The Spemann-Mangold organizer: the control of fate specification and morphogenetic rearrangements during gastrulation in Xenopus

Vertebrate embryonic development is controlled by sequentially operating signalling centres that organize spatial pattern by inductive interactions. The embryonic body plan is established during gastrulation through the action of the Spemann-Mangold or gastrula organizer, a signalling source discovered 75 years ago by Hans Spemann and Hilde Mangold. Transplantation of the organizer to a heterotopic location in a recipient embryo results in the formation of a secondary embryonic body axis, in which several tissue types, most notably somites and the neural tube, are derived from ventral host cells. Because of these non-cell autonomous recruiting or inducing activities the organizer has become a paradigm for studying intercellular communication in the vertebrate embryo. Here, I review some of the recent advances in understanding 1) the initiation of the Spemann-Mangold organizer, 2) its function in pattern formation along the dorsal-ventral and anterior-posterior axes and 3) the integration of cell fate specification events and downstream execution of morphogenetic movements during gastrulation in Xenopus laevis.     

Developmental biology of amphibians after Hans Spemann in Germany

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).     

Spemann's influence on Japanese developmental biology

The discovery of the organizer by H. Spemann and Hilde Mangold, prompted a number of studies of embryonic induction in Japan. C.O. Whitman, N. Yatsu, T. Sato, H. Oka, T. Yamada, and Y.K. Okada were the pioneers in the field of embryonic induction. T. Yamada postulated the double potential theory for embryonic induction. O. Nakamura has modified the fate map of Vogt using newt and Xenopusblastulae. T.S. Okada and G. Eguchi proposed the new concept of "transdifferentiation" based on in vitro experiments in the retina and lens. T.S. Okada is not only an excellent scientist, but he has also nurtured many active developmental biologists. M. Takeichi, from his school, discovered the cell adhesion molecle, cadherin. Nakamura and colleagues tried to determine the origin and formation of the organizer. They performed recombination experiments using the ectoderm, endoderm and mesoderm, and concluded that the phenomenon in which various mesoderm tissues are formed by the recombination of the presumptive ectoderm with endoderm was "regulation of the vegetal-animal gradient". Some groups have also tried to purify specific inducing factors. T. Yamada and colleagues isolated two different types of ribonucleoproteins. I. Kawakami and colleagues showed that the ribosome fraction has neural inducing capacity, and that the extracellular matrix contains mesodermal inducing factors. Finally Asashima and colleagues isolated and identified activin A as a MIF factor. This finding had a great influence not only in the field of developmental biology, but also in molecular biology. Using activin, Asashima's group has successfully generated various organs, tissues, trunk-tail and head structures in vitro using animal caps (undifferentiated cells). Some other important molecules such as BMP, chordin and bFGF are also being studied by young Japanese scientists.     

Head in the WNT: the molecular nature of Spemann's head organizer.

Formation of the head during vertebrate embryogenesis has been one of the most-studied topics in development, probably because we are such cephalized beings ourselves. Early experimenters found that the head is induced during gastrulation by Spemann's organizer. In 1999 we celebrate the 75th anniversary of the discovery of the organizer by Spemann and Mangold, a group of cells in amphibia that secretes powerful signalling molecules. Recently, advances have been made in identifying candidate head inducers. Not surprisingly, these inducers act in familiar molecular pathways, namely transforming growth factor beta (TGF-beta) and WNT signalling.     

Gene expression and pattern formation during early embryonic development in amphibians

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 Vgl and Activin (both belonging to the TGF-β 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 inDrosophila sog, homologous to chordin, determines the ventral side, whiledpp, homologous toBMP-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.     

Induction into the Hall of Fame: tracing the lineage of Spemann's organizer

The grafting experiments of Spemann and Mangold have been a textbook classic for years, but as with many conclusions from experimental embryology, the idea that the dorsal lip of the blastopore ;organized' the early patterning of the embryo has sometimes come under question. In their 1983 paper in JEEM, Smith and Slack extended these classical experiments in newts to the now-standard amphibian model Xenopus laevis. By using injected lineage tracers, they distinguished the fates of graft and host, and showed unambiguously that the organizer is responsible for neural induction and that it dorsalizes the mesoderm.     

Neural induction. A bird's eye view

Since the discovery of the phenomenon of neural induction by Spemann and Mangold in 1924, considerable effort has been invested in identifying the signals produced by the organizer that are responsible for diverting the fate of cells from epidermal to neural. Substantial progress has been made only recently by the finding in amphibians that BMP4 is a neural inhibitor and epidermal inducer, and that endogenous antagonists of BMPs are secreted by the organizer. However, recent results in the chick point to the existence of other, upstream events required before BMP inhibition stabilizes neural fates. Here we take a critical view of the evidence for and against the view that BMP inhibition is a sufficient trigger for neural induction in different vertebrates.     

Organizing chordates with an organizer

Understanding how the chordate body plan originated and evolved is still controversial. The discovery by Spemann and Mangold in 1924 of the vertebrate organizer and its inductive properties in patterning the AP and DV axis was followed by a long gap until the 1960s when scientists started characterizing the molecular events responsible for such inductions. However, the evolutionary origin of the organizer itself remained obscure until very recently; did it appear together with the origin and radiation of vertebrates, or was it a chordate affair? A recent study by Yu and collaborators, 1 which analyses the expression of several organizer-specific genes in amphioxus together with recent phylogenetic data that reversed the position of invertebrate extant chordates (e.g. urochordates and cephalochordates), indicates that the organizer probably appeared in early chordates. It likely had separate signalling centres generating BMP and Wnt signalling gradients along the DV and AP axis. The organizer was then lost in the urochordate lineage, most probably as an adaptation to a rapid and determinate development. BioEssays 29:619–624, 2007. © 2007 Wiley Periodicals, Inc.     

Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.     

Evolution of the organizer and the chordate body plan

The discovery of the organizer by Spemann and Mangold in 1924 raised two kinds of questions: those about the means of patterning the chordate body axis and those about the mechanisms of cell determination by induction. Some researchers, stressing the second, have suggested over the years that the organizer is poorly named and doesn't really organize because inducers act permissively, because they are not unique to the organizer, and because multipotent responsive cells develop complex local differentiations under artificial conditions. Furthermore, with the discovery of meso-endoderm induction in 1969, the possibility arose that this earlier induction generates as much organization as, or more than, does the organizer itself. Evidence is summarized in this article that the organizer does fulfill its title with regard to pattern formation: it adds greatly to embryonic organization by providing information about time, place, scale, and orientation for development by nearby members of the large multipotent competence groups surrounding the organizer. Embryos having smaller or larger organizers due to experimental intervention develop defective axial organization. Without an organizer the embryo develops no body axis and none of the four chordate characters: the notochord, gill slits, dorsal hollow nerve chord, and post-anal tail. For normal axis formation, the organizer's tripartite organization is needed. Each part differs in inducers, morphogenesis, and self-differentiation. The organizer is a trait of development of all members of the chordate phylum. In comparison to hemichordates, which constitute a phylum with some similarities to chordates, the chordamesoderm part is unique to the chordate organizer (the trunk-tail organizer). Its convergent extension displaces the gastrula posterior pole from alignment with the animal-vegetal axis and generates a new anteroposterior axis orthogonal to this old one. Once it has extended to full length, its signaling modifies the dorsoventral dimension. This addition to the organizer is seen as a major event in chordate evolution, bringing body organization beyond that achieved by oocyte organization and meso-endoderm induction in other groups.     

Embryonic Induction

"Spemann's" lecture treats experiments on the separation ofthe first two cells of a frog, or sea urchin, or salamanderembryo; the induction of a lens in a frog embryo by an opticvesicle (primordium of the eye); and the primary organizer thatis a dynamic center, establishing the basic organization ofthe embryo and inducing the nervous system and sense organs."Spemann" goes beyond science in speaking poetically of thebeauty and order in the universe, and to illustrate how a goodscholar should work he uses a lovely metaphor of piecing togetherthe fragments of a broken vase. "Spemann" concludes with a stirringplea for academic freedom.     

Overexpression of the secreted factor Mig30 expressed in the Spemann organizer impairs morphogenetic movements during Xenopus gastrulation

The Spemann organizer secretes several antagonists of growth factors during gastrulation. We describe a novel secreted protein, Mig30, which is expressed in the anterior endomesoderm of the Spemann organizer. Mixer-inducible gene 30 (Mig30) was isolated as a target of Mixer, a homeobox gene required for endoderm development. The Mig30 gene encodes a secreted protein containing a cysteine-rich domain and an immunoglobulin-like domain that belongs to the insulin-like growth factor-binding protein family. Overexpression of Mig30 in the dorsal region results in the retardation of morphogenetic movements during gastrulation and leads to microcephalic embryos. Overexpression of Mig30 also inhibits activin-induced elongation of ectodermal explants without affecting gene expression patterns in mesoderm and endoderm. These results suggest that Mig30 is involved in the regulation of morphogenetic movements during gastrulation in the extracellular space of the Spemann organizer.     

Spemann's organizer and self-regulation in amphibian embryos

In 1924, Spemann and Mangold demonstrated the induction of Siamese twins in transplantation experiments with salamander eggs. Recent work in amphibian embryos has followed their lead and uncovered that cells in signalling centres that are located at the dorsal and ventral poles of the gastrula embryo communicate with each other through a network of secreted growth-factor antagonists, a protease that degrades them, a protease inhibitor and bone-morphogenic-protein signals.     

Nodal signaling and vertebrate germ layer formation

The understanding of germ layer formation in vertebrates began with classical experimental embryology. Early in the 20th century, Spemann and Mangold (1924) identified a region of the early embryo capable of inducing an entire embryonic axis. Termed the dorsal organizer, the tissue and the activity have been shown to exist in all vertebrates examined. In mice, for example, the activity resides in a region of the gastrula embryo known as the node. Experiments by the Dutch embryologist Nieuwkoop (1967a, 1967b, 1973, 1977) showed that a signal derived from the vegetal half of the amphibian embryo is responsible for the formation of mesoderm. Nieuwkoop's results allowed the development of in vitro assays that led, in the late 1980s and early 1990s, to the identification of growth factors essential for germ layer formation. Through more recent genetic investigations in mice and zebrafish, we now know that one class of secreted growth factor, called Nodal because of its localized expression in the mouse node, is essential for formation of mesoderm and endoderm and for the morphological rearrangements that occur during gastrulation.     

FGF signaling restricts the primary blood islands to ventral mesoderm

According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. It is proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. We present evidence here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. We further observed that inhibition of FGF signaling with a dominant negative receptor resulted in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories was observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. On the other hand, we found no evidence that manipulation of BMP signaling, either positively or negatively, altered the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm.     

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

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

Requirement for anti-dorsalizing morphogenetic protein in organizer patterning

The amphibian Spemann organizer is subdivided in trunk and head organizer and it is unclear how this division is regulated. The Xenopus trunk organizer expresses anti-dorsalizing morphogenetic protein (ADMP), a potent organizer antagonist. We show that ADMP represses head formation during gastrulation and that its expression is activated by BMP antagonists. A specifically acting dominant-negative ADMP anteriorizes embryos and its coexpression with BMP antagonists induces secondary embryonic axes with heads as well as expression of head inducers. Unlike other BMPs, ADMP is not inhibited by a dominant-negative BMP type I receptor, Noggin, Cerberus and Chordin but by Follistatin, suggesting that it utilizes a distinct TGF-β receptor pathway and displays differential sensitivity to BMP antagonists. The results indicate that ADMP functions in the trunk organizer to antagonize head formation, thereby regulating organizer patterning.