Cellular and molecular bases of biomineralization in sea urchin embryos

Sea urchin embryos construct their skeleton following a precise gene-regulated time- and space-dependent programme, in concert with factors promoting cell adhesion and differentiation. The biomineral is deposited in a privileged extracellular space produced by the fused filopodia processes of the primary mesenchyme cells, the only cells producing a set of necessary matrix proteins. More than ten years ago we showed for the first time that signals from ectoderm cells promoted the expression of one of the major skeleton matrix genes by the primary mesenchyme cells. Since then, many of the crucial steps of this complex activation cascade, from ectoderm cells to embryonic spicules, have been elucidated. The experimental production of skeleton malformations, induced by the exposure to toxic metals or ionizing radiations, served as model to dissect the molecular mechanisms leading to biomineralization. With the aim of understanding the sea urchin skeleton physiology, we analysed the expression of well-known and newly-identified biomineral-related genes, including those coding for growth and transcription factors as well as for skeleton matrix proteins. This review summarizes our recent findings on sea urchin embryo skeletogenesis, with a particular attention to the role played by cellular and molecular signaling, approached by the use of experimentally induced skeleton malformations

Defensome against Toxic Diatom Aldehydes in the Sea Urchin Paracentrotus lividus

Many diatom species produce polyunsaturated aldehydes, such as decadienal, which compromise embryonic and larval development in benthic organisms. Here newly fertilized Paracentrotus lividus sea urchins were exposed to low concentration of decadienal and the expression levels of sixteen genes, implicated in a broad range of functional responses, were followed by Real Time qPCR in order to identify potential decadienal targets. We show that at low decadienal concentrations the sea urchin Paracentrotus lividus places in motion different classes of genes to defend itself against this toxic aldehyde, activating hsp60 and two proteases, hat and BP10, at the blastula stage and hsp56 and several other genes (14-3-3ε, p38 MAPK, MTase, and GS) at the prism stage. At this latter stage all genes involved in skeletogenesis (Nec, uni, SM50 and SM30) were also down-expressed, following developmental abnormalities that mainly affected skeleton morphogenesis. Moreover, sea urchin embryos treated with increasing concentrations of decadienal revealed a dose-dependent response of activated target genes. Finally, we suggest that this orchestrated defense system against decadienal represents part of the chemical defensome of P. lividus affording protection from environmental toxicants

Understanding How Microplastics Affect Marine Biota on the Cellular Level Is Important for Assessing Ecosystem Function: A Review

Plastic has become indispensable for human life. When plastic debris is discarded into waterways, these items can interact with organisms. Of particular concern are microscopic plastic particles (microplastics) which are subject to ingestion by several taxa. This review summarizes the results of cutting-edge research about the interactions between a range of aquatic species and microplastics, including effects on biota physiology and secondary ingestion. Uptake pathways via digestive or ventilatory systems are discussed, including (1) the physical penetration of microplastic particles into cellular structures, (2) leaching of chemical additives or adsorbed persistent organic pollutants (POPs), and (3) consequences of bacterial or viral microbiota contamination associated with microplastic ingestion. Following uptake, a number of individual-level effects have been observed, including reduction of feeding activities, reduced growth and reproduction through cellular modifications, and oxidative stress. Microplastic-associated effects on marine biota have become increasingly investigated with growing concerns regarding human health through trophic transfer. We argue that research on the cellular interactions with microplastics provide an understanding of their impact to the organisms’ fitness and, therefore, its ability to sustain their functional role in the ecosystem. The review summarizes information from 236 scientific publications. Of those, only 4.6% extrapolate their research of microplastic intake on individual species to the impact on ecosystem functioning. We emphasize the need for risk evaluation from organismal effects to an ecosystem level to effectively evaluate the effect of microplastic pollution on marine environments. Further studies are encouraged to investigate sublethal effects in the context of environmentally relevant microplastic pollution conditions

Manganese overload affects p38 MAPK phosphorylation and metalloproteinase activity during sea urchin embryonic development.

In the marine environment, manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. In earlier reports we found that the exposure of Paracentrotus lividus sea urchin embryos to manganese produced phenotypes with no skeleton. In addition, manganese interfered with calcium uptake, perturbed extracellular signal-regulated kinase (ERK) signaling, affected the expression of skeletogenic genes, and caused an increase of the hsc70 and hsc60 protein levels. Here, we extended our studies focusing on the temporal activation of the p38 mitogen-activated protein kinase (p38 MAPK) and the proteolytic activity of metalloproteinases (MMPs). We found that manganese affects the stage-dependent dynamics of p38 MAPK activation and inhibits the total gelatin-auto-cleaving activity of MMPs, with the exclusion of the 90-85 kDa and 68-58 kDa MMPs, whose levels remain high all throughout development. Our findings correlate, for the first time to our knowledge, an altered activation pattern of the p38 MAPK with an aberrant MMP proteolytic activity in the sea urchin embryo

Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects.

Manganese (Mn), one of the most abundant metals in nature present in rocks, soil and water, is also found in soft bottom sediments of the oceans. It represents a trace element that is accumulated and utilized by all forms of life and plays multiple roles ranging from bone mineralization to cellular protection. Although Mn is an essential nutrient, exposure of cells/organisms to high levels of Mn cause toxicity. In the marine environment, increased concentrations of bio-available Mn often result from anthropogenic activities, and consequently, Mn represents a new important factor in environmental contamination. Emission of Mn into the marine environment occurs from metallurgic and chemical industries including municipal wastewater discharges, sewage sludge, mining and mineral processing, emissions from alloy, steel, and iron production. Thus, a sharp Mn solubility gradient exists along the oxic/anoxic interfaces in such water columns. During hypoxia, that in many costal areas occurs as a result of eutrophication, Mn is released in a divalent ionic form (Mn2+) and reaches high concentrations in bottom waters. In this study, we investigated the effects of manganese chloride (MnCl2) on embryos of the sea urchin Paracentrotus lividus used as a model of Mn-induced toxicity. Embryos were continuously exposed to different MnCl2 concentrations from fertilization to the pluteus stage and examined after 6,12, 20, 24, 48, 72 hours. We found a severe dose-dependent inhibition of embryonic development, with consequent specific malformations. The EC50 value was determined in 7,7 mg/l, defined as the concentration causing 50% abnormal embryo development. These effects were rapidly reversed upon Mn washout, except after 40 hours of treatment. Major developmental defects consisted in the absence or reduced elongation of skeletal rods (spicules), suggesting a key role for Mn in embryonic skeleton development. Mn accumulation was determined in exposed embryos by AAS analysis and compared to physiological calcium (Ca) concentration found in the same specimens. We found that the Mn is accumulated into embryos in a time and dose-dependent manner, with a drastic increase 24 hrs post-fertilization. In contrast, Ca concentration is reduced in an opposite proportional way, consistent with its poor detection in primary mesenchyme cells, observed by in vivo labelling with cell permeable fluorescent calcein. A direct correlation has been observed between malformations, accumulation of Mn ions and the regulation of key stress proteins that provide the major protection against stressors. We found that Mn exposure caused an increase in HSC70 and HSC60 levels, but not in HSC90 levels and did not induce synthesis of the HSPs inducible forms. Analysis by 2D gel electrophoresis showed different patterns of protein spots in control and exposed embryos highlighting qualitative protein expression differences in response to Mn exposure. No increase in apoptosis in comparison to controls was measured by the TUNEL assay, as well as no reactive oxygen species (ROS) production was found by a fluorescent detection assay on live embryos. To conclude, developmental and stress response effects observed could be explained as a Mn toxicity linked to the accumulation and bio-concentration of Mn into the embryos tissues and a possible competition between Mn and Ca for membrane transport sites or other specific molecular targets

Impacts of UV-B radiation on short-term cultures of sea urchin coelomocytes

Three specialized cell types constitute the heterogeneous population present in the coelomic cavity fluid of sea urchins. The list includes: phagocytes, which undergo a stress-induced petaloid-filopodial transition, white or red amoebocytes and vibratile cells. As a whole, they act as the immune defense system of the sea urchin and respond to environmental and experimental challenge triggering specific stress markers. Here we extended our studies on coelomocytes short-term cultures by describing the morphology and occurrence of each cell type and analyzing their response to UV-B radiation at the biochemical level and with respect to DNA damage. The effects of different doses, ranging from 500 to 2,000 J/m2, on cells, which were then cultured in vitro for 1-6 h were tested. As early as 1 h after irradiation we found an increase in the levels of the heat shock protein70 (hsp) stress marker. A peak at 1,000 J/m2 corresponding to a 3.4-fold hsp70 increase over the levels of control coelomocytes was observed. Concurrently, we found an increase in the number of apoptotic nuclei detected by Hoechst staining, which varied from 7.1 to 15.6% in cells that were exposed to 500 and 2,000 J/m2, respectively. On the basis of our findings we confirm sea urchin coelomocytes as a sensitive cell culture system for environmental studies and propose their use for the analysis on the effects of UV-B radiation

Manganese Interferes with Calcium, Perturbs ERK Signaling, and Produces Embryos with No Skeleton

Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found that Mn exposure produced embryos with no skeleton, by preventing the deposition of the triradiate calcitic spicules usually produced only by specialized mesoderm cells. On the contrary, ectoderm and endoderm differentiation was not impaired. Endogenous Ca content in whole embryos and its localization in Golgi regions of skeletogenic cells was strongly reduced, as measured by atomic absorption spectrometry and in vivo calcein labeling. Spicule-lacking embryos showed persistent ERK activation by immunocytochemistry and immunoblotting, contrary to the physiological oscillations observed in normal embryos. The expression of the skeletogenic genes, Pl-msp130 and Pl-sm30, was also differentially affected if compared with controls. Here, we showed for the first time the ability of Mn to interfere with Ca uptake and internalization into skeletogenic cells and demonstrate that Ca content regulates ERK activation/inactivation during sea urchin embryo morphogenesis. The use of Mn-exposed sea urchin embryos as a new model to study signaling pathways occurring during skeletogenesis will provide new insights into the mechanisms involved in Mn embryo toxicity and underlie the role of calcium in the biomineralization process in vertebrate

Long-term environmental exposure to metals (Cu, Cd, Pb, Zn) activates the immune cell stress response in the common European sea star (Asterias rubens)

The common sea star Asterias rubens represents a key-species of the North-Eastern Atlantic macro benthic community. The cells of their immune system, known as coelomocytes, are the first line of defence against environmental hazards. Here, we report the results of investigations on the immune cells response of sea stars exposed to marine environmental pollution for long periods. We show that levels of the heat shock cognate protein 70 (HSC70) in coelomocytes from A. rubens, which were collected during a field study in the Sǿrfjord (North Sea, SW coast of Norway) along a contamination gradient, are directly associated with the long-term accumulation of Cd, Cu heavy metals exclusively in the tegument. Conversely, Pb and Zn accumulation in the tegument did not relate to HSC70 levels and none of the metals were found accumulated in the pyloric coeca. In addition the coelomocytes from A. rubens, collected in high and low metal impacted stations were examined by a proteomic approach using two-dimensional electrophoresis (2DE). By comparison of the proteomic maps, we observed that 31 protein spots differed in their relative abundance, indicating a gene expression response to the metal mixture exposure. All together, our results confirm that the echinoderm immune cells are a suitable model for the assessment of long-term exposure to environmental pollution, moreover that the increased level of HSC70 can be considered a signal of an acquired tolerance within a large spectrum of protein profile changes occurring in response to metal contamination. © 2011 Elsevier Ltd.IF: 1,953SCOPUS: ar.jinfo:eu-repo/semantics/publishe