Strongylocentrotus Purpuratus Research Articles

Manipulation of developing juvenile structures in purple sea urchins (Strongylocentrotus purpuratus) by morpholino injection into late stage larvae.

Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.

Single sea urchin phagocytes express messages of a single sequence from the diverse Sp185/333 gene family in response to bacterial challenge.

Immune systems in animals rely on fast and efficient responses to a wide variety of pathogens. The Sp185/333 gene family in the purple sea urchin, Strongylocentrotus purpuratus, consists of an estimated 50 (±10) members per genome that share a basic gene structure but show high sequence diversity, primarily due to the mosaic appearance of short blocks of sequence called elements. The genes show significantly elevated expression in three subpopulations of phagocytes responding to marine bacteria. The encoded Sp185/333 proteins are highly diverse and have central effector functions in the immune system. In this study we report the Sp185/333 gene expression in single sea urchin phagocytes. Sea urchins challenged with heat-killed marine bacteria resulted in a typical increase in coelomocyte concentration within 24 h, which included an increased proportion of phagocytes expressing Sp185/333 proteins. Phagocyte fractions enriched from coelomocytes were used in limiting dilutions to obtain samples of single cells that were evaluated for Sp185/333 gene expression by nested RT-PCR. Amplicon sequences showed identical or nearly identical Sp185/333 amplicon sequences in single phagocytes with matches to six known Sp185/333 element patterns, including both common and rare element patterns. This suggested that single phagocytes show restricted expression from the Sp185/333 gene family and infers a diverse, flexible, and efficient response to pathogens. This type of expression pattern from a family of immune response genes in single cells has not been identified previously in other invertebrates.

Cis-regulatory control of the nuclear receptor Coup-TF gene in the sea urchin Paracentrotus lividus embryo.

Coup-TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well-studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup-TF gene (PlCoup-TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup-TF, were isolated from a genomic library. The transcription initiation site was determined and 5′ deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (−532 to −232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratus upstream Coup-TF sequences, revealed considerable conservation, but none within module a. 5′ and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis-acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site-specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup-TF gene at pluteus stage sea urchin embryos. These findings lead to the future identification of these factors and to the hierarchical positioning of PlCoup-TF within the embryonic GRN.

Phylogenomic Resolution of the Hemichordate and Echinoderm Clade

Ambulacraria, comprising Hemichordata and Echinodermata, is closely related to Chordata, making it integral to understanding chordate origins and polarizing chordate molecular and morphological characters. Unfortunately, relationships within Hemichordata and Echinodermata have remained unresolved, compromising our ability to extrapolate findings from the most closely related molecular and developmental models outside of Chordata (e.g., the acorn worms Saccoglossus kowalevskii and Ptychodera flava and the sea urchin Strongylocentrotus purpuratus). To resolve long-standing phylogenetic issues within Ambulacraria, we sequenced transcriptomes for 14 hemichordates as well as 8 echinoderms and complemented these with existing data for a total of 33 ambulacrarian operational taxonomic units (OTUs). Examination of leaf stability values revealed rhabdopleurid pterobranchs and the enteropneust Stereobalanus canadensis were unstable in placement; therefore, analyses were also run without these taxa. Analyses of 185 genes resulted in reciprocal monophyly of Enteropneusta and Pterobranchia, placed the deep-sea family Torquaratoridae within Ptychoderidae, and confirmed the position of ophiuroid brittle stars as sister to asteroid sea stars (the Asterozoa hypothesis). These results are consistent with earlier perspectives concerning plesiomorphies of Ambulacraria, including pharyngeal gill slits, a single axocoel, and paired hydrocoels and somatocoels. The resolved ambulacrarian phylogeny will help clarify the early evolution of chordate characteristics and has implications for our understanding of major fossil groups, including graptolites and somasteroideans.

Characterization of the highly variable immune response gene family, He185/333, in the sea urchin, Heliocidaris erythrogramma.

This study characterizes the highly variable He185/333 genes, transcripts and proteins in coelomocytes of the sea urchin, Heliocidaris erythrogramma. Originally discovered in the purple sea urchin, Strongylocentrotus purpuratus, the products of this gene family participate in the anti-pathogen defenses of the host animals. Full-length He185/333 genes and transcripts are identified. Complete open reading frames of He185/333 homologues are analyzed as to their element structure, single nucleotide polymorphisms, indels and sequence repeats and are subjected to diversification analyses. The sequence elements that compose He185/333 are different to those identified for Sp185/333. Differences between Sp185/333 and He185/333 genes are also evident in the complexity of the sequences of the introns. He185/333 proteins show a diverse range of molecular weights on Western blots. The observed sizes and pIs of the proteins differ from predicted values, suggesting post-translational modifications and oligomerization. Immunofluorescence microscopy shows that He185/333 proteins are mainly located on the surface of coelomocyte subpopulations. Our data demonstrate that He185/333 bears the same substantial characteristics as their S. purpuratus homologues. However, we also identify several unique characteristics of He185/333 (such as novel element patterns, sequence repeats, distribution of positively-selected codons and introns), suggesting species-specific adaptations. All sequences in this publication have been submitted to Genbank (accession numbers JQ780171-JQ780321) and are listed in table S1.

Signal-dependent regulation of the sea urchin skeletogenic gene regulatory network

The endoskeleton of the sea urchin embryo is produced by primary mesenchyme cells (PMCs). Maternal inputs activate a complex gene regulatory network (GRN) in the PMC lineage in a cell-autonomous fashion during early development, initially creating a uniform population of prospective skeleton-forming cells. Previous studies showed that at post-blastula stages of development, several effector genes in the network exhibit non-uniform patterns of expression, suggesting that their regulation becomes subject to local, extrinsic cues. Other studies have identified the VEGF and MAPK pathways as regulators of PMC migration, gene expression, and biomineralization. In this study, we used whole mount in situ hybridization (WMISH) to examine the spatial expression patterns of 39 PMC-specific/enriched mRNAs in Strongylocentrotus purpuratus embryos at the late gastrula, early prism and pluteus stages. We found that all 39 mRNAs (including several regulatory genes) showed non-uniform patterns of expression within the PMC syncytium, revealing a global shift in the regulation of the skeletogenic GRN from a cell-autonomous to a signal-dependent mode. In general, localized regions of elevated gene expression corresponded to sites of rapid biomineral deposition. We used a VEGFR inhibitor (axitinib) and a MEK inhibitor (U0126) to show that VEGF signaling and the MAPK pathway are essential for maintaining high levels of gene expression in PMCs at the tips of rods that extend from the ventral region of the embryo. These inhibitors affected gene expression in the PMCs in similar ways, suggesting that VEGF acts via the MAPK pathway. In contrast, axitinib and U0126 did not affect the localized expression of genes in PMCs at the tips of the body rods, which form on the dorsal side of the embryo. Our results therefore indicate that multiple signaling pathways regulate the skeletogenic GRN during late stages of embryogenesis-VEGF/MAPK signaling on the ventral side and a separate, unidentified pathway on the dorsal side. These two signaling pathways appear to be activated sequentially (ventral followed by dorsal) and many effector genes are subject to regulation by both pathways.

Characterization of the Antarctic sea urchin (Sterechinus neumayeri) transcriptome and mitogenome: a molecular resource for phylogenetics, ecophysiology and global change biology.

This is the first de novo transcriptome and complete mitochondrial genome of an Antarctic sea urchin species sequenced to date. Sterechinus neumayeri is an Antarctic sea urchin and a model species for ecology, development, physiology and global change biology. To identify transcripts important to ocean acidification (OA) and thermal stress, this transcriptome was created pooling, and 13 larval samples representing developmental stages on day 11 (late gastrula), 19 (early pluteus) and 30 (mid pluteus) maintained at three CO2 levels (421, 652, and 1071 μatm) as well as four additional heat-shocked samples. The normalized cDNA pool was sequenced using emulsion PCR (pyrosequencing) resulting in 1.34M reads with an average read length of 492 base pairs. 40,994 isotigs were identified, averaging 1188 bp with a median coverage of 11×. Additional primer design and gap sequencing were required to complete the mitochondrial genome. The mitogenome of S. neumayeri is a circular DNA molecule with a length of 15 684 bp that contains all 37 genes normally found in metazoans. We detail the main features of the transcriptome and the mitogenome architecture and investigate the phylogenetic relationships of S. neumayeri within Echinoidea. In addition, we provide comparative analyses of S. neumayeri with its closest relative, Strongylocentrotus purpuratus, including a list of potential OA gene targets. The resources described here will support a variety of quantitative (genomic, proteomic, multistress and comparative) studies to interrogate physiological responses to OA and other stressors in this important Antarctic calcifier.

Effects of climate change on the physiology of giant kelp, Macrocystis pyrifera, and grazing by purple urchin, Strongylocentrotus purpuratus

Copyright © 2014 The Korean Society of Phycology 203 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860 Effects of climate change on the physiology of giant kelp, Macrocystis pyrifera, and grazing by purple urchin, Strongylocentrotus purpuratus Matthew B. Brown*, Matthew S. Edwards and Kwang Young Kim Department of Biology, San Diego State University, San Diego, CA 92182, USA Department of Oceanography, Chonnam National University, Gwangju 500-757, Korea

A novel fatty acid-binding protein-like carotenoid-binding protein from the gonad of the New Zealand sea urchin Evechinus chloroticus.

A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9′-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid.

A method for microinjection of Patiria miniata zygotes.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

In silico determination of the effect of multi-target drugs on calcium dynamics signaling network underlying sea urchin spermatozoa motility.

The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca2+]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca2+]i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca2+] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na+ channels (HCN), [Ca2+]-dependent Cl− channels (CaCC) and [Ca2+]-dependent K+ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca2+]-dependent K+ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca2+] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.

Spine reorientation influences drift particle capture efficiency in sea urchins

Many marine organisms use behavior to navigate hydrodynamic landscapes. The subtidal sea urchin Strongylocentrotus franciscanus reorients its spines as water velocity increases to reduce drag and remain attached to the substratum. Streamlining may be advantageous in this regard, but it is unclear how this change in drag profile will affect particle capture, a feeding strategy employed by these organisms. Streamlining in urchins results in a “spines down” posture while particle capture benefits from spines remaining erect, a difference that could potentially lead to decreased feeding rates in high water velocities. To investigate this we ran flow tank experiments with three species of urchin (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, and Strongylocentrotus purpuratus) which differ in size and spine length. All urchins studied displayed some degree of streamlining, although the threshold water velocity at which the behavior occurred varied among species. Particle capture was highly dependent on urchin size, with S. franciscanus, the largest of the three species studied, capturing the highest total mass across water velocities. However, taking into account urchin size and the changes in particle flux at each water velocity, S. purpuratus was significantly more efficient at capturing particles with “spines up” — an advantage which disappeared once spines were lowered. These results show that size and spine orientation affect how particles interact with urchins in flow and imply that spine morphology plays a role in whether or not an individual adopts a streamlined posture.

The effects of copper and nickel on the embryonic life stages of the purple sea urchin (Strongylocentrotus purpuratus).

The aim of this research was to generate data on the mechanisms of toxicity of copper [Cu (4-12µg/L)] and nickel [Ni (33-40µg/L)] during continuous sublethal exposure in seawater (32ppt, 15°C) in a sensitive test organism (Strongylocentrotus purpuratus) at its most sensitive life stage (developing embryo). Whole-body ions [calcium (Ca), sodium (Na), potassium (K), and magnesium (Mg)], metal burdens, Ca uptake, and Ca ATPase activity were measured every 12h during the first 72-84h of development. Ionoregulatory disruption was clearly an important mechanism of toxicity for both metals and occurred with minimal metal bioaccumulation. Most noteworthy was a significant disruption of Ca homeostasis, which was evident from an inhibition of unidirectional Ca uptake rates, whole-body Ca accumulation, and Ca ATPase activity intermittently during 72-84h of development. At various times, Cu- and Ni-exposed embryos also displayed lower levels of K and increased levels of Na suggesting inhibition of Na/K ATPase activity. Greater levels of Mg during initial stages of development in Cu-exposed embryos were also observed and were considered a possible compensatory mechanism for disruptions to Ca homeostasis because both of these ions are important constituents of the developing spicule. Notably, most of these effects occurred during the initial stages of development but were reversed by 72-84h. We therefore propose that it is of value to study the toxic impacts of contaminants periodically during development before the traditional end point of 48-72h.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

Modular evolution of DNA-binding preference of a Tbrain transcription factor provides a mechanism for modifying gene regulatory networks.

Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites.

Characterization of insulin-like peptides (ILPs) in the sea urchin Strongylocentrotus purpuratus: Insights on the evolution of the insulin family

The evolutionary history of the insulin-like peptides (ILPs), members of the insulin family, is still a matter of debate. Although ILPs structure and expression have been described in different metazoans, little is known about these molecules in non-chordate deuterostomes, such as the echinoderms. In order to fill this gap in the current literature, we have characterized two members of the insulin family found in the sea urchin Strongylocentrotus purpuratus genome (SpIgf1 and SpIgf2 that, after our analysis, we suggest to rename SpILP1 and SpILP2, respectively) together with their putative receptor (SpInsr). We found that SpILP1 gene structure is more similar to the cephalochordate amphioxus ILP, while the SpILP2 gene shows a completely different organization. In addition, we have revealed that SpILP1 and SpILP2 transcripts are expressed in different compartments during embryo/larva development and that the SpILP1 protein mature form differs in the egg and the larva, suggesting different biological roles. Finally, we have analyzed SpILP1 transcript and protein expression in response to different feeding regimes through real-time quantitative PCR, Western blot and immunohistochemistry methodologies, and found that its expression and localization are feeding-dependent. We discuss our findings in a comparative evolutionary perspective including data available in other animal models and provide new insights into the evolution of the insulin family molecules. In the model we put forward, the last common ancestor of all deuterostomes presented an ILP composed of the B–C–A–D–E domains, and successive lineage specific independent gene duplication events resulted in the presence of several ILPs in vertebrates and in echinoderms.

Effects of seawater acidification on gene expression: resolving broader-scale trends in sea urchins.

Sea urchins are ecologically and economically important calcifying organisms threatened by acidification of the global ocean caused by anthropogenic CO2 emissions. Propelled by the sequencing of the purple sea urchin (Strongylocentrotus purpuratus) genome, profiling changes in gene expression during exposure to high pCO2 seawater has emerged as a powerful and increasingly common method to infer the response of urchins to ocean change. However, analyses of gene expression are sensitive to experimental methodology, and comparisons between studies of genes regulated by ocean acidification are most often made in the context of major caveats. Here we perform meta-analyses as a means of minimizing experimental discrepancies and resolving broader-scale trends regarding the effects of ocean acidification on gene expression in urchins. Analyses across eight studies and four urchin species largely support prevailing hypotheses about the impact of ocean acidification on marine calcifiers. The predominant expression pattern involved the down-regulation of genes within energy-producing pathways, a clear indication of metabolic depression. Genes with functions in ion transport were significantly over-represented and are most plausibly contributing to intracellular pH regulation. Expression profiles provided extensive evidence for an impact on biomineralization, epitomized by the down-regulation of seven spicule matrix proteins. In contrast, expression profiles provided limited evidence for CO2-mediated developmental delay or induction of a cellular stress response. Congruence between studies of gene expression and the ocean acidification literature in general validates the accuracy of gene expression in predicting the consequences of ocean change and justifies its continued use in future studies.

Sequencing and analysis of the transcriptome of the acorn worm Ptychodera flava, an indirect developing hemichordate

Hemichordates are the sister group of echinoderms, and together they are closely related to chordates within the deuterostome lineage. Therefore, hemichordates represent an important animal group for the understanding of both the evolution of developmental mechanisms in deuterostome animals and the origin of chordates. Recently, the majority of studies investigating hemichordates have focused on the direct-developing enteropneust hemichordate Saccoglossus kowalevskii; few have focused on the indirect-developing hemichordates, partly because of the lack of extensive genomic resources in these animals. In this study, we report the sequencing and analysis of a transcriptome from an indirect-developing enteropneust hemichordate Ptychodera flava. We sequenced a mixed cDNA library from six developmental stages using the Roche GS FLX Titanium System to generate more than 879,000 reads. These reads were assembled into 17,990 contigs with an average length of 1316bp. We found that 60% of the assembled contigs, along with 28% of the unassembled singleton reads, had significant hits to sequences in the NCBI database by a BLASTx search, and we also annotated these sequences and obtained Gene Ontology (GO) terms for 6744 contigs and 5802 singletons. We further identified candidate P. flava transcripts corresponding to genes involved in major developmental signaling pathways, including the Wnt, Notch and TGF-β signaling pathways. Using available genome/transcriptome datasets from the direct-developing hemichordate S. kowalevskii, the echinoderm Strongylocentrotus purpuratus and the chordate Branchiostoma floridae, we found that 90%, 80% and 73% of the annotated protein sequences in these respective species matched our P. flava transcriptome in a homology search. We also constructed a database for the P. flava transcriptome, and researchers can easily access this dataset online. Our dataset significantly increases the amount of available P. flava sequence data and can serve as a reference transcriptome for future studies using this species.

Hox expression in the direct‐type developing sand dollar Peronella japonica

Echinoderms are a curious group of deuterostomes that forms a clade with hemichordates but has a pentameral body plan. Hox complex plays a pivotal role in axial patterning in bilaterians and often occurs in a cluster on the chromosome. In contrast to hemichordates with an organized Hox cluster, the sea urchin Strongylocentrotus purpuratus has a Hox cluster with an atypical organization. However, the current data on hox expression in sea urchin rudiments are fragmentary. We report a comprehensive examination of hox expression in a sand dollar echinoid. Nine hox genes are expressed in the adult rudiment, which are classified into two groups, but hox11/13b belongs to both: one with linear expression in the coelomic mesoderm and another with radial expression around the adult mouth. The linear genes may endow the coelom/mesentery with axial information to direct postmetamorphic transformation of the digestive tract, whereas the radial genes developmentally correlate with the morphological novelties of echinoderms and/or sea urchins. Recruitment of the radial genes except hox11/13b appears to be accompanied by the loss of ancestral/axial roles. This in toto co-option of the hox genes provides insight into the molecular mechanisms underlying the evolution of echinoderms from a bilateral ancestor.

Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs and their expression analysis under thermal stress in the sea cucumber Apostichopus japonicus

Hsp10 and Hsp60 are important heat shock proteins (HSPs), which might be indispensable in the heat shock response and many other physiological processes. We obtained full-length cDNAs of genes hsp10 and hsp60 and classified their mRNA expression levels under thermal stress in the sea cucumber Apostichopus japonicus. Full-length hsp10 cDNA was 1528 bp containing a 5' untranslated region (UTR) of 83 bp, a 3' UTR of 1133 bp and an open reading frame (ORF) of 312 bp encoding 103 amino acid residues. Full-length hsp60 cDNA was 2560 bp containing a 118-bp 5' UTR, a 678-bp 3' UTR and a 1764-bp ORF encoding 586 amino acid residues. The deduced amino acid sequences of Hsp10 and Hsp60 shared the highest identity with sequences of the sea urchin Strongylocentrotus purpuratus, and phylogenetic trees showed that the evolution of Hsp10 and Hsp60 was almost in accord with the evolution of species. Further analysis by real-time PCR showed that the expression of hsp10 and hsp60 mRNA was highly up-regulated at 26 °C compared with other three groups (20, 22, 24 °C), and their expression in the intestine was in a time-dependent manner at 26 °C. The results suggested that hsp10 and hsp60 were involved in the heat-shock response in the sea cucumber A. japonicus.