The role of the Spemann organizer in anterior-posterior patterning of the trunk

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.     

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

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

Cytoplasmic and molecular reconstruction of Xenopus embryos: synergy of dorsalizing and endo-mesodermalizing determinants drives early axial patterning

Ablation of vegetal cytoplasm from newly fertilized Xenopus eggs results in the development of permanent blastula-type embryos (PBEs). PBEs cleave normally and develop into a very simple tissue consisting only of atypical epidermis. We tried to restore complete embryonic development in PBEs by cytoplasmic transplantation or by mRNA injection. We show a two-step reconstruction of the body plan. In the first step, PBEs injected with either marginal cytoplasm or synthetic VegT RNA restored gastrulation and mesoderm formation, but not axial patterning. Injection of Xwnt8 mRNA (acting upstream of beta-catenin and thus substitutes for the dorsal determinant) did not restore axial development in PBEs. Simultaneous injections of Xwnt8 and VegT into PBEs resulted in dorsal axis development, showing the synergy of these molecules in axial development. These results suggest that the mixing of two cytoplasmic determinants, i.e. the dorsal determinant in the vegetal pole and the endo-mesodermal determinant in the whole vegetal half, triggers the early axial developmental process in Xenopus embryos.     

Depletion of three BMP antagonists from Spemann's organizer leads to a catastrophic loss of dorsal structures

Transplanted Spemann's organizer induces dorsal embryonic cell fates such as the nervous system and somites, but in normal development, elimination of individual organizer signals (such as the bone morphogenetic protein [BMP] antagonists) has surprisingly modest effects on these tissues. Thus, the role of BMP antagonists may be limited to fine tuning the size of the dorsal domain. However, at least five BMP antagonists are specifically expressed in the organizer, and all can mimic aspects of organizer function, suggesting overlapping functions. Here, we deplete the function of three BMP antagonists, chordin, noggin, and follistatin, in Xenopus tropicalis. We demonstrate that this results in catastrophic failure of dorsal development and expansion of ventral and posterior fates. We conclude that BMP antagonists are required for formation of the neural plate and dorsal mesoderm. In addition, our results show that neural specification requires the continuous activity of BMP antagonists from blastula through gastrula stages.     

BMP antagonism by Spemann's organizer regulates rostral-caudal fate of mesoderm

Recent revisions to the Xenopus fate map challenge the interpretation of previous maps and current models of amphibian axial patterning (Lane, M.C., Smith, W.C., 1999. The origins of primitive blood in Xenopus: implications for axial patterning. Development 126 (3), 423-434.; Lane, M.C., Sheets, M.D., 2000. Designation of the anterior/posterior axis in pregastrula Xenopus laevis. Dev. Biol. 225, 37-58). We determined the rostralmost contributions to both dorsal and ventral mesoderm concomitantly from marginal zone progenitors in stage 6 embryos. Data reveal an unequivocal rostral-to-caudal progression of both dorsal and ventral mesoderm across the pre-gastrula axis historically called the dorsal-ventral axis, and a dorsal-to-ventral progression from animal-to-vegetal in the marginal zone. These findings support the proposed revisions to the fate and axis orientation maps. Most importantly, these results raise questions about the role of the organizer grafts and organizer-derived BMP antagonists in the "induction" of secondary axes. We re-examine both phenomena, and find that organizer grafts and BMP antagonists evoke caudal-to-rostral mesodermal fate transformations, and not ventral-to-dorsal transformations as currently believed. We demonstrate that BMP antagonism evokes a second axis because it stimulates precocious mediolateral intercalation of caudal, dorsal mesoderm. The implications of these findings for models of organizer function in vertebrate axial patterning are discussed.     

Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging

The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction. Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ, which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). "Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus." Development. 126, 3703-3713.]. We present a revised model of gastrulation movements in Xenopus laevis.     

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.     

Designation of the anterior/posterior axis in pregastrula Xenopus laevis

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

Induction and patterning of the telencephalon in Xenopus laevis

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.     

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

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

Xenopus ADAMTS1 negatively modulates FGF signaling independent of its metalloprotease activity

We have isolated the Xenopus ortholog of ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motifs), XADAMTS1, which is expressed in the presumptive ectoderm, then the Spemann organizer, and later in the trunk organizer region and posterior ectoderm in the Xenopus embryo. We show that, when overexpressed in the dorsal marginal zone or in the anterior ectoderm by mRNA injection, XADAMTS1 inhibits gastrulation or generates embryos with an enlarged cement gland, respectively. XADAMTS1 also reduces the expression of Xbra in both whole embryos and FGF-treated animal caps. These effects of XADAMTS1 are likely to be due to its inhibition of the Ras-MAPK cascade because XADAMTS1 inhibits the phosphorylation of ERK by FGF4 in animal caps. Deletion analysis of XADAMTS1 revealed that a combination of the signal peptide and the C-terminal region containing the thrombospondin type 1 repeats is necessary and sufficient for this function, whereas the metalloprotease domain is dispensable. In addition, loss-of-function analysis with antisense morpholino oligos showed that knockdown of XADAMTS1 sensitizes animal caps to Xbra induction by FGF2. These data suggest that secreted XADAMTS1 negatively modulates FGF signaling in the Xenopus embryo.     

In synergy with noggin and follistatin, Xenopus nodal-related gene induces sonic hedgehog on notochord and floor plate

In early development of vertebrates, sonic hedgehog functions in dorsal-ventral patterning of dorsal tissue (nervous system and somites). In Xenopus, sonic hedgehog (Xshh) is first expressed in the Spemann organizer/notochord and floor plate. We report here the mechanism governing Xshh mRNA induction in these regions. In animal cap assays, the antagonizing BMPs signal was not sufficient to induce Xshh mRNA expression; however, it could induce Xshh mRNA expression in the presence of Xnr-1. In whole embryos, when secondary axes were induced by coexpressing noggin and Xnr-1 or follistatin and Xnr-1, Xshh mRNA expression was observed in the notochord and floor plate within the induced axes. It seems apparent that spatially restricted Xshh mRNA expression is determined as intersection of the two signals.     

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

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

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.     

The establishment of Spemann's organizer and patterning of the vertebrate embryo

Molecular studies have begun to unravel the sequential cell-cell signalling events that establish the dorsal-ventral, or 'back-to-belly', axis of vertebrate animals. In Xenopus and zebrafish, these events start with the movement of membrane vesicles associated with dorsal determinants. This mediates the induction of mesoderm by generating gradients of growth factors. Dorsal mesoderm then becomes a signalling centre, the Spemann's organizer, which secretes several antagonists of growth-factor signalling. Recent studies have led to new models for the regulation of cell-cell signalling during development, which may also apply to the homeostasis of adult tissues.