Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

Capture and return of sexual genomes by hybridogenetic frogs provide clonal genome enrichment in a sexual species.

Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.

New data from Monoplacophora and a carefully-curated dataset resolve molluscan relationships.

Relationships among the major lineages of Mollusca have long been debated. Morphological studies have considered the rarely collected Monoplacophora (Tryblidia) to have several plesiomorphic molluscan traits. The phylogenetic position of this group is contentious as morphologists have generally placed this clade as the sister taxon of the rest of Conchifera whereas earlier molecular studies supported a clade of Monoplacophora + Polyplacophora (Serialia) and phylogenomic studies have generally recovered a clade of Monoplacophora + Cephalopoda. Phylogenomic studies have also strongly supported a clade including Gastropoda, Bivalvia, and Scaphopoda, but relationships among these taxa have been inconsistent. In order to resolve conchiferan relationships and improve understanding of early molluscan evolution, we carefully curated a high-quality data matrix and conducted phylogenomic analyses with broad taxon sampling including newly sequenced genomic data from the monoplacophoran Laevipilina antarctica. Whereas a partitioned maximum likelihood (ML) analysis using site-homogeneous models recovered Monoplacophora sister to Cephalopoda with moderate support, both ML and Bayesian inference (BI) analyses using mixture models recovered Monoplacophora sister to all other conchiferans with strong support. A supertree approach also recovered Monoplacophora as the sister taxon of a clade composed of the rest of Conchifera. Gastropoda was recovered as the sister taxon of Scaphopoda in most analyses, which was strongly supported when mixture models were used. A molecular clock based on our BI topology dates diversification of Mollusca to ~546 MYA (+/- 6 MYA) and Conchifera to ~540 MYA (+/- 9 MYA), generally consistent with previous work employing nuclear housekeeping genes. These results provide important resolution of conchiferan mollusc phylogeny and offer new insights into ancestral character states of major mollusc clades.

The developmental transcriptome for Lytechinus variegatus exhibits temporally punctuated gene expression changes.

Embryonic development is arguably the most complex process an organism undergoes during its lifetime, and understanding this complexity is best approached with a systems-level perspective. The sea urchin has become a highly valuable model organism for understanding developmental specification, morphogenesis, and evolution. As a non-chordate deuterostome, the sea urchin occupies an important evolutionary niche between protostomes and vertebrates. Lytechinus variegatus (Lv) is an Atlantic species that has been well studied, and which has provided important insights into signal transduction, patterning, and morphogenetic changes during embryonic and larval development. The Pacific species, Strongylocentrotus purpuratus (Sp), is another well-studied sea urchin, particularly for gene regulatory networks (GRNs) and cis-regulatory analyses. A well-annotated genome and transcriptome for Sp are available, but similar resources have not been developed for Lv. Here, we provide an analysis of the Lv transcriptome at 11 timepoints during embryonic and larval development. Temporal analysis suggests that the gene regulatory networks that underlie specification are well-conserved among sea urchin species. We show that the major transitions in variation of embryonic transcription divide the developmental time series into four distinct, temporally sequential phases. Our work shows that sea urchin development occurs via sequential intervals of relatively stable gene expression states that are punctuated by abrupt transitions.

Mitigating Anticipated Effects of Systematic Errors Supports Sister-Group Relationship between Xenacoelomorpha and Ambulacraria.

Xenoturbella and the acoelomorph worms (Xenacoelomorpha) are simple marine animals with controversial affinities. They have been placed as the sister group of all other bilaterian animals (Nephrozoa hypothesis), implying their simplicity is an ancient characteristic [1, 2]; alternatively, they have been linked to the complex Ambulacraria (echinoderms and hemichordates) in a clade called the Xenambulacraria [3-5], suggesting their simplicity evolved by reduction from a complex ancestor. The difficulty resolving this problem implies the phylogenetic signal supporting the correct solution is weak and affected by inadequate modeling, creating a misleading non-phylogenetic signal. The idea that the Nephrozoa hypothesis might be an artifact is prompted by the faster molecular evolutionary rate observed within the Acoelomorpha. Unequal rates of evolution are known to result in the systematic artifact of long branch attraction, which would be predicted to result in an attraction between long-branch acoelomorphs and the outgroup, pulling them toward the root [6]. Other biases inadequately accommodated by the models used can also have strong effects, exacerbated in the context of short internal branches and long terminal branches [7]. We have assembled a large and informative dataset to address this problem. Analyses designed to reduce or to emphasize misleading signals show the Nephrozoa hypothesis is supported under conditions expected to exacerbate errors, and the Xenambulacraria hypothesis is preferred in conditions designed to reduce errors. Our reanalyses of two other recently published datasets [1, 2] produce the same result. We conclude that the Xenacoelomorpha are simplified relatives of the Ambulacraria.

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins.

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.

The complete mitochondrial genome of Telmatobufo australis (Amphibia: Anura: Calyptocephalellidae).

The mitochondrial (mt) genome of Telmatobufo australis is a circular molecule of 17,989 bp in length, comprising 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. Gene order and content are identical to those previously reported from other neobatrachian mt genomes. Two protein-coding genes (COI and ATP6) presumably used GTG as start codons while COIII possessed an incomplete stop codon.

The nervous system of Xenacoelomorpha: a genomic perspective.

Xenacoelomorpha is, most probably, a monophyletic group that includes three clades: Acoela, Nemertodermatida and Xenoturbellida. The group still has contentious phylogenetic affinities; though most authors place it as the sister group of the remaining bilaterians, some would include it as a fourth phylum within the Deuterostomia. Over the past few years, our group, along with others, has undertaken a systematic study of the microscopic anatomy of these worms; our main aim is to understand the structure and development of the nervous system. This research plan has been aided by the use of molecular/developmental tools, the most important of which has been the sequencing of the complete genomes and transcriptomes of different members of the three clades. The data obtained has been used to analyse the evolutionary history of gene families and to study their expression patterns during development, in both space and time. A major focus of our research is the origin of 'cephalized' (centralized) nervous systems. How complex brains are assembled from simpler neuronal arrays has been a matter of intense debate for at least 100 years. We are now tackling this issue using Xenacoelomorpha models. These represent an ideal system for this work because the members of the three clades have nervous systems with different degrees of cephalization; from the relatively simple sub-epithelial net of Xenoturbella to the compact brain of acoels. How this process of 'progressive' cephalization is reflected in the genomes or transcriptomes of these three groups of animals is the subject of this paper.

LTR retroelements are intrinsic components of transcriptional networks in frogs.

BACKGROUND: LTR retroelements (LTR REs) constitute a major group of transposable elements widely distributed in eukaryotic genomes. Through their own mechanism of retrotranscription LTR REs enrich the genomic landscape by providing genetic variability, thus contributing to genome structure and organization. Nonetheless, transcriptomic activity of LTR REs still remains an obscure domain within cell, developmental, and organism biology. RESULTS: Here we present a first comparative analysis of LTR REs for anuran amphibians based on a full depth coverage transcriptome of the European pool frog, Pelophylax lessonae, the genome of the African clawed frog, Silurana tropicalis (release v7.1), and additional transcriptomes of S. tropicalis and Cyclorana alboguttata. We identified over 1000 copies of LTR REs from all four families (Bel/Pao, Ty1/Copia, Ty3/Gypsy, Retroviridae) in the genome of S. tropicalis and discovered transcripts of several of these elements in all RNA-seq datasets analyzed. Elements of the Ty3/Gypsy family were most active, especially Amn-san elements, which accounted for approximately 0.27% of the genome in Silurana. Some elements exhibited tissue specific expression patterns, for example Hydra1.1 and MuERV-like elements in Pelophylax. In S. tropicalis considerable transcription of LTR REs was observed during embryogenesis as soon as the embryonic genome became activated, i.e. at midblastula transition. In the course of embryonic development the spectrum of transcribed LTR REs changed; during gastrulation and neurulation MuERV-like and SnRV like retroviruses were abundantly transcribed while during organogenesis transcripts of the XEN1 retroviruses became much more active. CONCLUSIONS: The differential expression of LTR REs during embryogenesis in concert with their tissue-specificity and the protein domains they encode are evidence for the functional roles these elements play as integrative parts of complex regulatory networks. Our results support the meanwhile widely accepted concept that retroelements are not simple "junk DNA" or "harmful genomic parasites" but essential components of the transcriptomic machinery in vertebrates.

Massively parallel RNA sequencing identifies a complex immune gene repertoire in the lophotrochozoan Mytilus edulis.

The marine mussel Mytilus edulis and its closely related sister species are distributed world-wide and play an important role in coastal ecology and economy. The diversification in different species and their hybrids, broad ecological distribution, as well as the filter feeding mode of life has made this genus an attractive model to investigate physiological and molecular adaptations and responses to various biotic and abiotic environmental factors. In the present study we investigated the immune system of Mytilus, which may contribute to the ecological plasticity of this species. We generated a large Mytilus transcriptome database from different tissues of immune challenged and stress treated individuals from the Baltic Sea using 454 pyrosequencing. Phylogenetic comparison of orthologous groups of 23 species demonstrated the basal position of lophotrochozoans within protostomes. The investigation of immune related transcripts revealed a complex repertoire of innate recognition receptors and downstream pathway members including transcripts for 27 toll-like receptors and 524 C1q domain containing transcripts. NOD-like receptors on the other hand were absent. We also found evidence for sophisticated TNF, autophagy and apoptosis systems as well as for cytokines. Gill tissue and hemocytes showed highest expression of putative immune related contigs and are promising tissues for further functional studies. Our results partly contrast with findings of a less complex immune repertoire in ecdysozoan and other lophotrochozoan protostomes. We show that bivalves are interesting candidates to investigate the evolution of the immune system from basal metazoans to deuterostomes and protostomes and provide a basis for future molecular work directed to immune system functioning in Mytilus.

Acoelomorph flatworms are deuterostomes related to Xenoturbella.

Xenoturbellida and Acoelomorpha are marine worms with contentious ancestry. Both were originally associated with the flatworms (Platyhelminthes), but molecular data have revised their phylogenetic positions, generally linking Xenoturbellida to the deuterostomes and positioning the Acoelomorpha as the most basally branching bilaterian group(s). Recent phylogenomic data suggested that Xenoturbellida and Acoelomorpha are sister taxa and together constitute an early branch of Bilateria. Here we assemble three independent data sets-mitochondrial genes, a phylogenomic data set of 38,330 amino-acid positions and new microRNA (miRNA) complements-and show that the position of Acoelomorpha is strongly affected by a long-branch attraction (LBA) artefact. When we minimize LBA we find consistent support for a position of both acoelomorphs and Xenoturbella within the deuterostomes. The most likely phylogeny links Xenoturbella and Acoelomorpha in a clade we call Xenacoelomorpha. The Xenacoelomorpha is the sister group of the Ambulacraria (hemichordates and echinoderms). We show that analyses of miRNA complements have been affected by character loss in the acoels and that both groups possess one miRNA and the gene Rsb66 otherwise specific to deuterostomes. In addition, Xenoturbella shares one miRNA with the ambulacrarians, and two with the acoels. This phylogeny makes sense of the shared characteristics of Xenoturbellida and Acoelomorpha, such as ciliary ultrastructure and diffuse nervous system, and implies the loss of various deuterostome characters in the Xenacoelomorpha including coelomic cavities, through gut and gill slits.

Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix.

BACKGROUND: The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously. RESULTS: Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components. CONCLUSIONS: The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins.

Data integration using scanners with SQL output--the bioscanners project at sourceforge.

There is currently no standardized approach for parsing output that the numerous bioinformatical tools generate. Because the framework approach of the Bio-toolkits has some shortcomings, we searched for alternative approaches. To this end, we evaluated scanner generators for various programming languages with respect to their potential of standalone, small and fast applications that can easily delivered on any modern and many ancient operating system. We developed sample applications that generate standard SQL database code and thereby greatly simplify the parsing work of data integration and data analysis. At the sourceforge project page the source code and some binaries for a selection of our applications are freely available at http://bioscanners.sourceforge.net.

Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin.

BACKGROUND: Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. RESULTS: The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. CONCLUSIONS: The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental phosphoproteins as phosphoryn or dentin matrix protein-1, phosphodontin may perform similar functions in the sea urchin tooth. More than half of the detected proteins were not previously identified at the protein level, thus confirming the existence of proteins only known as genomic sequences previously.

Monte Carlo analysis of an ODE Model of the Sea Urchin Endomesoderm Network.

BACKGROUND: Gene Regulatory Networks (GRNs) control the differentiation, specification and function of cells at the genomic level. The levels of interactions within large GRNs are of enormous depth and complexity. Details about many GRNs are emerging, but in most cases it is unknown to what extent they control a given process, i.e. the grade of completeness is uncertain. This uncertainty stems from limited experimental data, which is the main bottleneck for creating detailed dynamical models of cellular processes. Parameter estimation for each node is often infeasible for very large GRNs. We propose a method, based on random parameter estimations through Monte-Carlo simulations to measure completeness grades of GRNs. RESULTS: We developed a heuristic to assess the completeness of large GRNs, using ODE simulations under different conditions and randomly sampled parameter sets to detect parameter-invariant effects of perturbations. To test this heuristic, we constructed the first ODE model of the whole sea urchin endomesoderm GRN, one of the best studied large GRNs. We find that nearly 48% of the parameter-invariant effects correspond with experimental data, which is 65% of the expected optimal agreement obtained from a submodel for which kinetic parameters were estimated and used for simulations. Randomized versions of the model reproduce only 23.5% of the experimental data. CONCLUSION: The method described in this paper enables an evaluation of network topologies of GRNs without requiring any parameter values. The benefit of this method is exemplified in the first mathematical analysis of the complete Endomesoderm Network Model. The predictions we provide deliver candidate nodes in the network that are likely to be erroneous or miss unknown connections, which may need additional experiments to improve the network topology. This mathematical model can serve as a scaffold for detailed and more realistic models. We propose that our method can be used to assess a completeness grade of any GRN. This could be especially useful for GRNs involved in human diseases, where often the amount of connectivity is unknown and/or many genes/interactions are missing.

Deeply conserved chordate noncoding sequences preserve genome synteny but do not drive gene duplicate retention.

Animal genomes possess highly conserved cis-regulatory sequences that are often found near genes that regulate transcription and development. Researchers have proposed that the strong conservation of these sequences may affect the evolution of the surrounding genome, both by repressing rearrangement, and possibly by promoting duplicate gene retention. Conflicting data, however, have made the validity of these propositions unclear. Here, we use a new computational method to identify phylogenetically conserved noncoding elements (PCNEs) in a manner that is not biased by rearrangement and duplication. This method is powerful enough to identify more than a thousand PCNEs that have been conserved between vertebrates and the basal chordate amphioxus. We test 42 of our PCNEs in transgenic zebrafish assays--including examples from vertebrates and amphioxus--and find that the majority are functional enhancers. We find that PCNEs are enriched around genes with ancient synteny conservation, and that this association is strongest for extragenic PCNEs, suggesting that cis-regulatory interdigitation plays a key role in repressing genome rearrangement. Next, we classify mouse and zebrafish genes according to association with PCNEs, synteny conservation, duplication history, and presence in bidirectional promoter pairs, and use these data to cluster gene functions into a series of distinct evolutionary patterns. These results demonstrate that subfunctionalization of conserved cis-regulation has not been the primary determinate of gene duplicate retention in vertebrates. Instead, the data support the gene balance hypothesis, which proposes that duplicate retention has been driven by selection against dosage imbalances in genes with many protein connections.

Chordin is required for neural but not axial development in sea urchin embryos.

The oral-aboral (OA) axis in the sea urchin is specified by the TGFbeta family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.

In-depth, high-accuracy proteomics of sea urchin tooth organic matrix.

BACKGROUND: The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of genes potentially coding for matrix proteins, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy proteomic analysis. RESULTS: We identified 138 proteins in the matrix of tooth powder. Only 56 of these proteins were previously identified in the matrices of test (shell) and spine. Among the novel components was an interesting group of five proteins containing alanine- and proline-rich neutral or basic motifs separated by acidic glycine-rich motifs. In addition, four of the five proteins contained either one or two predicted Kazal protease inhibitor domains. The major components of tooth matrix were however largely identical to the set of spicule matrix proteins and MSP130-related proteins identified in test (shell) and spine matrix. Comparison of the matrices of crushed teeth to intact teeth revealed a marked dilution of known intracrystalline matrix proteins and a concomitant increase in some intracellular proteins. CONCLUSION: This report presents the most comprehensive list of sea urchin tooth matrix proteins available at present. The complex mixture of proteins identified may reflect many different aspects of the mineralization process. A comparison between intact tooth matrix, presumably containing odontoblast remnants, and crushed tooth matrix served to differentiate between matrix components and possible contributions of cellular remnants. Because LC-MS/MS-based methods directly measures peptides our results validate many predicted genes and confirm the existence of the corresponding proteins. Knowledge of the components of this model system may stimulate further experiments aiming at the elucidation of structure, function, and interaction of biomineral matrix components.

Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites.

Cyclic ADP-ribose is an important Ca(2+)-mobilizing cytosolic messenger synthesized from beta-NAD(+) by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCbeta and ARCgamma are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a beta-NAD(+) transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca(2+) wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.

The sea urchin (Strongylocentrotus purpuratus) test and spine proteomes.

BACKGROUND: The organic matrix of biominerals plays an important role in biomineral formation and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin, which is an important model organism for developmental biology and biomineralization, only few matrix components have been identified and characterized at the protein level. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of possible matrix proteins at the gene level, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy, proteomic analysis. RESULTS: We identified 110 proteins as components of sea urchin test and spine organic matrix. Fourty of these proteins occurred in both compartments while others were unique to their respective compartment. More than 95% of the proteins were detected in sea urchin skeletal matrices for the first time. The most abundant protein in both matrices was the previously characterized spicule matrix protein SM50, but at least eight other members of this group, many of them only known as conceptual translation products previously, were identified by mass spectrometric sequence analysis of peptides derived from in vitro matrix degradation. The matrices also contained proteins implicated in biomineralization processes previously by inhibition studies using antibodies or specific enzyme inhibitors, such as matrix metalloproteases and members of the mesenchyme-specific MSP130 family. Other components were carbonic anhydrase, collagens, echinonectin, a alpha2-macroglobulin-like protein and several proteins containing scavenger receptor cysteine-rich domains. A few possible signal transduction pathway components, such as GTP-binding proteins, a semaphorin and a possible tyrosine kinase were also identified. CONCLUSION: This report presents the most comprehensive list of sea urchin skeletal matrix proteins available at present. The complex mixture of proteins identified in matrices of the sea urchin skeleton may reflect many different aspects of the mineralization process. Because LC-MS/MS-based methods directly measures peptides our results validate many predicted genes and confirm the existence of the corresponding proteins. Considering the many newly identified matrix proteins, this proteomic study may serve as a road map for the further exploration of biomineralization processes in an important model organism.