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PubMed2005

Nutritive phagocyte incubation chambers provide a structural and nutritive microenvironment for germ cells of Strongylocentrotus droebachiensis, the green sea urchin.

Here we characterize the germinal epithelia of both sexes of Strongylocentrotus droebachiensis, the green sea urchin, throughout its annual gametogenic cycle, using light and electron microscopy and cytochemistry. In both sexes, germinal epithelia include two interacting cellular populations: nutritive phagocytes (NPs) and germ cells. After spring spawning, NPs accumulate nutrients; amitotic oogonia and often mitotic spermatogonia occur in clusters beneath NPs; and subsequent gametogenic stages are residual or absent. During the summer, NP nutrients are mobilized for use in vitellogenesis by residual primary oocytes or to support limited spermatogenesis. In addition, some residual primary oocytes may degenerate and be phagocytized by NPs. Significant nutrient mobilization from NPs and substantial gonial cell mitoses (indicative of new gametogenesis) occur in the fall. In both sexes, all of these changes are facilitated by NPs that form basal incubation chambers near the gonadal wall and within which germ cells are surrounded by nutrients released from the NPs. In females, germ cells at several stages of gametogenesis may be housed in separate chambers in the same NP. Primary oocytes also carry out jelly coat formation, meiosis, and cortical granule translocation within NP incubation chambers. In males, many NPs cooperate to provide large continuous chambers that contain spermatogenic cells at diverse stages. In both sexes these chambers persist throughout the year and isolate gametogenesis from the gonadal lumen. NPs become slender and shorten as their nutrients are depleted. Ova or spermatozoa are stored in the gonadal lumen. Post-spawning, NPs phagocytize differentiated germ cells while simultaneously enclosing intact gonial and residual gametogenic cells in basal chambers near the gonadal wall. In light of our observations, we suggest investigating proteins that may be important in the structural, phagocytic, and nutritive functions of NPs and for which corresponding genes have already been identified in the genome of S. purpuratus, the closely related purple sea urchin.

Animals↗

PubMed2005

Colonization, dispersal, and hybridization influence phylogeography of North Atlantic sea urchins (Strongylocentrotus droebachiensis).

We used frequency-based and coalescent-based phylogeographic analysis of sea urchin (Strongylocentrotus droebachiensis) mitochondrial DNA (mtDNA) sequences and previously published microsatellite data to understand the relative influence of colonization and gene flow from older (north Pacific) and younger (northeast Atlantic) sea urchin populations on genetic variation in the northwest Atlantic. We found strong evidence of survival of northwestern Atlantic populations in local Pleistocene glacial refugia: most haplotypes were the same as or closely related to Pacific haplotypes, with deep gene genealogies that reflect divergence times within the northwestern Atlantic that are much older than the last glacial maximum. We detected gene flow across the North Atlantic in the form of haplotypes shared with or recently descended from European populations. We also found evidence of significant introgression of haplotypes from a closely related species (S. pallidus). The relative magnitude of gene flow estimated by coalescent methods (and the effective population size differences among oceanic regions) depended on the genetic marker used. In general, we found very small effective population size in the northeastern Atlantic and high trans-Arctic gene flow between the Pacific and northwestern Atlantic. Both analyses suggested significant back-migration to the Pacific. However, microsatellites more strongly reflected older Pacific migration (with similar effective population sizes across the Arctic), whereas mtDNA sequences appeared to be more sensitive to recent trans- Atlantic dispersal (with larger differences in effective population size). These differences across marker types might have several biological or methodological causes, and they suggest caution in interpretation of the results from a single locus or class of markers.

Animals↗

PubMed1996

SpHbox7, a new Abd-B class homeobox gene from the sea urchin Strongylocentrotus purpuratus: insights into the evolution of hox gene expression and function.

Hox genes, by virtue of their key functions in axial patterning, have long been thought to be pivotal players in the evolution of developmental mechanisms. Despite their potential importance in evolution, there is little information about Hox genes in animal groups that are most closely related to ancestral Chordates. Accordingly, we have taken the step of analyzing Hox gene expression and function in the sea urchin embryo, whose simple bilateral body plan is thought to resemble that of a stem organism in the Chordate lineage. Here we describe the isolation, sequences analysis and spatiotemporal expression pattern of a sea urchin (Strongylocentrotus purpuratus) Abd-B-like gene, designated SpHbox7. We show that this gene is one of at least two Abd-B-like genes in the S. purpuratus genome, a result that argues against the simple hypothesis that Hox gene duplications occurred only during the evolution of the chordates. SpHbox7 transcripts are first detectable in midblastula stage embryos, increase in amount during gastrulation, decline slightly by the pluteus stage, and are not detectable in any tissue of the adult. Whole mount in situ hybridization and antibody staining with an SpHbox7-specific antibody reveal that both SpHbox7 mRNA and protein are present throughout the embryo in the blastula. Subsequently, they are localized in the invaginating archenteron, secondary mesenchyme, and oral ectoderm. By the pluteus larva stage, SpHbox7 protein and mRNA are present in the gut, larval arms, and portions of the oral ectoderm. This complex and dynamic expression pattern suggests that SpHbox7 has a role in the patterning of the gut, the mesoderm, and the oral surface.

Aging↗

PubMed2006

Neuron-specific expression of a synaptotagmin gene in the sea urchin Strongylocentrotus purpuratus.

Interest in chordate evolution has emphasized a need for a better understanding of the comparative neuroanatomy of invertebrate deuterostomes. However, molecular and genetic approaches to neurobiological studies in these groups are hampered by a lack of neuron-specific molecular markers. A monoclonal antibody, 1E11, is neuron specific and is useful in identification of neural structures in larvae and adults of echinoderms, hemichordates, and urochordates. To identify a neuron-specific gene product, we have characterized the antigen recognized by 1E11. In immunoblots and immunoprecipitations of neural tissue from adult Strongylocentrotus purpuratus, 1E11 recognizes a 57-kDa band. Tandem mass spectrometry of trypsin digests of the 57-kDa band permitted peptide mass mapping and sequencing of five peptides. All of the sequenced peptides, and 12 additional mass-mapped peptides, are found within the open reading frame of a cDNA encoding synaptotagmin B (Sp-SynB). In situ RNA hybridizations with synaptotagmin B probes with S. purpuratus larvae reveal a pattern of expression that is similar to that revealed by the antibody 1E11. Antibodies produced against a bacterially expressed Sp-SynB protein recognize a 57-kDa protein and colocalize with 1E11. When a full-length Sp-SynB cDNA is expressed in chicken embryonic cells, the cells become immunoreactive to 1E11. We conclude that synaptotagmin B is a gene expressed in neurons that has conserved epitopes in other invertebrate deuterostomes.

Amino Acid Sequence↗

PubMed1993

Improved preservation of ultrastructure in difficult-to-fix organisms by high pressure freezing and freeze substitution: I. Drosophila melanogaster and Strongylocentrotus purpuratus embryos.

In this study, we have applied the techniques of high pressure freezing and freeze substitution to embryonic cell types which are usually difficult to fix properly for electron microscopy. In both Drosophila and Strongylocentrotus purpuratus, we see improved preservation of both membrane systems and cytoskeleton when compared to published results on the same cells using conventional electron microscope (EM) fixation methods. Finally, we have seen that postembedding labelling of sections is possible even after light osmium fixation during freeze substitution.

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PubMed1975

Ultrastructural studies of regenerating spines of the sea urchin Strongylocentrotus purpuratus. I. Cell types without spherules.

The fine structure of regenerating tips of spines of the sea urchin Strongylocentrotus purpuratus was investigated. Each conical tip consisted of an inner dermis, which deposits and contains the calcite skeleton, and an external layer of epidermis. Although cell types termed spherulecytes containing large, intracellular membrane bound spherules were also present in spine tissues, only epidermal and dermal cell types lacking such spherules are described in this paper. The epidermis was composed largely of free cells representing several functional types. Over the apical portion of the tip these cells occurred in groups, while proximally they were distributed within longitudinal grooves present along the periphery of the spine from the base to the tip. The terminal portions of apical processes extending from some of the epidermal cells formed a thin, contiguous outer layer consisting of small individual islands of cytoplasm bearing microvilli. Adjacent islands were connected around the periphery by a junctional complex extending roughly 200 A in depth in which the opposing plasma membranes were separated by a narrow gap about 145 A in width bridged by amorphous material. Other epidermal cells were closely associated with the basal lamina, which was 900 A in thickness and delineated the dermoepidermal junction; some of these cells appeared to synthesize the lamina, while others may be sensory nerve cells. The dermis at the spine tip also consisted of several functional types of free cells; the most interesting of these was the calcoblast, which deposits the skeleton. Calcoblasts extended a thin, cytoplasmic skeletal sheath which surrounded the tips and adjacent proximal portions of each of the longitudinally oriented microspines comprising the regenerating skeleton, and distally, formed a conical extracellular channel ahead of the mineralizing tip. The intimate relationship between calcoblasts and the growing mineral surface strongly suggests that these cells directly control both the kinetics of mineral deposition and morphogenesis of the skeleton. Other cell types in the dermis were precalcoblasts and phagocytes. Precalcoblasts may function as fibroblasts and are possible precursors of calcoblasts. Closely associated with the basal lamina at the dermoepidermal junction were extracellular unbanded anchoring fi0rils 150 A to 200 A51 in diameter. Scattered proximally among dermal cells were other extracellular fibrils, presumably collagenous, about 300 A in diameter wit

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PubMed1975

Ultrastructural studies of regenerating spines of the sea urchin Strongylocentrotus purpuratus. II. Cell types with spherules.

The fine structure of spherulecytes, cell types with large, intracellular membrane-bound vacuoles termed spherules, was investigated in regenerating tips of spines of the sea urchin Strongylocentrotus purpuratus. Two categories of cell types were observed: red spherulecytes and colorless spherulecytes. Red spherulecytes were represented by a single cell type, the eleocyte, while colorless spherulecytes consisted of three morphologically distinct cell types termed morula cells, granulocytes, and vacuolecytes. Eleocytes and morula cells were distributed in both the epidermis and dermis, while granulocytes and vacuolecytes were present only in the dermis. After processing for light and electron microscopy, the spherules of eleocytes typically appeared empty, having lost their content of the red pigment, echinochrome. In contrast, the spherules of morula cell, granulocytes, and vacuolecytes enclosed a variety of granular and other material. The cell types reported in this paper resembled, to various degrees, spherulecytes in the coelomic fluid of echinoids described by other investigators.

Animals↗

PubMed1989

Expression of two actin genes during larval development in the sea urchin Strongylocentrotus purpuratus.

We report the first measurements of cell number, total RNA, and transcript accumulations for two actin genes during larval development of the sea urchin Strongylocentrotus purpuratus. At 5 weeks of feeding, when development of laboratory-raised larvae is completed, the cell number has increased about 100-fold with respect to the pluteus-stage embryo to about 150,000 +/- 50,000, and the total RNA has increased 46-fold to about 130 ng per larva. The transcripts of the Cylla cytoskeletal actin gene, which is expressed in adult tissues, continue to accumulate throughout larval development. A contrasting pattern of transcript accumulation is observed for Cyllla, a different cytoskeletal actin gene that in the embryo is expressed only in aboral ectoderm. These transcripts increase in number early in larval development, when the larval epidermis is differentiating, and then decline in quantity. It is known that at metamorphosis the larval epidermis is largely histolyzed and that the Cyllla gene is not expressed in the juvenile or adult.

Actins↗

PubMed1989

Expression and structure of the CyIIIb actin gene of the sea urchin Strongylocentrotus purpuratus.

The developmental pattern of expression of the Strongylocentrotus purpuratus CyIIIb actin gene was determined by RNA blot hybridizations carried out with a gene-specific probe and total embryonic RNA isolated from various stages of development. The results indicate that the CyIIIb mRNA is not detected in the maternal pool, and, although the gene is activated at the early stages (about 10 hr postfertilization), considerable amounts of mRNA do not accumulate until well into the pluteus stage 3 days later. These results suggest either a post-transcriptional regulatory mechanism that governs early embryonic expression of the CyIIIb actin or a late embryonic transcriptional enhancement of this gene. We present here the complete nucleotide sequence of the CyIIIb gene, which lies within the 10,361 base pairs of the sequenced region. The entire transcription unit is 7,455 nt long and shares structural similarities with the other cytoskeletal-type actin genes from this sea urchin. Sequence comparisons of CyIIIb to the CyIIIa actin gene, to which it is linked, reveals extensive homology even in the introns. The deduced amino acid sequence of the CyIIIb actin shows five amino acid substitutions compared with the CyIIIa actin and nine when compared with the CyI, the endodermal embryonic cytoskeletal-type actin. Five out of these nine amino acid differences occur within a small peptide (position 257 to 267). The 5' flanking sequence of the CyIIIb gene shows a remarkable homology (approximately 75-80%) with the CyIIIa upstream region up to the position -200 and a lack of any obvious similarity further upstream. This observation suggests that the two genes possibly share some common regulatory factors.

Actins↗

PubMed1990

Gastrulation in the sea urchin Strongylocentrotus purpuratus is blocked by the fluorescein dye erythrosin B.

Erythrosin B, a food, drug, and cosmetic dye, arrested gastrulation in embryos of the sea urchin Strongylocentrotus purpuratus. A 30 min pulse of 5 microM erythrosin B added at 18 hr postinsemination blocked gastrulation scored at 50 hr postinsemination when control embryos had completed gastrulation. Dye addition at later times had no detectable effects on development through 50 hr postinsemination. The dye may block primary invagination via its known effects on plasma membrane permeability and fluidity.

Animals↗

PubMed1990

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus).

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.

Animals↗

PubMed1988

Ovostatin, an endogenous trypsin inhibitor of sea urchin eggs: purification and characterization of ovostatin from eggs of the sea urchin, Strongylocentrotus intermedius.

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K-21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC50 (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 +/- 0.13 micrograms/ml (4.55 +/- 0.65 x 10(-8)M), 3.0 +/- 0.28 microgram/ml (1.5 +/- 0.14 x 10(-7)M), and 4.8 +/- 0.2 microgram/ml (2.4 +/- 0.1 x 10(-7)M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025-0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca2+ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.

Animals↗

PubMed1988

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus.

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.

Animals↗

PubMed2000

Cloning and characterization of alphaP integrin in embryos of the sea urchin Strongylocentrotus purpuratus.

Differentially expressed integrins have been shown to be involved in the intricate cell movements that occur during early development. Because the migration and movement of cells have been well characterized in sea urchin embryos, we searched for alpha-integrin subunits in this organism. An alpha integrin subunit, alphaP, was cloned from Strongylocentrotus purpuratus mesenchyme blastula stage mRNA by RT-PCR and RACE and found to exhibit 74-77% sequence similarity to mammalian alpha(5), alpha(8), alpha(IIb), and alpha(v) integrin. The 8-kb transcript was most abundant at the prism stage, although low levels could be detected at all stages by Northern blot analysis and RT-PCR. A polyclonal antibody to this novel integrin was generated against a 100-amino-acid alphaP fragment fused to glutathione S-transferase and shown to recognize a 180-kDa alpha-integrin in the egg and in all stages of embryogenesis studied.

Amino Acid Sequence↗

PubMed1993

The embryonic ciliated band of the sea urchin, Strongylocentrotus purpuratus derives from both oral and aboral ectoderm.

The ciliated band of the Strongylocentrotus purpuratus embryo consists of a columnar epithelium, 3-5 cells wide, which gives rise to a small number of neuroblasts. It arises late in development, as a border separating the squamous epithelial cells of the oral and aboral ectoderm. To determine the lineage origins of this structure, we performed double labeling experiments at the 2-cell stage and the 16-cell stage, which were designed to reveal clonal boundaries in the ciliated band. The ciliated band forms in a region of the ectoderm derived from descendants of the following blastomeres: No, VO, Na1u, Na2u, right and left NL1u, and right and left NL2u. In contrast to the lineage contributions of the embryonic territories established early in development, lineage origins of the ciliated band are variable. Specification of the ciliated band thus depends on intercellular interaction rather than lineage.

Animals↗

PubMed1994

Multiple Otx binding sites required for expression of the Strongylocentrotus purpuratus Spec2a gene.

The control region of the aboral ectoderm-specific Spec2a gene of Strongylocentrotus purpuratus contains a 188-bp enhancer element, the RSR enhancer, required for temporal activation and aboral ectoderm/mesenchyme cell expression. Within the enhancer is a positive cis-regulatory element with the core consensus sequence TAATCC, which is capable of binding the sea urchin orthodenticle-related homeobox protein SpOtx. In this report, we extend our analysis of the RSR enhancer by dissecting it into smaller pieces and testing these pieces in an enhancer activation assay. The 188-bp enhancer region could not be divided without partial loss of activity, and two of the three pieces tested exhibited some activity. Using site-directed mutagenesis, we showed that three Otx consensus binding sites were responsible for the activity of the enhancer, acting in a non-cooperative manner to yield full activity. Mutagenizing the three Otx sites and a fourth one just upstream abolished all activity in the context of the complete Spec2a control region. Bandshift analysis revealed that the Otx sites were able to bind SpOtx, suggesting that this transcription factor mediates positive control at these sites. Non-SpOtx binding sites overlapping two of the Otx sites may also play a role in Spec2a expression. Using a lacZ reporter gene, we showed that a 76-bp DNA fragment containing two of the Otx sites was sufficient for aboral ectoderm/mesenchyme cell expression. These results suggest that the RSR enhancer plus an upstream DNA element required for mesenchyme cell repression are necessary and sufficient for the proper temporal activation and aboral ectoderm expression of the Spec2a gene and that the Otx elements play a positive role in this process.

Animals↗

PubMed1995

An orthodenticle-related protein from Strongylocentrotus purpuratus.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus. The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

Amino Acid Sequence↗

PubMed1996

Twins raised from separated blastomeres develop into sexually mature Strongylocentrotus purpuratus.

The first two blastomeres of Strongylocentrotus purpuratus embryos were separated and the resulting twins raised in pairs through larval life and to sexual maturity. We derive two conclusions from this study: changes in the pattern of specification of embryonic cell fates following blastomere separation result in the creation of two sets of fully functional coelomic pouches and imaginal rudiments, and sex determination in sea urchins is chromosomal, since the pairs of twins were always of the same sex.

Animals↗