Spatial expression of Hox cluster genes in the ontogeny of a sea urchin

The Hox cluster of the sea urchin Strongylocentrous purpuratus contains ten genes in a 500 kb span of the genome. Only two of these genes are expressed during embryogenesis, while all of eight genes tested are expressed during development of the adult body plan in the larval stage. We report the spatial expression during larval development of the five 'posterior' genes of the cluster: SpHox7, SpHox8, SpHox9/10, SpHox11/13a and SpHox11/13b. The five genes exhibit a dynamic, largely mesodermal program of expression. Only SpHox7 displays extensive expression within the pentameral rudiment itself. A spatially sequential and colinear arrangement of expression domains is found in the somatocoels, the paired posterior mesodermal structures that will become the adult perivisceral coeloms. No such sequential expression pattern is observed in endodermal, epidermal or neural tissues of either the larva or the presumptive juvenile sea urchin. The spatial expression patterns of the Hox genes illuminate the evolutionary process by which the pentameral echinoderm body plan emerged from a bilateral ancestor.     

The anteroposterior axis in echinoderms and displacement of the mouth in their phylogeny and ontogeny

The difference in the position of the anteroposterior axis in larval and adult echinoderms is related to the displacement of the mouth from the anterior end of the body to the posterior end in the phylogeny of echinoderms, which occurred in the course of the reorganization of their body plan from bilateral asymmetrical to radiosymmetrical. Traces of this phylogenetic process have been especially fully preserved in the ontogeny of crinoids. Other recent echinoderms have largely lost such traces. Dislocation of Hox-genes in sea urchins, resulting from the translocation of these genes to the 5′ end of the chromosome and inversion of the anterior Hox-genes, is explained by the necessity to preserve the spatial and temporal colinearity in the course of the convergence of the starting and final stages of the mouth displacement process, similar to the elevation process in crinoids, and inclusion in the basic body plan of the structure of a rudiment now regulated directly by the anterior Hox-genes.     

Gene expression analysis of Six3, Pax6, and Otx in the early development of the stalked crinoid Metacrinus rotundus

The stalked crinoid, Metacrinus rotundus, is one of the most basal extant echinoderms. Here, we show the expression patterns of Six3, Pax6, and Otx in the early development of M. rotundus. All three genes are highly expressed in stages from the gastrula to the auricularia larval stage. Ectodermal expression of MrOtx appears to be correlated with development of the ciliary band. These three genes are expressed sequentially along the embryonic body axis in the anterior and middle walls of the archenteron in the order of MrPax6, MrSix3, and MrOtx. The anterior, middle, and posterior parts of the archenteron in the late gastrula differentiate into the axo-hydrocoel, the enteric sac, and somatocoels at later stages, respectively. The three genes are expressed sequentially from the tip of the axo-hydrocoel to the bottom of enteric sac in the order of MrSix3, MrPax6, and MrOtx at the later stages. This suggests that these genes are involved in patterning of the larval endo-mesoderm in stalked crinoids. The present results suggest that radical alterations have occurred in the expression and function of homeobox genes in basal echinoderms.     

Axial patterning of the pentaradial adult echinoderm body plan

Adult echinoderms possess a highly diverged, pentaradial body plan. Developmental mechanisms underlying this body plan are completely unknown, but are critical in understanding how echinoderm pentamery evolved from bilateral ancestors. These mechanisms are difficult to study in indirect-developing species; in this study, we use the direct-developing sea urchin Heliocidaris erythrogramma, whose accelerated adult development can be perturbed by NiCl₂. We introduce a new nomenclature for the adult echinoderm axes to facilitate discussion of the radially symmetric body plan and the events required to pattern it. In sea urchins, the adult oral–aboral axis is often conflated with the long axes of the five rays; we identify these as distinct body axes, the proximodistal (PD). In addition, we define a circular axis, the circumoral (CO), along which the division into five sectors occurs. In NiCl₂-treated larvae, aspects of normal PD pattern were retained, but CO pattern was abolished. Milder treatments resulted in relatively normal juveniles ranging from biradial to decaradial. NiCl₂ treatment had no effect either on mesodermal morphology or on the ectodermal gene expression response to an inductive mesodermal signal. This suggests that the mesoderm does not mediate the disruption of CO patterning by NiCl₂. In contrast, mesodermal signaling may explain the presence of PD pattern in treated larvae. However, variations in appendage pattern suggest that ectodermal signals are also required. We conclude that CO patterning in both germ layers is dependent on ectodermal events and PD patterning is controlled by mutual ectoderm–mesoderm signaling.     

The <Emphasis Type="Italic">Hox8</Emphasis> of the hemichordate <Emphasis Type="Italic">Balanoglossus misakiensis</Emphasis>

Deuterostomes comprise a monophyletic group of animals that include chordates, xenoturbellids, and the Ambulacraria, which consists of echinoderms and hemichordates. The ancestral chordate probably had 14 Hox genes aligned linearly along the chromosome, with the posterior six genes showing an independent duplication compared to protostomes. In contrast, ambulacrarians are characterized by a duplication of the posterior Hox genes, resulting in three genes known as Hox11/13a, Hox11/13b, and Hox11/13c. Here, we isolated 12 Hox genes from the hemichordate Balanoglossus misakiensis and found an extra Hox gene that has not been reported in hemichordates. The extra B. misakiensis gene was suggested to be Hox8 from paralog-characteristic residues in its hexapepetide motif and homeodomain and a comparison with Strongylocentrotus purpuratus Hox genes. Our data suggest that the ancestor of echinoderms and hemichordates may have had a full complement of 12 Hox genes.     

PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.     

Expression of amino acid transporter genes in developmental stages and adult tissues of Antarctic echinoderms

Epithelial cells in the body wall of adult and developmental stages of marine invertebrates absorb dissolved organic material directly from seawater. Despite over a century of study, little is known about the molecular biological mechanisms responsible for this transport process. Previous studies on embryonic and larval Antarctic echinoderms show that amino acid uptake could provide an important supplement of metabolic substrates. In the present study, partial cDNA sequences of 11 putative amino acid transporter genes were isolated from six species of Antarctic echinoderms including the Antarctic sea stars Acodontaster hodgsoni, Diplasterias brucei, Odontaster meridionalis, Odontaster validus, and Perknaster fuscus, and the Antarctic sea urchin Sterechinus neumayeri. Conserved domains of cDNA-deduced amino acid sequences characterized these genes as being members of a family of amino acid transporters (solute carrier family 6). Expression of these genes was detected throughout embryonic and larval development of two species that have contrasting developmental modes (A. hodgsoni: lecithotrophic; O. meridionalis: planktotrophic). In all six species studied, the expression of amino acid transporter genes was detected in tube feet and digestive organs of adult animals, demonstrating that members of a single amino acid transporter gene family are expressed during the entire life history of a marine invertebrate. The identification of these genes is an important step toward developing a mechanistic understanding of amino acid transport capacities in Antarctic marine invertebrates.     

Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.     

Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa)

The bilaterian animals are divided into three great branches: the Deuterostomia, Ecdysozoa, and Lophotrochozoa. The evolution of developmental mechanisms is less studied in the Lophotrochozoa than in the other two clades. We have studied the expression of Hox genes during larval development of two lophotrochozoans, the polychaete annelids Nereis virens and Platynereis dumerilii. As reported previously, the Hox cluster of N. virens consists of at least 11 genes (de Rosa R, Grenier JK, Andreeva T, Cook CE, Adoutte A, Akam M, Carroll SB, Balavoine G, Nature, 399:772–776, 1999; Andreeva TF, Cook C, Korchagina NM, Akam M, Dondua AK, Ontogenez 32:225–233, 2001); we have also cloned nine Hox genes of P. dumerilii. Hox genes are mainly expressed in the descendants of the 2d blastomere, which form the integument of segments, ventral neural ganglia, pre-pygidial growth zone, and the pygidial lobe. Patterns of expression are similar for orthologous genes of both nereids. In Nereis, Hox2, and Hox3 are activated before the blastopore closure, while Hox1 and Hox4 are activated just after this. Hox5 and Post2 are first active during the metatrochophore stage, and Hox7, Lox4, and Lox2 at the late nectochaete stage only. During larval stages, Hox genes are expressed in staggered domains in the developing segments and pygidial lobe. The pattern of expression of Hox cluster genes suggests their involvement in the vectorial regionalization of the larval body along the antero-posterior axis. Hox gene expression in nereids conforms to the canonical patterns postulated for the two other evolutionary branches of the Bilateria, the Ecdysozoa and the Deuterostomia, thus supporting the evolutionary conservatism of the function of Hox genes in development. Milana Kulakova, Nadezhda Bakalenko and Elena Novikova contributed equally to this work.     

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Hox genes in a pentameral animal

There is renewed interest in how the different body plans of extant phyla are related. This question has traditionally been addressed by comparisons between vertebrates and Drosophila. Fortunately, there is now increasing emphasis on animals representing other phyla. Pentamerally symmetric echinoderms are a bilaterian metazoan phylum whose members exhibit secondarily derived radial symmetry. Precisely how their radially symmetric body plan originated from a bilaterally symmetric ancestor is unknown, however, two recent papers address this subject. Peterson et al. propose a hypothesis on evolution of the anteroposterior axis in echinoderms, and Arenas-Mena et al. examine expression of five posterior Hox genes during development of the adult sea urchin.     

Heterochronic activation of VEGF signaling and the evolution of the skeleton in echinoderm pluteus larvae

The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well‐organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.     

Arrays in rays: terminal addition in echinoderms and its correlation with gene expression

The echinoderms are deuterostomes that superimpose radial symmetry upon bilateral larval morphology. Consequently, they are not the first animals that come to mind when the concepts of segmentation and terminal addition are being discussed. However, it has long been recognized that echinoderms have serial elements along their radii formed in accordance with the ocular plate rule (OPR). The OPR is a special case of terminal growth, forming elements of the ambulacra that define the rays in echinoderms. New elements are added at the terminus of the ray, which may or may not be marked by a calcified element called the terminal plate (the "ocular" of sea urchins). The OPR operates in every echinoderm, from the occasionally bizarre fossils of the Cambrian to the most familiar extant taxa. Using the OPR and other criteria of recognition, echinoderm body wall can be divided into two main regions: extraxial components are associated with the somatocoels, axial components (formed in accordance with the OPR) with the hydrocoel. We compare patterns of development in axial regions of echinoderms with those found in the anterior-posterior axes of the earliest echinoderms as well as other invertebrates. Although axial and extraxial skeletons appear to be composed of the same biomineral matrix, the genes involved in patterning these two skeletal components are likely distinct. During development of the axial skeleton, for instance, the genes engrailed and orthodenticle are expressed in spatial and temporal patterns consistent with the OPR. Other genes such as distal-less seem to demarcate early ontogenetic boundaries between the axial rudiment and the extraxial larval body. There is a complex and pervasive reorganization of gene expression domains to produce the highly divergent morphologies seen in the Echinodermata. We integrate morphological and genetic information, particularly with respect to the origins of radial symmetry in the rudiment, and the concomitant development of the rays.     

<Emphasis Type="Italic">Engrailed</Emphasis> is expressed in larval development and in the radial nervous system of <Emphasis Type="Italic">Patiriella</Emphasis> sea stars

We documented expression of the pan-metazoan neurogenic gene engrailed in larval and juvenile Patiriella sea stars to determine if this gene patterns bilateral and radial echinoderm nervous systems. Engrailed homologues, containing conserved En protein domains, were cloned from the radial nerve cord. During development, engrailed was expressed in ectodermal (nervous system) and mesodermal (coeloms) derivatives. In larvae, engrailed was expressed in cells lining the larval and future adult coeloms. Engrailed was not expressed in the larval nervous system. As adult-specific developmental programs were switched on during metamorphosis, engrailed was expressed in the central nervous system and peripheral nervous system (PNS), paralleling the pattern of neuropeptide immunolocalisation. Engrailed was first seen in the developing nerve ring and appeared to be up-regulated as the nervous system developed. Expression of engrailed in the nerve plexus of the tube feet, the lobes of the hydrocoel along the adult arm axis, is similar to the reiterated pattern of expression seen in other animals. Engrailed expression in developing nervous tissue reflects its conserved role in neurogenesis, but its broad expression in the adult nervous system of Patiriella differs from the localised expression seen in other bilaterians. The role of engrailed in patterning repeated PNS structures indicates that it may be important in patterning the fivefold organisation of the ambulacrae, a defining feature of the Echinodermata.     

Building divergent body plans with similar genetic pathways

Deuterostome animals exhibit widely divergent body plans. Echinoderms have either radial or bilateral symmetry, hemichordates include bilateral enteropneust worms and colonial pterobranchs, and chordates possess a defined dorsal-ventral axis imposed on their anterior-posterior axis. Tunicates are chordates only as larvae, following metamorphosis the adults acquire a body plan unique for the deuterostomes. This paper examines larval and adult body plans in the deuterostomes and discusses two distinct ways of evolving divergent body plans. First, echinoderms and hemichordates have similar feeding larvae, but build a new adult body within or around their larvae. In hemichordates and many direct-developing echinoderms, the adult is built onto the larva, with the larval axes becoming the adult axes and the larval mouth becoming the adult mouth. In contrast, indirect-developing echinoderms undergo radical metamorphosis where adult axes are not the same as larval axes. A second way of evolving a divergent body plan is to become colonial, as seen in hemichordates and tunicates. Early embryonic development and gastrulation are similar in all deuterostomes, but, in chordates, the anterior-posterior axis is established at right angles to the animal-vegetal axis, in contrast to hemichordates and indirect-developing echinoderms. Hox gene sequences and anterior-posterior expression patterns illuminate deuterostome phylogenetic relationships and the evolution of unique adult body plans within monophyletic groups. Many genes that are considered vertebrate 'mesodermal' genes, such as nodal and brachyury T, are likely to ancestrally have been involved in the formation of the mouth and anus, and later were evolutionarily co-opted into mesoderm during vertebrate development.     

Isolation and expression analysis of a poriferan <Emphasis Type="Italic">Antp</Emphasis>-class <Emphasis Type="Italic">Bar-/Bsh-</Emphasis>like homeobox gene

A novel non-Hox Antp-class gene (BarBsh-Hb) was isolated from the marine sponge Halichondria sp. This gene shares high sequence identity with eumetazoan genes from the Bsh and Bar gene families and can be distinguished from other non-Hox Antp-class genes by diagnostic residues. We also present an alignment of all known (full-length) poriferan non-Hox Antp-class genes. Maximum likelihood methods were employed to estimate phylogenetic relationships among non-Hox genes and BarBsh-Hb. We employed RT-PCR techniques to look at expression across different developmental stages (larval to rhagon). BarBsh-Hb product was present in newly released larvae, but expression was not detected 8–16 h post-release. Expression of BarBsh-Hb was detected in later-stage (>16 h post-release), free-swimming larvae until they settled and attached to the substratum, after which expression was down-regulated. In a separate set of experiments, low levels of expression were observed in normal adult tissue and disaggregated adult tissue, but BarBsh-Hb expression increased during tissue re-aggregation. These data increase the number of non-Hox homeobox genes identified in sponges and provide evidence of regulation of this non-Hox gene during sponge development. While the Bar and Bsh genes play important roles in the development of nervous tissue—especially visual systems—in metazoans, the specific role(s) BarBsh-Hb play(s) in sponge development is unclear and deserves greater attention.Edited by C. Desplan     

Ion flow regulates left–right asymmetry in sea urchin development

The degree of conservation among phyla of early mechanisms that pattern the left–right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H⁺/K⁺-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H⁺/K⁺-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H⁺ and/or K⁺, and also of Ca²⁺, functions in the establishment of LR asymmetry in sea urchin development.Electronic Supplementary Material Supplementary material is available for this article at     

Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.