Lefty proteins are long-range inhibitors of squint-mediated nodal signaling

The regulation of signaling pathways by feedback inhibitors has become an emerging theme in the control of pattern formation during development. Nodal and Lefty proteins belong to divergent subfamilies of the TGF-beta family. Nodal signals promote mesendoderm induction in vertebrates, and Lefty proteins antagonize it. In zebrafish, Squint functions as a long-range Nodal signal during mesoderm induction. We report that the range over which Squint induces mesoderm is reduced by Lefty proteins. In contrast, the activity range of the short-range Nodal signal Cyclops is not regulated by Lefty activity. We present three lines of evidence that Lefty proteins diminish the range of Squint signaling by acting not only as antagonists of Squint autoregulation but also as long-range inhibitors of Squint activity. First, Lefty can block Nodal signaling at a distance. Second, Lefty regulates the range of Squint signaling before regulating squint expression. Third, Lefty restricts the range of Squint activity in squint mutant embryos, in which the endogenous gene is not subject to autoregulation. We also find that Lefty restricts the response to both high and low levels of Nodal signaling. These results indicate that Lefty proteins restrict the activity range of Nodal signals by dampening Nodal signaling in surrounding cells.     

Opposing Nodal and BMP Signals Regulate Left–Right Asymmetry in the Sea Urchin Larva

Nodal and BMP signals are important for establishing left-right (LR) asymmetry in vertebrates. In sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed that BMP signaling is activated in the left coelomic pouch, specifically in the veg2 lineage, but not in the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is required for activating the expression of the left-sided genes and the formation of the left-sided structures. On the other hand, Nodal signals on the right side inhibit BMP signaling and control LR asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP signaling is the positive signal for left-sided development in sea urchins, suggesting that the opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in deuterostomes.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.     

Genome sequencing in the sea urchin embryo: what is new concerning the cell cycle?

Sea urchin is a classical research model system in developmental biology; moreover, the external fertilization and growth of embryos, their rapid division cycle, their transparency and the accessibility of these embryos to molecular visualization methods, made them good specimens to analyze the regulatory mechanisms of cell division. These features as well as the phylogenetic position of sea urchin, close to vertebrates but in an outgroup within the deuterostomes, led scientists working on this model to sequence the genome of the species S. purpuratus. The genome contains a full repertoire of cell cycle control genes. A comparison of this toolkit with those from vertebrates, nematodes, drosophila, as well as tunicates, provides new insight into the evolution of cell cycle control. While some gene subtypes have undergone lineage-specific expansions in vertebrates (i.e. cyclins, mitotic kinases,...), others seem to be lost in vertebrates, for instance the novel cyclin B identified in S. purpuratus. On the other hand, some genes which were previously thought to be vertebrate innovations, are also found in sea urchins (i.e. MCM9). To note is also the absence of cell cycle inhibitors of the INK type, which are apparently confined to vertebrates. The uncovered genomic repertoire of cell-cycle regulators will thus provide molecular tools that should further enhance future research on cell cycle control and developmental regulation in this model.     

GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses

Within the TGF-β superfamily, there are approximately forty ligands divided into two major branches: the TGF-β/Activin/Nodal ligands and the BMP/GDF ligands. We studied the ligand GDF3 and found that it inhibits signaling by its co-family members, the BMPs; however, GDF3 has been described by others to have Nodal-like activity. Here, we show that GDF3 can activate Nodal signaling, but only at very high doses and only upon mRNA over-expression. In contrast, GDF3 inhibits BMP signaling upon over-expression of GDF3 mRNA, as recombinant protein, and regardless of its dose. We therefore further characterized the mechanism through which GDF3 protein acts as a specific BMP inhibitor and found that the BMP inhibitory activity of GDF3 resides redundantly in the unprocessed, predominant form and in the mature form of the protein. These results confirm and extend the activity that we described for GDF3 and illuminate the experimental basis for the different observations of others. We suggest that GDF3 is either a bi-functional TGF-β ligand, or, more likely, that it is a BMP inhibitor that can artificially activate Nodal signaling under non-physiological conditions.     

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.     

The immune gene repertoire encoded in the purple sea urchin genome

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology.     

Molecular characterization and phylogenetic analysis of SpBMP5-7, a new member of the TGF-beta superfamily expressed in sea urchin embryos.

TGF-beta ligands are probably pan-bilaterian in phylogenetic distribution. The family appears to have diversified greatly with the evolution of the vertebrates, but only a few invertebrate deuterostome TGF-beta molecules have so far been isolated. A search for members of this family expressed in sea urchin embryos, using canonical PCR primers, revealed a single-copy gene encoding a new TGF-beta protein. The sequence which it encodes is closely related to those of vertebrate bone morphogenetic proteins (BMPs) 5-7. No additional TGF-beta family members were uncovered other than univin, which had previously been reported.     

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral–aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral–aboral gene regulatory network. A model for oral–aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Lefty's function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral–aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.     

The amphioxus genome enlightens the evolution of the thyroid hormone signaling pathway

Thyroid hormones (THs) have pleiotropic effects on vertebrate development, with amphibian metamorphosis as the most spectacular example. However, developmental functions of THs in non-vertebrate chordates are largely hypothetical and even TH endogenous production has been poorly investigated. In order to get better insight into the evolution of the thyroid hormone signaling pathway in chordates, we have taken advantage of the recent release of the amphioxus genome. We found amphioxus homologous sequences to most of the genes encoding proteins involved in thyroid hormone signaling in vertebrates, except the fast-evolving thyroglobulin: sodium iodide symporter, thyroid peroxidase, deiodinases, thyroid hormone receptor, TBG, and CTHBP. As only some genes encoding proteins involved in TH synthesis regulation were retrieved (TRH, TSH receptor, and CRH receptor but not their corresponding receptors and ligands), there may be another mode of upstream regulation of TH synthesis in amphioxus. In accord with the notion that two whole genome duplications took place at the base of the vertebrate tree, one amphioxus gene often corresponded to several vertebrate homologs. However, some amphioxus specific duplications occurred, suggesting that several steps of the TH pathway were independently elaborated in the cephalochordate and vertebrate lineages. The present results therefore indicate that amphioxus is capable of producing THs. As several genes of the TH signaling pathway were also found in the sea urchin genome, we propose that the thyroid hormone signaling pathway is of ancestral origin in chordates, if not in deuterostomes, with specific elaborations in each lineage, including amphioxus.     

Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo

In the sea urchin embryo, the oral-aboral axis is specified after fertilization by mechanisms that are largely unknown. We report that early sea urchin embryos express Nodal and Antivin in the presumptive oral ectoderm and demonstrate that these genes control formation of the oral-aboral axis. Overexpression of nodal converted the whole ectoderm into oral ectoderm and induced ectopic expression of the orally expressed genes goosecoid, brachyury, BMP2/4, and antivin. Conversely, when the function of Nodal was blocked, by injection of an antisense Morpholino oligonucleotide or by injection of antivin mRNA, neither the oral nor the aboral ectoderm were specified. Injection of nodal mRNA into Nodal-deficient embryos induced an oral-aboral axis in a largely non-cell-autonomous manner. These observations suggest that the mechanisms responsible for patterning the oral-aboral axis of the sea urchin embryo may share similarities with mechanisms that pattern the dorsoventral axis of other deuterostomes.