Aboral Ectoderm Research Articles

Spatial variability of and effect of light on the cœlenteron pH of a reef coral

Coral reefs, the largest bioconstruction on Earth, are formed by calcium carbonate skeletons of corals. Coral skeleton formation commonly referred to as calcification occurs in a specific compartment, the extracellular calcifying medium (ECM), located between the aboral ectoderm and the skeleton. Calcification models often assume a direct link between the surrounding seawater and the ECM. However, the ECM is separated from the seawater by several tissue layers and the cœlenteron, which contains the cœlenteric fluid found in both polyps and cœnosarc (tissue connecting the polyps). Symbiotic dinoflagellate-containing cells line the cœlenteron and their photosynthetic activity contributes to changes in the chemistry of the cœlenteric fluid, particularly with respect to pH. The aim of our study is to compare cœlenteron pH between the cœnosarc and polyps and to compare areas of high or low dinoflagellate density based on tissue coloration. To achieve this, we use liquid ion exchange (LIX) pH microsensors to profile pH in the cœlenteron of polyps and the cœnosarc in different regions of the coral colony in light and darkness. We interpret our results in terms of what light and dark exposure means for proton gradients between the ECM and the coelenteron, and how this could affect calcification.

A single-cell RNA-seq analysis of early larval cell-types of the starfish, Patiria pectinifera: Insights into evolution of the chordate body plan

Ambulacrarians (echinoderms and hemichordates) are a sister group to chordates; thus, their larval cell-types may provide clues about evolution of chordate body plans. Although most genic information accumulated to date pertains to sea urchin embryogenesis, starfish embryogenesis represents a more ancestral mode than that of sea urchins. We performed single-cell RNA-seq analysis of cell-types from gastrulae and bipinnarial larvae of the starfish, Patiria pectinifera, and categorized them into 22 clusters, each of which is composed of cells with specific, shared profiles of development-relevant gene expression. Oral and aboral ectoderm, apical plate, hindgut or archenteron, midgut or intestine, pharynx, endomesoderm, stomodeum, and mesenchyme of the gastrulae, and neurons, ciliary bands, enterocoel and muscle of larvae were characterized by expression profiles of at least two relevant transcription factor genes and signaling molecular genes. Expression of Hox2, Hox7, Hox9/10, and Hox11/13b was detected in cells of clusters that form the larval enterocoel. By comparing homologous gene expression profiles in chordate embryos, we discuss and propose how the chordate body plan evolved from a deuterostome ancestor, from which the echinoderm body plan also evolved.

Expression Analysis of Cnidarian-Specific Neuropeptides in a Sea Anemone Unveils an Apical-Organ-Associated Nerve Net That Disintegrates at Metamorphosis.

Neuropeptides are ancient neuronal signaling molecules that have diversified across Cnidaria (e.g., jellyfish, corals, and sea anemones) and its sister group Bilateria (e.g., vertebrates, insects, and worms). Over the course of neuropeptide evolution emerged lineage-specific neuropeptides, but their roles in the evolution and diversification of nervous system function remain enigmatic. As a step toward filling in this knowledge gap, we investigated the expression pattern of a cnidarian-specific neuropeptide—RPamide—during the development of the starlet sea anemone Nematostella vectensis, using in situ hybridization and immunohistochemistry. We show that RPamide precursor transcripts first occur during gastrulation in scattered epithelial cells of the aboral ectoderm. These RPamide-positive epithelial cells exhibit a spindle-shaped, sensory-cell-like morphology, and extend basal neuronal processes that form a nerve net in the aboral ectoderm of the free-swimming planula larva. At the aboral end, RPamide-positive sensory cells become integrated into the developing apical organ that forms a bundle of long cilia referred to as the apical tuft. Later during planula development, RPamide expression becomes evident in sensory cells in the oral ectoderm of the body column and pharynx, and in the developing endodermal nervous system. At metamorphosis into a polyp, the RPamide-positive sensory nerve net in the aboral ectoderm degenerates by apoptosis, and RPamide expression begins in ectodermal sensory cells of growing oral tentacles. In addition, we find that the expression pattern of RPamide in planulae differs from that of conserved neuropeptides that are shared across Cnidaria and Bilateria, indicative of distinct functions. Our results not only provide the anatomical framework necessary to analyze the function of the cnidarian-specific neuropeptides in future studies, but also reveal previously unrecognized features of the sea anemone nervous system—the apical organ neurons of the planula larva, and metamorphosis-associated reorganization of the ectodermal nervous system.

SoxB2 in sea urchin development: implications in neurogenesis, ciliogenesis and skeletal patterning

BackgroundCurrent studies in evolutionary developmental biology are focused on the reconstruction of gene regulatory networks in target animal species. From decades, the scientific interest on genetic mechanisms orchestrating embryos development has been increasing in consequence to the fact that common features shared by evolutionarily distant phyla are being clarified. In 2011, a study across eumetazoan species showed for the first time the existence of a highly conserved non-coding element controlling the SoxB2 gene, which is involved in the early specification of the nervous system. This discovery raised several questions about SoxB2 function and regulation in deuterostomes from an evolutionary point of view.ResultsDue to the relevant phylogenetic position within deuterostomes, the sea urchin Strongylocentrotus purpuratus represents an advantageous animal model in the field of evolutionary developmental biology. Herein, we show a comprehensive study of SoxB2 functions in sea urchins, in particular its expression pattern in a wide range of developmental stages, and its co-localization with other neurogenic markers, as SoxB1, SoxC and Elav. Moreover, this work provides a detailed description of the phenotype of sea urchin SoxB2 knocked-down embryos, confirming its key function in neurogenesis and revealing, for the first time, its additional roles in oral and aboral ectoderm cilia and skeletal rod morphology.ConclusionsWe concluded that SoxB2 in sea urchins has a neurogenic function; however, this gene could have multiple roles in sea urchin embryogenesis, expanding its expression in non-neurogenic cells. We showed that SoxB2 is functionally conserved among deuterostomes and suggested that in S. purpuratus this gene acquired additional functions, being involved in ciliogenesis and skeletal patterning.

Cis-Regulatory control of the initial neurogenic pattern of onecut gene expression in the sea urchin embryo

Specification of the ciliated band (CB) of echinoid embryos executes three spatial functions essential for postgastrular organization. These are establishment of a band about 5 cells wide which delimits and bounds other embryonic territories; definition of a neurogenic domain within this band; and generation within it of arrays of ciliary cells that bear the special long cilia from which the structure derives its name. In Strongylocentrotus purpuratus the spatial coordinates of the future ciliated band are initially and exactly determined by the disposition of a ring of cells that transcriptionally activate the onecut homeodomain regulatory gene, beginning in blastula stage, long before the appearance of the CB per se. Thus the cis-regulatory apparatus that governs onecut expression in the blastula directly reveals the genomic sequence code by which these aspects of the spatial organization of the embryo are initially determined. We screened the entire onecut locus and its flanking region for transcriptionally active cis-regulatory elements, and by means of BAC recombineered deletions identified three separated and required cis-regulatory modules that execute different functions. The operating logic of the crucial spatial control module accounting for the spectacularly precise and beautiful early onecut expression domain depends on spatial repression. Previously predicted oral ectoderm and aboral ectoderm repressors were identified by cis-regulatory mutation as the products of goosecoid and irxa genes respectively, while the pan-ectodermal activator SoxB1 supplies a transcriptional driver function.

Expession patterns of mesenchyme specification genes in two distantly related echinoids, Glyptocidaris crenularis and Echinocardium cordatum

The molecular mechanism of the larval mesenchyme cell specification in echinoids has been well analyzed. However, most of the data have been provided by studies of a single group of echinoids, the order Camarodonta. Little is known about this mechanism in other echinoid orders. We examined the expression patterns of mesenchyme specification genes, micro1, hesC, alx1, tbr, ets1, cyp1, and gcm, in the two non-Camarodonta echinoids, Glyptocidaris crenularis and Echinocardium cordatum. We found that the expression patterns of some genes contained characteristics that were unique to one of the species; others were shared by the two species. Some of the shared characteristics of G. crenularis and E. cordatum are not found in the species belonging to Camarodonta, suggesting the derived status of this order. The expression of ets1 in E. cordatum aboral ectoderm is one of the molecular level modifications possibly related to an evolutionarily novel larval structure, the posterior process. Our results suggest that a considerable number of modifications in the mesenchyme specification mechanisms have been introduced during the echinoid evolution.

Climate policy rather than politics should be the result

The molecular mechanism of the larval mesenchyme cell specification in echinoids has been well analyzed. However, most of the data have been provided by studies of a single group of echinoids, the order Camarodonta. Little is known about this mechanism in other echinoid orders. We examined the expression patterns of mesenchyme specification genes, micro1, hesC, alx1, tbr, ets1, cyp1, and gcm, in the two non-Camarodonta echinoids, Glyptocidaris crenularis and Echinocardium cordatum. We found that the expression patterns of some genes contained characteristics that were unique to one of the species; others were shared by the two species. Some of the shared characteristics of G. crenularis and E. cordatum are not found in the species belonging to Camarodonta, suggesting the derived status of this order. The expression of ets1 in E. cordatum aboral ectoderm is one of the molecular level modifications possibly related to an evolutionarily novel larval structure, the posterior process. Our results suggest that a considerable number of modifications in the mesenchyme specification mechanisms have been introduced during the echinoid evolution.

Geometric control of ciliated band regulatory states in the sea urchin embryo.

The trapezoidal ciliated band (CB) of the postgastrular sea urchin embryo surrounds the oral ectoderm, separating it from adjacent embryonic territories. Once differentiated, the CB is composed of densely arranged cells bearing long cilia that endow the larva with locomotion and feeding capability. The spatial pattern from which the CB will arise is first evidenced during pregastrular stages by expression of the pioneer gene onecut. Immediately after gastrulation, the CB consists of four separate regulatory state domains, each of which expresses a unique set of transcription factors: (1) the oral apical CB, located within the apical neurogenic field; (2) the animal lateral CB, which bilaterally separates the oral from aboral ectoderm; (3) the vegetal lateral CB, which bilaterally serves as signaling centers; and (4) the vegetal oral CB, which delineates the boundary with the underlying endoderm. Remarkably, almost all of the regulatory genes specifically expressed within these domains are downregulated by interference with SoxB1 expression, implying their common activation by this factor. Here, we show how the boundaries of the CB subdomains are established, and thus ascertain the design principle by which the geometry of this unique and complex regulatory state pattern is genomically controlled. Each of these boundaries, on either side of the CB, is defined by spatially confined transcriptional repressors, the products of regulatory genes operating across the border of each subdomain. In total this requires deployment of about ten different repressors, which we identify in this work, thus exemplifying the complexity of information required for spatial regulatory organization during embryogenesis.

Proton-transfer reaction dynamics and energetics in calcification and decalcification.

CaCO3 -saturated saline waters at pH values below 8.5 are characterized by two stationary equilibrium states: reversible chemical calcification/decalcification associated with acid dissociation, Ca(2+) +HCO3 (-) ⇌CaCO3 +H(+) ; and reversible static physical precipitation/dissolution, Ca(2+) +CO3 (2-) ⇌CaCO3 . The former reversible reaction was determined using a strong base and acid titration. The saturation state described by the pH/PCO2 -independent solubility product, [Ca(2+) ][CO3 (2-) ], may not be observed at pH below 8.5 because [Ca(2+) ][CO3 (2-) ]/([Ca(2+) ][HCO3 (-) ]) ≪1. Since proton transfer dynamics controls all reversible acid dissociation reactions in saline waters, the concentrations of calcium ion and dissolved inorganic carbon (DIC) were expressed as a function of dual variables, pH and PCO2 . The negative impact of ocean acidification on marine calcifying organisms was confirmed by applying the experimental culture data of each PCO2 /pH-dependent coral polyp skeleton weight (Wskel) to the proton transfer idea. The skeleton formation of each coral polyp was performed in microspaces beneath its aboral ectoderm. This resulted in a decalcification of 14 weight %, a normalized CaCO3 saturation state Λ of 1.3 at PCO2 ≈400 ppm and pH ≈8.0, and serious decalcification of 45 % and Λ 2.5 at PCO2 ≈1000 ppm and pH ≈7.8.

Gene regulatory control in the sea urchin aboral ectoderm: Spatial initiation, signaling inputs, and cell fate lockdown

The regulation of oral–aboral ectoderm specification in the sea urchin embryo has been extensively studied in recent years. The oral–aboral polarity is initially imposed downstream of a redox gradient induced by asymmetric maternal distribution of mitochondria. Two TGF-β signaling pathways, Nodal and BMP, are then respectively utilized in the generation of oral and aboral regulatory states. However, a causal understanding of the regulation of aboral ectoderm specification has been lacking. In this work control of aboral ectoderm regulatory state specification was revealed by combining detailed regulatory gene expression studies, perturbation and cis-regulatory analyses. Our analysis illuminates a dynamic system where different factors dominate at different developmental times. We found that the initial activation of aboral genes depends directly on the redox sensitive transcription factor, hypoxia inducible factor 1α (HIF-1α). Two BMP ligands, BMP2/4 and BMP5/8, then significantly enhance aboral regulatory gene transcription. Ultimately, encoded feedback wiring lockdown the aboral ectoderm regulatory state. Our study elucidates the different regulatory mechanisms that sequentially dominate the spatial localization of aboral regulatory states.

Direct and indirect control of oral ectoderm regulatory gene expression by Nodal signaling in the sea urchin embryo

The Nodal signaling pathway is known from earlier work to be an essential mediator of oral ectoderm specification in the sea urchin embryo, and indirectly, of aboral ectoderm specification as well. Following expression of the Nodal ligand in the future oral ectoderm during cleavage, a sequence of regulatory gene activations occur within this territory which depend directly or indirectly on nodal gene expression. Here we describe additional regulatory genes that contribute to the oral ectoderm regulatory state during specification in Strongylocentrotus purpuratus, and show how their spatial expression changes dynamically during development. By means of system wide perturbation analyses we have significantly improved current knowledge of the epistatic relations among the regulatory genes of the oral ectoderm. From these studies there emerge diverse circuitries relating downstream regulatory genes directly and indirectly to Nodal signaling. A key intermediary regulator, the role of which had not previously been discerned, is the not gene. In addition to activating several genes earlier described as targets of Nodal signaling, the not gene product acts to repress other oral ectoderm genes, contributing crucially to the bilateral spatial organization of the embryonic oral ectoderm.

The dynamic gene expression patterns of transcription factors constituting the sea urchin aboral ectoderm gene regulatory network

The temporal and spatial expression patterns of regulatory genes are required for building a gene regulatory network (GRN). The current ectoderm GRN model for the sea urchin embryo includes pregastrular specification functions in the oral (OE) and aboral ectoderm (AE). Unlike the OE, which is resolved into several subdomains, the AE is considered a simpler territory due to the lack of detailed gene expression studies in this territory. Here, we perform temporal and spatial gene expression studies on the eight transcription factor genes constituting the AE GRN. Based on the differential gene expression patterns, we conclude that the AE contains at least three subdomains at the mesenchyme blastula stage. We also performed immunostaining for pSmad1/5/8 to monitor the activation of the BMP signaling pathway. The dynamic changes in the expression patterns of these transcription factor genes and the nuclearization of pSmad1/5/8 may provide a foundation for resolving the AE GRN.

TGFβ signaling positions the ciliary band and patterns neurons in the sea urchin embryo

The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGFβ signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning.

Spatiotemporal expression pattern of an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus during early development, and its potential role in larval vertical migration

We have cloned and studied Hp-ECPN, an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus. Hp-ecpn cDNA was produced and found to contain a 1461-bp open reading frame that encodes 486 amino acids. Accumulation of Hp-ecpn mRNA and protein expression occurred at the 14 h postfertilization (hpf) swimming blastula stage and thereafter. The Hp-ECPN protein was N-glycosylated, and the amino acid sequence was similar to that of vertebrate encephalopsins. Whole-mount immunohistochemistry revealed the presence of Hp-ECPN in cells (ECPN cells) that appeared initially around the tip of the archenteron in 20 hpf early gastrulae. By the 54 hpf pluteus stage, ECPN cells had spread through the aboral ectoderm, and, by the eight-arm pluteus stage, were restricted to the tips of the larval arms and the posterior end of the body. The number of ECPN cells increased under conditions of continuous light, but decreased under continuous dark. Knockdown of Hp-ecpn mRNA using morpholino antisense oligonucleotides decreased the number of ECPN cells considerably, and inhibited the vertical swimming of the larvae. This suggested that Hp-ECPN plays a role in photosensitive larval swimming vertical migration. In adult tissues, the ECPN cells were detected exclusively in tube feet.

Reduced O2 and elevated ROS in sea urchin embryos leads to defects in ectoderm differentiation

The sea urchin oral-aboral (OA) axis is established in part by Nodal signaling. The OA axis is also influenced by treatments affecting respiration and Nodal transcription is influenced by redox-dependent transcription factors. This suggests that intracellular redox state plays a role in OA axis specification. Since cellular redox state can be altered by the formation of excess reactive oxygen species (ROS), and hypoxia and paraquat generate ROS in cells, we asked whether these treatments affected specification of the OA axis and Nodal expression. Embryos cultured under conditions that elevate ROS, demonstrate perturbed ectoderm specification, but other territories are not affected. Immunohistochemical and Q-RT-PCR analyses revealed that both oral and aboral ectoderm genes are downregulated. Our results argue that elevating ROS in sea urchin embryos by these treatments blocks early steps in ectoderm differentiation preceding the polarization of the ectoderm into oral and aboral territories.

A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo

The current gene regulatory network (GRN) for the sea urchin embryo pertains to pregastrular specification functions in the endomesodermal territories. Here we extend gene regulatory network analysis to the adjacent oral and aboral ectoderm territories over the same period. A large fraction of the regulatory genes predicted by the sea urchin genome project and shown in ancillary studies to be expressed in either oral or aboral ectoderm by 24 h are included, though universally expressed and pan-ectodermal regulatory genes are in general not. The loci of expression of these genes have been determined by whole mount in situ hybridization. We have carried out a global perturbation analysis in which expression of each gene was interrupted by introduction of morpholino antisense oligonucleotide, and the effects on all other genes were measured quantitatively, both by QPCR and by a new instrumental technology (NanoString Technologies nCounter Analysis System). At its current stage the network model, built in BioTapestry, includes 22 genes encoding transcription factors, 4 genes encoding known signaling ligands, and 3 genes that are yet unknown but are predicted to perform specific roles. Evidence emerged from the analysis pointing to distinctive subcircuit features observed earlier in other parts of the GRN, including a double negative transcriptional regulatory gate, and dynamic state lockdowns by feedback interactions. While much of the regulatory apparatus is downstream of Nodal signaling, as expected from previous observations, there are also cohorts of independently activated oral and aboral ectoderm regulatory genes, and we predict yet unidentified signaling interactions between oral and aboral territories.

Neural development of the brittlestar Amphiura filiformis

Comparative features of the development of the larval nervous system of ophiuroids have the potential for resolving aspects of echinoderm evolution. In Amphiura filiformis serotonergic neural progenitors appear in the animal plate of late gastrulae. The serotonergic progenitors increase in number and become displaced to the aboral ectoderm side of the developing ciliary band. The ciliary band neurons appear as irregularly spaced neural progenitors on the oral side of the ciliary band lateral to the mouth. These cells extend neurites along the axis of the ciliary band, which meet at the center of the ventral transverse ciliary band. The larval nervous system begins as a U-shaped tract of axons that surrounds the oral field and tracts of axons and neurons in the ciliary bands of the larval arms are added. In addition, the larval nervous system has an extensive pre-oral neuropil, rings of nerves surrounding the anus and pyloric sphincters, and a plexus of axons that surround the esophagus. The nervous system of the juvenile develops beneath the oral ectoderm. The components of the adult nervous system: five segments of radial nerve, commissures that form the nerve ring, and podial nerves all appear as the juvenile develops. The larval nervous system begins to fragment and degenerate as the juvenile grows. The complete description of neural development of an ophiuroid reveals that the four classes so far investigated are consistent with phylogenies based on adult features and comparisons of neural organization help rationalize conflicting hypotheses of the evolution of larval forms in echinoderms.

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.

Spatio-temporal expression of a Netrin homolog in the sea urchin Hemicentrotus pulcherrimus (HpNetrin) during serotonergic axon extension

A netrin homolog of the sea urchin, Hemicentrotus pulcherrimus (HpNetrin) was characterized in terms of its expression behavior. The predicted amino acid sequence was an ortholog of hemichordate netrin-1. Reverse transcriptase-PCR, immunoblotting, and whole mount immunohistochemistry showed that gene transcription and protein expression occurred from 15-hour post-fertilization (hpf) swimming blastula stage to, at least 53-hpf pluteus stage. HpNetrin was detected on the entire basal surface of the ectoderm in swimming and 16-hpf mesenchyme blastulae. However, by 24-hpf prism stage, immunoreaction on the oral ectoderm decreased, whereas it increased in the aboral ectoderm including near the animal plate ectoderm (area-I). By 48-hpf pluteus stage, the HpNetrin-rich area-I comprised a 40mm wide dorsal midline belt (DMB) that stretched from the dorsal posterior edge of the animal plate to the posterior end of the larval body. Serotonergic cells were first detected in the HpNetrin-moderate area between the anterior DMB and the HpNetrin-poor oral ectoderm near the animal plate in 24-hpf prism larvae. By 48-hpf pluteus stage, these cells extended axons toward the middle-ridge to form a neural plexus of the apical ganglion. At 53-hpf pluteus stage, these axons extended further away from the apical ganglion directly or through the DMB toward the HpNetrin-poor contralateral ectoderm. The protein expression and axon extension pattern were reproduced in embryonic cell-aggregates formed from artificially dissociated 20-hpf gastrulae and resembled small blastula. In Hpnetrin morpholino anti-sense oligonucleotide-injected plutei, serotonergic axon extension was severely inhibited. Thus, HpNetrin functions as a serotonergic axon guidance cue in this basal deuterostome.

Sp-Smad2/3 mediates patterning of neurogenic ectoderm by nodal in the sea urchin embryo

Nodal functions in axis and tissue specification during embryogenesis. In sea urchin embryos, Nodal is crucial for specification of oral ectoderm and is thought to pattern neurogenesis in the animal plate. To determine if Nodal functions directly in suppressing neuron differentiation we have prepared mutant forms of Sp-Smad2/3. Expressing an activated form produces embryos similar to embryos overexpressing Nodal, but with fewer neurons. In chimeras in which Nodal is suppressed, cells expressing activated Sp-Smad2/3 form oral ectoderm, but not neurons. In embryos with vegetal signaling blocked, neurons do not form if activated Smad2/3 is co-expressed. Expression of dominant negative mutants produces embryos identical to those resulting from blocking Nodal expression. In chimeras overexpressing Nodal, cells expressing dominant negative Sp-Smad2/3 form aboral ectoderm and give rise to neurons. In permanent blastula chimeras dominant negative Sp-Smad2/3 is able to suppress the effects of Nodal permitting neuron differentiation. In these chimeras Nodal expression in one half suppresses neural differentiation across the interface. Anti-phospho-Smad3 reveals that the cells adjacent to cells expressing Nodal have nuclear immunoreactivity. We conclude Sp-Smad2/3 is a component of the Nodal signaling pathway in sea urchins and that Nodal diffuses short distances to suppress neural differentiation.