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In human history, many key points have characterized technological progress, such as the use of metals, which began in prehistoric times and continues to the present day, with many industrial uses [...].
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Autism spectrum disorder (ASD) is a heterogeneous collection of neurodevelopmental disorders, difficult to diagnose and currently lacking treatment options. The possibility of finding reliable biomarkers useful for early identification would offer the opportunity to intervene with treatment strategies to improve the life quality of ASD patients. To date, there are many recognized risk factors for the development of ASD, both genetic and non-genetic. Although genetic and epigenetic factors may play a critical role, the extent of their contribution to ASD risk is still under study. On the other hand, non-genetic risk factors include pollution, nutrition, infection, psychological states, and lifestyle, all together known as the exposome, which impacts the mother's and fetus's life, especially during pregnancy. Pathogenic and non-pathogenic maternal immune activation (MIA) and autoimmune diseases can cause various alterations in the fetal environment, also contributing to the etiology of ASD in offspring. Activation of monocytes, macrophages, mast cells and microglia and high production of pro-inflammatory cytokines are indeed the cause of neuroinflammation, and the latter is involved in ASD's onset and development. In this review, we focused on non-genetic risk factors, especially on the connection between inflammation, macrophage polarization and ASD syndrome, MIA, and the involvement of microglia.
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Trastorno del Espectro Autista , Efectos Tardíos de la Exposición Prenatal , Embarazo , Femenino , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Microglía/patología , Efectos Tardíos de la Exposición Prenatal/patología , Inflamación/patología , Macrófagos/patologíaRESUMEN
Carbonic anhydrases (CANs) are conserved metalloenzymes catalysing the reversible hydration of carbon dioxide into protons and bicarbonate, with important roles in cells physiology. Some CAN-coding genes were found in sea urchin genome, although only one involved in embryonic skeletogenesis was described in Paracentrotus lividus. Here, we investigated gene expression patterns of P. lividus embryos cultured in the presence of acetazolamide (AZ), a CAN inhibitor, to combine morphological defects with their molecular underpinning. CAN inhibition blocked skeletogenesis, affected the spatial/temporal expression of some biomineralization-related genes, inhibited embryos swimming. A comparative analysis on the expression of 127 genes in control and 3â h/24â h AZ-treated embryos, using NanoString technology, showed the differential expression of genes encoding for structural/regulatory proteins, with different embryonic roles: biomineralization, transcriptional regulation, signalling, development and defence response. The study of the differentially expressed genes and the signalling pathways affected, besides in silico analyses and a speculative 'interactomic model', leads to predicting the presence of various CAN isoforms, possibly involved in different physiological processes/activities in sea urchin embryo, and their potential target genes/proteins. Our findings provide new valuable molecular data for further studies in several biological fields: developmental biology (biomineralization, axes patterning), cell differentiation (neural development) and drug toxicology (AZ effects on embryos/tissues).
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Anhidrasas Carbónicas , Paracentrotus , Animales , Acetazolamida/farmacología , Acetazolamida/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/farmacología , Paracentrotus/genética , Perfilación de la Expresión Génica , Transducción de Señal , Regulación del Desarrollo de la Expresión Génica , Embrión no Mamífero/metabolismoRESUMEN
Many anthropogenic pollutants such as metals are discharged into the marine environment through modern sources. Among these, lithium (Li), nickel (Ni), and zinc (Zn) can interfere with biological processes in many organisms when their concentration rises. These metals are toxic to sea urchin embryos, affecting their development. Indeed, animal/vegetal and dorso/ventral embryonic axes are differently perturbed: Li is a vegetalizing agent, Ni can disrupt dorso-ventral axis, Zn can be animalizing. To address the molecular response adopted by embryos to cope with these metals or involved in the gene networks regulating embryogenesis, and to detect new biomarkers for evaluating hazards in polluted environments in a well-known in vivo model, we applied a high-throughput screening approach to sea urchin embryos. After fertilization, Paracentrotus lividus embryos were exposed to Li, Ni, and Zn for 24/48 h. At both endpoints, RNAs were analyzed by NanoString nCounter technology. By in silico analyses, we selected a panel of 127 transcripts encoding for regulatory and structural proteins, ranked in categories: Apoptosis, Defense, Immune, Nervous, Development, and Biomineralization. The data analysis highlighted the dysregulation of many genes in a metal-dependent manner. A functional annotation analysis was performed by the KEEG Orthology database. This study provides a platform for research on metals biomarkers in sea urchins.
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Invertebrates represent about 95% of existing species, and most of them belong to aquatic ecosystems. Marine invertebrates are found at intermediate levels of the food chain and, therefore, they play a central role in the biodiversity of ecosystems. Furthermore, these organisms have a short life cycle, easy laboratory manipulation, and high sensitivity to marine pollution and, therefore, they are considered to be optimal bioindicators for assessing detrimental chemical agents that are related to the marine environment and with potential toxicity to human health, including neurotoxicity. In general, albeit simple, the nervous system of marine invertebrates is composed of neuronal and glial cells, and it exhibits biochemical and functional similarities with the vertebrate nervous system, including humans. In recent decades, new genetic and transcriptomic technologies have made the identification of many neural genes and transcription factors homologous to those in humans possible. Neuroinflammation, oxidative stress, and altered levels of neurotransmitters are some of the aspects of neurotoxic effects that can also occur in marine invertebrate organisms. The purpose of this review is to provide an overview of major marine pollutants, such as heavy metals, pesticides, and micro and nano-plastics, with a focus on their neurotoxic effects in marine invertebrate organisms. This review could be a stimulus to bio-research towards the use of invertebrate model systems other than traditional, ethically questionable, time-consuming, and highly expensive mammalian models.
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Paracentrotus lividus is a Mediterranean and Eastern Atlantic sea urchin species, very sensitive to chemical and physical environmental changes and widely used in eco-toxicological studies. Here, we applied a high throughput screening approach on P. lividus embryos exposed to UVB radiation (UV), Cadmium Chloride (Cd) and their combination (Cd/UV), to deeply characterize the molecular responses adopted by embryos to cope with these stressors. in vitro eco-toxicological assays were performed by exposing embryos to Cd (10-4 M) soon after fertilization, to UV (200 and 400J/m2) at early stage of development, while in co-exposure experiments, Cd-exposed embryos were irradiated with UV at 200 J/m2. By NanoString nCounter technology, custom-made probes were developed and hybridized on total RNA extracted from exposed embryos at 51h after fertilization. By in silico analyses, we selected and retrieved at the NCBI nucleotide database a panel of P. lividus transcripts encoding for many regulatory and structural proteins that we ranked in categories, i.e., Apoptosis, Biomineralization, Defense, Development, Immunity, Signaling and Transcription Factors. The analysis of 127 transcripts highlighted the dysregulation of many genes, some specifically activated to cope with stress agents, others involved in the complex molecular network of genes that regulate embryo development. We revealed the downregulation of Biomineralization and Development genes and the upregulation of Defensive genes in Cd and Cd/UV embryos. Our approach, using sea urchin embryo as an in vivomodel, contributes to advance our knowledge about cellular responses to UV, Cd and their combination.
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Management of dredged materials disposal is regulated by several environmental normative requirements, and it is often supported by the integration of chemical data with ecotoxicological characterization. The reliability of a bioassay to assess the potential toxicity of dredged sediments requires the selection of quality criteria that should be based on simple analytical methods and easily understandable hazard for politicians and environmental managers. The sea urchin embryo-toxicity bioassay is considered an essential component for evaluating the quality of sediments in harbour areas but its use, when based exclusively on the observation of normal vs. abnormal embryos, may alter the interpretation of the results, overestimating the risk assessment. To improve the reliability of this assay in establishing a causative relationship between quality of sediments and sea urchin embryonic development, here we developed and validated three Integrative Toxicity Indexes (ITI 2.0, ITI 3.0, ITI 4.0), modifying the already-known ITI (here ITI 1.0). Based on this aim, we used Taranto harbour as a model pilot-study to compare results to those obtained from standard criteria. Among the tested indexes, the ITI 4.0, discriminating strictly developmental delay and morphological defects from fertilized egg to gastrula stage, resulted in the most promising.
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Sedimentos Geológicos , Erizos de Mar , Animales , Bioensayo , Embrión no Mamífero , Proyectos Piloto , Reproducibilidad de los ResultadosRESUMEN
The sea urchin embryo develops a well-defined biomineralized endoskeleton, synthesized exclusively by the skeletogenic cells, supported by ectodermal cues for the correct skeleton patterning. The biomineralization process is tightly regulated via a hierarchical order of gene expression, including transcription and growth factors, biomineralization proteins. Recently, the role of kinases and intracellular signaling pathways in sea urchin skeletogenesis has been addressed, although the downstream components still remain unknown. In this study, we investigated the role of phosphatidylinositide 3-kinase (PI3K)-mediated signaling pathway in Paracentrotus lividus, to identify its genes/proteins targets. The effects of LY294002 (LY), a PI3K-specific inhibitor, were evaluated at morphological and molecular levels. Treatment with 40⯵M LY from the blastula stage completely blocked skeleton deposition, which was reversed by wash out experiments. Besides, LY caused a slight delay in the tripartite gut development. Despite the skeleton absence, a few skeleton-specific proteins/mRNAs were regularly expressed and localized in LY-treated embryos, as shown for MSP130 and SM50 by immunofluorescence and in situ hybridization experiments. QPCR analyses showed that LY differently affected the expression of genes coding for other biomineralization proteins, transcription and growth factors. SM30 and carbonic anhydrase expression was severely downregulated, while almost all the transcription factors analyzed were upregulated. Based on the present results and in silico analyses, we propose an "interactomic" model simulating PI3K connections in P. lividus embryos. Our findings define a novel regulatory step in the embryonic skeletogenesis, and provide valuable molecular data for further studies on the role of PI3K signaling in invertebrate biomineralization.
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Desarrollo Óseo/efectos de los fármacos , Cromonas/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Morfolinas/farmacología , Paracentrotus/embriología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Animales , Huesos/efectos de los fármacos , Huesos/embriología , Huesos/metabolismo , Biología Computacional , Embrión no Mamífero , Epistasis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Paracentrotus/efectos de los fármacos , Paracentrotus/genética , Paracentrotus/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Many industrial activities release Nickel (Ni) in the environment with harmful effects for terrestrial and marine organisms. Despite many studies on the mechanisms of Ni toxicity are available, the understanding about its toxic effects on marine organisms is more limited. We used Paracentrotus lividus as a model to analyze the effects on the stress pathways in embryos continuously exposed to different Ni doses, ranging from 0.03 to 0.5â¯mM. We deeply examined the altered embryonic morphologies at 24 and 48â¯h after Ni exposure. Some different phenotypes have been classified, showing alterations at the expenses of the dorso-ventral axis as well as the skeleton and/or the pigment cells. At the lowest dose used, Ni mainly induced a multi-spicule phenotype observed at 24â¯h after treatment. On the contrary, at the highest dose of Ni (0.5â¯mM), 90% of embryos showed no skeleton and no pigment cells. Therefore, we focused on this dose to study protein and gene expression patterns at 24 and 48â¯h after exposure. Among the proteins analyzed, i.e. p38MAPK, Grp78 and Mn-SOD, only p38MAPK was induced by Ni treatment. Moreover, we analyzed the mRNA profiles of a pool of genes that are involved in stress response and in development mechanisms, i.e. the transcription factors Pl-NFkB and Pl-FOXO; a marker of DNA repair, Pl-XPB/ERCC3; a mitogen-activated protein kinase (MAPK), Pl-p38; an ER stress gene, Pl-grp78; an adapter protein, Pl-14-3-3ε; two markers of pigment cells, Pl-PKS1 and Pl-gcm. The spatial expression of mesenchymal marker genes has been evaluated in Ni-treated embryos at both 24 and 48â¯h after exposure. Our results indicated that Ni acts at several levels in P. lividus sea urchin, by affecting embryo development, influencing the embryonic immune response and activating stress response pathways to counteract the suffered injury and to promote embryos surviving.
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Embrión no Mamífero/efectos de los fármacos , Níquel/toxicidad , Paracentrotus/embriología , Contaminantes Químicos del Agua/toxicidad , Animales , Embrión no Mamífero/fisiología , Desarrollo Embrionario , Expresión Génica , Paracentrotus/fisiologíaRESUMEN
Lithium (Li), Nickel (Ni), and Zinc (Zn) are metals normally present in the seawater, although they can have adverse effects on the marine ecosystem at high concentrations by interfering with many biological processes. These metals are toxic for sea urchin embryos, affecting their morphology and developmental pathways. In particular, they perturb differently the correct organization of the embryonic axes (animal-vegetal, dorso-ventral): Li is a vegetalizing agent and Ni disrupts the dorso-ventral axis, while Zn has an animalizing effect. To deeply address the response of Paracentrotus lividus embryos to these metals, we studied the expression profiling of Pl-Fra transcription factor (TF), relating it to Pl-jun, a potential partner for AP-1 complex formation, and to Pl-MT, known to be an AP-1 target and to have a protective role against heavy metals. The AP-1 TFs are found throughout the animal kingdom and are involved in many cellular events, i.e. cell proliferation and differentiation, immune and stress responses, cancer growth. Here, we isolated the complete Pl-Fra cDNA and showed that Pl-Fra transcript, already present in the unfertilized eggs, was newly synthesized from the blastula stage, while its spatial distribution was mainly observed in skeletogenic cells, similarly to Pl-jun. Interestingly, Pl-Fra expression was induced by the different metals and the induction kinetics revealed its persistent expression during treatments. Moreover, its temporal and spatial behavior in response to the three metals was comparable to that of Pl-jun and Pl-MT. The understanding of AP-1 functions in invertebrates may provide new knowledge about the mechanisms of response to metal injuries, as well as it might lead to acknowledge the TFs as new type of biomarkers for the evaluation of hazards in polluted environment.
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Metales/toxicidad , Paracentrotus/embriología , Factor de Transcripción AP-1/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/fisiología , Regulación de la Expresión Génica , Paracentrotus/fisiología , Erizos de MarRESUMEN
Gadolinium (Gd), a metal of the lanthanide series used as contrast agent for magnetic resonance imaging, is released into the aquatic environment. We investigated the effects of Gd on the development of four sea urchin species: two from Europe, Paracentrotus lividus and Arbacia lixula, and two from Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Exposure to Gd from fertilization resulted in inhibition or alteration of skeleton growth in the plutei. The similar morphological response to Gd in the four species indicates a similar mechanism underlying abnormal skeletogenesis. Sensitivity to Gd greatly varied, with the EC50 ranging from 56 nM to 132 µM across the four species. These different sensitivities highlight the importance of testing toxicity in several species for risk assessment. The strong negative effects of Gd on calcification in plutei, together with the plethora of marine species that have calcifying larvae, indicates that Gd pollution is urgent issue that needs to be addressed.
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Gadolinio/toxicidad , Paracentrotus/fisiología , Filogeografía , Contaminantes Químicos del Agua/toxicidad , Animales , Arbacia , Monitoreo del Ambiente , Paracentrotus/efectos de los fármacos , Erizos de MarRESUMEN
The sea urchin embryo is a well-recognized developmental biology model and its use in toxicological studies has been widely appreciated. Many studies have focused on the evaluation of the effects of chemical stressors and their mixture in marine ecosystems using sea urchin embryos. These are well equipped with defense genes used to cope with chemical stressors. Recently, ultraviolet radiation (UVR), particularly UVB (280-315 nm), received more attention as a physical stressor. Mainly in the Polar Regions, but also at temperate latitudes, the penetration of UVB into the oceans increases as a consequence of the reduction of the Earth's ozone layer. In general, UVR induces oxidative stress in marine organisms affecting molecular targets such as DNA, proteins, and lipids. Depending on the UVR dose, developing sea urchin embryos show morphological perturbations affecting mainly the skeleton formation and patterning. Nevertheless, embryos are able to protect themselves against excessive UVR, using mechanisms acting at different levels: transcriptional, translational and post-translational. In this review, we recommend the sea urchin embryo as a suitable model for testing physical stressors such as UVR and summarize the mechanisms adopted to deal with UVR. Moreover, we review UV-induced apoptotic events and the combined effects of UVR and other stressors.
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Adaptación Fisiológica , Embrión no Mamífero/fisiología , Erizos de Mar/embriología , Rayos Ultravioleta , Animales , Daño del ADN , Estrés Oxidativo , Erizos de Mar/fisiología , Estrés FisiológicoRESUMEN
Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue.
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Nanopartículas , Fagocitosis/efectos de los fármacos , Erizos de Mar/fisiología , Transducción de Señal/efectos de los fármacos , Titanio/farmacología , Receptores Toll-Like/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Regulación de la Expresión Génica , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Fagocitos/efectos de los fármacos , Fagocitos/inmunología , Fagocitos/metabolismo , Fosforilación , Receptores Toll-Like/genéticaRESUMEN
Human and natural activities release many pollutants in the marine environment. The mixture of pollutants can affect many organisms concurrently. We used Paracentrotus lividus as a model to analyze the effects on signal transduction pathways and stress gene expression in embryos exposed continuously to double stress, i.e., cadmium (Cd) from fertilization and UVB at cleavage (Cd/UVB-embryos). By microscopical inspection, we evaluated embryonic morphology after 72 h of development. Tissue-specific markers were used to assess mesoderm differentiation by immunofluorescence. We analyzed p38MAPK, ERK1/2, and JNK activation by Western blot and mRNA profiles of Pl-MT, Pl-14-3-3epsilon, and Pl-jun genes by real-time quantitative polymerase chain reaction (qPCR) and the localization of their transcripts by whole mount in situ hybridization (WMISH). We found that the Cd/UVB combined exposure induced morphological malformations in 76% of pluteus embryos, mainly affecting the development of the skeleton, including the normal branching of skeletal roads. In Cd/UVB-embryos, p38MAPK was activated 1 h after UVB exposure and a remarkable overexpression of the Pl-MT, Pl-14.3.3epsilon, and Pl-jun genes 24 h after UVB exposure. Pl-MT and Pl-14.3.3epsilon mRNAs were misexpressed as they were localized in a position different from that observed in wild-type embryos, i.e., the intestine. On the contrary, Pl-jun mRNA has remained localized in the skeletogenic cells despite their displacement in exposed embryos. In conclusion, Cd/UVB exposure affected skeletal patterning producing alternative morphologies in which p38MAPK activation and Pl-MT, Pl-14.3.3epsilon, and Pl-jun gene overexpression seem linked to a protective role against the stress response induced by Cd/UVB.
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Cadmio/toxicidad , Embrión no Mamífero/anomalías , Embrión no Mamífero/efectos de la radiación , Paracentrotus/embriología , Paracentrotus/efectos de la radiación , Animales , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/embriología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Paracentrotus/efectos de los fármacos , Paracentrotus/genética , ARN Mensajero/genética , Esqueleto/anomalías , Esqueleto/efectos de los fármacos , Esqueleto/embriología , Esqueleto/efectos de la radiación , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Growing evidence suggests that the transcription factors belonging to the Jun family are involved in many important cellular events, such as the control of bone development in mammalians. We have characterized, for the first time, a member of the Jun family from embryos of the sea urchin Paracentrotus lividus. The Pl-jun protein sequence includes all the functional domains characteristic of members of the Jun family (i.e. the basic leucine zipper, the basic DNA-binding and the c-Jun N-terminal kinase docking-like domains), which are evolutionarily conserved. Moreover, all the key serine and threonine residues, which are phosphorylation targets for different kinases necessary for jun activation, appear to be well preserved. A model of the monomeric protein provides a simulation of the three-dimensional structure and shows the potential sites for dimerization and DNA binding. Pl-jun mRNA is expressed in the unfertilized egg and throughout sea urchin embryo development. As the development proceeds, Pl-jun mRNA becomes exclusively expressed in the skeletogenic cells. Intriguingly, these cells contain significant amounts of the phosphorylated active protein entirely localized into their nuclei. These findings strengthen our hypothesis that suggests an active role for Pl-jun in skeletogenic cells, thus indicating that this transcription factor is a novel component of the gene regulatory networks controlling skeletogenesis. Database: Nucleotide sequence data have been deposited in the EMBL databases under the accession number: HE817756.
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Cartílago/embriología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Organogénesis/fisiología , Paracentrotus/embriología , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Filogenia , ARN Mensajero/metabolismo , Factores de Transcripción/genéticaRESUMEN
The increase of UVB radiation reaching marine environment has harmful effects on living organisms. Paracentrotus lividus sea urchin embryos living in shallow water are exposed to radiations, providing a good model for studying the molecular mechanisms activated upon UV stress. Here, we report the modulated time- and dose-dependent expression of six genes, known to be involved in stress response, in embryos exposed at cleavage to 400 and 800 J/m(2) UVB, and collected at early (morula) and later (gastrula) stages. We analyzed their mRNA levels by QPCR and found that Pl-14-3-3 showed a dose-dependent induction, both early and late in development; Pl-c-jun was up-regulated proportionally to the UVB dose at early stages and only at 800 J/m(2) UVB at later stages; Pl-XPB-ERCC3, Pl-MT and Pl-NF-kB were highly up-regulated later in development at the high dose, with the exception of Pl-XPB-ERCC3 whose mRNA levels were high also at the lower dose; Pl-FOXO expression was not affected by UVB radiation. We believe that the identification of UVB-responsive genes in irradiated sea urchin embryos, reported for the first time in this study, will be helpful for the understanding of the involved molecular pathways. The correlation between the impaired morphogenesis, affecting endo-mesoderm differentiation, and gene modulations described herewith is also discussed.
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Expresión Génica/efectos de la radiación , Paracentrotus/efectos de la radiación , Rayos Ultravioleta , Proteínas 14-3-3/genética , Animales , Biomarcadores/análisis , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Embrión no Mamífero/efectos de la radiación , Desarrollo Embrionario/efectos de la radiación , Metalotioneína/genética , FN-kappa B/genética , Paracentrotus/embriología , Paracentrotus/genética , Proteínas Proto-Oncogénicas c-jun/genéticaRESUMEN
Echinoderms have an extensive endoskeleton composed of magnesian calcite, a form of calcium carbonate that contains small amounts of magnesium carbonate and occluded matrix proteins. Adult sea urchins have several calcified structures, including test, teeth, and spines, composed of numerous ossicles which form a three-dimensional meshwork of mineral trabeculae, the stereom. The biomineral development begins in 24-hour-old embryos within the primary mesenchyme cells (PMCs), the only cells producing a set of necessary matrix proteins. The deposition of the biomineral occurs in a privileged extracellular space produced by the fused filopodial processes of the PMCs. We showed for the first time that signals from ectoderm cells overlying PMCs play an important role in the regulation of biomineralization-related genes. It is believed that growth factors are produced by ectoderm cells and released into the blastocoel where they interact with cognate receptor tyrosine kinases restricted to PMCs, which activate signaling cascades regulating the expression of biomineralization-related genes. We demonstrated the implication of a TGF-beta family factor by a perturbation model in which skeleton elongation was indirectly blocked by monoclonal antibodies to an extracellular matrix (ECM) protein located on the apical surface of ectoderm. Thus, it was inferred that interfering with the binding of the ECM ligand, a member of the discoidin family, to its cell surface receptor, a ßC integrin, disrupts the ectodermal cell signaling cascade, resulting in reduced or aberrant skeletons. During the last few years, we analyzed the expression of biomineralization-related genes in other examples of experimentally induced skeleton malformations, produced by the exposure to toxic metals, such as Cd and Mn or ionizing radiations, such as UV-B and X-rays. Besides the obvious toxicological implication, since the mis-expression of spicule matrix genes paralleled skeleton defects, we believe that by means of these studies we can dissect the molecular steps taking place and possibly understand the physiological events regulating embryonic biomineralization.
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Regulación del Desarrollo de la Expresión Génica , Erizos de Mar , Animales , Anticuerpos Monoclonales/genética , Carbonato de Calcio/metabolismo , Ectodermo/citología , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Mesodermo/citología , Erizos de Mar/embriología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
We used Paracentrotus lividus sea urchin embryos, a well-established model in developmental biology and ecotoxicology, for investigation on stress/anti-apoptotic protein expression elicited in response to harmful ionizing radiation, such as X-rays. We evaluated the acute effects of a high-dose exposure (5 Gy) on P. lividus analyzing by Western blotting the accumulation levels of HSP60, HSP70, BAG3 and a putative p63 at 24 and 48 h after irradiation. We found an increase in the HSP70, BAG3, and p63 protein levels only 48 h after irradiation, whereas no HSP60 increase was detected either at 24 or 48 h. Levels of the mRNA coding for HSP70 and p63 were also investigated by relative RT-PCR and were found to increase 24 h after irradiation, returning to their initial levels at 48 h. Results demonstrate the presence of an adaptive regulatory mechanism operating at the transcriptional level at 24 h, followed by a translational activation at 48 h post-irradiation. In conclusion, our findings confirm the sea urchin embryo as a sensible bioindicator of cell damage and we propose this model for studies on the protective pathways activated in response to X-rays. The novel result of the involvement of BAG3 and p63 in the response to X-rays, never tested so far in any other embryonic system, opens the way for their use as biomarkers of X-ray hazards.
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Embrión no Mamífero/efectos de la radiación , Erizos de Mar/embriología , Activación Transcripcional , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Embrión no Mamífero/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas de la Membrana/genética , Rayos XRESUMEN
Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m(2)) and a 2.7-fold (at 800 J/m(2)) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo.