the animal kingdom have developed independently during evolution. So the eye of squids (cephalopoda), which shows in the adult a similar morphology and physiology as the eye of vertebrates, was described in textbooks of biology and zoology as a typical example of convergent development and as an analogous organ (same function, but different embryonic origin and development) (Tomarev et al 1997). In contrast the molecular data suggest that an evolutionarily conserved control gene (Gruss and Walther 1992) is responsible for the first steps of eye development in all animals with eyes (figure 2). The hierarchy which follows consists of the expression and regulation of a large number of secondary and tertiary genes, responsible for the specialization of the various eye types in different species.

2. Historical background

In 1935 Hans Spemann received the Nobel prize for medicine and physiology for the famous organizer experiment (Spemann and Mangold 1924) (figures 1A, 4A). He was the second biologist (and the first zoologist) after the American geneticist Thomas Hunt Morgan, to receive this award. The interesting point is that there was a strict separation between the research fields of embryology, developmental biology and genetics at that time. Spemann could never be convinced that genetics plays a central role in embryogenesis, although he and his colleagues performed xenoplastic transplantations of the presumptive urodelian belly epidermis into the mouth area of anurans and vice versa indicating the importance of the genome of the reacting tissue (Spemann and Schotté 1932; Rotmann 1935). Exactly 60 years after Spemann three "molecular" developmental biologists received the Nobel prize in 1995 for experimental medicine and physiology, showing that today there exists a close correlation between embryo-logy, cell biology, developmental biology (physiology), molecular and classical genetics and evolution. In fact in many laboratories of developmental biologists the strategies and methods of all the research fields mentioned above are used simultaneously. By molecular techniques it could be shown that homologous genes and their products are expressed in different animal phyla. This opened new perspectives in groups ranging from coelenterates to vertebrates in the discussion of evolutionarily conserved mechanisms of ontogeny and phylogeny.

The Spemann organizer experiment, one of the best-known experiments in biology, initiated many of the recent research activities mentioned above. It could be shown that a special area, i.e., the dorsal blastopore lip of the early amphibian gastrula, is able to "organize" a secondary axis when transplanted to the ventral side of a host embryo (figure 1A). Therefore Spemann called this area an "organizer" (German: Organisator). The experiment was a milestone in developmental biology and has influenced many studies up to now. Since organizer-like areas are also found in other vertebrates like zebrafish, chicken, mice and humans, the interest of many laboratories concentrated on the study of the role of the organizer in early embryogenesis. The traditional view was that the Spemann organizer had an instructive role for the rest of the embryo, which was thought to react as an inert and uncommitted entity in a permissive way on the signals emanating from this unique centre (figure 3A). Molecular data from many laboratories have shown that the organizer area (dorsal blastopore lip) can still be considered as a unique area of the embryo. The knowledge of the molecular mechanisms responsible for the formation of the central nervous system and the whole embryological body plan has substantially improved. It could be shown that a number of secreted proteins responsible for neural induction are synthesized exclusively or predominantly on the presumptive dorsal side including the organizer area. However, in contrast to the traditional view, they interact as inhibitors with antagonists localized mainly on the ventral and vegetal side of the embryo (figure 3B).

As a basis for this new concept, we showed that disaggregation of presumptive ectoderm (figure 4D) results in the formation of brain structures instead of epidermis (Grunz and Tacke 1989, 1990). These results contradict the view that the ectoderm is triggered via special receptors by inducing factors, localized in the dorsal blastopore lip including Spemann’s organizer, to form neural derivatives (see below).

2.1 The search for early embryonic inducers

The search for inducing factors started shortly after the discovery of the organizer in the 30s and 40s. However, there were many shortcomings in that many chemical substances for example methylene blue, urea, etc., could induce brain structures in competent amphibian ectoderm (see details in Saxén and Toivonen 1962; Nakamura and Toivonen 1978; Gilbert and Saxén 1993). As a result many scientists lost interest in the topic of early embryonic induction. Only a few groups, in Japan (Yamada), Finland (Toivonen, Saxén) and Germany (Tiedemann), continued to work on the problem.

2.2 The vegetalizing factor (non recombinant Activin); the first inducing factor isolated in highly purified form

Tiedemann’s group isolated (using sophisticated biochemical methods) an inducer in highly purified form, the so-called vegetalizing factor (Tiedemann 1959; Tiedemann et al 1961; Asashima et al 1991; Grunz 1996b). This factor, now known as Activin, induces mesoderm and endoderm derivatives in competent ectoderm in a concentration and incubation time dependent manner (Grunz 1983). Already in the 70s and 80s we had began to work on the mechanism of action of vegetalizing factor (Grunz 1970, 1976, 1979, 1983; Grunz and Staubach 1979a, b), which were confirmed and extended with XTC-MIF or recombinant Activin about 10–20 years later (Smith 1987; Grunz et al 1989; Asashima et al 1990, 1991; Smith et al 1990; Ariizumi et al 1991). The importance of different threshold concentrations for differential gene expression was demonstrated subsequently using recombinant Activin (Green and Smith 1990; Green et al 1992; Shimizu and Gurdon 1999).

2.3 Experiments showing the importance of secondary cell interactions and the initial cell mass for pattern formation – the prerequisite for the discovery
of the neural default status of the ectoderm

If ectoderm of Triturus alpestris (two animal caps) is treated with highly purified vegetalizing factor (non recombinant Activin) in the sandwich-technique (figure 4B), it differentiated into unspecific endoderm only. However, when a pellet of vegetalizing factor was placed at the edge of six animal caps, mesodermal and neural structures could be observed (Grunz 1979). The results suggested that endoderm induced in the very vicinity of the inducer stimulated the more distal cells to form mesoderm and neural derivatives by secondary cell interactions. Similar results were recently received with ectoderm of Cynops pyrrhogaster (Ariizumi et al 1999). There are still speculations about the exact molecular mechanism underlying this pattern formation (Reilly and Melton 1996; McDowell et al 1997; Gurdon and Dyson 1998). In a small sandwich the inducer reaches all cells with similar threshold concentrations, which causes the differentiation of one cell type only. This is also the case when disaggregated cells are treated with moderate Activin concentrations: all cells differentiate into notochord only (Grunz and Tacke 1989). If disaggregated cells are treated with high Activin concentrations they differentiate into endoderm derivatives. However, if they can interact with non induced Triturus ectodermal cells, mesodermal derivatives will differentiate by secondary cell interactions (Minuth and Grunz 1980). To confirm these results we performed similar experiments with Xenopus ectoderm using our original disaggregation protocol with minor modifications (Grunz 1969). However, it turned out that the control series was the more interesting one. Disaggregated ectodermal cells kept single for more than one hour differentiated into neural structures without any inducer (Grunz and Tacke 1989). These unexpected results were the basis for the neural default model of the ectoderm and the discovery of the importance of BMPs as "antiorganizers" (see below).

Although the Einsteck-experiment of Spemann and Hilde Mangold (figures 1A, 4A) stimulated many scientists to search for neuralizing factors, those could only be isolated in a partially purified form and could not be characterized as specific molecules (Grunz et al 1986; Janeczek et al 1992; Mikhailov et al 1995). Their synthesis in homogeneous form was first possible after the introduction of the new molecular techniques of modern developmental genetics. The first neural specific genes expressed in the developing nervous system were isolated by the group of Dawid (Sargent and Dawid 1983; Richter et al 1988).

It turned out that as a prerequisite for the formation of the Spemann organizer, first the marginal zone including the so-called Nieuwkoop center must be established by mesodermalizing and endodermalizing factors, including vg1 and Activin (Melton 1991; Kessler and Melton 1995; Dohrmann et al 1993; Regabgliati and Dawid 1993). A very important factor for axis formation located in the Spemann organizer and in the corresponding areas in zebrafish and mouse is nodal (Conlon et al 1994; Smith
et al 1995; Varlet et al 1997; Rebagliati et al 1998). Nodal is an important factor for trunk formation, which must be inhibited in rostral regions (Piccolo et al 1999).

2.4 The first genes and their products localized in the dorsal blastopore lip including Spemann organizer

The first gene which participates in the formation of the Spemann organizer, goosecoid (gsc), was characterized by molecular screening methods starting from cDNA libraries (Cho et al 1991). This gene is described as an early response gene, activated after formation of the Nieuwkoop center on the presumptive dorsal side of the embryo. The formation of the Nieuwkoop center is the final result of the so-called cortical rotation initiated by the sperm entry and fertilization process (Gimlich and Gerhart 1984; Gerhart et al 1989; Moon and Kimelman 1998). Very early in development (up till the eight cell stage) the presumptive dorsal side of the embryo is determined by the Wnt/b-catenin-pathway (Grunz 1977, 1994, 1997; Larabell et al 1997; Schneider et al 1993, 1996) (figure 5).

After the expression of gsc several genes, coding for secreted proteins, are expressed in the dorsal blastopore lip. These turn out to be highly active in inducing secondary axis after injection of the mRNA into the ventral blastomeres of an early embryo. In contrast to the homeobox-containing gene gsc, these recently isolated genes code for secreted neuralizing factors. The determination of the animal/vegetal, dorsal/ventral and anterior/posterior polarities leading to pattern and axis formation takes place via complex interactions of maternal factors and distinct spatial and temporal activated genes and their products. Cho and coworkers (Watabe et al 1995) found that the activation of goosecoid is correlated with the activation of the Wnt-pathway. Goosecoid contains two elements, a distal Activin/Vg-1 response element and a proximal element that responds to Wnt signals (figure 5). While Activin is ubiquitously distributed in the vegetal half, the Wnt-associated b -catenin pathway is already asymmetrically activated at the dorsal side starting from the two-cell stage (Grunz 1994; Larabell et al 1997). Further steps leading to dorsalization and Spemann organizer formation include frizbee (antagonist of Wnt), the receptor Frizzled, glycogen synthase kinase-3, b -catenin, APC and the homeobox genes siamois and goosecoid. In this model these genes activate in concert with Vg1 and Activin secreted proteins like chordin (figure 5). However, we will see below that the mechanism by which they exert their biological activity differs significantly from the traditional view.

2.5 The neural default model (autonomous neural differentiation of the ectoderm) – the basis for
the understanding of neural induction

Dissociated early gastrula ectoderm differentiates into neural tissue (figure 4D), when the cells are kept single for more than 1 h prior to reaggregation (Grunz and Tacke 1989). If the supernatant of disaggregated early gastrulae is added to the cells, neuralization can be prevented and the reaggregated cells form epidermis (Grunz and Tacke 1990). Already at that time we assumed that the extracellular space between inducing and responding tissue contained factors which were responsible for the inhibition of neural differentiation (Grunz and Tacke 1990). The addition of Activin, a member of the TGFb-superprotein-family, did not inhibit neuralization of dissociated ectodermal cells. After treatment with low concentrations of Activin, ectoderm still formed neural tissue together with mesodermal derivatives; higher concentrations of Activin caused the formation of large amounts of notochord and somites (Grunz and Tacke 1989; Grunz 1996a). Activin never shifted the pathway of differentiation from neural structures to epidermis. However, it was shown five years later that another member of the TGFb-superprotein-family, BMP-4 was the key molecule, which could prevent neuralization and could induce epidermis (Wilson and Hemmati-Brivanlou 1995). These findings with dissociated ectodermal cells resulted in the "neural default" model of the ectoderm (Hemmati-Brivanlou and Melton 1997). Furthermore it turned out that BMP-2/4, which has low (ventral) mesodermal inducing activity (Plessow et al 1991; Clement et al 1995), acts as an antagonist to genes (and their products) located in the dorsal blastopore lip including the area of the Spemann organizer. Inhibition of Activin receptors and injection of Follistatin mRNA results in the differentiation of neural tissues. Follistatin is an antagonist of Activin and is expressed in the dorsal blastopore lip (Hemmati-Brivanlou et al 1994). Also the expression of a dominant-negative BMP-4 receptor, non cleavable forms of BMP4/7 or antisense BMP-4 RNA leads to neuralization of animal caps instead of epidermal determination (Hemmati-Brivanlou and Melton 1992, 1994; Maeno et al 1994; Suzuki et al 1994; Hawley et al 1995; Ishikawa et al 1995; Sasai et al 1995; Xu et al 1995). On the other hand treatment of ectoderm with Follistatin protein did not result in the induction of neural structures (Grunz 1996a; Kablar 1999).

Traditionally the early gastrula animal cap (presumptive ectoderm and neuroectoderm) was considered as an uncommitted omnipotent tissue that needed to receive instructive (neuralizing) signals from the organizer and the involuting dorsal blastopore lip. At first sight it appeared paradoxical that disaggregated ectodermal cells kept single for up to 4 h prior to reaggregation formed neural tissue without any inducer treatment (Grunz and Tacke 1989). Thus, in contrast to the traditional view, the ectoderm forms neural structures unless ‘told’ otherwise. So the ‘default status’ of the ectoderm must be considered neural and not epidermal (Hemmati-Brivanlou and Melton 1997; Honore and Hemmati-Brivanlou 1997). On the other hand when animal caps are isolated and cultured as intact tissue, they form ciliated epidermis (Grunz et al 1975). This is also true for isolated outer and sensorial layer of Xenopus ectoderm (Asashima and Grunz 1983). Activin cannot shift neural determination into epidermis; depending on the concentration used, the ectoderm will form ventral and dorsal mesoderm (Grunz 1983). In the animal cap assay (figure 4C) BMP-4 or BMP-2 induces ventral mesodermal structures. It turned out that BMP-4 plays a central role as antagonist to neuralizing secreted proteins located in the organizer area.

2.6 Secreted proteins expressed in the zone of the Spemann organizer

Of central importance is the fact that all secreted proteins so far identified in or close to the Spemann organizer (chordin, follistatin, noggin, cerberus, dickkopf) interact with BMP-4 and form a complex in the extracellular space between inducing endomesoderm and overlaying ectoderm (Ueno et al 1987; Smith and Harland 1992; Sasai et al 1994; Bouwmeester et al 1996; Glinka et al 1998). With the exception of the organizer area, BMP-4 is located mainly on the ventral side with a decreasing concentration gradient to the dorsal side (figure 6A). The finding is quite different from the traditional expectation that neuralizing factors act via specific receptors on the plasma membrane. This means that neuralizing factors must be considered as inhibitors, secreted into the extracellular space between inducing chorda-endomesoderm and the ectodermal target cells, that prevent BMP-4 from binding to its receptor.

A very potent neural inducer (chordin) was isolated and characterised by Sasai et al (1994). It forms complexes with BMP-4 in the extracellular space and prevents the BMP-4 interaction with its receptor on ectodermal target cells (figure 6A). In contrast to BMP-4, chordin possesses no receptor on the plasma membrane of the ectodermal target cells. BMP-4, a member of the TGFb -protein superfamily, has in contrast to secreted neuralizing factors like chordin, noggin, follistatin and cerberus specific receptors on the plasmamembrane. This enables us to explain the mechanism of neuralization of disaggregated ectodermal cells. During culture as single cells the BMP-4 present in the extracellular matrix will be diluted out into the culture medium. Therefore the binding of BMP-4 to its receptor is prevented because the concentration falls below the biologically active threshold concentration.

To summarize, chordin causes neural induction by preventing BMP-4 from binding to its receptor. A new dimension towards the understanding of the formation of anteroposterior structures and the importance of signalling in gradient-like formation came from the observation that the BMP-4/chordin-complex can be cleaved by a secreted metalloprotease, a homologue of the Drosophila gene Tolloid, named Xolloid. It has been suggested that his enzyme cleaves the BMP-4/chordin complex in intermediate areas between presumptive ventral and dorsal most zones, which results in the liberation of BMP-4 and a gradient of BMP-4-concentrations (figure 6A). These interactions between chordin and BMP-4 are likely to be of evolutionary importance. Homologous molecules of BMP-4, chordin and Xolloid are also found in Drosophila [decapentaplegic (dpp), short gastrulation (sog) and Tolloid]. While in Drosophila sog participates in the determination of the ventral side of the embryo, chordin takes part in determining the dorsal side in Xenopus. Opposite roles are played by dpp (dorsal side in Drosophila) and the homologous molecule BMP-4 (ventral side in Xenopus). Interestingly, similar genes are also found in zebrafish. Chordino (formely dino) mutants with a defect in the homologue of the Xenopus gene chordin show a ventralized phenotype with a reduced neural plate. On the other hand the mutation swirl with a mutation of the zebrafish BMP-2b (associated with the activity of BMP-4) is characterized by extremely dorsalized phenotype in which the neural plate is expanded at the cost of lateral and ventral derivatives (Schulte-Merker et al 1997; Miller-Bertoglio et al 1997; Hammerschmidt et al 1996a, b; Kishimoto et al 1997; Nguyen et al 1998). Double mutants swirl^–/–/chordino^–/– have a swirl phenotype (i.e., swirl is epistatic to chordino). This indicates that the function of the protein chordin is to antagonize BMPs in both vertebrates Xenopus and zebrafish. However it should be pointed out that there also exist distinct species differences. Apparently the inhibition of BMP is not a sufficient trigger for neural induction in the chick (Streit and Stern 1999). Taken together the data so far available suggest that basic principles of body plan organisation are more similar between invertebrates and vertebrates (both bilateralia) than assumed in former days (Hammerschmidt and Nüsslein-Volhard 1993). On the basis of the characterization of these evolutionary conserved genes it has been concluded that a common ancestor (urbilateralia) existed about 300 million years ago. At the beginning of the 19th century the French anatomist Geoffry-St-Hillaire dissected a lobster upside-down. Under these conditions the lobster (invertebrate) shows a similar organization to vertebrates, i.e., the circulating system on the ventral side and the central nervous system on the dorsal side (DeRobertis and Sasai 1996). What at one time appeared to be curiosities and contradictory hypotheses have turned out to be of unexpected value and seem supported by the molecular data today.

2.7 Planar versus vertical signalling during the early steps of neural induction

Although we have come to know much about neuralizing factors and their antagonists, the exact molecular mechanisms of signalling during the early steps of neural induction are not yet well understood. In the classical view neural inducing signals are transmitted from the involuting mesoderm to the overlaying ectoderm by vertical signals. Recent data show that head-endomesoderm plays an important role in the determination of the head and forebrain area (Ninomiya et al 1998; Piccolo et al 1999). An extracellular gap exists between the underlying endomesoderm and the overlaying ectoderm (Grunz and Staubauch 1979a). From recent data we know that in this extracellular space secreted proteins interact by protein–protein-binding and short distance diffusion (see below). The seldom observed cell contacts between inducing and ectodermal target cells are important for cell guidance rather than for neuralizing signals. On the basis of the classical total exogastrulation experiment Holtfreter (1933b) suggested that neural induction takes place by vertical signalling. Early blastulae of the Mexican axolotl (newts, urodeles) placed in hypertonic medium show a total exogastrulation instead of the involution of the endomesoderm and resulting in the separation of the ectoderm from the endomesoderm. An important conclusion was that neural structures are induced by the interaction of involuting endomesoderm and the overlaying ectoderm via vertical signalling. Contradictory results were obtained more recently with Xenopus embryos (frogs, anurans) (Altaba 1992; Doniach 1992). If Xenopus embryos were treated in a similar way as Axolotl embryos in hypertonic medium, at first sight they form similar exogastrulae. However, in histological sections and by whole mount in situ hybridization with neural specific markers of neural structures are found in the most distal part of the ectoderm. These data suggest that instead of vertical signals planar signals might be transmitted in the plane of the ectoderm emanating from the dorsal blastopore (Spemann organizer) prior to the involution of the ectoderm. Similar results indicating an anterior/posterior pattern of neural markers were obtained with so-called Keller-sandwiches (Doniach et al 1992). We have performed similar experiments using Triturus (urodeles) and Xenopus (anurans). In agreement with the data of Altaba (1992) and Doniach (1992) we could show that exogastrulae show neural marker expression in the distal part of the ectoderm, suggesting an important role of planar signalling during the early steps of neural induction (Grunz et al 1995).

In our opinion the data from Xenopus embryos must be explained in another way. It is well known that the morphological organization of the Xenopus (anuran) embryo is quite different from Axolotl or Triturus (both urodeles). In contrast to urodeles a part of the endomesoderm is already located inside of the early frog (Xenopus) gastrula, which allows vertical signalling prior to involution. Furthermore in hypertonic culture medium Xenopus gastrulae first show a partial involution prior to the exogastrulation, thereby allowing vertical signalling. Therefore in comparative studies with Triturus and Xenopus embryos we used a special microsurgery method to separate the endomesoderm from the ectoderm in very early gastrulae (Chen et al 1999). This experimental protocol combines the advantages of the Holtfreter type exogastrula and the Keller-sandwich technique, and which we refer to as pseudoexogastrulae (figure 4E). In contrast to normal exogastrulae obtained by placing embryos in high salt, we found neural structures (normal histology) and signals of neural markers in the intermediate zone between endomesoderm and ectoderm only rather than in the most distal part of the ectoderm. So we think that normal exogastrulae of Xenopus embryos and also Keller-sandwiches are not suitable to decide the question of planar or vertical signals. Therefore our data suggest that planar signals are of minor importance for the early steps of neural induction. Planar signals over long distances are also unlikely, because all neuralizing factors so far identified act by interaction with BMP-4 in the intercellular space between involuting endomesoderm and reacting neuroectoderm by short distance migration.

An excellent candidate to support our hypothesis that planar signalling is of minor importance during the primary steps of neural induction is the gene cerberus and its products (Bouwmeester et al 1996). The head inducer cerberus, a multifunctional antagonist of nodal, BMP and Wnt signals, is expressed in the very early Xenopus gastrula already at the most anterior part of the endomesoderm inside of the embryo; this permits vertical signalling. If cerberus mRNA is injected into early cleavage stages, ectopic heads are induced. Cerberus protein could be obtained by injection of the mRNA in early embryos and isolation from animal caps (ectoderm) in late blastulae. After cultivation of ectodermal cells disaggregated in Ca²⁺- and Mg²⁺-free medium the supernatant contained cerberus proteins – a short and a long form (Piccolo et al 1999). A short cerberus-like protein could also be isolated from transfected human 293 T cells. In a sensitized dissociation-reaggregation animal cap assay the Cerberus-Long (Cerl-L) has neural inducing activity. Of interest is the fact that similar to chordin, cerberus binds BMP-4 in the extracellular space. Still more important is the observation that Cer-L binds not only to BMP-4 but also to nodal, expressed in the dorsal mesoderm. In contrast Cerberus-Short (Cer-S) does not bind nodal (figure 7). BMP-4 binding to Cer-L is specific since 1 nM can be competed by 10 nM BMP-2 but not by TGFb 1, EGF or PDGF. Furthermore it is of interest that cer binds to nodal, but not to Vg1 and Activin. The differential binding of Cer-L and Cer-S to different molecules responsible for axis formation confirms again the importance of the establishment of gradients (figure 6B) and boundaries for the anteroposterior and dorsal/ ventral organization of the embryo (see below: Chd, BMP-4 and Xolloid). This statement is corroborated by the finding that Cer-L but not Cer-S binds to Wnt-8, which is thought to be important for trunk formation during gastrulation. Injection of Frzb-1 mRNA (Leyns et al 1997) together with Cer-L into early embryos causes an enlargement of ectopic heads in contrast to the series with Cer-L alone. Frz-1 is expressed in the organizer area and acts as an antagonist to Wnt-8. Taken together, this means that signals involved in the trunk development including BMP, nodal and Wnt must be inhibited in the rostral zone in order to form the head area (Piccolo et al 1999).

3. The antiorganizers

In addition to BMP-4 a cascade of different genes have been analysed to date, all working as antiorganizers (figure 3). Effectors of BMP-4 like msx1 and smad could also suppress neural differentiation in animal caps, when the RNA was injected into early embryos (Suzuki et al 1997; Wilson et al 1997; Bhushan et al 1998; Nakayama et al 1998). Furthermore homeobox-containing genes (vent1, vent2, vox) correlated with the BMP-4 pathway could be identified on the ventral side of the embryo (Onichtchouk et al 1996, 1998).

4. The importance of gradients

Already in the oocyte certain distinct animal-vegetal gradients are established (Melton 1987, 1991). After fertilization cortical rotation causes the formation of a dorsal/ ventral gradient (Gerhart et al 1989). In early cleavage stages maternal factors are translocated to the dorsal side of the embryo (Grunz 1977, 1994; Miller and Moon 1996; Larabell et al 1997), which leads to the formation of the Nieuwkoop centre, followed by the organization of the dorsal blastopore lip including the zone of the Spemann organizer. The establishment of the anterior–posterior axis takes place during gastrulation. The interaction of secreted inducers like noggin, chordin or cerberus with BMP-4 alone cannot explain the exact mechanism of the establishment of an anterior posterior gradient. However, it could be shown that in addition to the neuralizing factors a metalloprotease is secreted into the extracellular space between inducing and reacting tissue. This enzyme, Xolloid, a homologue of the Drosophila Tolloid, cleaves chordin in the chordin/BMP4-complex resulting again in a release of BMP-4 in the intermediate zone between presumptive head and tail area. BMP-4 could bind in concentration dependent manner to its receptor causing among other outcomes, the establishment of a cranial/ caudal organization. The presence of Xolloid in Xenopus and its homologue in Drosophila supports the view that genes important for dorsal/ventral and anterior/posterior organization of the embryo are closely related and evolutionarily conserved in both invertebrates and vertebrates (Anderson et al 1985; DeRobertis and Sasai 1996).

Cerberus could point to an additional mechanism leading to anterior–posterior pattern formation. Head and trunk organization could depend on specific spatial and temporal interaction of the short or long form of cerberus with BMP-4, nodal and Wnt (Piccolo et al 1999). Furthermore retinoic acids, their receptors and degrading enzymes must be considered as important mechanisms for the organization of anterior–posterior pattern formation (Durston et al 1989; Hollemann et al 1998; DeRoos et al 1999).

5. Amphibian embryos – a model for
organ engineering

We were able to show many years ago that amphibian ectoderm is able to differentiate along different pathways when treated with specific inducing factors (Grunz 1983): the ectoderm of the late blastula/early gastrula is an omnipotent tissue similar to the early embryonic human inner cell mass (so-called stem-cells). In competent ectoderm, Activin is able to induce dorsal and ventral mesoderm depending on the concentration and incubation time. Nearly all mesodermal derivatives like notochord, somites, pronephros and blood can be induced. Very high concentrations cause the differentiation of endoderm derivatives like gut and liver.

If animal caps (competent ectoderm) are treated simultaneously with retinoic acid and Activin, pronephros are induced in high percentages (Moriya et al 1993). If such an in vitro induced kidney tissue is implanted in nephrotectonized host embryos, a rescue of kidney function is observed (Chan et al 1999). We have shown that liver tissue too can be produced under in vitro conditions (Minuth and Grunz 1980). Recently we could replace the heart anlage of a host embryo by heart tissue produced under in vitro conditions (Grunz 1999). Heart structures can be produced under in vitro conditions, when the Spemann organizer is treated with Suramin (Grunz 1992). When this tissue is implanted into host embryos with an extirpated heart anlage a rescue of the heart primordium can be observed (figure 8); we have obtained larvae with functionally intact hearts. These methods could be valuable tools for further studies on in vitro organ engineering.

6. Conclusions and perspectives

Particularly in the last five years, substantial progress followed the finding that BMP-4 is a neural inhibitor and epidermal inducer in amphibians. The Spemann organizer secretes antagonists of BMPs. In contrast to the traditional view and expectations these neural inducers like Noggin, Chordin, Follistatin and Cerberus have no receptor ‘of their own’. They form a complex with BMPs in the extracellular space and in this way prevent the binding of BMP to its receptor. A distinct interaction between inducers and antagonists results in the formation of head (anterior) and trunk/tail (posterior) structures. Comparative studies show that several genes and their products are evolutionarily conserved and that the basic mechanisms of pattern formation are quite similar in invertebrates and vertebrates. This along with data on genes expressed on the future dorsal or ventral side of the embryo, is the basis for the urbilateralia-hypothesis (DeRobertis 1997).

The findings that homologous genes like Pax6 can be found in more than seven animal phyla ranging from coelenterates to vertebrates opened new perspectives for the understanding of (eye) evolution (Gruss and Walther 1992; Halder et al 1995a). In contrast to the prevailing view of convergence (a well known example being the squid and vertebrate eyes), Gehring (1997) postulates that the different eye types in various animal phyla have a monophyletic origin.

Exact knowledge concerning the molecular mechanisms of pattern and tissue formation will be very important for the gold rush-like activities that are certain to occur in
the field of organ engineering. The formation of 3-dimensional organ structures such as brain, heart, liver, etc., depends on a cascade of molecular steps including cell-to-cell signalling and pattern formation. Since competent amphibian ectoderm is an omnipotent tissue, the basic mechanisms of differentiation can be considered as closely related to those involved in the diversification of human stem cells (inner cell mass). The information obtained from amphibia will be significant for the study of developmental mechanisms in invertebrates and vertebrates including humans.

Acknowledgements

This work was financially supported by the Deutsche Forschungsgemeinschaft. I would like to thank Vidynand Nanjundiah, Bangalore for critical reading of the manuscript.

References

Altaba A R I 1992 Planar and Vertical Signals in the Induction and Patterning of the Xenopus Nervous System; Development 116 67–80

Anderson K V, Bokla L and Nüsslein-Volhard Ch 1985 Establishment of dorsal–ventral polarity in the Drosophila embryo: The induction of polarity by the toll gene product; Cell 42 791–798

Ariizumi T, Komazaki S and Asashima M 1999 Activin-treated urodele animal caps: II. Inductive interactions in newt animal caps after treatment with Activin A; Zool. Sci. 16 115–124

Ariizumi T, Moriya N, Uchiyama H and Asashima M 1991 Concentration-Dependent Inducing Activity of Activin-A; Roux’s Arch. Dev. Biol. 200 230–233

Asashima M and Grunz H 1983 Effects of inducers on inner and outer gastrula ectoderm layers of Xenopus laevis; Differentiation 23 157–159

Asashima M, Nakano H and Uchiyama H 1990 The vegetalizing factor belongs to a family of mesoderm-inducing proteins related to erythroid differentiation factor; Naturwissenschaften 77 389–391

Asashima M, Uchiyama H, Nakano H, Eto Y, Ejima D, Sugino H, Davids M, Plessow S, Born J, Hoppe P, Tiedemann H and Tiedemann H 1991 The vegetalizing factor from chicken embryos: its EDF (Activin A)-like activity; Mech. Dev. 34 135–141

Bhushan A, Chen Y and Vale W 1998 Smad7 inhibits mesoderm formation and promotes neural cell fate in Xenopus embryos; Dev. Biol. 200 260–268

Bouwmeester T, Kim S H, Sasai Y, Lu B and Derobertis E M 1996 Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann’s organizer; Nature (London) 382 595–601

Chan T C, Ariizumi T and Asashima M 1999 A model system for organ engineering: Transplantation of in vitro induced embryonic kidney; Naturwissenschaften 86 224–227

Chen Y, Hollemann T, Pieler T and Grunz H 1999 Planar signalling is not sufficient to generate a specific anterior/ posterior neural pattern in pseudoexogastruale explants from Xenopus and Triturus; Mech. Dev. (in press)

Cho K W Y, Blumberg B, Steinbeisser H and Derobertis E M 1991 Molecular Nature of Spemanns Organizer – The Role of the Xenopus Homeobox Gene goosecoid; Cell 67 1111–1120

Clement J H, Fettes P, Knöchel S, Lef J and Knöchel W 1995 Bone morphogenetic protein 2 (BMP-2) in the early development of Xenopus laevis; Mech. Dev. 52 357–370

Conlon F L, Lyons K M, Takaesu N, Barth K S, Kispert A, Herrmann B and Robertson E J 1994 A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse; Development 120 1919–1928

DeRobertis E M 1997 Evolutionary biology – The ancestry of segmentation; Nature (London) 387 25–26

DeRobertis E M and Sasai Y 1996 A common plan for dorsoventral patterning in Bilateria; Nature (London) 380 37–40

DeRoos K, Sonneveld E, Compaan P, tenBerge D, Durston A J and van der Saag P T 1999 Expression of retinoic acid 4-hydroxylase (CYP26) during mouse and Xenopus laevis embryogenesis; Mech. Dev. 82 205–211

Dohrmann C E, Hemmati-Brivanlou A, Thomsen G H, Fields A, Woolf T M and Melton D A 1993 Expression of Activin Messenger RNA During Early Development in Xenopus laevis; Dev. Biol. 157 474–483

Doniach T 1992 Induction of Anteroposterior Neural Pattern in Xenopus by Planar Signals; Development (Suppl.) 183–193

Doniach T, Phillips C R and Gerhart J C 1992 Planar induction of antererposterior pattern in the developing central nervous system of Xenopus laevis; Science 257 542–545

Durston A J, Timmermans J P M, Hage W J, Hendriks H F J, DeVries N J, Heideveld M and Nieuwkoop P D 1989 Retinoic acid causes an anteroposterior transformation in the developing central nervous system; Nature (London) 340 140–144

Gehring W 1997 Die genetische Steuerung von Morphogenese und Evolution der Augen; Nova Acta Leopold. 76 303–312

Gerhart J, Danilchik M, Doniach T, Roberts S, Rowning B and Stewart R 1989 Cortical rotation of the Xenopus egg: consequences for the anteroposterior pattern of embryonic dorsal development; Development (Suppl.) 107 37–51

Gilbert S F and Saxén L 1993 Spemann’s organizer: models and molecules; Mech. Dev. 41 73–89

Gimlich R L and Gerhart J C 1984 Early cellular interactions promote embryonic axis formation in Xenopus laevis; Dev. Biol. 104 117–130

Glinka A, Wu W, Delius H, Monaghan A P, Blumenstock C and Niehrs C 1998 Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction; Nature (London) 391 357–362

Green J B A, New H V and Smith J C 1992 Responses of Embryonic Xenopus Cells to Activin and FGF Are Separated by Multiple Dose Thresholds and Correspond to Distinct Axes of the Mesoderm; Cell 71 731–739

Green J B A and Smith J C 1990 Graded Changes in Dose of a Xenopus Activin-A Homologue Elicit Stepwise Transitions in Embryonic Cell Fate; Nature (London) 347 391–394

Grunz H 1969 Hemmung der Reaggregation dissoziierter Amphibienzellen durch Inhibitoren der RNS- und Proteinsynthese; Roux’s Arch. Dev. Biol. 163 184–196

Grunz H 1970 Abhängigkeit der Kompetenz des Amphibien-Ektoderms von der Proteinsynthese; Roux’s Arch. Dev. Biol. 165 91–102

Grunz H 1976 Changes of cell surface and determination of amphibian ectoderm after induction; in Proceedings of the Sixth European Congress on Electron Microscopy Jerusalem, Biological Vol. II, 571–573, Biological Vol. II, 571–573 G098

Grunz H 1977 The differentiation of the four animal and the four vegetal blastomeres of the eight-cell-stage of Triturus alpestris; Roux´s Arch. Dev. Biol. 181 267–277

Grunz H 1979 Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass; Roux’s Arch. Dev. Biol. 187 49–57

Grunz H 1983 Change in the differentiation pattern of Xenopus laevis ectoderm by variation of the incubation time and concentration of vegetalizing factor; Roux’s Arch. Dev. Biol. 192 130–137

Grunz H 1992 Suramin Changes the Fate of Spemann’s Organizer and Prevents Neural Induction in Xenopus laevis; Mech. Dev. 38 133–142

Grunz H 1994 The four animal blastomeres of the eight-cell stage of Xenopus laevis are intrinsically capable of differentiating into dorsal mesodermal derivatives; Int. J. Dev. Biol. 38 69–76

Grunz H 1996a Factors responsible for the establishment of the body plan in the amphibian embryo; Int. J. Dev. Biol. 40 279–289

Grunz H 1996b The long road to chemical and molecular embryology. What amphibians can teach us on differentiation – An interview with professor Heinz Tiedemann; Int. J. Dev. Biol. 40 113–122

Grunz H 1997 Neural Induction in Amphibians; Curr. Topics Dev. Biol. 35 191–228

Grunz H 1999 Amphibian embryos as a model system for organ engineering: in vitro induction and rescue of the heart anlage; Int. J. Dev. Biol. 43 361–364

Grunz H and Staubach J 1979a Cell contacts between chorda-mesoderm and the overlaying neuroectoderm (presumptive central nervous system) during the period of primary embryonic induction in amphibians; Cell Diff. 14 59–65

Grunz H and Staubach J 1979b Change of the surface charge of ectodermal cells after induction; Roux´s Arch. Dev. Biol. 186 77–80

Grunz H and Tacke L 1989 Neural differentiation of Xenopus laevis ectoderm takes place after disaggregation and delayed reaggregation without inducer; Cell Diff. Dev. 28 211–
218

Grunz H and Tacke L 1990 Extracellular Matrix Components Prevent Neural Differentiation of Disaggregated Xenopus Ectoderm Cells; Cell Diff. Dev. 32 117–124

Grunz H, Born J, Tiedemann H and Tiedemann H 1986 The activation of a neuralizing factor in the neural plate is correlated with its homoiogenetic-inducing activity; Roux’s Arch. Dev. Biol. 195 464–466

Grunz H, Born J, Davids M, Hoppe P, Loppnow-Blinde B, Tacke L, Tiedemann H and Tiedemann H 1989 A mesoderm-inducing factor from a Xenopus laevis cell line: Chemical properties and relation to the vegetalizing factor from chicken embryos; Roux’s Arch. Dev. Biol. 198 8–13

Grunz H, Multier A M, Herbst R and Arkenberg G 1975 The differentiation of isolated amphibian ectoderm with or without treatment of an inductor. A scanning electron microscop study; Roux’s Arch. Dev. Biol. 178 277–284

Grunz H, Schüren C and Richter K 1995 The role of vertical and planar signals during the early steps of neural induction; Int. J. Dev. Biol. 39 539–543

Gruss P and Walther C 1992 Pax in Development; Cell 69 719–722

Gurdon J B and Dyson S 1998 Cells’ perception of position in a concentration gradient; Cell 95 159–162

Halder G, Callaerts P and Gehring W J 1995a Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila; Science 267 1788–1792

Halder G, Callaerts P and Gehring W J 1995b New perspectives on eye evolution; Curr. Opinion Genet. Dev. 5 602–609

Hammerschmidt M and Nüsslein-Volhard C 1993 The Expression of a Zebrafish Gene Homologous to Drosophila-snail Suggests a Conserved Function in Invertebrate and Vertebrate Gastrulation; Development 119 1107–1118

Hammerschmidt M, Pelegri F, Mullins M C, Kane D A, vanEeden F J M, Granato M, Brand M, FurutaniSeiki M, Haffter P, Heisenberg C P, Jiang Y J, Kelsh R N, Odenthal J, Warga R M and Nüsslein-Volhard C 1996a dino and mercedes, two genes regulating dorsal development in the zebrafish embryo; Development 123 95–102

Hammerschmidt M, Serbedzija G N and Mcmahon A P 1996b Genetic analysis of dorsoventral pattern formation in the zebrafish: Requirement of a BMP-like ventralizing activity and its dorsal repressor; Genes Dev. 10 2452–2461

Hawley S H B, Wunnenbergstapleton K, Hashimoto C, Laurent M N, Watabe T, Blumberg B W and Cho K W Y 1995 Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction; Genes Dev. 9 2923–2935

Hemmati-Brivanlou A and Melton D A 1997 Vertebrate embryonic cells will become nerve cells unless told otherwise; Cell 88 13–17

Hemmati-Brivanlou A and Melton D A 1992 A Truncated Activin Receptor Inhibits Mesoderm Induction and Formation of Axial Structures in Xenopus Embryos. Nature (London) 359 609–614

Hemmati-Brivanlou A and Melton D A 1994 Inhibition of Activin receptor signaling promotes neuralization in Xenopus; Cell 77 273–281

Hemmati-Brivanlou A, Kelly O G and Melton D A 1994 Follistatin, an antagonist of Activin, is expressed in the Spemann organizer and displays direct neuralizing activity; Cell 77 283–295

Hollemann T, Chen Y L, Grunz H and Pieler T 1998 Regionalized metabolic activity establishes boundaries of retinoic acid signalling; EMBO J. 17 7361–7372

Holtfreter J 1933a Nachweis der Induktionsfähigkeit abgetöteter Keimteile. Isolations- und Transplantationsversuche; Roux’s Arch. Dev. Biol. 128 584–633

Holtfreter J 1933b Die totale Exogastrulation, eine Selbstablösung des Ektoderms vom Entomesoderm. Kaiser Wilhelm-Institut Biologie, Berlin-Dahlem 669–793

Honore E and Hemmati-Brivanlou A 1997 The "default model" of vertebrate neural specification; M S Med. Sci. 13 192–200

Ishikawa T, Yoshioka H, Ohuchi H, Noji S and Nohno T 1995 Truncated type II receptor for BMP-4 induces secondary axial structures in Xenopus embryos; Biochem. Biophys. Res. Commun. 216 26–33

Janeczek J, Born J, Hoppe P and Tiedemann H 1992 Partial Characterization of Neural-Inducing Factors from Xenopus gastrulae – Evidence for a Larger Protein Complex Containing the Factor; Roux’s Arch. Dev. Biol. 201 30–35

Kablar B 1999 Follistain possesses trunk and tail organizer activity and lacks head organizer activity; Tissue Cell 31
28–33

Kessler D S and Melton D 1995 Induction of dorsal mesoderm by soluble, mature Vg1 protein; Development 121 2155–2164

Kishimoto Y, Lee K H, Zon L, Hammerschmidt M and SchulteMerker S 1997 The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning; Development 124 4457–4466

Knöchel W, Born J, Hoppe P, Loppnow-Blinde B, Tiedemann H, Tiedemann H, McKeehan W and Grunz H 1987 Mesoderm inducing factors: Their possible relationship to heparin binding growth factors and transforming growth factor-b; Naturwissenschaften 74 604–606

Larabell C A, Torres M, Rowning B A, Yost C, Miller J R, Wu M, Kimelman D and Moon R T 1997 Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway; J. Cell Biol. 136 1123–1136

Lemaire P and Yasuo H 1998 Developmental signalling: A careful balancing act; Curr. Biol. 8 228–231

Leyns L, Bouwmeester T, Kim S H, Piccolo S and DeRobertis E M 1997 Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer; Cell 88 747–756

Maeno M, Ong R C, Suzuki A, Ueno N and Kung H F 1994 A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: Roles of animal pole tissue in the development of ventral mesoderm; Proc Natl. Acad. Sci. USA 91 10260–10264

Mansouri A, Goudreau G and Gruss P 1999 Pax genes and their role in organogenesis; Cancer Res. 59 1707S–1709S

McDowell N, Zorn A M, Crease D J and Gurdon J B 1997 Activin has direct long-range signalling activity and can form a concentration gradient by diffusion; Curr. Biol. 7 671–681

Melton D A 1987 Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes; Nature (London) 328 80–82

Melton D A 1991 Pattern Formation During Animal Development; Science 252 234–241

Mikhailov A T, Gorgolyuk N A, Tacke L, Mykhoyan M M and Grunz H 1995 Partially purified factor from embryonic chick brain can provoke neuralization of Rana temporaria and Triturus alpestris but not Xenopus laevis early gastrula ectoderm; Int. J. Dev. Biol. 39 317–325

Miller J R and Moon R T 1996 Signal transduction through beta-catenin and specification of cell fate during embryogenesis; Genes Dev. 10 2527–2539

Miller-Bertoglio V E, Fisher S, Sanchez A, Mullins M C and Halpern M E 1997 Differential regulation of chordin expression domains in mutant zebrafish; Dev. Biol. 192 537–550

Minuth M and Grunz H 1980 The formation of mesodermal derivates after induction with vegetalizing factor depends on secondary cell interactions; Cell Diff. 3 229–238

Moon R T and Kimelman D 1998 From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus; BioEssays 20 536–545

Moriya N, Uchiayama H and Asashima M 1993 Induction of pronephric tubules by Activin and retinoic in presumptive ectoderm of Xenopus laeveis; Dev. Growth Diff. 35 123–128

Nakamura O and Toivonen S 1978 Organizer. A milestone of a half-century from Spemann (Amsterdam, Oxford, New York: Elsevier/North-Holland Biomedical Press)

Nakayama T, Snyder M A, Grewal S S, Tsuneizumi K, Tabata T and Christian J L 1998 Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning; Development 125 857–867

Nguyen V H, Schmid B, Trout J, Connors S A, Ekker M and Mullins M C 1998 Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes; Dev. Biol. 199 93–110

Nieuwkoop P D and Faber J 1956 Normal table of Xenopus laevis (Daudin) (Amsterdam: North Holland)

Ninomiya H, Ariizumi T and Asashima M 1998 Activin-treated ectoderm has complete organizing center activity in Cynops embryos; Dev. Growth Diff. 40 199–208

Onichtchouk D, Gawantka V, Dosch R, Delius H, Hirschfeld K, Blumenstock C and Niehrs C 1996 The Xvent-2 homeobox gene is part of the BMP-4 signalling pathway controlling dorsoventral patterning of Xenopus mesoderm; Development 122 3045–3053

Onichtchouk D, Glinka A and Niehrs C 1998 Requirement for Xvent-1 and Xvent-2 gene function in dorsoventral patterning of Xenopus mesoderm; Development 125 1447–1456

Piccolo S, Agius E, Leyns L, Bhattacharyya S, Grunz H, Bouwmeester T and DeRobertis E M 1999 The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals; Nature (London) 397 707–710

Plessow S, Köster M and Knöchel W 1991 cDNA Sequence of Xenopus laevis Bone Morphogenetic Protein-2 (BMP-2); Biochim. Biophys. Acta 1089 280–282

Rebagliati M R, Toyama R, Haffter P and Dawid I B 1998 Cyclops encodes a nodal-related factor involved in midline signaling; Proc. Natl. Acad. Sci. USA 95 9932–9937

Regabgliati M R and Dawid I B 1993 Expression of Activin Transcripts in Follicle Cells and Oocytes of Xenopus laevis; Dev. Biol. 159 574–580

Reilly K M and Melton D A 1996 Short-range signaling by candidate morphogens of the TGF beta family and evidence for a relay mechanism of induction; Cell 86 743–754

Richter K, Grunz H and Dawid I B 1988 Gene expression in the embryonic nervous system of Xenopus laevis; Dev. Biol. 85 8086–8090

Rotmann 1935 Reiz und Reizbeantwortung in der Amphibienentwicklung; Verh. Dtsch. Zool. Ges. (Zool. Anz. Suppl.) 76–83

Sargent T D and Dawid I B 1983 Differential gene expression in the gastrula of Xenopus laevis; Science 222 135–139

Sasai Y, Lu B, Steinbeisser H and Derobertis E M 1995 Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus; Nature (London) 376 333–
336

Sasai Y, Lu B, Steinbeisser H, Geissert D, Gont L K and Derobertis E M 1994 Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes; Cell 79 779–790

Saxén L and Toivonen S 1962 Primary embryonic induction (London: Logos Press–Academic Press)

Schneider S, Herrenknecht K, Butz S, Kemler R and Hausen P 1993 Catenins in Xenopus Embryogenesis and Their Relation to the Cadherin-Mediated Cell–Cell Adhesion System; Development 118 629–640

Schneider S, Steinbeisser H, Warga R M and Hausen P 1996 Beta-Catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos; Mech. Dev. 57
191–198

Schulte-Merker S, Lee K J, McMahon A P and Hammerschmidt M 1997 The zebrafish organizer requires chordino; Nature (London) 387 862–863

Shimizu K and Gurdon J 1999 A quantitative analysis of
signal transduction from Activin receptor to nucleus and its relevance to morphogen gradient interpretation; Proc. Natl. Acad. Sci. USA 96 6791–6796

Slack J M W, Darlington B G, Heath J K and Godsave S F 1987 Mesoderm induction in early Xenopus embryos by heparin-binding growth factors; Nature (London) 326 197–200

Smith J C 1987 A mesoderm inducing factor is produced by a Xenopus cell line; Development 99 3–14

Smith J C, Price B M J, Vannimmen K and Huylebroeck D 1990 Identification of a potent Xenopus mesoderm-inducing factor as a homologue of Activin A; Nature (London) 345 729–731

Smith W C and Harland R M 1992 Expression Cloning of noggin, a New Dorsalizing Factor Localized to the Spemann Organizer in Xenopus Embryos; Cell 70 829–840

Smith W C, Mckendry R, Ribisi S and Harland R M 1995 A nodal-related gene defines a physical and functional domain within the Spemann organizer; Cell 82 37–46

Spemann H and Mangold H 1924 Über Induktion von Embryonalanlagen durch Implantation artfremder Organisatoren; Roux’s Arch. Dev. Biol. 100 599–638

Spemann H and Schotté O 1932 Über xenoplastische Translantation als Mittel zur zur Analyse der embryonalen Induktion; Naturwissenschaften 20 463–467

Streit A and Stern C D 1999 Neural induction – a bird’s eye view; Trends Genet. 15 20–24

Suzuki A, Chang C B, Yingling J M, Wang W F and Hemmati-Brivanlou A 1997 Smad5 induces ventral fates in Xenopus embryo; Dev. Biol. 184 402–405

Suzuki A, Thies R S, Yamaji N, Song J J, Wozney J M, Murakami K and Ueno N 1994 A truncated bone morphogenetic protein receptor affects dorsal–ventral patterning in the early Xenopus embryo; Proc. Natl. Acad. Sci. USA 91 10255–10259

Suzuki A, Ueno N and Hemmati-Brivanlou A 1997 Xenopus msx1 mediates epidermal induction and neural inhibition by BMP4; Development 124 3037–3044

Tiedemann H 1959 Neue Ergebnisse zur Frage nach der chemischen Natur der Induktionsstoffe beim Organisatoreffekt Spemanns; Die Naturwissenschaften 22 613–623

Tiedemann H, Becker U and Tiedemann H 1961 Aktivie-rung des mesodermalen Induktionsfaktors in Huehnermuskulatur durch Behandlung mit Phenol; Embryologia 6
185–203

Tomarev S I, Callaerts P, Kos L, Zinovieva R, Halder G, Gehring W and Piatigorsky J 1997 Squid Pax-6 and eye development; Proc. Natl. Acad. Sci. USA 94 2421–2426

Ueno N, Ling N, Ying S-Y, Esch F, Shimasaki S and Guillemin R 1987 Isolation and partial characterization of follistatin: A single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone; Proc. Natl. Acad. Sci. USA 84 8282–8286

Varlet I, Collignon J and Robertson E J 1997 nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation; Development 124 1033–1044

Watabe T, Kim S, Candia A, Rothbacher U, Hashimoto C, Inoue K and Cho K W Y 1995 Molecular mechanisms of Spemann’s organizer formation: Conserved growth factor synergy between Xenopus and mouse; Genes Dev. 9 3038–3050

Weinmaster G 1998 Reprolysins and Astacins . . . Alive, Alive-O; Science 279 336–337

Weinstein D C and Hemmati-Brivanlou A 1997 Neural induction in Xenopus laevis: Evidence for the default model; Curr. Opnion Neurobiol. 7 7–12

Wilson P A and Hemmati-Brivanlou A 1995 Induction of epidermis and inhibition of neural fate by Bmp-4; Nature (London) 376 331–333

Wilson P A, Lagna G, Suzuki A and Hemmati-Brivanlou A 1997 Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer smad1; Development 124 3177–3184

Xu R H, Kim J B, Taira M, Zhan S I, Sredni D and Kung H F 1995 A dominant negative bone morphogenetic protein 4 receptor causes neuralization in Xenopus ectoderm; Biochem. Biophys. Res. Commun. 212 212–219

Source: http://www.ias.ac.in/jbiosci/december1999/article16.htm