Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2−/− or Chd−/− mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2−/− mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2−/− embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2−/−vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.
BMP signaling; CV2; Chd; Chdl-1; Chdl-2; long-range signaling; morphogenetic field; vertebral development; Tolloid; Twisted gastrulation
A growing body of work indicates that neural induction may be initiated prior to the establishment of the gastrula mesodermal organizer. Here we examine neural induction in Xenopus embryos in which mesoderm formation has been blocked by Cerberus-short, a reagent that specifically inhibits Nodal-related (Xnr) signals. We find that extensive neural structures with cyclopic eyes and brain tissue are formed despite the absence of mesoderm. This neural induction correlates with the expression of chordin and other BMP inhibitors - such as noggin, follistatin and Xnr3 - at the blastula stage, and requires β-Catenin signaling. Activation of the β-Catenin pathway by mRNA microinjections or by treatment with LiCl leads to differentiation of neurons, as well as neural crest, in ectodermal explants. Xnr signals are required for the maintenance, but not for the initiation, of BMP antagonist expression. Recent work has demonstrated a role for β-Catenin signaling in neural induction mediated by the transcriptional down-regulation of BMP-4 expression. The present results suggest an additional function for β-Catenin, the early activation of expression of secreted BMP antagonists, such as Chordin, in a pre-organizer region in the dorsal side of the Xenopus blastula.
Xenopus laevis; neural induction; Spemann's organizer; β-Catenin; Chordin; Noggin; Follistatin; Xnr3; Lefty; Nodal-related
The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.
Crossveinless-2; Chordin; Twisted gastrulation; Short gastrulation; BMP; Dpp; Tolloid; CR domain; von Willebrand factor type D domain; TIL domain; Extracellular matrix; Organogenesis; Somite; Sclerotome; Notochord; Neural crest; Dorsal root ganglion; Branchial arch; Cartilage; Vertebra; Intervertebral disc; Lung
The patterning of the CNS relies on the interaction of multiple signaling molecules such as Sonic Hedgehog, Wnts, and BMPs and their antagonists Chordin and Noggin. The identification of the secreted molecule Tiarin (Tsuda et al., 2002, this issue of Neuron), produced by the nonneural ectoderm at border of the anterior and lateral neural plate, now introduces a novel signaling pathway participating in CNS development.
The intensity of the BMP signal is determined by cell surface receptors that phosphorylate Smad1/5/8 at the C-terminus. In addition to this BMP-activated phosphorylation, recent studies have shown that sequential phosphorylations by MAPK and GSK3 kinases can negatively regulate the activity of the pSmad1Cter signal. These phosphorylations in the linker region cause Smad1 to be transported to the centrosomal region, polyubiquitinylated and degraded by the proteasomal machinery. In Xenopus embryos, Wnt signals, which regulate GSK3, induce ectoderm to adopt an epidermal fate and this Wnt effect requires an active BMP-Smad1/5/8 signaling pathway. These findings have profound implications for understanding how dorsal-ventral and anterior-posterior patterning are seamlessly integrated in the early embryonic morphogenetic field.
Embryos and developing organs have the remarkable ability of self-regenerating after experimental manipulations. In the Xenopus blastula half-embryos can regenerate the missing part, producing identical twins. Studies on the molecular nature of Spemann’s organizer have revealed that self-regulation results from the battle between two signaling centers under reciprocal transcriptional control. Long-range communication between the dorsal and ventral sides is mediated by the action of growth factor antagonists – such as the BMP antagonist Chordin – that regulate the flow of BMPs within the embryonic morphogenetic field. BMPs secreted by the dorsal Spemann organizer tissue are released by metalloproteinases of the Tolloid family, which cleave Chordin at a distance of where they were produced. The dorsal center secretes Chordin, Noggin, BMP2 and ADMP. The ventral center of the embryo secretes BMP4, BMP7, Sizzled, Crossveinless-2 and Tolloid-related. Crossveinless-2 binds Chordin/BMP complexes, facilitating their flow towards the ventral side, where BMPs are released by Tolloid allowing peak BMP signaling. Self-regulation occurs because transcription of ventral genes is induced by BMP while transcription of dorsal genes is repressed by BMP signals. This assures that for each action of Spemann’s organizer there is a reaction in the ventral side of the embryo. Because both dorsal and ventral centers express proteins of similar biochemical activities, they can compensate for each other. A novel biochemical pathway of extracellular growth factor signaling regulation has emerged from these studies in Xenopus. This remarkable dorsal-ventral positional information network has been conserved in evolution and is ancestral to all bilateral animals.
Morphogenetic fields; Embryonic induction; Dorsal-Ventral patterning; BMP; Chordin; Crossveinless-2; Tolloid; Sizzled; Hox genes; Urbilateria
BMPs pattern the dorsal-ventral axis of vertebrate embryos. Smad1/5/8 transduces the BMP signal, and receives phosphorylation inputs from both MAPK and GSK3. Phosphorylation of Smad1 by MAPK and GSK3 result in its polyubiquitination and transport to the centrosome where it is degraded by the proteasome. These linker phosphorylations inhibit BMP/Smad1 signaling by shortening its duration. Wnt, which negatively regulates GSK3 activity, prolongs the BMP/Smad1 signal. Remarkably, linker-phosphorylated Smad1 has been shown to be inherited asymmetrically during cell division. Drosophila contains a single Smad1/5/8 homologue, Mad, and is stabilized by phosphorylation-resistant mutations at GSK3 sites, causing Wingless-like effects. We summarize here the significance of linker-phosphorylated Smad1/Mad in relation to signal intensity and duration, and how this integrates the Wnt and BMP pathways during cell differentiation.
BMP; Wnt; FGF; Smad; mitosis asymmetry
Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2;Tsg and Cv2;Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.
BMP; Crossveinless-2; Chordin; Twisted Gastrulation; Tolloid; vertebra; morphogenetic field; cartilage; pattern formation
A key question in developmental biology is how growth factor signals are integrated to generate pattern. In this study we investigated the integration of the Drosophila BMP and Wingless/GSK3 signaling pathways via phosphorylations of the transcription factor Mad. Wingless was found to regulate the phosphorylation of Mad by GSK3 in vivo. In epistatic experiments, the effects of Wingless on wing disc molecular markers (senseless, distalless and vestigial) were suppressed by depletion of Mad with RNAi. Wingless overexpression phenotypes, such as formation of ectopic wing margins, were induced by Mad GSK3 phosphorylation-resistant mutant protein. Unexpectedly, we found that Mad phosphorylation by GSK3 and MAPK occurred in segmental patterns. Mad depletion or overexpression produced Wingless-like embryonic segmentation phenotypes. In Xenopus embryos, segmental border formation was disrupted by Smad8 depletion. The results show that Mad is required for Wingless signaling and for the integration of gradients of positional information.
The morphogenetic field concept was proposed by experimental embryologists to account for the self-regulative behavior of embryos. Such fields have remained an abstract concept until the recent identification of their molecular components using a combination of genetics, biochemistry, and theoretical modeling. One of the best studied models of a morphogenetic field is the Dorsal-Ventral (D-V) patterning of the early frog embryo. This patterning system is regulated by the bone morphogenetic protein (BMP) signaling pathway and an intricate network of secreted protein antagonists. This biochemical pathway of interacting proteins functions in the extracellular space to generate a D-V gradient of BMP signaling, which is maintained during extensive morphogenetic movements of cell layers during gastrulation. The D-V field is divided into a dorsal and a ventral center, in regions of low and high BMP signaling respectively, under opposite transcriptional control by BMPs. The robustness of the patterning is assured at two different levels. First, in the extracellular space by secreted BMP antagonists that generate a directional flow of BMP ligands to the ventral side. The flow is driven by the regulated proteolysis of the Chordin inhibitor and by the presence of a molecular sink on the ventral side that concentrates BMP signals. The tolloid metalloproteinases and the Chordin-binding protein Crossveinless-2 (CV2) are key components of this ventral sink. Second, by transcriptional feedback at the cellular level: The dorsal and ventral signaling centers adjust their size and level of BMP signaling by transcriptional feedback. This allows cells on one side of a gastrula containing about 10,000 cells to communicate with cells in the opposite pole of the embryo.
A network of BMP ligands and antagonists regulates embryonic patterning in frogs. Proteolysis of inhibitors, a molecular sink, and transcriptional feedback loops ensure its robustness.
Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: 1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; 2) CV2 complexes with Twisted gastrulation and BMP4; 3) CV2 is not a substrate for tolloid proteinases; 4) CV2 binds to purified Chordin protein with high affinity (KD in the 1 nM range); 5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; 6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.
Synaptic activity induces changes in the number of dendritic spines. Here, we report a pathway of regulated endocytosis triggered by arcadlin, a protocadherin induced by electroconvulsive and other excitatory stimuli in hippocampal neurons. The homophilic binding of extracellular arcadlin domains activates TAO2β, a splice variant of the thousand and one amino acid protein kinase 2, cloned here by virtue of its binding to the arcadlin intracellular domain. TAO2β is a MAPKKK that activates the MEK3 MAPKK, which phosphorylates the p38 MAPK. Activation of p38 feeds-back on TAO2β, phosphorylating a key serine required for triggering endocytosis of N-cadherin at the synapse. Arcadlin knockout increases the number of dendritic spines, and the phenotype is rescued by siRNA knockdown of N-cadherin. This pathway of regulated endocytosis of N-cadherin via protocadherin/TAO2β/MEK3/p38 provides a molecular mechanism for transducing neuronal activity into changes in synaptic morphologies.
Most animals evolved from a common ancestor, Urbilateria, which already had in place the developmental genetic networks for shaping body plans. Comparative genomics has revealed rather unexpectedly that many of the genes present in bilaterian animal ancestors were lost by individual phyla during evolution. Reconstruction of the archetypal developmental genomic tool-kit present in Urbilateria will help to elucidate the contribution of gene loss and developmental constraints to the evolution of animal body plans.
Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl ┤ Xlr ┤ Chd ┤ BMP → P-Smad1 → Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10−8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.
Research on the mechanisms of embryonic induction had a great setback in the 1940s when Barth discovered and Holtfreter confirmed that ectoderm of Ambystoma maculatum salamander embryos could form brain tissue when cultured in a simple saline solution. We have revisited this classical experiment and found that when cultured animal cap ectoderm attaches to a glass substratum, it can self-organize to form complex organs such as brain vesicles, eyes, lens and olfactory placodes. Only anterior neural organs were generated. Under these culture conditions ERK became diphosphorylated, indicating a sustained activation of the Ras/MAPK pathway. Using sand particles as an heterologous neural inducer similar results were obtained. Addition of U0126, a specific antagonist of MEK, the enzyme that phosphorylates ERK/MAPK, inhibited neural differentiation. The closely related control compound U0124 had no effect. We conclude that neural induction in the absence of organizer in Ambystoma maculatum requires Ras/MAPK activation. These findings provide a molecular explanation for the activity of heterologous neural inducers that dominated thinking in amphibian experimental embryology for many decades.
Ambystoma maculatum; Xenopus; Ras; MAPK; ERK; BMP; FGF; brain differentiation; Smad1; embryonic induction
The blastula Chordin- and Noggin-expressing (BCNE) center located in the dorsal animal region of the Xenopus blastula embryo contains both prospective anterior neuroectoderm and Spemann organizer precursor cells. Here we show that, contrary to previous reports, the canonical Wnt target homeobox genes, Double knockdown of these genes using antisense morpholinos in Xenopus laevis blocked head formation, reduced the expression of the other BCNE center genes, upregulated Bmp4 expression, and nullified hyperdorsalization by lithium chloride. Moreover, gain- and loss-of-function experiments showed that Siamois and Twin expression is repressed by the vegetal transcription factor VegT. We propose that VegT expression causes maternal β-Catenin signals to restrict Siamois and Twin expression to the BCNE region. A two-step inhibition of BMP signals by Siamois and Twin – first by transcriptional repression of Bmp4 and then by activation of the expression of the BMP inhibitors Chordin and Noggin – in the BCNE center is required for head formation.
Cell signaling; Spemann organizer; Siamois; Twin; Chordin; Noggin; BMP; Wnt; Xenopus
A network of secreted proteins that interact with each other in the extracellular space regulates embryonic morphogenesis. Mathematical modeling offers an excellent opportunity to understand how morphogens signal and self-regenerate pattern.
BMP Receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1Cter signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38 and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1GSK3 antigen levels and redistributed it from the centrosome to cytoplasmic LRP6-signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis, and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorso-ventral (BMP) and antero-posterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.
Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing1. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins2, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-β binding domains3-6. Here we show that CTGF directly binds BMP4 and TGF-β1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-β1. These results show that CTGF inhibits BMP and activates TGF-β signals by direct binding in the extracellular space.
The head inducer Cerberus is a multivalent extracellular inhibitor
Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants.
Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk–tail development1–6. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures1. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.
In Xenopus, zygotic transcription starts 6 hours after fertilization at the midblastula transition and therefore the first steps in embryonic development are regulated by maternally inherited proteins and mRNAs. While animal-vegetal polarity is already present in the oocyte, the dorsoventral axis is only established upon fertilization by the entry of the sperm and the subsequent rotation of the egg cortex. In a screen for maternal mRNAs whose stability is regulated by this cortical rotation, we isolated the Xenopus homologue of the Drosophila gene Bicaudal-C (xBic-C). It encodes a putative RNA-binding molecule expressed maternally and localized predominantly to the vegetal half of the egg. Upon fertilization and cortical rotation, xBic-C mRNA is displaced together with the heavy yolk towards the future dorsal side of the embryo. In UV-ventralized embryos, xBic-C is polyadenylated less than in untreated embryos that undergo cortical rotation. Overexpression of xBic-C by injection of synthetic mRNA in whole embryos or in ectodermal explants leads to ectopic endoderm formation. This endoderm-inducing activity is dependent on the presence of the RNA-binding domain of the protein. In contrast to the two other known maternally encoded endoderm inducers, Vg1 and VegT, xBic-C ectopic expression leads specifically to endoderm formation in the absence of mesoderm induction.
Bicaudal; Egg determinant; Endoderm; Induction; Polyadenylation; VegT; Vg1; Xenopus
Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.
In Xenopus, mesoderm induction by endoderm at the blastula stage is well documented, but the molecular nature of the endogenous inductive signals remains unknown. The carboxy-terminal fragment of Cerberus, designated Cer-S, provides a specific secreted antagonist of mesoderm-inducing Xenopus Nodal-Related (Xnr) factors. Cer-S does not inhibit signalling by other mesoderm inducers such as Activin, Derrière, Vg1 and BMP4, nor by the neural inducer Xnr3. In the present study we show that Cer-S blocks the induction of both dorsal and ventral mesoderm in animal-vegetal Nieuwkoop-type recombinants. During blastula stages Xnr1, Xnr2 and Xnr4 are expressed in a dorsal to ventral gradient in endodermal cells. Dose-response experiments using cer-S mRNA injections support the existence of an endogenous activity gradient of Xnrs. Xnr expression at blastula can be activated by the vegetal determinants VegT and Vg1 acting in synergy with dorsal β-catenin. The data support a modified model for mesoderm induction in Xenopus, in which mesoderm induction is mediated by a gradient of multiple Nodal-related signals released by endoderm at the blastula stage.
Mesoderm induction; Nodal; Xnr; Cerberus; TGF-β; Derrière; Activin; VegT; β-catenin; Vg1; Cer-S