Seguiment temporal de la garota comuna Paracentrotus lividus en les Illes Medes. Exercici 1995

Podeu consultar l'Informe complet a: http://hdl.handle.net/2445/24663

Seguiment temporal de la garota comuna Paracentrotus lividus en les Illes Medes. Exercici 1997

Podeu consultar l'Informe complet a: http://hdl.handle.net/2445/23662

Seguiment temporal de la garota comuna Paracentrotus lividus en les Illes Medes. Exercici 1999

Podeu consultar l'Informe complet a: http://hdl.handle.net/2445/23667

Seguiment temporal de la garota comuna Paracentrotus Lividus en les Illes Medes. Exercici 2000

Podeu consultar l'Informe complet a: http://hdl.handle.net/2445/23669

Seguiment temporal de la garota comuna Paracentrotus Lividus en les Illes Medes. Exercici 2001

Podeu consultar l'Informe complet a: http://hdl.handle.net/2445/23672

Characterization of sea urchin yolk glycoproteins

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, S. purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein was prepared. lmmunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa egg yolk glycoprotein during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified and peptide mapping revealed that they were cleavage products derived from the major yolk glycoprotein. The antibody identified a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: L. pictus, A. punctulata, and D. excentricus. However, eggs from other echinoderm classes and from chicken, frog, fruit fly, and nematode did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked, polymannose carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed, revealing striking similarities in pI and in amino acid and monosaccharide composition. Peptide mapping showed that the 160-kDa glycoprotein from the four echinoids are structurally homologous. The major yolk glycoprotein appeared to be proteolyzed by a thiol protease, which could be activated in yolk particles prepared from unfertilized eggs by low pH. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout embryonic development, and that externalization of the glycoproteins was not detectable. The yolk glycoprotein precursor began to be synthesized in premetamorphosis larvae, and continued in adult males and females. Both the yolk glycoproteins and the yolk platelets disappeared during larval development. This disappearance has special significance because there were no yolk proteins in the direct developing sea urchin, H. erthryogramma, which bypasses larval development and metamorphoses directly into an adult. ^

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 µM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 µM) and furosemide (IC50 = 315.4 µM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

Seawater carbonate chemistry and early development of the sea urchin Lytechinus variegatus in a laboratory experiment using artificial seawater

Land-based aquaculture facilities often utilize additional bicarbonate sources such as commercial sea salts that are designed to boost alkalinity in order to buffer seawater against reductions in pH. Despite these preventative measures, many facilities are likely to face occasional reductions in pH and corresponding reductions in carbonate saturation states due to the accumulation of metabolic waste products. We investigated the impact of reduced carbonate saturation states (Omega Ca, Omega Ar) on embryonic developmental rates, larval developmental rates, and echinoplutei skeletal morphometrics in the common edible sea urchin Lytechinus variegatus under high alkalinity conditions. Commercial artificial seawater was bubbled with a mixture of air and CO2 gas to reduce the carbonate saturation state. Rates of embryonic and larval development were significantly delayed in both the low and extreme low carbonate saturation state groups relative to the control at a given time. Although symmetry of overall skeletal body lengths was not affected, allometric relationships were significantly different between treatment groups. Larvae reared under ambient conditions had significantly greater postoral arm and overall body lengths relative to body lengths than larvae grown under extreme low carbonate saturation state conditions, indicating that extreme changes in the carbonate system affected not only developmental rates but also larval skeletal shape. Reduced rates of embryonic development and delayed and altered larval skeletal growth are likely to negatively impact larval culturing of L. variegatus in land-based, intensive culture situations where calcite and aragonite saturation states are lowered by the accumulation of metabolic waste products.

Expression of the Hox gene complex in the indirect development of a sea urchin

Hox complex genes control spatial patterning mechanisms in the development of arthropod and vertebrate body plans. Hox genes are all expressed during embryogenesis in these groups, which are all directly developing organisms in that embryogenesis leads at once to formation of major elements of the respective adult body plans. In the maximally indirect development of a large variety of invertebrates, the process of embryogenesis leads only to a free-living, bilaterally organized feeding larva. Maximal indirect development is exemplified in sea urchins. The 5-fold radially symmetric adult body plan of the sea urchin is generated long after embryogenesis is complete, by a separate process occurring within imaginal tissues set aside in the larva. The single Hox gene complex of Strongylocentrotus purpuratus contains 10 genes, and expression of eight of these genes was measured by quantitative methods during both embryonic and larval developmental stages and also in adult tissues. Only two of these genes are used significantly during the entire process of embryogenesis per se, although all are copiously expressed during the stages when the adult body plan is forming in the imaginal rudiment. They are also all expressed in various combinations in adult tissues. Thus, development of a microscopic, free-living organism of bilaterian grade, the larva, does not appear to require expression of the Hox gene cluster as such, whereas development of the adult body plan does. These observations reflect on mechanisms by which bilaterian metazoans might have arisen in Precambrian evolution.

Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors

“TKO” is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an αP3A2⋅TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the αP3A2⋅TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. αP3A2⋅TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of αP3A2⋅TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

Male-by-female interactions influence fertilization success and mediate the benefits of polyandry in the sea urchin Heliocidaris erythrogramma

Numerous studies have reported that females benefit from mating with multiple males (polyandry) by minimizing the probability of fertilization by genetically incompatible sperm. Few, however, have directly attributed variation in female reproductive success to the fertilizing capacity of sperm. In this study we report on two experiments that investigated the benefits of polyandry and the interacting effects of males and females at fertilization in the free-spawning Australian sea urchin Heliocidaris erythrogramma. In the first experiment we used a paired (split clutch) experimental design and compared fertilization rates within female egg clutches under polyandry (eggs exposed to the sperm from two males simultaneously) and monandry (eggs from the same female exposed to sperm from each of the same two males separately). Our analysis revealed a significant fertilization benefit of polyandry and strong interacting effects of males and females at fertilization. Further analysis of these data strongly suggested that the higher rates of fertilization in the polyandry treatment were due to an overrepresentation of fertilizations due to the most compatible male. To further explore the interacting effects of males and females at fertilization we performed a second factorial experiment in which four mates were crossed with two females (in all eight combinations). In addition to confirming that fertilization success is influenced by male X female interactions, this latter experiment revealed that both sexes contributed significant variance to the observed patterns of fertilization. Taken together, these findings highlight the importance of male X female interactions at fertilization and suggest that polyandry will enable females to reduce the cost of fertilization by incompatible gametes.

A sea urchin (Diadema africanum Rodriguez et al., 2013) and a pipe fish (Syngnathus phlegon Risso, 1827) : two new records for the Azores

Maciel & Costa (2010) reported the presence of 17 species of costal sea urchins (Echinoidea) in the Azores, while Porteiro et al. (2010) reported six species of pipefishes (Syngnathidae) for the area. During SCUBA diving at Santa Maria Island, the first author detected a sea urchin and a pipe fish which are new to the marine fauna of the Azores.