Nieuwkoop Center Research Articles

Induction of primitive streak and Hensen's node by the posterior marginal zone in the early chick embryo.

In the preprimitive streak chick embryo, the search for a region capable of inducing the organizer, equivalent to the Nieuwkoop Center of the amphibian embryo, has focused on Koller's sickle, the hypoblast and the posterior marginal zone. However, no clear evidence for induction of an organizer without contribution from the inducing tissue has been provided for any of these structures. We have used DiI/DiO labeling to establish the fate of midline cells in and around Koller's sickle in the normal embryo. In the epiblast, the boundary between cells that contribute to the streak and those that do not lies at the posterior edge of Koller's sickle, except at stage X when it lies slightly more posteriorly in the epiblast. Hypoblast and endoblast (a second lower layer formed under the streak) have distinct origins in the lower layer, and goosecoid expression distinguishes between them. We then used anterior halves of chick prestreak embryos as recipients for grafts of quail posterior marginal zone; quail cells can be identified subsequently with a quail-specific antibody. Anterior halves alone usually formed a streak, most often from the posterior edge. Quail posterior marginal zones without Koller's sickle were grafted to the anterior side of anterior halves. These grafts were able to increase significantly the frequency of streaks arising from the anterior pole of stage X-XI anterior halves without contributing to the streak or node. Stage XII anterior halves no longer responded. A goosecoid-expressing hypoblast did not form under the induced streak, indicating that it is not required for streak formation. We conclude that the marginal zone posterior to Koller's sickle can induce a streak and node, without contributing cells to the induced streak.

De novo induction of the organizer and formation of the primitive streak in an experimental model of notochord reconstitution in avian embryos.

We have developed a model system for analyzing reconstitution of the notochord using cultured blastoderm isolates lacking Hensen's node and the primitive streak. Despite lacking normal notochordal precursor cells, the notochord still forms in these isolates during the 36 hours in culture. Reconstitution of the notochord involves an inducer, which acts upon a responder, thereby inducing a reconstituted notochord. To better understand the mechanism of notochord reconstitution, we asked whether formation of the notochord in the model system was preceded by reconstitution of Hensen's node, the organizer of the avian neuraxis. Our results show not only that a functional organizer is reconstituted, but that this organizer is induced from the responder. First, fate mapping reveals that the responder forms a density, morphologically similar to Hensen's node, during the first 10-12 hours in culture, and that this density expresses typical markers of Hensen's node. Second, the density, when fate mapped or when labeled and transplanted in place of Hensen's node, forms typical derivatives of Hensen's node such as endoderm, notochord and the floor plate of the neural tube. Third, the density, when transplanted to an ectopic site, induces a secondary neuraxis, identical to that induced by Hensen's node. And fourth, the density acts as a suppressor of notochord reconstitution, as does Hensen's node, when transplanted to other blastoderm isolates. Our results also reveal that the medial edge of the isolate forms a reconstituted primitive streak, which gives rise to the normal derivatives of the definitive primitive streak along its rostrocaudal extent and which expresses typical streak markers. Finally, our results demonstrate that the notochordal inducer also induces the reconstituted Hensen's node and, therefore, acts like a Nieuwkoop Center. These findings increase our understanding of the mechanism of notochord reconstitution, provide new information and a novel model system for studying the induction of the organizer and reveal the potential of the epiblast to regulate its cell fate and patterns of gene expression during late gastrula/early neurula stage in higher vertebrates.

Only the endophyll-Rauber's sickle complex and not cells derived from the caudal marginal zone induce a primitive streak in the upper layer of avian blastoderms.

From unincubated quail blastoderms, we have excised, caudal marginal zones (caudally from Rauber's sickle), upper layer fragments covering Rauber's sickle or Rauber's sickle fragments alone (as controls), and placed them on the ventral side of the cranial quadrant of unincubated chicken blastoderms. Also, quail Rauber's sickle fragments, all or not associated with quail endophyll, were placed on the ventral side of isolated central upper layer discs of prestreak chicken blastoderms from which the deep layer was previously removed. Only the Rauber's sickle-derived cells (sickle endoblast cells), placed on unincubated or shortly incubated blastoderms induce, after culture, a primitive streak (PS) and a normal embryo. This indicates, together with previous experimental evidence, that even in the presence of endophyll, neither the deep part of the caudal marginal zone nor the upper layer above it can induce a primitive streak. This experimental study affords further evidence that the function of the avian Rauber's sickle is homologous to the function (mesoderm induction) of the vegetal dorsalizing cells (Nieuwkoop centre) in amphibian blastulas.

Pre-MBT patterning of early gene regulation in Xenopus: the role of the cortical rotation and mesoderm induction

Patterning events that occur before the mid-blastula transition (MBT) and that organize the spatial pattern of gene expression in the animal hemisphere have been analyzed in Xenopus embryos. We present evidence that genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16–128-cell stage) cultured until gastrula stages, we demonstrate by RT-PCR analysis that the expression of goosecoid ( gsc), wnt-8 and brachyury ( bra) is dependent on mesoderm induction. In contrast, nodal-related 3 ( nr3) and siamois ( sia) are expressed in a manner that is independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. Our data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo.

1 Pattern Formation in Zebrafish–Fruitful Liaisons between Embryology and Genetics

Vertebrate embryos, despite quite diverse early morphologies, appear to employ similar cellular strategies and conserved biochemical pathways in their development (Eyal-Giladi, 1997). In the past decade, a small tropical teleost, zebrafish (Danio rerio), became an important model system in which to study development (Streisinger et al., 1981). By combining embryology with molecular and classical genetic methods, our understanding of early inductive and morphogenetic events during vertebrate embryogenesis significantly advanced. In zebrafish, dorsal-ventral polarity is established during early cleavage and is dependent on microtubular transport of determinants from the vegetal pole to the blastomeres positioned on top of the yolk cell. The syncytium forming from these marginal blastomeres in the early blastula exhibits dorsal-ventral asymmetry with beta-catenin localized to the nuclei on the presumptive dorsal side of the syncytium. The yolk cell is a source of signals that induce and pattern overlying blastoderm. Therefore, the dorsal yolk syncytial layer is equivalent to the Nieuwkoop center of the amphibian embryo. The embryonic shield, a thickening of the dorsal blastoderm margin, exhibits properties similar to the amphibian Spemann organizer. However, certain inductive and patterning signals from the organizer might be produced before the shield forms or might originate outside of the shield. Similar to the amphibian embryo, the key patterning functions of the fish dorsal organizer (i.e., dorsalization of mesoderm, ectoderm, and coordination of gastrulation movements) are performed by secreted molecules that antagonize the ventralizing activity of the swil (zbmp-2) and zbmp-4 gene products expressed on the ventral side of the embryo. These functions of the dorsal organizer require the activity of the chordino gene (a zebrafish homologue of chordin), bozozok, mercedes and ogon loci.

Antagonism of cell adhesion by an alpha-catenin mutant, and of the Wnt-signaling pathway by alpha-catenin in Xenopus embryos.

In Xenopus laevis development, beta-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell-cell adhesion and can modulate beta-catenin signaling. alpha-catenin links beta-catenin to the actin-based cytoskeleton. To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin. The binding domain of beta-catenin has been mapped to the NH2-terminal 210 amino acids of alphaN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length alphaN-catenin or a mutant of beta-catenin that lacks the internal armadillo repeats. We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, alpha-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular beta-catenin signaling pathway in vivo.

Formation and function of Spemann's organizer.

The organizer is formed in an equatorial sector of the blastula stage amphibian embryo by cells that have responded to two maternal agents: a general mesoendoderm inducer (involving the TFG-beta signaling pathway) and a dorsal modifier (probably involving the Wnt signaling pathway). The meso-endoderm inducer is secreted by most vegetal cells, those containing maternal materials that had been localized in the vegetal hemisphere of the oocyte during oogenesis. As a consequence of the inducer's distribution and action, the competence domains of prospective ectoderm, mesoderm, and endoderm are established in an animal-to-vegetal order in the blastula. The dorsal modifier signal is secreted by a sector of cells of the animal and vegetal hemispheres on one side of the blastula. These cells contain maternal materials transported there in the first cell cycle from the vegetal pole of the egg along microtubules aligned by cortical rotation. The Nieuwkoop center is the region of blastula cells secreting both maternal signals, and hence specifying the organizer in an equatorial sector. Final steps of organizer formation at the late blastula or early gastrula stage may involve locally secreted zygotic signals as well. At the gastrula stage, the organizer secretes a variety of zygotic proteins that act as antagonists to various members of the BMP and Wnt families of ligands, which are secreted by cells of the competence domains surrounding the organizer. BMPs and Wnts favor ventral development, and cells near the organizer are protected from these agents by the organizer's inducers. The nearby cells are derepressed in their inherent capacity for dorsal development, which is apparent in the neural induction of the ectoderm, dorsalization of the mesoderm, and anteriorization of the endoderm. The organizer also engages in extensive specialized morphogenesis, which brings it within range of responsive cell groups. It also self-differentiates to a variety of axial tissues of the body.

Head induction by simultaneous repression of Bmp and Wnt signalling in Xenopus.

The Spemann organizer of the amphibian embryo can be subdivided into two discrete activities, namely trunk organizer and head organizer. Several factors secreted from the organizer that are involved in trunk organization are thought to act by repressing Bmp signalling. With the exception of the secreted factor cerberus, little is known about head-organizer inducers. Here we show that co-expression of a dominant-negative Bmp receptor with inhibitors of the Wnt-signalling pathway in Xenopus leads to the induction of complete secondary axes, including a head. This induction does not require expression of the siamois marker of Nieuwkoop centre signalling, suggesting that cells are directly shifting to head-organizer fate. Furthermore, we find that cerberus is a potent inhibitor of Wnt signalling. Our results indicate that head-organizer activity results from the simultaneous repression of Bmp and Wnt signalling and they suggest a mechanism for region-specific induction by the organizer.

Establishment of polarities in the oocyte of Xenopus laevis: the provisional axial symmetry of the full-grown oocyte of Xenopus laevis.

We aimed at understanding of formation and function of the "Nieuwkoop Centre" in embryonic pattern formation. Discussed are data on genesis of cytoplasmic localizations in ovarian oocytes, transient modifications of cytoskeletal structures creating cytoplasmic asymmetries in fertilized eggs, the axis determining "vegetal cortical rotation" and fate of distinct cells, as shown by injection of specific molecular markers into particular blastomeres at specific times. Egg rotation and centrifugation suggested that sperm that gravity cooperate in symmetrization of the axially symmetrical anuran egg. After fertilization in space or in a fast rotating clinostate, axis formation and embryonic development were normal although the blastocoel was transiently abnormal. Normal tadpoles came back on Earth after ovulation, fertilization and culture in space. They metamorphosed normally and got healthy Earth-born F1 offspring. We conclude that neither sperm nor gravity are required for determination of the bilateral symmetry in the embryo of Xenopus laevis. In normal development sperm and gravity, either alone or in collaboration, may overrule an initial bilaterality inherent to, the full-grown oocyte, residing in some still unidentified component(s)/or mechanisms.

Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/beta-catenin signaling pathway, but not by Vg1, Activin or Noggin.

The molecular nature of the primary dorsalizing inducing event in Xenopus is controversial and several secreted factors have been proposed as potential candidates: Wnts, Vg1, Activin and Noggin. Recent studies, however, have provided new insight into the activity of the dorsalizing region, called the Nieuwkoop Center. (1) The activity of this dorsalizing center involves an entire signal transduction pathway that requires maternal beta-catenin (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994) Cell 79, 791-803). (2) A transcription factor with potent dorsalizing activity, Siamois, is expressed within the Nieuwkoop Center (Lemaire, P., Garrett, N. and Gurdon, J. B. (1995) Cell 81, 85-94). We have used these two properties of the Nieuwkoop Center to evaluate the dorsalizing activity of the four secreted factors Wnt8, Vg1, Activin and Noggin. The requirement for beta-catenin was tested by coexpressing a cadherin, which sequesters beta-catenin at the cell membrane and specifically blocks its intracellular signaling activity (Fagotto, F., Funayama, N., Gluck, U. and Gumbiner, B. M. (1996) J. Cell Biol. 132, 1105-1114). Induction of Siamois expression was detected by RT-PCR. Of the four growth factors, only Wnt was sensitive to inhibition of beta-catenin activity and only Wnt could induce Siamois expression. Therefore, Wnt is able to induce a bonafide Nieuwkoop Center, while Vg1, Activin and Noggin probably induce dorsal structures by a different mechanism. To order the steps in the Nieuwkoop Center signaling cascade, we have tested the relationship between beta-catenin and GSK, a serine-threonine kinase that has been implicated in axis formation in a step downstream of Wnt. We found that GSK acts upstream of beta-catenin, similar to the order of these components in the Wingless pathway in Drosophila. We have also examined the relationship between the Wnt/beta-catenin pathway and Siamois. We show that beta-catenin induces expression of Siamois and that the free signaling pool of beta-catenin is required for normal expression of endogenous Siamois. We conclude that the sequence of steps in the signaling pathway is Wnt-->GSK-->beta-catenin-->Siamois.

The homeobox gene Siamois is a target of the Wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm.

Siamois, a Xenopus zygotic homeobox gene with strong dorsalising activity, is expressed in the dorsal-vegetal organiser known as the Nieuwkoop centre. We show that, in contrast to Spemann organiser genes such as goosecoid, chordin and noggin, Siamois gene expression is not induced following overexpression of mesoderm inducers in ectodermal (animal cap) cells. However, Siamois is induced by overexpressing a dorsalising Wnt molecule. Furthermore, like Wnt, Siamois can dorsalise ventral mesoderm and cooperate with Xbrachyury to generate dorsal mesoderm. These results suggest that Siamois is a mediator of the Wnt-signalling pathway and that the synergy between the Wnt and mesoderm induction pathways occurs downstream of the early target genes of these two pathways. Overexpression of Siamois in animal cap cells reveals that this gene can act in a non vegetal or mesodermal context. We show the following. (1) Animal cap cells overexpressing Siamois secrete a factor able to dorsalise ventral gastrula mesoderm in tissue combination experiments. (2) The Spemann organiser-specific genes goosecoid, Xnr-3 and chordin, but not Xlim.1, are activated in these caps while the ventralising gene Bmp-4 is repressed. However, the dorsalising activity of Siamois-expressing animal caps is significantly different from that of noggin- or chordin-expressing animal caps, suggesting the existence of other dorsalising signals in the embryo. (3) Ectodermal cells overexpressing Siamois secrete a neuralising signal and can differentiate into cement gland and, to a lesser extent, into neural tissue. Hence, in the absence of mesoderm induction, overexpression of Siamois is sufficient to confer organiser properties on embryonic cells.

β-Catenin Has Wnt-like Activity and Mimics the Nieuwkoop Signaling Center inXenopusDorsal–Ventral Patterning

β-Catenin is a protein known to associate with the cytoplasmic domains of members of the cadherin family of cell adhesion molecules. Recently, Funayamaet al.(Funayamaet al.(1995).J. Cell Biol.128, 959–968.) demonstrated that overexpression of β-catenin causes the formation of a secondary axis inXenopus laevisembryos. In order to understand the role of β-catenin in axis formation, we examined its biological activity in further detail. β-Catenin is effective at inducing a secondary axis when overexpressed in the vegetal ventral region of early cleavage stage (4–32 cell) embryos. β-Catenin may act as part of the Nieuwkoop center because cells overexpressing β-catenin do not contribute directly to axial structures. Overexpression of β-catenin can specifyde novoaxis formation, as shown by its ability to rescue UV-ventralized embryos. Overexpression of β-catenin alone is not sufficient to cause elongation of animal caps or to induce mesodermal markers in animal caps. In these assays, overexpression of β-catenin behaves like ectopic expression of certain members of theWntgene family. Like Wnts, overexpression of β-catenin was also found to increase gap junctional communication in cells of the ventral animal cap. Overexpression of β-catenin causes a small increase in the rate of aggregation ofXenopusblastomeres. Overexpression of C-cadherin causes a more dramatic increase in the rate of aggregation ofXenopusblastomeres, but does not enhance gap junction communication or induce axis duplication; hence, we argue that increased adhesion is not sufficient to account for β-catenin's ability to regulate patterning or gap junction communication. We propose a signaling role for β-catenin during axis formation inXenopus.

Axis formation in zebrafish

Recent advances in our understanding of axis formation and patterning in zebrafish relate the developmental mode of this aspiring genetic model organism to higher vertebrates. The effect of UV irradiation and lithium treatment, as well as detailed early lineage analyses, have shed some light on dorsoventral axis formation. However, the molecular mechanism of axis formation, as well as the identity of a fish Nieuwkoop center, are still open issues. A Vg1 homolog is expressed in zebrafish, and activin as well as the mouse nodal gene product have been demonstrated to induce mesoderm and ectopic axes, respectively, in zebrafish. The zebrafish organizer is defined by the expression domains of goosecoid, axial and lim1. The cyclops gene is involved in maintaining goosecoid expression in axial mesoderm of the head. Large mutageneis screens provide the basis for a genetic analysis of axis formation.

Autonomous Endodermal Determination in Xenopus: Regulation of Expression of the Pancreatic Gene XlHbox 8

In neural plate stage Xenopus embryos, XlHbox 8 expression marks anterior endodermal cells fated to develop into pancreas/duodenum, and expression continues in adult pancreas in exocrine duct, acinar, and islet cells. Here, XlHbox 8 is used as a marker in experiments addressing the mechanisms of early endodermal patterning, particularly with respect to the role of specific polypeptide growth factors. When mesoderm-free vegetal explants (VEs) from early blastula stage embryos are cultured in isolation, XlHbox 8 expression develops autonomously in the dorsal region, strongly suggesting that endodermal region-specific determination occurs before MBT. Data from microinjection experiments using RNA encoding the activin and FGF dominant negative receptors and growth factor treatments of isolated VEs suggest that activin positively regulates XlHbox 8 expression, whereas bFGF is a potent negative regulator. Moreover, bFGF induces mesodermal marker expression in VEs. This suggests that the early endodermal determination state is plastic and that elevated levels of bFGF may convert vegetal (endodermal) cells into mesoderm. We propose a model for XlHbox 8 regulation in which an early signal from the Nieuwkoop center (whose eventual fate is endoderm) predisposes dorsovegetal cells for autonomous XlHbox 8 expression, in an area of high local activin (or activin-like) ligand concentration, and low relative concentrations of bFGF.

Expression cloning of Siamois, a xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis

Using an expression cloning strategy that relies on a functional assay, we have cloned a novel Xenopus homeobox-containing gene, Siamois. Embryos injected in a ventral-vegetal blastomere with as little as 5 pg of Siamois mRNA develop a complete secondary axis, but the progeny of the injected cells do not participate in the secondary axis formation. In normal development, Siamois mRNA is first detected shortly after the midblastula transition, which is earlier than mRNAs for goosecoid or Xbrachyury, and is present most abundantly in the dorsal endoderm of early gastrulae. The activation of this gene can be obtained cell autonomously in dispersed embryo cells. These results indicate that Siamois may play an important role in the formation of the Nieuwkoop center.

Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands

To increase our understanding of haematopoiesis during early vertebrate development, we have studied the expression pattern of the transcription factor GATA-2 in Xenopus embryos, and asked how this is regulated. We show that the blood island precursors of the ventral mesoderm express GATA-2 RNA at neural tube stages, some 5 hours before globin RNA is detected in their derivatives. Prior to this however, GATA-2 is expressed much more widely within the embryo. Maternal transcripts are uniformly distributed, and zygotic transcription is activated during gastrulation throughout ventral and lateral regions of the embryo, with expression highest in the sensorial ectoderm and only weak in the ventral mesoderm. The domain of GATA-2 expression in neurulae outlines the region of the neural plate and suggests a possible wider role in dorsoventral patterning. To identify the signals involved in regulating this pattern of expression, we performed experiments with embryo explants. GATA-2 is activated autonomously in isolated animal caps and this activation is suppressed by the mesoderm-inducing factor activin, but not by FGF. Thus, the down-regulation of GATA-2 observed in the region of the Spemann organiser may be a response to an activin-like signal emanating from the dorsal-vegetal region or Nieuwkoop centre. GATA-2 activation in animal caps and ventral marginal zones was suppressed by co-culturing with dorsal marginal zones, suggesting that a signal from the Spemann organiser is involved in suppression of GATA-2 in the dorsal region of the embryo. Expression of a candidate for this signal, noggin, had the same effect. Taken together, the observations presented here suggest that GATA-2 activation occurs by default in the absence of signals, that the restriction of its expression within the early embryo is controlled by negative signals emanating from the Nieuwkoop centre and the organiser, and that noggin and activin-like molecules play a role in these signalling pathways.

Suramin prevents transcription of dorsal marker genes in Xenopus laevis embryos, isolated dorsal blastopore lips and activin A induced animal caps

Suramin, a polyanionic compound which is known to interact with the receptors of growth factors inhibits the expression of dorsal marker genes in whole embryos and isolated dorsal blastopore lips. Suramin also prevents activin A induced dorsalization of animal cap explants from blastula stage embryos, but it simultaneously evokes a shift of the differentiation pattern from dorsal mesodermal structures (notochord, somites) to ventral mesodermal derivatives (mesothelium and erythroid precursor cells). The results are consistent with the assumption that the dorsal vegetal zone (Nieuwkoop center) primarily releases more general/ventral mesodermalization signals. They further suggest a dual role of activin A in early embryogenesis. While the maternal component may contribute to a more general/ventral type of induction, increasing concentrations of the zygotic component along with the activation of primary response genes may contribute to the dorsalization of the organizer.

Processed Vg1 protein is an axial mesoderm inducer in xenopus

Vg1 is a TGF beta-related growth factor encoded by a maternal mRNA localized to vegetal blastomeres in Xenopus embryos. Vg1 precursor protein is abundant in vegetal cells, but the processed mature form has not been readily detected and no activity has been demonstrated for the putative Vg1 mature protein. We have engineered a BMP2-Vg1 fusion (BVg1) that promotes formation of mature Vg1 protein in vivo. Injection of BVg1 mRNA induces dorsal mesoderm in animal cap cells, and BVg1 expression in ultraviolet-ventralized embryos fully restores a normal dorsal axis. Blastomeres expressing BVg1 act as a Nieuwkoop center, the region that induces the Spemann organizer. our results lead us to suggest that localized posttranslational processing of Vg1 precursor protein on the future dorsal side of the embryo is a key step in generating dorsal mesoderm and the body axis in Xenopus.

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos: (Spemann's organizer/dorsal mesoderm/neural induction/suramin/inhibition of notochord formation).

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.

Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.

This study analyzes the hierarchy of signals that spatially restrict expression of Xenopus Xwnt-8 to mesodermal cells outside of the Spemann organizer field and examines the potential role that endogenous Xwnt-8 may play in dorsoventral patterning of the embryonic mesoderm. The effects of ectopic introduction of a Nieuwkoop center-like activity or of ectopic expression of goosecoid, on the distribution of endogenous Xwnt-8 transcripts were analyzed. The results of these studies are consistent with the hypothesis that maternally derived signals from the Nieuwkoop center function to positively regulate expression of the homeo box gene goosecoid in Spemann organizer cells, leading to a subsequent repression of Xwnt-8 expression in these cells. This exclusion of Xwnt-8 from cells of the organizer field may be important for normal dorsal development, in that ectopic expression of Xwnt-8 in organizer cells after the midblastula stage, by injection of plasmid DNA, ventralizes the fate of these cells. This is distinct from the previously observed dorsalizing effect of Xwnt-8 when expressed prior to the midblastula stage by injection of RNA. The effects of plasmid-derived Xwnt-8 on isolated blastula animal cap ectoderm were also analyzed. Expression of Xwnt-8 in animal pole ectoderm after the midblastula stage ventralizes the response of dorsal animal pole cells to activin and allows naive ectodermal cells to differentiate as ventral mesoderm in the absence of added growth factors. Collectively, these data are consistent with the hypothesis that Xwnt-8 plays a role in the mesodermal differentiation of ventral marginal zone cells during normal development. Furthermore, endogenous Xwnt-8 may ventralize the response of lateral mesodermal cells to dorsalizing signals from the organizer, thus contributing to the graded nature of the final body pattern.