Showing papers on "Strongylocentrotus purpuratus published in 2012"

Gene structure in the sea urchin Strongylocentrotus purpuratus based on transcriptome analysis

Abstract: A comprehensive transcriptome analysis has been performed on protein-coding RNAs of Strongylocentrotus purpuratus, including 10 different embryonic stages, six feeding larval and metamorphosed juvenile stages, and six adult tissues. In this study, we pooled the transcriptomes from all of these sources and focused on the insights they provide for gene structure in the genome of this recently sequenced model system. The genome had initially been annotated by use of computational gene model prediction algorithms. A large fraction of these predicted genes were recovered in the transcriptome when the reads were mapped to the genome and appropriately filtered and analyzed. However, in a manually curated subset, we discovered that more than half the computational gene model predictions were imperfect, containing errors such as missing exons, prediction of nonexistent exons, erroneous intron/exon boundaries, fusion of adjacent genes, and prediction of multiple genes from single genes. The transcriptome data have been used to provide a systematic upgrade of the gene model predictions throughout the genome, very greatly improving the research usability of the genomic sequence. We have constructed new public databases that incorporate information from the transcriptome analyses. The transcript-based gene model data were used to define average structural parameters for S. purpuratus protein-coding genes. In addition, we constructed a custom sea urchin gene ontology, and assigned about 7000 different annotated transcripts to 24 functional classes. Strong correlations became evident between given functional ontology classes and structural properties, including gene size, exon number, and exon and intron size.

Dynamic Evolution of Toll-Like Receptor Multigene Families in Echinoderms

Abstract: The genome of the purple sea urchin, Strongylocentrotus purpuratus, was the first to be sequenced from a long-lived large invertebrate. Analysis of this genome uncovered a surprisingly complex immune system in which the moderately sized sets of pattern recognition receptors that form the core of vertebrate innate immunity are encoded in large multigene families. The sea urchin genome contains 253 Toll-like receptor (TLR) genes, more than 200 Nod-like receptors and 1095 scavenger receptor cysteine-rich domains, a ten-fold expansion relative to vertebrates. Given their stereotypic structure and simple intron-exon architecture, the TLRs are the most tractable of these families for more detailed analysis. An immune defense role for these receptors is suggested by their sequence diversity and expression in immunologically active tissues, including phagocytes. This complexity of the sea urchin TLR multigene families largely derives from expansions that are independent of those in vertebrates and protostomes, although a small family of TLRs with structure similar to that of Drosophila Toll likely originated in an ancient eumetazoan ancestor. Several other invertebrate deuterostome genomes have been sequenced, including the cephalochordate, Branchiostoma floridae and the sea urchin Lytechinus variegatus, as well as partial sequences from two other sea urchin species. Here, we present an analysis of the invertebrate deuterostome TLRs with emphasis on the echinoderms. Representatives of most of the S. purpuratus TLR subfamilies and homologs of the protostome-like sequences are found in L. variegatus. The phylogeny of these genes within sea urchins highlights lineage-specific expansions at higher resolution than is evident at the phylum level. These analyses identify quickly evolving TLR subfamilies that are likely to have novel functions and other, more stable, subfamilies that may function similarly to those of vertebrates.

Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO2 seawater conditions

Abstract: Ocean acidification, or the increased uptake of CO2 by the ocean due to elevated atmospheric CO2 concentrations, may variably impact marine early life history stages, as they may be especially susceptible to changes in ocean chemistry. Investigating the regulatory mechanisms of early development in an environmental context, or ecological development, will contribute to increased understanding of potential organismal responses to such rapid, large-scale environmental changes. We examined transcript-level responses to elevated seawater CO2 during gastrulation and the initiation of spiculogenesis, two crucial developmental processes in the purple sea urchin, Strongylocentrotus purpuratus . Embryos were reared at the current, accepted oceanic CO2 concentration of 380 microatmospheres (μatm), and at the elevated levels of 1000 and 1350 μatm, simulating predictions for oceans and upwelling regions, respectively. The seven genes of interest comprised a subset of pathways in the primary mesenchyme cell gene regulatory network (PMC GRN) shown to be necessary for the regulation and execution of gastrulation and spiculogenesis. Of the seven genes, qPCR analysis indicated that elevated CO2 concentrations only had a significant but subtle effect on two genes, one important for early embryo patterning, Wnt8 , and the other an integral component in spiculogenesis and biomineralization, SM30b . Protein levels of another spicule matrix component, SM50, demonstrated significant variable responses to elevated CO2. These data link the regulation of crucial early developmental processes with the environment that these embryos would be developing within, situating the study of organismal responses to ocean acidification in a developmental context.

ATP-binding cassette (ABC) transporter expression and localization in sea urchin development

Abstract: Background: ATP-binding cassette (ABC) transporters are membrane proteins that regulate intracellular concentrations of myriad compounds and ions. There are >100 ABC transporter predictions in the Strongylocentrotus purpuratus genome, including 40 annotated ABCB, ABCC, and ABCG “multidrug efflux” transporters. Despite the importance of multidrug transporters for protection and signaling, their expression patterns have not been characterized in deuterostome embryos. Results: Sea urchin embryos expressed 20 ABCB, ABCC, and ABCG transporter genes in the first 58 hr of development, from unfertilized egg to early prism. We quantified transcripts of ABCB1a, ABCB4a, ABCC1, ABCC5a, ABCC9a, and ABCG2b, and found that ABCB1a mRNA was 10–100 times more abundant than other transporter mRNAs. In situ hybridization showed ABCB1a was expressed ubiquitously in embryos, while ABCC5a was restricted to secondary mesenchyme cells and their precursors. Fluorescent protein fusions showed localization of ABCB1a on apical cell surfaces, and ABCC5a on basolateral surfaces. Conclusions: Embryos use many ABC transporters with predicted functions in cell signaling, lysosomal and mitochondrial homeostasis, potassium channel regulation, pigmentation, and xenobiotic efflux. Detailed characterization of ABCB1a and ABCC5a revealed that they have different temporal and spatial gene expression profiles and protein localization patterns that correlate to their predicted functions in protection and development, respectively. Developmental Dynamics 241:1111–1124, 2012. © 2012 Wiley Periodicals, Inc.

Phylogenetic analysis and expression patterns of p16 and p19 in Paracentrotus lividus embryos.

Abstract: P16 and P19 are two small acidic proteins involved in the formation of the biomineralized skeleton of sea urchin embryos and adults. Here, we describe the cloning and the embryonic temporal and spatial expression profiles of p16 and p19 mRNAs, identified for the first time in Paracentrotus lividus. Phylogenetic analysis showed a high degree of similarity of the deduced Pl-P16 and Pl-P19 sequences with the Lytechinus variegatus and Strongylocentrotus purpuratus orthologs. While only a reduced similarity with other phyla, including mammals, was detected, their implication in biomineralized tissues calls for their conservation in evolution. By comparative quantitative PCR and in situ hybridization, we found that Pl-p16 and Pl-p19 expression was restricted to skeletogenic cells throughout embryogenesis, with transcript levels peaking at the late gastrula stage. Dissimilar Pl-p16 and Pl-p19 spatial expression within the primary mesenchyme cell syncytium at the gastrula and pluteus stages suggests the occurrence of a different regulation of gene transcription.

Autonomy in specification of primordial germ cells and their passive translocation in the sea urchin.

Abstract: The process of germ line determination involves many conserved genes, yet is highly variable. Echinoderms are positioned at the base of Deuterostomia and are crucial to understanding these evolutionary transitions, yet the mechanism of germ line specification is not known in any member of the phyla. Here we demonstrate that small micromeres (SMics), which are formed at the fifth cell division of the sea urchin embryo, illustrate many typical features of primordial germ cell (PGC) specification. SMics autonomously express germ line genes in isolated culture, including selective Vasa protein accumulation and transcriptional activation of nanos; their descendants are passively displaced towards the animal pole by secondary mesenchyme cells and the elongating archenteron during gastrulation; Cadherin (G form) has an important role in their development and clustering phenotype; and a left/right integration into the future adult anlagen appears to be controlled by a late developmental mechanism. These results suggest that sea urchin SMics share many more characteristics typical of PGCs than previously thought, and imply a more widely conserved system of germ line development among metazoans.

Development Under Elevated pCO2 Conditions Does Not Affect Lipid Utilization and Protein Content in Early Life-History Stages of the Purple Sea Urchin, Strongylocentrotus purpuratus

Abstract: Ocean acidification (OA) is expected to have a major impact on marine species, particularly during early life-history stages. These effects appear to be species-specific and may include reduced survival, altered morphology, and depressed metabolism. However, less information is available regarding the bioenergetics of development under elevated CO(2) conditions. We examined the biochemical and morphological responses of Strongylocentrotus purpuratus during early development under ecologically relevant levels of pCO(2) (365, 1030, and 1450 μatm) that may occur during intense upwelling events. The principal findings of this study were (1) lipid utilization rates and protein content in S. purpuratus did not vary with pCO(2); (2) larval growth was reduced at elevated pCO(2) despite similar rates of energy utilization; and (3) relationships between egg phospholipid content and larval length were found under control but not high pCO(2) conditions. These results suggest that this species may either prioritize endogenous energy toward development and physiological function at the expense of growth, or that reduced larval length may be strictly due to higher costs of growth under OA conditions. This study highlights the need to further expand our knowledge of the physiological mechanisms involved in OA response in order to better understand how present populations may respond to global environmental change.

Innate Immune Complexity in the Purple Sea Urchin: Diversity of the Sp185/333 System

Abstract: The California purple sea urchin, Strongylocentrotus purpuratus, is a long-lived echinoderm with a complex and sophisticated innate immune system. There are several large gene families that function in immunity in this species including the Sp185/333 gene family that has ∼50 (±10) members. The family shows intriguing sequence diversity and encodes a broad array of diverse yet similar proteins. The genes have two exons of which the second encodes the mature protein and has repeats and blocks of sequence called elements. Mosaics of element patterns plus single nucleotide polymorphisms-based variants of the elements result in significant sequence diversity among the genes yet maintains similar structure among the members of the family. Sequence of a bacterial artificial chromosome insert shows a cluster of six, tightly linked Sp185/333 genes that are flanked by GA microsatellites. The sequences between the GA microsatellites in which the Sp185/333 genes and flanking regions are located, are much more similar to each other than are the sequences outside the microsatellites suggesting processes such as gene conversion, recombination, or duplication. However, close linkage does not correspond with greater sequence similarity compared to randomly cloned and sequenced genes that are unlikely to be linked. There are three segmental duplications that are bounded by GAT microsatellites and include three almost identical genes plus flanking regions. RNA editing is detectible throughout the mRNAs based on comparisons to the genes, which, in combination with putative post-translational modifications to the proteins, results in broad arrays of Sp185/333 proteins that differ among individuals. The mature proteins have an N-terminal glycine-rich region, a central RGD motif, and a C-terminal histidine-rich region. The Sp185/333 proteins are localized to the cell surface and are found within vesicles in subsets of polygonal and small phagocytes. The coelomocyte proteome shows full-length and truncated proteins, including some with missense sequence. Current results suggest that both native Sp185/333 proteins and a recombinant protein bind bacteria and are likely important in sea urchin innate immunity.

The protein precursors of peptides that affect the mechanics of connective tissue and/or muscle in the echinoderm Apostichopus japonicus.

Abstract: Peptides that cause muscle relaxation or contraction or that modulate electrically-induced muscle contraction have been discovered in the sea cucumber Apostichopus japonicus (Phylum Echinodermata; Class Holothuroidea). By analysing transcriptome sequence data, here the protein precursors of six of these myoactive peptides (the SALMFamides Sticho-MFamide-1 and -2, NGIWYamide, stichopin, GN-19 and GLRFA) have been identified, providing novel insights on neuropeptide and endocrine-type signalling systems in echinoderms. The A. japonicus SALMFamide precursor comprises eight putative neuropeptides including both L-type and F-type SALMFamides, which contrasts with previous findings from the sea urchin Strongylocentrotus purpuratus where L-type and F-type SALMFamides are encoded by different genes. The NGIWYamide precursor contains five copies of NGIWYamide but, unlike other NG peptide-type neuropeptide precursors in deuterostomian invertebrates, the NGIWYamide precursor does not have a C-terminal neurophysin domain, indicating loss of this character in holothurians. NGIWYamide was originally discovered as a muscle contractant, but it also causes stiffening of mutable connective tissue in the body wall of A. japonicus, whilst holokinins (PLGYMFR and derivative peptides) cause softening of the body wall. However, the mechanisms by which these peptides affect the stiffness of body wall connective tissue are unknown. Interestingly, analysis of the A. japonicus transcriptome reveals that the only protein containing the holokinin sequence PLGYMFR is an alpha-5 type collagen. This suggests that proteolysis of collagen may generate peptides (holokinins) that affect body wall stiffness in sea cucumbers, providing a novel perspective on mechanisms of mutable connective tissue in echinoderms.

Histamine is a modulator of metamorphic competence in Strongylocentrotus purpuratus (Echinodermata: Echinoidea)

Abstract: A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus. Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae. We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom - metamorphosis.

Iodine accumulation in sea urchin larvae is dependent on peroxide

Abstract: SUMMARY Iodine has many important biological functions and its concentrations vary with the environment. Recent research has provided novel insights into iodine uptake mechanisms in marine bacteria and kelp through hydrogen peroxide-dependent diffusion (PDD). This mechanism is distinct from sodium-dependent mechanisms known from vertebrates. In vertebrates, iodine accumulates in the thyroid gland by the action of the apical iodide transporter (AIT) and the sodium/iodide symporter (NIS). Neither of these proteins has, thus far, been identified outside of the chordates, and PDD (as an iodine uptake mechanism) has never been studied in animals. Using 125 I as a marker for total iodine influx, we tested iodine uptake via sodium-dependent transport versus PDD in embryos and larvae of the sea urchin Strongylocentrotus purpuratus . We found that iodine uptake in S. purpuratus is largely independent of NIS/AIT. Instead, we found that uptake is dependent on the presence and production of hydrogen peroxide, indicating that sea urchin larvae use PDD as a mechanism for iodine acquisition. Our data, for the first time, provide conclusive evidence for this mechanism in an animal. Furthermore, our data provide preliminary evidence that sodium-dependent iodine uptake via active transporter proteins is a synapomorphy of vertebrates.

Population genetics of cis-regulatory sequences that operate during embryonic development in the sea urchin Strongylocentrotus purpuratus.

Abstract: Despite the fact that noncoding sequences comprise a substantial fraction of functional sites within all genomes, the evolutionary mechanisms that operate on genetic variation within regulatory elements remain poorly understood. In this study, we examine the population genetics of the core, upstream cis-regulatory regions of eight genes (AN, CyIIa, CyIIIa, Endo16, FoxB, HE, SM30 a, and SM50) that function during the early development of the purple sea urchin, Strongylocentrotus purpuratus. Quantitative and qualitative measures of segregating variation are not conspicuously different between cis-regulatory and closely linked "proxy neutral" noncoding regions containing no known functional sites. Length and compound mutations are common in noncoding sequences; conventional descriptive statistics ignore such mutations, under-representing true genetic variation by approximately 28% for these loci in this population. Patterns of variation in the cis-regulatory regions of six of the genes examined (CyIIa, CyIIIa, Endo16, FoxB, AN, and HE) are consistent with directional selection. Genetic variation within annotated transcription factor binding sites is comparable to, and frequently greater than, that of surrounding sequences. Comparisons of two paralog pairs (CyIIa/CyIIIa and AN/HE) suggest that distinct evolutionary processes have operated on their cis-regulatory regions following gene duplication. Together, these analyses provide a detailed view of the evolutionary mechanisms operating on noncoding sequences within a natural population, and underscore how little is known about how these processes operate on cis-regulatory sequences.

Effects of Seawater Acidification on Cell Cycle Control Mechanisms in Strongylocentrotus purpuratus Embryos

Abstract: Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1) the onset of DNA synthesis, (2) production of a mitotic regulator, cyclin B, and (3) formation of the mitotic spindle We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ∼70, 75, and 80 by CO(2) infusion Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G(1)/S, G(2)/M and metaphase/anaphase transitions at relatively similar rates These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics

Sequencing and analysis of the gastrula transcriptome of the brittle star Ophiocoma wendtii

Abstract: The gastrula stage represents the point in development at which the three primary germ layers diverge. At this point the gene regulatory networks that specify the germ layers are established and the genes that define the differentiated states of the tissues have begun to be activated. These networks have been well-characterized in sea urchins, but not in other echinoderms. Embryos of the brittle star Ophiocoma wendtii share a number of developmental features with sea urchin embryos, including the ingression of mesenchyme cells that give rise to an embryonic skeleton. Notable differences are that no micromeres are formed during cleavage divisions and no pigment cells are formed during development to the pluteus larval stage. More subtle changes in timing of developmental events also occur. To explore the molecular basis for the similarities and differences between these two echinoderms, we have sequenced and characterized the gastrula transcriptome of O. wendtii. Development of Ophiocoma wendtii embryos was characterized and RNA was isolated from the gastrula stage. A transcriptome data base was generated from this RNA and was analyzed using a variety of methods to identify transcripts expressed and to compare those transcripts to those expressed at the gastrula stage in other organisms. Using existing databases, we identified brittle star transcripts that correspond to 3,385 genes, including 1,863 genes shared with the sea urchin Strongylocentrotus purpuratus gastrula transcriptome. We characterized the functional classes of genes present in the transcriptome and compared them to those found in this sea urchin. We then examined those members of the germ-layer specific gene regulatory networks (GRNs) of S. purpuratus that are expressed in the O. wendtii gastrula. Our results indicate that there is a shared ‘genetic toolkit’ central to the echinoderm gastrula, a key stage in embryonic development, though there are also differences that reflect changes in developmental processes. The brittle star expresses genes representing all functional classes at the gastrula stage. Brittle stars and sea urchins have comparable numbers of each class of genes and share many of the genes expressed at gastrulation. Examination of the brittle star genes in which sea urchin orthologs are utilized in germ layer specification reveals a relatively higher level of conservation of key regulatory components compared to the overall transcriptome. We also identify genes that were either lost or whose temporal expression has diverged from that of sea urchins.

Current Status of Echinoderm Genome Analysis - What do we Know?

Abstract: Echinoderms have long served as model organisms for a variety of biological research, especially in the field of developmental biology. Although the genome of the purple sea urchin Strongylocentrotus purpuratus has been sequenced, it is the only echinoderm whose whole genome sequence has been reported. Nevertheless, data is rapidly accumulating on the chromosomes and genomic sequences of all five classes of echinoderms, including the mitochondrial genomes and Hox genes. This blossoming new data will be essential for estimating the phylogenetic relationships among echinoderms, and also to examine the underlying mechanisms by which the diverse morphologies of echinoderms have arisen.

Proteomic Responses of Sea Urchin Embryos to Stressful Ultraviolet Radiation

Abstract: Solar ultraviolet radiation (UVR, 290-400 nm) penetrates into seawater and can harm shallow-dwelling and planktonic marine organisms. Studies dating back to the 1930s revealed that echinoids, especially sea urchin embryos, are powerful models for deciphering the effects of UVR on embryonic development and how embryos defend themselves against UV-induced damage. In addition to providing a large number of synchronously developing embryos amenable to cellular, biochemical, molecular, and single-cell analyses, the purple sea urchin, Strongylocentrotus purpuratus, also offers an annotated genome. Together, these aspects allow for the in-depth study of molecular and biochemical signatures of UVR stress. Here, we review the effects of UVR on embryonic development, focusing on the early-cleavage stages, and begin to integrate data regarding single-protein responses with comprehensive proteomic assessments. Proteomic studies reveal changes in levels of post-translational modifications to proteins that respond to UVR, and identify proteins that can then be interrogated as putative targets or components of stress-response pathways. These responsive proteins are distributed among systems upon which targeted studies can now begin to be mapped. Post-transcriptional and translational controls may provide early embryos with a rapid, fine-tuned response to stress during early stages, especially during pre-blastula stages that rely primarily on maternally derived defenses rather than on responses through zygotic gene transcription.

Influence of stocking density and temperature on early development and survival of the purple sea urchin, Strongylocentrotus purpuratus (Stimpson, 1857)

Abstract: We evaluated the effect of four densities (940, 1880, 3760, 7520 eggs cm−2 and 0.5, 1, 2, 4 ind mL−1 of embryos and larvae, respectively) and four temperatures (8, 11, 14, 17°C) on early growth and survival of the sea urchin Strongylocentrotus purpuratus. Prism-stage length was significantly greater in embryos initially held at 940 and 1880 eggs cm−2 than in those held at 3760 and 7520 eggs cm−2. Larvae grew significantly faster and had significantly greater survival when reared at 0.5 or 1 ind mL−1 than when held at 2 or 4 ind mL−1. Embryos had greater survival at 11 and 14°C than at 8 and 17°C, whereas embryo length was significantly smaller at 8°C than at 11, 14 or 17°C. Larvae grew significantly slower at 8°C than at 11, 14 or 17°C, whereas survival was significantly reduced at 8 and 17°C compared with 11 and 14°C. Per cent survival from prism to metamorphic competency in the best treatments was 48.9 ± 2.2% and 50.0 ± 3.6% (mean ± SE) for the 1 ind mL−1 and 11°C treatments, respectively. On the basis of these results, for rearing of S. purpuratus under static conditions, we recommend that fertilized eggs and larvae be held at ≤1880 eggs cm−2 and ≤1 ind mL−1, respectively, and at 11–14°C.

Seasonal variation in the effects of food availability on gametogenesis in the purple urchin (Strongylocentrotus purpuratus)

Abstract: The amount of food and when it is available affect both the timing of reproduction and the number of gametes produced by purple sea urchins, Strongylocentrotus purpuratus To investigate this further, the effects of food availability on feeding rates, gonad growth, and gamete development were examined in S purpuratus collected from the Point Loma kelp forest near San Diego, California, USA (3269° N, 11726° W) in September (Fall) 2007, and February (Spring) and July (Summer) 2008, using laboratory mesocosms Each seasonal laboratory feeding experiment lasted 3 months, and different levels of food availability were established with different feeding frequencies (from 1 to 7 days week−1) Gonad tissues of male and female urchins were staged at the end of each experiment using histological analyses Reduced food availability resulted in increased daily consumption rates, especially in the Fall when gamete development began Food limitation at this time resulted in failure to produce viable gametes, suggesting there is a critical period early in gonad development when food limitation affects reproductive competency Food limitation later in gonad development did not stop viable gamete production, although it did reduce gamete output

Nucleosome exclusion from the interspecies-conserved central AT-rich region of the Ars insulator

Abstract: The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC) These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator

Sex and microhabitat influence the uptake and allocation of mycosporine-like amino acids to tissues in the purple sea urchin, Strongylocentrotus purpuratus

Abstract: UVR-absorbing mycosporine-like amino acids (MAAs) were detected in tissues of Strongylocentrotus purpuratus and in ten species of Rhodophyte macroalgae (eight previously untested) collected from intertidal microhabitats in November and January 2006–2007 in Central California (35°09′N, 120°45′W). In sea urchins, MAA concentrations were higher in ovaries than testes, while epidermal concentrations were similar between sexes. Ovaries and epidermal tissues had similar MAA signatures and broadband UVA/UVB absorbance, while testes had a narrower absorption ranges shifted toward higher energy wavelengths. Sea urchins occupying pits in the substrate exhibited lower MAA concentrations than those outside pits, suggesting adult microhabitat may impact UV protection. Light levels did not influence gonadal MAA concentrations, but correlated with elevated epidermal MAA concentrations for males in the sunniest microhabitat. This study suggests sex and habitat strongly influence MAA concentrations among individual S. purpuratus and that allocation of MAA sunscreens to tissues in response to UVR is sex-dependent.

Calbindin-D32k Is Localized to a Subpopulation of Neurons in the Nervous System of the Sea Cucumber Holothuria glaberrima (Echinodermata)

Abstract: Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates.

Ocean conditions and bottom-up modifications of gonad development in the sea urchin Strongylocentrotus purpuratus over space and time

Abstract: Intraspecific life history attributes of growth, survival, and reproduction can vary in response to changes in the physical environment. These changes can induce a cascading effect across trophic levels. In marine systems, shifts in ocean conditions such as warm and cold phases of ENSO can change primary production in benthic algae, which in turn modify resources avail- able to benthic omnivores. Monthly gonad samples of the purple sea urchin Strongylocentrotus purpuratus were taken from 2007 to mid-2009 at sites from Vancouver Island, Canada, to Punta Baja, Mexico. Although mean sea surface temperature (SST) changed by ~8°C from north to south, latitudinal patterns of size-specific gonad weight were not detected, although differences existed across sites. At individual sites, mean SST changed by <1°C from 2007 to 2009. Gonad index data from Yankee Point, California (USA), from 1952 to 1964 included mean annual temper- atures that varied by 2.4°C and both strong El Nino and La Nina events. Yankee Point data showed significant (p ≤ 0.01) correlations with the Multivariate ENSO Index, SST, the Northern Oscillation Index, and the Oceanic Nino Index. Dissections at other sites along the coast from mid- 1960 to 2009 showed that size-specific gonad weights were lower during periods of El Nino than during periods of La Nina.

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

Abstract: To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript w...

Genomics in the Sea Urchin: New Perspectives on a Perennial Model System

Abstract: The first genome sequenced from an echinoderm was completed from the purple sea urchin, Strongylocentrotus purpuratus, as part of the Sea Urchin Genome Project. Here, we describe how this resource has synergized with the long-standing repertoire of experimental techniques developed for the sea urchin over the last century to position the sea urchin as a key model organism for studying modern genome biology. The Sea Urchin Genome Project continues to advance studies in this phylum, as genomes from six additional echinoderms are currently at various stages of sequencing. These species were selected to cover a broad phylogenetic distribution and comprise a set of sequences for powerful comparative analyses. In combination with its rich catalog of experimental methods, the genome sequence provides a framework for addressing question in gene regulatory network biology within a deuterostome model that has close affinities to vertebrates. Furthermore, this invertebrate genome sequence samples an entirely new branch of animal phylogeny, and the biology implied by the complexity of what it encodes reveals unprecedented directions of animal evolution.

Effects of increased pCO 2 and geographic origin on purple sea urchin (Strongylocentrotus purpuratus) calcite elemental composition

Abstract: We would like to thank Anonymous Reviewer 2 for his/her constructive review of our manuscript. The comments, questions, and suggestions raised in the interactive discussion have greatly improved the manuscript. In this study we examined (1) the geochemical composition of purple sea urchin (Strongylocentrotus purpuratus) skeleton precipitated during both adult and early life history stages; (2) potential differences in geochemical composition among individuals originating from regions spanning a broad latitudinal range encompassing a spectrum of oceanographic regimes; and (3) the impact of ocean acidification on Mg and Sr incorporation into larval and juvenile S. purpuratus skeleton in culture. Both reviewers identified the strengths of the manuscript as being (1) and (2) above, and raised important questions that have strengthened our interpretation of (3) in the revised manuscript.