Caterina Costa

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 vertebrates.

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

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

Echinoderms as Blueprints for Biocalcification: Regulation of Skeletogenic Genes and Matrices

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 βC 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.

Phylogenetic analysis and homology modelling of Paracentrotus lividus nectin.

The extracellular matrix protein Pl-nectin, a 210-kDa homodimer originally purified from sea urchin eggs, plays a crucial role in cell adhesion and embryonic morphogenesis. The compiled cDNA sequence, obtained by RT-PCR primer walking and 3′ RACE, identified a 984aa product containing a 23aa signal peptide and including all six internal peptides identified by protein microsequencing. The protein is a new member of the galactose-binding protein superfamily as it consists of six 151–156aa-long tandemly repeated domains (D1–D6), homologous to the discoidin-like domains, also known as F5/8-type C domains. Based on homology modelling, we present a three-dimensional structure (3D) for D5, identified as the prototype domain. The molecular modelling of the assembled Pl-nectin homodimer accounts for a Pl-nectin quaternary structure composed of two 105-kDa C-shaped monomers linked by a S–S bridge. The presence of an LDT motif between the first and the second exposed loops of the D2 domain suggests the binding of Pl-nectin to an integrin receptor. Altogether, the in silico analysis described here is consistent with previous biochemical reports and offers a basis for predictions to be experimentally tested.

Skeletogenesis by transfated secondary mesenchyme cells is dependent on extracellular matrix–ectoderm interactions in Paracentrotus lividus sea urchin embryos

In the sea urchin embryo, primary mesenchyme cells (PMCs) are committed early in development to direct skeletogenesis, provided that a permissive signal is conveyed from adjacent ectoderm cells. We showed that inhibition of extracellular matrix (ECM)–ectoderm cells interaction, by monoclonal antibodies (mAb) to Pl‐nectin, causes an impairment of skeletogenesis and reduced expression of Pl‐SM30, a spicule‐specific matrix protein. When PMCs are experimentally removed, some secondary mesenchyme cells (SMCs) switch to skeletogenic fate. Here, for the first time we studied SMC transfating in PMC‐less embryos of Paracentrotus lividus. We observed the appearance of skeletogenic cells within 10 h of PMCs removal, as shown by binding of wheat germ agglutinin (WGA) to cell surface molecules unique to PMCs. Interestingly, the number of WGA‐positive cells, expressing also msp130, another PMC‐specific marker, doubled with respect to that of PMCs present in normal embryos, though the number of SM30‐expressing cells remained constant. In addition, we investigated the ability of SMCs to direct skeletogenesis in embryos exposed to mAbs to Pl‐nectin after removal of PMCs. We found that, although phenotypic SMC transfating occurred, spicule development, as well as Pl‐SM30‐expression was strongly inhibited. These results demonstrate that ectoderm inductive signals are necessary for transfated SMCs to express genes needed for skeletogenesis.

Cell Adhesion and Communication: A Lesson from Echinoderm Embryos for the Exploitation of New Therapeutic Tools

In this chapter, we summarise fundamental findings concerning echinoderms as well as research interests on this phylum for biomedical and evolutionary studies. We discuss how current knowledge of echinoderm biology, in particular of the sea urchin system, can shed light on the understanding of important biological phenomena and in dissecting them at the molecular level. The general principles of sea urchin embryo development are summarised, mainly focusing on cell communication and interactions, with particular attention to the cell-extracellular matrix and cell-cell adhesion molecules and related proteins. Our purpose is not to review all the work done over the years in the field of cellular interaction in echinoderms. On the contrary, we will rather focus on a few arguments in an effort to re-examine some ideas and concepts, with the aim of promoting discussion in this rapidly growing field and opening new routes for research on innovative therapeutic tools.

The newly characterized Pl-jun is specifically expressed in skeletogenic cells of the Paracentrotus lividus sea urchin embryo.

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.

Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields.

Response to metals treatment of Fra1, a member of the AP-1 transcription factor family, in P. lividus sea urchin embryos

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.

Nickel toxicity in P. lividus embryos: Dose dependent effects and gene expression analysis

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 0.5 mM. We deeply examined the altered embryonic morphologies at 24 and 48 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 24 h after treatment. On the contrary, at the highest dose of Ni (0.5 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 48 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 48 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.