Ars insulator protects transgenes from long-term silencing in sea urchin larva

Reporter genes have been used as a powerful tool to analyze cis-regulatory elements responsible for temporal and spatial expression in the early development of sea urchin. However, here we show that the transgenes introduced into the sea urchin embryos undergo suppression in larval stage. The transgene silencing could be one of major obstacle in the analysis of regulatory regions in the late stages of development. We previously demonstrated that a DNA fragment (ArsI) located in the upstream region of sea urchin (Hemicentrotus pulcherrimus) arylsulfatase gene has the property of an insulator. We show that tandem ArsI prevents silencing of a transgene in sea urchin larvae when the ArsI is fused to the 5′ end of the reporter gene. Furthermore, we demonstrate that DNA of the reporter gene introduced into the sea urchin eggs is methylated during development and that the ArsI protects the transgene from the DNA methylation.     

HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo

The mechanism of micromere specification is one of the central issues in sea urchin development. In this study we have identified a sea urchin homologue of ets 1 + 2. HpEts, which is maternally expressed ubiquitously during the cleavage stage and which expression becomes restricted to the skeletogenic primary mesenchyme cells (PMC) after the hatching blastula stage. The overexpression of HpEts by mRNA injection into fertilized eggs alters the cell fate of non-PMC to migratory PMC. HpEts induces the expression of a PMC-specific spicule matrix protein, SM50, but suppresses of aboral ectoderm-specific arylsulfatase and endoderm-specific HpEndo16. The overexpression of dominant negative delta HpEts which lacks the N terminal domain, in contrast, specifically represses SM50 expression and development of the spicule. In the upstream region of the SM50 gene there exists an ets binding site that functions as a positive cis-regulatory element. The results suggest that HpEts plays a key role in the differentiation of PMCs in sea urchin embryogenesis.     

Molecular Cloning and Characterization of Protein Kinase C from the Sea Urchin Lytechinus pictus

Protein kinase C (PKC) has been shown to play a role in events involved in fertilization such as activation of the Na⁺/H⁺antiporter and an NADPH dependent oxidase. In addition, it is involved in cell fate programming later in development of the sea urchin embryo. In order to further address the role of PKC in sea urchin development, we have screened a Lytechinus pictus ovary tissue cDNA library and identified one clone for sea urchin protein kinase C (suPKC1). This clone encodes a deduced protein with a molecular mass of 72.4 kDa, which shows strong homology to invertebrate and mammalian protein kinase C (PKC) sequences. PKC has been partially purified from eggs of L. pictus. This kinase activity has been shown to be dependent upon phosphatidylserine, diacylglycerol and Ca²⁺. In agreement with this biochemical data, suPKC1 has a C2 or Ca²⁺-binding domain suggesting its activity would be Ca²⁺-dependent. Polyclonal antibodies raised against peptides of the suPKC1 sequence recognize an antigen of approximately 71 kDa in DE52 fractions that contain PKC activity; this reactivity is not observed in fractions that lack PKC activity. Using a ribonuclease protection assay, we have demonstrated the presence of suPKC1 message throughout developmental stages of the sea urchin embryo.     

Toxicity of landfill leachate to sea urchin development with a focus on ammonia

Sea urchin gametes and embryos serve as a model system to evaluate toxicity in the marine environment. In this study, the toxicity of complex chemical mixtures in leachate samples to sea urchin development was examined with a focus on ammonia, which was the main contaminant of concern in most samples. Two rapid tests, the submitochondrial particle function and bacterial luminescence tests, were also used. Ammonia is highly toxic to sea urchin embryos with an EC50 of 1.3 mg l⁻¹ for the embryos of the Australian sea urchin Heliocidaris tuberculata. Leachate ammonia levels were well above these EC50 concentrations. To assess the contribution of ammonia to leachate toxicity in sea urchin development, we compared the predicted toxic units (PTU) and observed toxic units (OTU) for ammonia for each sample. The PTU/OTU comparison revealed that the sensitivity of the sea urchin embryos to ammonia were altered (enhanced or decreased) by other chemicals in the leachates. This result emphasises the need for parallel chemical analyses and a suite bioassays for evaluating the toxicity of complex and variable chemical mixtures.     

The influence of herbivores on primary producers can vary spatially and interact with disturbance

Identifying the major drivers of ecosystem change remains a central area of ecological research. Although top–down drivers of change have received particular focus, we still have little understanding of how consistently these factors control an ecosystem's shift in both directions, between different ecosystem states. Using a crossed experiment in a shallow embayment in southeastern Australia, we investigated the roles of disturbance (kelp removal) and sea urchin herbivory (via increased density) to determine their contributions to shifts away from a kelp‐dominated community. In a second experiment, done in urchin barren areas at two sites, we tested whether reductions in ambient sea urchin densities allowed an algal shift in the reverse direction. In both experiments, we observed that high densities of sea urchins could negatively influence kelp and macroalgal abundance. However, in the kelp bed, a moderate or severe disturbance resulted in a comparable algal response, irrespective of urchin density. The influence of sea urchins also varied dramatically between the two urchin barren sites. Here, reducing urchin densities resulted in algal recovery at one site, but at the other site, substantial colonisation of barren areas by canopy‐forming brown algae and Ulvales occurred across all (low, medium, and high) urchin density treatments. Our findings illustrate multiple pathways of urchin barren creation and algal recovery, and reveal that shifts both to and from an urchin barren state can occur irrespective of herbivore pressure. These alternate pathways can operate over short spatial distances or with different regimes of disturbance.     

Mathematical model for early development of the sea urchin embryo

In Xenopus and Drosophila, the nucleocytoplasmic ratio controls many aspects of cell-cycle remodeling during the transitory period that leads from fast and synchronous cell divisions of early development to the slow, carefully regulated growth and divisions of somatic cells. After the fifth cleavage in sea urchin embryos, there are four populations of differently sized blastomeres, whose interdivision times are inversely related to size. The inverse relation suggests nucleocytoplasmic control of cell division during sea urchin development as well. To investigate this possibility, we developed a mathematical model based on molecular interactions underlying early embryonic cell-cycle control. Introducing the nucleocytoplasmic ratio explicitly into the molecular mechanism, we are able to reproduce many physiological features of sea urchin development.     

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions.     

Lack of age-associated telomere shortening in long- and short-lived species of sea urchins

The red sea urchin, Strongylocentrotus franciscanus, can live in excess of 100 years while the sea urchin Lytechinus variegatus has an estimated lifespan of only 3-4 years. In an effort to understand the molecular mechanism underlying the difference in their longevity we characterized telomere biology in these species of sea urchins. Telomerase activity was found throughout early stages of development in L. variegatus and is maintained in adult tissues of L. variegatus and S. franciscanus. Terminal restriction fragment analysis indicated a lack of age-associated telomere shortening. These data suggest that long- and short-lived sea urchins do not utilize telomerase repression as a mechanism to suppress neoplastic transformation.     

Participatory Management, Popular Knowledge, and Community Empowerment: The Case of Sea Urchin Harvesting in the Vieux-Fort Area of St. Lucia

Participatory management approaches are increasingly recognized as an effective strategy for enabling the sustainable use of natural resources. The southeast coast of St. Lucia is one of the sites where a particular form of participatory management, a co-management regime, was recently developed to control the sea urchin fishery. The Caribbean Natural Resources Institute (CANARI), a NGO based in Vieux-Fort, St. Lucia, played a key role in the development of this co-management arrangement. This case study of the sea urchin fishery in Vieux-Fort examines the extent of the devolution of authority to locally-based sea urchin harvesters, explores the potential contribution of local knowledge to the understanding of sea urchin behavior, and points to elements of a strategy aimed at strengthening the organizational capacity of the core group of sea urchin harvesters. The study addresses both present practice and future possibilities in response to concrete questions raised by participants in the study.     

The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs

We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Effects of 5 azacytidine on DNA methylation and early development of sea urchins and ascidia

5-azacytidine (5-azaCR), an analogue of cytidine, inhibits nuclear DNA methylation in early sea urchin embryos. This inhibition is specific and dose-dependent. Exposure of sea urchin embryos at any stage between one-cell and blastula, to micromolar quantities of 5-azaCR invariably inhibits development beyond the blastula stage. In a substantial number of embryos arrested at the blastula stage, spicule formation proceeds although other morphological differentiation is lacking. No significant effect on development is seen if sea urchin embryos are exposed to 5-azaCR at post-blastula stages. 5-azaCR also inhibits the development of a mosaic egg such as the ascidian Phallusia mammilata at the blastula stage, indicating that both regulative (sea urchin) and mosaic (ascidian) embryos respond more or less similarly to 5-azaCR treatment.     

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

A Century of Sea Urchin Development

SYNOPSIS. In the last quarter of the nineteenth century severalinvestigators including Richard and Oskar Hertwig, Theodor Boveri,Hans Driesch, Curt Herbst, T. H. Morgan and others turned theirattention to sea urchin eggs and early embryos. This favorablecombination of outstanding investigators and the sea urchinembryo as an experimental organism contributed to a fundamentalunderstanding of the cell, fertilization and heredity. The advantagesof the sea urchin continued to be recognized as experimentalembryologists used these embryos to develop the concepts ofgradients, regulative development and inductive interactions.Then, as developmental biology arose from chemical embryology,the sea urchin embryo once again emerged as an ideal experimentalanimal, pivotal in the understanding of the molecular and developmentalbiology of eukaryotic organisms.