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.     

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.     

Neural induction and patterning in the mouse in the absence of the node and its derivatives

The signals which induce vertebrate neural tissue and pattern it along the anterior-posterior (A-P) axis have been proposed to emanate from Spemann's organizer, which in mammals is a structure termed the node. However, mouse embryos mutant for HNF3 beta lack a morphological node and node derivatives yet undergo neural induction. Gene expression domains occur at their normal A-P axial positions along the mutant neural tubes in an apparently normal temporal manner, including the most anterior and posterior markers. This neural patterning occurs in the absence of expression of known organizer genes, including the neural inducers chordin and noggin. Other potential signaling centers in gastrulating mutant embryos appear to express their normal constellation of putative secreted factors, consistent with the possibility that neural-inducing and -patterning signals emanate from elsewhere or at an earlier time. Nevertheless, we find that the node and the anterior primitive streak, from which the node derives, are direct sources of neural-inducing signals, as judged by expression of the early midbrain marker Engrailed, in explant-recombination experiments. Similar experiments showed the neural-inducing activity in HNF3 beta mutants to be diffusely distributed. Our results indicate that the mammalian organizer is capable of neural induction and patterning of the neural plate, but that maintenance of an organizer-like signaling center is not necessary for either process.     

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or beta-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.     

The FGFR pathway is required for the trunk-inducing functions of Spemann's organizer.

Xenopus laevis embryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. Here, we report that the fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway was required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. Since the organizer influences the differentiation of these tissues by emitting signals that pattern the mesodermal germ layer, our data indicate that the FGFR regulates the production of these signals. In addition, the FGFR pathway was required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway had a minimal effect on the function of the head organizer. We propose that the FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells. The implications of our findings for the evolutionarily conserved role of the FGFR pathway in the functions of Spemann's organizer and other vertebrate-signaling centers are discussed.     

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.     

Computational analysis of BMP gradients in dorsal-ventral patterning of the zebrafish embryo

The genetic network controlling early dorsal-ventral (DV) patterning has been extensively studied and modeled in the fruit fly Drosophila. This patterning is driven by signals coming from bone morphogenetic proteins (BMPs), and regulated by interactions of BMPs with secreted factors such as the antagonist short gastrulation (Sog). Experimental studies suggest that the DV patterning of vertebrates is controlled by a similar network of BMPs and antagonists (such as Chordin, a homologue of Sog), but differences exist in how the two systems are organized, and a quantitative comparison of pattern formation in them has not been made. Here, we develop a computational model in three dimensions of the zebrafish embryo and use it to study molecular interactions in the formation of BMP morphogen gradients in early DV patterning. Simulation results are presented on the dynamics BMP gradient formation, the cooperative action of two feedback loops from BMP signaling to BMP and Chordin synthesis, and pattern sensitivity with respect to BMP and Chordin dosage. Computational analysis shows that, unlike the case in Drosophila, synergy of the two feedback loops in the zygotic control of BMP and Chordin expression, along with early initiation of localized Chordin expression, is critical for establishment and maintenance of a stable and appropriate BMP gradient in the zebrafish embryo.     

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.     

Induction and axial patterning of the neural plate: Planar and vertical signals

In this review I summarize recent findings on the contributions of different cell groups to the formation of the basic plan of the nervous system of vertebrate embryos. Midline cells of the mesoderm—the organizer, notochord, and prechordal plate—and midline cells of the neural ectoderm—the notoplate and floor plate—appear to have a fundamental role in the induction and patterning of the neural plate. Vertical signals acting across tissue layers and planar signals acting through the neural epithelium have distinct roles and cooperate in induction and pattern formation. Whereas the prechordal plate and notochord have distinct vertical signaling properties, the initial anteroposterior (A-P) pattern of the neural plate may be induced by planar signals originating from the organizer region. Planar signals from the notoplate may also contribute to the mediolateral (M-L) patterning of the neural plate. These and other findings suggest a general view of neural induction and axial patterning. © 1993 John Wiley & Sons, Inc.     

The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative regulation of BMP2/4 and Wnt pathways

The neuroectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be regionalized into forebrain, midbrain, hindbrain and spinal cord by a two-step process. In the activation step, the Spemann gastrula organizer induces neuroectoderm with anterior character, followed by posteriorization by a transforming signal. Recently, simultaneous inhibition of BMP and Wnt signaling was shown to induce head formation in frog embryos. However, how the inhibition of BMP and Wnt signaling pathways specify a properly patterned head, and how they are regulated in vivo, is not understood. Here we demonstrate that the loss of anterior neural fates observed in zebrafish bozozok (boz) mutants occurs during gastrulation due to a reduction and subsequent posteriorization of neuroectoderm. The neural induction defect was correlated with decreased chordino expression and consequent increases in bmp2b/4 expression, and was suppressed by overexpression of BMP antagonists. Whereas expression of anterior neural markers was restored by ectopic BMP inhibition in early boz gastrulae, it was not maintained during later gastrulation. The posteriorization of neuroectoderm in boz was correlated with ectopic dorsal wnt8 expression. Overexpression of a Wnt antagonist rescued formation of the organizer and anterior neural fates in boz mutants. We propose that boz specifies formation of anterior neuroectoderm by regulating BMP and Wnt pathways in a fashion consistent with Nieuwkoop's two-step neural patterning model. boz promotes neural induction by positively regulating organizer-derived chordino and limiting the antineuralizing activity of BMP2b/4 morphogens. In addition, by negative regulation of Wnt signaling, boz promotes organizer formation and limits posteriorization of neuroectoderm in the late gastrula.     

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.     

Evolution of BMP signaling in Drosophila oogenesis: a receptor-based mechanism

The bone morphogenetic protein (BMP) signaling pathway is a conserved regulator of cellular and developmental processes in animals. The mechanisms underlying BMP signaling activation differ among tissues and mostly reflect changes in the expression of pathway components. BMP signaling is one of the major pathways responsible for the patterning of the Drosophila eggshell, a complex structure derived from a layer of follicle cells (FCs) surrounding the developing oocyte. Activation of BMP signaling in the FCs is dynamic. Initially, signaling is along the anterior-posterior (A/P) axis; later, signaling acquires dorsal-ventral (D/V) polarity. These dynamics are regulated by changes in the expression pattern of the type I BMP receptor thickveins (tkv). We recently found that signaling dynamics and TKV patterning are highly correlated in the FCs of multiple Drosophila species. In addition, we showed that signaling patterns are spatially different among species. Here, we use a mathematical model to simulate the dynamics and differences of BMP signaling in numerous species. This model predicts that qualitative and quantitative changes in receptor expression can lead to differences in the spatial pattern of BMP signaling. We tested these predications experimentally in three different Drosophila species and through genetic perturbations of BMP signaling in D. melanogaster. On the basis of our results, we concluded that changes in tkv patterning can account for the experimentally observed differences in the patterns of BMP signaling in multiple Drosophila species.     

The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures

During vertebrate development, an organizing signaling center, the isthmic organizer, forms at the boundary between the midbrain and hindbrain. This organizer locally controls growth and patterning along the anteroposterior axis of the neural tube. On the basis of transplantation and ablation experiments in avian embryos, we show here that, in the caudal midbrain, a restricted dorsal domain of the isthmic organizer, that we call the isthmic node, is both necessary and sufficient for the formation and positioning of the roof plate, a signaling structure that marks the dorsal midline of the neural tube and that is involved in its dorsoventral patterning. This is unexpected because in other regions of the neural tube, the roof plate has been shown to form at the site of neural fold fusion, which is under the influence of epidermal ectoderm derived signals. In addition, the isthmic node contributes cells to both the midbrain and hindbrain roof plates, which are separated by a boundary that limits cell movements. We also provide evidence that mid/hindbrain roof plate formation involves homeogenetic mechanisms. Our observations indicate that the isthmic organizer orchestrates patterning along the anteroposterior and the dorsoventral axis.     

A balance of FGF, BMP and WNT signalling positions the future placode territory in the head

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). Here, we identify the head mesoderm as the source of PPR inducing signals, reinforced by factors from the neural plate. We show that several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in na?ve ectoderm. We also show that WNT signalling plays a crucial role in restricting placode formation to the head. Finally, we demonstrate that the decision of multipotent cells to become placode or neural crest precursors is mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify.     

Timed interactions between the Hox expressing non-organiser mesoderm and the Spemann organiser generate positional information during vertebrate gastrulation

We report a novel developmental mechanism. Anterior-posterior positional information for the vertebrate trunk is generated by sequential interactions between a timer in the early non-organiser mesoderm and the organiser. The timer is characterised by temporally colinear activation of a series of Hox genes in the early ventral and lateral mesoderm (i.e., the non-organiser mesoderm) of the Xenopus gastrula. This early Hox gene expression is transient, unless it is stabilised by signals from the Spemann organiser. The non-organiser mesoderm and the Spemann organiser undergo timed interactions during gastrulation which lead to the formation of an anterior-posterior axis and stable Hox gene expression. When separated from each other, neither non-organiser mesoderm nor the Spemann organiser is able to induce anterior-posterior pattern formation of the trunk. We present a model describing that convergence and extension continually bring new cells from the non-organiser mesoderm within the range of organiser signals and thereby create patterned axial structures. In doing so, the age of the non-organiser mesoderm, but not the age of the organiser, defines positional values along the anterior-posterior axis. We postulate that the temporal information from the non-organiser mesoderm is linked to mesodermal Hox expression.     

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Cell-autonomous and inductive processes among three embryonic domains control dorsal-ventral and anterior-posterior development of Xenopus laevis

This review aims to propose an integrated model for dorsal-ventral and anterior-posterior development of Xenopus. Fertilized Xenopus eggs contain two determinants, a vegetal half endomesodermal determinant and a vegetal pole dorsal determinant (DD). The organizer forms in the specific intersection of the determinants, in a cell-autonomous manner. At late blastula, different combinations of the determinants form three embryonic domains, the competent animal domain, the organizer domain, and the entire vegetal half domain. These three domains cooperatively form dorsal-ventral and anterior-posterior axes: the organizer domain secrets dorsal inducing signals which induce or 'activate' the competent animal domain to form anterior-most neural tissues. The vegetal non-dorsal-marginal domain secrets posteriorizing signals, which 'transform' the anterior properties of the neural tissue to posterior properties.