Maternal TGF-ß ligand Panda breaks the radial symmetry of the sea urchin embryo by antagonizing the Nodal type II receptor ACVRII.

In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of the TGF-ß nodal in the ventral ectoderm. nodal expression is first induced ubiquitously in the 32-cell embryo and becomes progressively restricted to the presumptive ventral ectoderm by the early blastula stage. This early spatial restriction of nodal expression is independent of Lefty, and instead relies on the activity of Panda, a maternally expressed TGF-ß ligand related to Lefty and Inhibins, which is required maternally for D/V axis specification. However, the mechanism by which Panda restricts the early nodal expression has remained enigmatic and it is not known if Panda works like a BMP ligand by opposing Nodal and antagonizing Smad2/3 signaling, or if it works like Lefty by sequestering an essential component of the Nodal signaling pathway. In this study, we report that Panda functions as an antagonist of the TGF-ß type II receptor ACVRII (Activin receptor type II), which is the only type II receptor for Nodal signaling in the sea urchin and is also a type II receptor for BMP ligands. Inhibiting translation of acvrII mRNA disrupted D/V patterning across all 3 germ layers and caused acvrII morphants to develop with a typical Nodal loss-of-function phenotype. In contrast, embryos overexpressing acvrII displayed strong ectopic Smad1/5/8 signaling at blastula stages and developed as dorsalized larvae, a phenotype very similar to that caused by over activation of BMP signaling. Remarkably, embryos co-injected with acvrII mRNA and panda mRNA did not show ectopic Smad1/5/8 signaling and developed with a largely normal dorsal-ventral polarity. Furthermore, using an axis induction assay, we found that Panda blocks the ability of ACVRII to orient the D/V axis when overexpressed locally. Using co-immunoprecipitation, we showed that Panda physically interacts with ACVRII, as well as with the Nodal co-receptor Cripto, and with TBR3 (Betaglycan), which is a non-signaling receptor for Inhibins in mammals. At the molecular level, we have traced back the antagonistic activity of Panda to the presence of a single proline residue, conserved with all the Lefty factors, in the ACVRII binding motif of Panda, instead of a serine as in most of TGF-ß ligands. Conversion of this proline to a serine converted Panda from an antagonist that opposed Nodal signaling and promoted dorsalization to an agonist that promoted Nodal signaling and triggered ventralization when overexpressed. Finally, using phylogenomics, we analyzed the emergence of the agonist and antagonist form of Panda in the course of evolution. Our data are consistent with the idea that the presence of a serine at that position, like in most TGF-ß, was the ancestral condition and that the initial function of Panda was possibly in promoting and not in antagonizing Nodal signaling. These results highlight the existence of key functional and structural elements conserved between Panda and Lefty, allow to draw an intriguing parallel between sea urchin Panda and mammalian Inhibin α and raise the unexpected possibility that the original function of Panda may have been in activation of the Nodal pathway rather than in its inhibition.

RAS-independent ERK activation by constitutively active KSR3 in non-chordate metazoa.

During early development of the sea urchin embryo, activation of ERK signalling in mesodermal precursors is not triggered by extracellular RTK ligands but by a cell-autonomous, RAS-independent mechanism that was not understood. We discovered that in these cells, ERK signalling is activated through the transcriptional activation of a gene encoding a protein related to Kinase Suppressor of Ras, that we named KSR3. KSR3 belongs to a family of catalytically inactive allosteric activators of RAF. Phylogenetic analysis revealed that genes encoding kinase defective KSR3 proteins are present in most non-chordate metazoa but have been lost in flies and nematodes. We show that the structure of KSR3 factors resembles that of several oncogenic human RAF mutants and that KSR3 from echinoderms, cnidarians and hemichordates activate ERK signalling independently of RAS when overexpressed in cultured cells. Finally, we used the sequence of KSR3 factors to identify activating mutations of human B-RAF. These findings reveal key functions for this family of factors as activators of RAF in RAS-independent ERK signalling in invertebrates. They have implications on the evolution of the ERK signalling pathway and suggest a mechanism for its co-option in the course of evolution.

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

Deciphering and modelling the TGF-ß signalling interplays specifying the dorsal-ventral axis of the sea urchin embryo.

During sea urchin development, secretion of Nodal and BMP2/4 ligands and their antagonists Lefty and Chordin from a ventral organiser region specifies the ventral and dorsal territories. This process relies on a complex interplay between the Nodal and BMP pathways through numerous regulatory circuits. To decipher the interplay between these pathways, we used a combination of treatments with recombinant Nodal and BMP2/4 proteins and a computational modelling approach. We assembled a logical model focusing on cell responses to signalling inputs along the dorsal-ventral axis, which was extended to cover ligand diffusion and enable multicellular simulations. Our model simulations accurately recapitulate gene expression in wild-type embryos, accounting for the specification of ventral ectoderm, ciliary band and dorsal ectoderm. Our model simulations further recapitulate various morphant phenotypes, reveal a dominance of the BMP pathway over the Nodal pathway and stress the crucial impact of the rate of Smad activation in dorsal-ventral patterning. These results emphasise the key role of the mutual antagonism between the Nodal and BMP2/4 pathways in driving early dorsal-ventral patterning of the sea urchin embryo.

Maternal factors regulating symmetry breaking and dorsal-ventral axis formation in the sea urchin embryo.

Specification of the main axes of polarity of the embryo is an essential process during embryonic development. In many species, this process is achieved by the localization of maternal factors into discrete regions of the egg. However, in other animals, like in amniotes and in echinoderms, the considerable plasticity of the early blastomeres seems to preclude the existence of maternal determinants and the mechanisms by which the radial symmetry of the egg is broken remain largely enigmatic. In this chapter, we review recent progress on the identification of maternal components involved in symmetry breaking and dorsal-ventral (D/V) axis formation of the sea urchin embryo. We will first review some key experiments on D/V axis formation from classical embryologists that provided evidence for a weak maternal D/V prepattern. We will then detail more recent molecular analyses that established the critical role played by Nodal signaling in allocating cell fates along the secondary axis and led to the discovery that maternal transcription factors such as the Sry-related HMG box B1 (SoxB1), the Octamer binding factor1/2 (Oct1/2), the T-cell factor/Lymphoid enhancer-binding factor (TCF/LEF) and the Erythroblastosis virus E26 Oncogene Homolog (ETS) domain transcriptional repressor Translocation-Ets-Leukemia virus protein (Yan/Tel) as well as maternal signaling molecules like Univin are essential for the initiation of nodal expression. Finally, we will describe recent advances that uncovered a role in symmetry breaking and dorsal-ventral axis orientation for the transforming growth factor beta (TGF-beta)-like factor Panda, which appears to be both necessary and sufficient for D/V axis orientation. Therefore, even in the highly regulative sea urchin embryo, the activity of localized maternal factors provides the embryo with a blueprint of the D/V axis.

A wnt2 ortholog in the sea urchin Paracentrotus lividus.

Members of the wnt gene family encode secreted glycoproteins that mediate critical intercellular communications in metazoans. Large-scale genome and transcriptome analyses have shown that this family is composed of 13 distinct subfamilies. These analyses have further established that the number of wnt genes per subfamily varies significantly between metazoan phyla, highlighting that gene duplication and gene loss events have shaped the complements of wnt genes during evolution. In sea urchins, for example, previous work reported the absence of representatives of both the WNT2 and WNT11 subfamilies in two different species, Paracentrotus lividus and Strongylocentrotus purpuratus. Recently, however, we identified a gene encoding a WNT2 ortholog in P. lividus and, based on that finding, we also reanalyzed the genome of S. purpuratus. Yet, we found no evidence of a bona fide wnt2 gene in S. purpuratus. Furthermore, we established that the P. lividus wnt2 gene is selectively expressed in vegetal tissues during embryogenesis, in a pattern that is similar, although not identical, to that of other P. lividus wnt genes. Taken together, this study amends previous work on the P. lividus wnt complement and reveals an unexpected variation in the number of wnt genes between closely related sea urchin species.

Expression of exogenous mRNAs to study gene function in echinoderm embryos.

With the completion of the genome sequencing projects, a new challenge for developmental biologists is to assign a function to the thousands of genes identified. Expression of exogenous mRNAs is a powerful, versatile and rapid technique that can be used to study gene function during development of the sea urchin. This chapter describes how this technique can be used to analyze gene function in echinoderm embryos, how it can be combined with cell transplantation to perform mosaic analysis and how it can be applied to identify downstream targets genes of transcription factors and signaling pathways. We describe specific examples of the use of overexpression of mRNA to analyze gene function, mention the benefits and current limitations of the technique and emphasize the importance of using different controls to assess the specificity of the effects observed. Finally, this chapter details the different steps, vectors and protocols for in vitro production of mRNA and phenotypic analysis.

MAPK and GSK3/ß-TRCP-mediated degradation of the maternal Ets domain transcriptional repressor Yan/Tel controls the spatial expression of nodal in the sea urchin embryo.

In the sea urchin embryo, specification of the dorsal-ventral axis critically relies on the spatially restricted expression of nodal in the presumptive ventral ectoderm. The ventral restriction of nodal expression requires the activity of the maternal TGF-ß ligand Panda but the mechanism by which Panda restricts nodal expression is unknown. Similarly, what initiates expression of nodal in the ectoderm and what are the mechanisms that link patterning along the primary and secondary axes is not well understood. We report that in Paracentrotus lividus, the activity of the maternally expressed ETS-domain transcription factor Yan/Tel is essential for the spatial restriction of nodal. Inhibiting translation of maternal yan/tel mRNA disrupted dorsal-ventral patterning in all germ layers by causing a massive ectopic expression of nodal starting from cleavage stages, mimicking the phenotype caused by inactivation of the maternal Nodal antagonist Panda. We show that like in the fly or in vertebrates, the activity of sea urchin Yan/Tel is regulated by phosphorylation by MAP kinases. However, unlike in the fly or in vertebrates, phosphorylation by GSK3 plays a central role in the regulation Yan/Tel stability in the sea urchin. We show that GSK3 phosphorylates Yan/Tel in vitro at two different sites including a ß-TRCP ubiquitin ligase degradation motif and a C-terminal Ser/Thr rich cluster and that phosphorylation of Yan/Tel by GSK3 triggers its degradation by a ß-TRCP/proteasome pathway. Finally, we show that, Yan is epistatic to Panda and that the activity of Yan/Tel is required downstream of Panda to restrict nodal expression. Our results identify Yan/Tel as a central regulator of the spatial expression of nodal in Paracentrotus lividus and uncover a key interaction between the gene regulatory networks responsible for patterning the embryo along the dorsal-ventral and animal-vegetal axes.

p38 MAPK as an essential regulator of dorsal-ventral axis specification and skeletogenesis during sea urchin development: a re-evaluation.

Dorsal-ventral axis formation in the sea urchin embryo relies on the asymmetrical expression of the TGFß Nodal. The p38-MAPK pathway has been proposed to be essential for dorsal-ventral axis formation by acting upstream of nodal expression. Here, we report that, in contrast to previous studies that used pharmacological inhibitors of p38, manipulating the activity of p38 by genetic means has no obvious impact on morphogenesis. Instead, we discovered that p38 inhibitors strongly disrupt specification of all germ layers by blocking signalling from the Nodal receptor and by interfering with the ERK pathway. Strikingly, while expression of a mutant p38 that is resistant to SB203580 did not rescue dorsal-ventral axis formation or skeletogenesis in embryos treated with this inhibitor, expression of mutant Nodal receptors that are resistant to SB203580 fully restored nodal expression in SB203580-treated embryos. Taken together, these results establish that p38 activity is not required for dorsal-ventral axis formation through nodal expression nor for skeletogenesis. Our results prompt a re-evaluation of the conclusions of several recent studies that linked p38 activity to dorsal-ventral axis formation and to patterning of the skeleton.

A minimal molecular toolkit for mineral deposition? Biochemistry and proteomics of the test matrix of adult specimens of the sea urchin Paracentrotus lividus.

UNLABELLED: The sea urchin endoskeleton consists of a magnesium-rich biocalcite comprising a small amount of occluded organic macromolecules. This structure constitutes a key-model for understanding the mineral--organics interplay, and for conceiving in vitro bio-inspired materials with tailored properties. Here we employed a deep-clean technique to purify the occluded proteins from adult Paracentrotus lividus tests. We characterized them by 1- and 2D-electrophoreses, ELISA and immunoblotting, and using liquid chromatography coupled with Mass Spectrometry (nanoLC-MS/MS), we identified two metalloenzymes (carbonic anhydrase and MMP), a set of MSP130 family members, several C-type lectins (SM29, SM41, PM27) and cytoskeletal proteins. We demonstrate the effect of the protein extract on the crystals, with an in vitro crystallization assay. We suggest that this small set of biomineralization proteins may represent a 'minimal molecular crystallization toolkit'. SIGNIFICANCE: Biominerals often exhibit superior chemical properties, when compared to their inorganic counterparts. This is due pro parte to the proteins that are occluded in the mineral. However, the limited available studies on biomineralization have not yet succeeded in identifying a minimal set of proteins directly involved in the formation of the biomineral in vivo and sufficiently required for in vitro precipitation. Indeed, the high number of proteins identified by high-throughput screening in the recent years does not encourage the possibility of recreating or tailoring the mineral in vitro. Thus, the identification of biomineralization proteins involved in protein-mineral interactions is highly awaited. In the present study, we used the sea urchin, Paracentrotus lividus (P. lividus), to identify the native proteins directly taking part in protein-mineral interactions. We employed an improved deep-clean technique to extract and purify the native occluded skeletal matrix proteins from the test and identified them by the highly sensitive technique of nanoLC-MS/MS. We show that this minimal set of proteins has a shaping effect on the formation of biocalcite in vitro. This work gives insights on the biomineralization of the sea urchin, while it paves the way for the identification of biomineralization proteins in other biomineralizing systems. Understanding the 'biologically controlled mineralization' will facilitate the in vitro formation and tailoring of biominerals in mild conditions for applications in medicine and materials science.

A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms.

During development of chordates, establishment of the body plan relies on the activity of an organizing centre located on the dorsal side of the embryo that patterns the embryo and induces neural tissue. Intriguingly, the evolutionary origin of this crucial signalling centre remains unclear and whether analogous organizers regulate D/V patterning in other deuterostome or protostome phyla is not known. Here we provide evidence that the ventral ectoderm of the sea urchin embryo is a long-range organizing centre that shares several fundamental properties with the Spemann organizer: the ability to induce duplicated embryonic axes when ectopically induced, the ability to induce neural fate in neighbouring tissues and the ability to finely regulate the level of BMP signalling by using an autoregulatory expansion-repression mechanism. These findings suggest that the evolutionary origin of the Spemann organizer is more ancient than previously thought and that it may possibly be traced back to the common ancestor of deuterostomes.

The Maternal Maverick/GDF15-like TGF-ß Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo.

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-ß) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-ß distantly related to Inhibins, Lefty, and TGF-ß that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-ß may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.

Recent studies suggest that specification of the dorsal-ventral and left-right axes of the sea urchin embryo relies on Nodal-expressing signalling centres located in the ventral ectoderm and in the archenteron that share striking similarities with vertebrate organising centres. Nodal and its downstream target BMP2/4 pattern all three germ layers along the dorsal-ventral axis, repress neural fates and control morphogenesis of the larva. Moreover, Nodal establishes left-right asymmetry by repressing formation of the adult rudiment and inhibiting germline cells differentiation on the right side, while BMP2/4 promotes expression of mesodermal genes on the left side. These findings provide a framework for future studies and raise new questions regarding the events upstream and downstream of Nodal and BMP signalling during axis formation.

Reciprocal signaling between the ectoderm and a mesendodermal left-right organizer directs left-right determination in the sea urchin embryo.

During echinoderm development, expression of nodal on the right side plays a crucial role in positioning of the rudiment on the left side, but the mechanisms that restrict nodal expression to the right side are not known. Here we show that establishment of left-right asymmetry in the sea urchin embryo relies on reciprocal signaling between the ectoderm and a left-right organizer located in the endomesoderm. FGF/ERK and BMP2/4 signaling are required to initiate nodal expression in this organizer, while Delta/Notch signaling is required to suppress formation of this organizer on the left side of the archenteron. Furthermore, we report that the H(+)/K(+)-ATPase is critically required in the Notch signaling pathway upstream of the S3 cleavage of Notch. Our results identify several novel players and key early steps responsible for initiation, restriction, and propagation of left-right asymmetry during embryogenesis of a non-chordate deuterostome and uncover a functional link between the H(+)/K(+)-ATPase and the Notch signaling pathway.

Maternal Oct1/2 is required for Nodal and Vg1/Univin expression during dorsal-ventral axis specification in the sea urchin embryo.

The TGFß family member Nodal is expressed early in the presumptive ventral ectoderm of the early sea urchin embryo and its activity is crucial for dorsal-ventral (D/V) axis specification. Analysis of the nodal promoter identified a number of critical binding sites for transcription factors of different families including Sox, Oct, TCF and bZIP, but in most cases the specific factors that regulate nodal expression are not known. In this study, we report that the maternal factor Oct1/2 functions as a positive regulator of nodal and that its activity is essential for the initiation of nodal expression. Inhibition of Oct1/2 mRNA translation produced embryos with severe axial defects similar to those observed following inhibition of Nodal function. We show that perturbing Oct1/2 function specifically disrupted specification of the ventral and dorsal ectodermal regions and that these effects were caused by the failure of nodal to be expressed early in development. Furthermore, we identified the key gene vg1/univin, which is also necessary for nodal expression, as an additional factor that was completely dependent on Oct1/2 for its zygotic expression. These data demonstrate that the maternal Oct1/2 protein plays an early and essential role in D/V axis specification by initiating the expression of nodal and vg1/univin, two genes that act at the top of the D/V ectoderm gene regulatory network.

Wnt6 activates endoderm in the sea urchin gene regulatory network.

In the sea urchin, entry of ß-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of ß-catenin, of a dominant-negative variant of GSK-3ß or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.

Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm.

Echinoderms, which are phylogenetically related to vertebrates and produce large numbers of transparent embryos that can be experimentally manipulated, offer many advantages for the analysis of the gene regulatory networks (GRN) regulating germ layer formation. During development of the sea urchin embryo, the ectoderm is the source of signals that pattern all three germ layers along the dorsal-ventral axis. How this signaling center controls patterning and morphogenesis of the embryo is not understood. Here, we report a large-scale analysis of the GRN deployed in response to the activity of this signaling center in the embryos of the Mediterranean sea urchin Paracentrotus lividus, in which studies with high spatial resolution are possible. By using a combination of in situ hybridization screening, overexpression of mRNA, recombinant ligand treatments, and morpholino-based loss-of-function studies, we identified a cohort of transcription factors and signaling molecules expressed in the ventral ectoderm, dorsal ectoderm, and interposed neurogenic ("ciliary band") region in response to the known key signaling molecules Nodal and BMP2/4 and defined the epistatic relationships between the most important genes. The resultant GRN showed a number of striking features. First, Nodal was found to be essential for the expression of all ventral and dorsal marker genes, and BMP2/4 for all dorsal genes. Second, goosecoid was identified as a central player in a regulatory sub-circuit controlling mouth formation, while tbx2/3 emerged as a critical factor for differentiation of the dorsal ectoderm. Finally, and unexpectedly, a neurogenic ectoderm regulatory circuit characterized by expression of "ciliary band" genes was triggered in the absence of TGF beta signaling. We propose a novel model for ectoderm regionalization, in which neural ectoderm is the default fate in the absence of TGF beta signaling, and suggest that the stomodeal and neural subcircuits that we uncovered may represent ancient regulatory pathways controlling embryonic patterning.

Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo.

Nodal factors play fundamental roles in induction and patterning of the mesoderm and endoderm in vertebrates, but whether this reflects an ancient role or one that evolved recently in vertebrates is not known. Here, we report that in addition to its primary role in patterning the ectoderm, sea urchin Nodal is crucial for patterning of the endoderm and skeletogenic mesoderm through the regulation of the expression of key transcription factors and signalling molecules, including BMP2/4 and FGFA. In addition, we uncovered an essential role for Nodal and BMP2/4 in the formation and patterning of the non-skeletogenic mesoderm. By comparing the effects of misexpressing Nodal or an activated Nodal receptor in clones of cells, we provide evidence that Nodal acts over a long range in the endomesoderm and that its effects on the blastocoelar cell precursors are likely to be direct. The activity of Nodal and BMP2/4 are antagonistic, and although bmp2/4 is transcribed in the ventral ectoderm downstream of Nodal, the BMP2/4 ligand is translocated to the dorsal side, where it activates signalling in the dorsal primary mesenchyme cells, the dorsal endoderm and in pigment cell precursors. Therefore, correct patterning of the endomesoderm depends on a balance between ventralising Nodal signals and dorsalising BMP2/4 signals. These experiments confirm that Nodal is a key regulator of dorsal-ventral polarity in the sea urchin and support the idea that the ventral ectoderm, like the Spemann organiser in vertebrates, is an organising centre that is required for patterning all three germ layers of the embryo.

Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network.

Formation of the dorsal-ventral axis of the sea urchin embryo relies on cell interactions initiated by the TGFbeta Nodal. Intriguingly, although nodal expression is restricted to the ventral side of the embryo, Nodal function is required for specification of both the ventral and the dorsal territories and is able to restore both ventral and dorsal regions in nodal morpholino injected embryos. The molecular basis for the long-range organizing activity of Nodal is not understood. In this paper, we provide evidence that the long-range organizing activity of Nodal is assured by a relay molecule synthesized in the ventral ectoderm, then translocated to the opposite side of the embryo. We identified this relay molecule as BMP2/4 based on the following arguments. First, blocking BMP2/4 function eliminated the long-range organizing activity of an activated Nodal receptor in an axis rescue assay. Second, we demonstrate that BMP2/4 and the corresponding type I receptor Alk3/6 functions are both essential for specification of the dorsal region of the embryo. Third, using anti-phospho-Smad1/5/8 immunostaining, we show that, despite its ventral transcription, the BMP2/4 ligand triggers receptor mediated signaling exclusively on the dorsal side of the embryo, one of the most extreme cases of BMP translocation described so far. We further report that the pattern of pSmad1/5/8 is graded along the dorsal-ventral axis and that two BMP2/4 target genes are expressed in nested patterns centered on the region with highest levels of pSmad1/5/8, strongly suggesting that BMP2/4 is acting as a morphogen. We also describe the very unusual ventral co-expression of chordin and bmp2/4 downstream of Nodal and demonstrate that Chordin is largely responsible for the spatial restriction of BMP2/4 signaling to the dorsal side. Thus, unlike in most organisms, in the sea urchin, a single ventral signaling centre is responsible for induction of ventral and dorsal cell fates. Finally, we show that Chordin may not be required for long-range diffusion of BMP2/4, describe a striking dorsal-ventral asymmetry in the expression of Glypican 5, a heparin sulphated proteoglycan that regulates BMP mobility, and show that this asymmetry depends on BMP2/4 signaling. Our study provides new insights into the mechanisms by which positional information is established along the dorsal-ventral axis of the sea urchin embryo, and more generally on how a BMP morphogen gradient is established in a multicellular embryo. From an evolutionary point of view, it highlights that although the genes used for dorsal-ventral patterning are highly conserved in bilateria, there are considerable variations, even among deuterostomes, in the manner these genes are used to shape a BMP morphogen gradient.