Lividus Sea Urchin Embryos Research Articles

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

In the marine environment increasing concentrations of bio-available compounds often result from anthropogenic activities. Among metal ions, manganese represents a new emergent factor in environmental contamination. Here, we studied the effects of manganese on Paracentrotus lividus sea urchin embryos using biological and biochemical approaches for the analysis of impact on development, tissue accumulation and stress markers. Embryos were continuously exposed from fertilization to manganese at concentrations ranging from 1.0 to 61.6 mg l(-1), monitored for developmental abnormalities at 48 h after fertilization, and used for atomic spectrometric analysis at various times from 6 to 72 h. We found that concentration- and time-dependent increases in morphological abnormalities were directly correlated to manganese accumulation, with major defects in skeleton formation at 48 h. Concurrently, we found an upregulation of the hsc70 and hsc60 stress proteins detected by immunoblotting, whereas no induction of apoptosis or ROS production was observed by TUNEL and live tests, respectively. Taken together, our findings demonstrate that the observed manganese embryo-toxicity is related to both its intracellular accumulation and misregulated homeostasis, and confirm the importance of stress proteins as protective agents in the acquisition of tolerance and resistance to apoptosis.

Cadmium induces the expression of specific stress proteins in sea urchin embryos

Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72–70, 56, 28, and 25 kDa) was induced, with an increase in the rate of synthesis of 72–70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure.

Expression of univin, a TGF-β growth factor, requires ectoderm–ECM interaction and promotes skeletal growth in the sea urchin embryo

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM–ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-β superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.

A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) alpha, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1 alpha) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70 has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.

A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed

In the present study, double immunofluorescence and immunoblot analysis have been used to show that centrosomes, isolated from Paracentrotus lividus sea urchin embryos at the first mitotic metaphase, contain the constitutive chaperone, heat-shock protein (HSP) 70. More specifically, we demonstrate that centrosomes contain only the HSP70-d isoform, which is one of the four isoforms identified in P. lividus. We also provide evidence that p34(cell division control kinase-2) and t complex polypeptide-1 (TCP-1) α, a subunit of the TCP-1 complex, are localized on the centrosomes. Furthermore, inhibition of TCP-1 in vivo, via microinjecting an anti-(TCP-1α) antibody into P. lividus eggs before fertilization, either impaired mitosis or induced severe malformations in more than 50% of embryos. In addition, we have isolated the whole mitotic apparatus and shown that HSP70 localizes on the fibres of spindles and asters, and binds them in an ATP-dependent manner. These observations suggest that HSP70has a chaperone role in assisting the TCP-1 complex in tubulin folding, when localized on centrosomes, and during the assembling and disassembling of the mitotic apparatus, when localized on the fibres of spindles and asters.

Sea-urchin Embryo Bioassay for in situ Evaluation of the Biological Quality of Coastal Seawater

The Paracentrotus lividus sea-urchin embryo bioassay, consisting of incubation of fertilized eggs in test water and measurement of the percentage of four-armed plutei larvae developed after the incubation period (2–3 days), has been adapted for in situ evaluation of seawater quality in coastal areas. Mature sea-urchins are dissectedin situ and fertilization is performed in the field; fertilized eggs are delivered into screw lid 50-ml cylinders with 20μm nylon mesh in both ends filled with sieved local seawater. The cylinders, tied to 60-cm ropes with weights on one end and buoys in the other one, are placed by scuba divers in the test sites at subtidal level and recovered after the incubation period. The contents of each cylinder are then transferred into a vial, fixed with formalin and observed directly under an inverted microscope to record the percentage (N=100) and size (length, N=25) of four-arm pluteus larvae. Our results show that the bioassay can discriminate between well known polluted and unpolluted sites, but further improvement is needed in order to: (1) take into account differences of temperature between sites; (2) minimize larval mortality due to reasons other than pollution.

Regulative specification of ectoderm in skeleton disrupted sea urchin embryos treated with monoclonal antibody to Pl-nectin.

Pl-nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl-nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl-nectin-coated substrates was calcium dependent, and reached its optimum at 10 mM Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl-nectin, which prevent ectoderm cell-Pl-nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.

THE OLIGOMERIC INTEGRITY OF TOPOSOME IS ESSENTIAL FOR ITS MORPHOGENETIC FUNCTION

Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex fromParacentrotus lividussea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non-reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180–160kDa and a lower band of 80kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus-like structures with skeletal elements.

The S.//.A.IG amino acid motif is present in a replication dependent late H3 histone variant of P. lividus sea urchin

A novel gene encoding a new H3 histone variant (H3L) has been identified in P. lividus sea urchin embryo. It encodes a H3 histone protein showing the S.//.A.IG amino acid motif typical of the replication independent H3.3 variants but in a mRNA showing the 3′ terminal stem-loop nucleotide sequence that is typical of the replication dependent variants. The gene is intronless, the corresponding short transcript is non-polyadenylated and its expression is replication dependent with a timing of a late variant. The new H3 variant is expressed as a minor component with respect to a major replication dependent late H3 histone here identified by partial cDNA sequence. These results show that classification of histones in replication dependent and independent variants only on the basis of their amino acid sequences should be reconsidered.

Spatial expression of alpha and beta tubulin genes in the late embryogenesis of the sea urchin Paracentrotus lividus.

In Paracentrotus lividus sea urchin embryos, at blastula stage, there is an abrupt increase in the abundance of alpha and beta tubulin transcripts in particular of the PI beta 1, PI beta 2 and PI alpha 2 forms. In order to assign specific functions to the various embryonic tubulin genes, we have used whole-mount in situ hybridization to determine spatial patterns of expression of five different alpha and beta tubulin embryonic genes. The PI beta 3 transcripts, as previously shown for PI alpha 2, start to localize in a few founder cells from which the neurogenic territory differentiates. The other four embryonic tubulin mRNAs (PI beta 1/2 and PI alpha 1/10), are localized in the ciliated band- and gut-territory. These territories originate by morphogenetic processes, which occur in late embryogenesis in the sea urchin and depend on cellular interactions. In particular, the interactions between the oral and aboral ectoderm specify the position of the ciliated band, whereas the invagination of the vegetal plate forms the gut territory. We suppose that the increase in alpha and beta tubulin transcripts could be functionally related to these two morphogenetic events. Our results show in fact that specific tubulin isotypes, or a mix of them, are expressed in and mark the ciliated band and the neighboring oral/aboral ectoderm cells of the ciliated band, in addition to the cells of the gut territory. The same localization of all these tubulin transcripts has been confirmed by whole-mount in situ hybridization experiments performed on embryos treated with agents able to induce deciliation or exogastrulation. Furthermore a putative correlation of PI beta 2 with cilium formation has been shown by the results obtained on deciliated embryos.

Isolation of cDNA clones encoding DNA methyltransferase of sea urchin P. lividus: Expression during embryonic development

A full-length cDNA, encoding a DNA (cytosine-5)-methyltransferase (DNA MTase), has been assembled from a series of overlapping cDNA clones isolated from P. lividus sea urchin embryo cDNA libraries. The cDNA contains 103 bp 5′-UTR, 4839 bp open reading frame corresponding to a 1612 amino acids (aa) protein and 2240 bp 3′-UTR including a terminal 18-bp poly(A) tail. Both the cDNA and the encoded protein are the longest so far reported for DNA MTases. The protein shows five distinct and sequential regions of identity with the other animal DNA MTases, with values of identity from zero to 80%. Northern blot analyses reveal a single RNA band of about 7.5 kb in length showing a highly regulated concentration pattern during development with peak value at the four blastomere stage.

Cloning and characterization of a developmentally regulated sea urchin cDNA encoding glutamine synthetase

A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5′ untranslated region (5′-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3′-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical residues, as compared with human and lobster class-II GS. The sequence contains the Mn2+-binding aa and the highly conserved aa regions observed in other GS. Northern blot analyses show that the GS mRNA is present in the sea urchin egg and is developmentally regulated in the embryo.

Isolation of a new H3.3 histone variant cDNA of P. lividus sea urchin: Sequence and embryonic expression

A cDNA encoding a new H3 histone variant has been isolated from a Paracentrotus lividus sea urchin embryo cDNA library. The encoded protein is identical to the H3.3 histone subtype identified in other species, with the difference that E replaces D at position 81. The clone corresponds to a transcript of about 1.6 kb, not dependent on DNA replication, present in the unfertilized egg and at all stages of embryonic development. The coding part of the cDNA cross-reacts also with a 0.5 kb H3 late histone mRNA.