Mathematical model for early development of the sea urchin embryo

In Xenopus and Drosophila, the nucleocytoplasmic ratio controls many aspects of cell-cycle remodeling during the transitory period that leads from fast and synchronous cell divisions of early development to the slow, carefully regulated growth and divisions of somatic cells. After the fifth cleavage in sea urchin embryos, there are four populations of differently sized blastomeres, whose interdivision times are inversely related to size. The inverse relation suggests nucleocytoplasmic control of cell division during sea urchin development as well. To investigate this possibility, we developed a mathematical model based on molecular interactions underlying early embryonic cell-cycle control. Introducing the nucleocytoplasmic ratio explicitly into the molecular mechanism, we are able to reproduce many physiological features of sea urchin development.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

Effects of the volatile anesthetic halothane on fertilization and early development in the sea urchin Lytechinus variegatus: evidence that abnormal development is due to polyspermy

The volatile anesthetic halothane, when present at fertilization, dose-dependently increases the incidence of abnormally developing sea urchin embryos at the first cell division. Microscopic examinations of eggs stained with aceto-orcein or the DNA fluorochrome bisbenzimide and direct observations on isolated sperm aster complexes show that halothane induces polyspermy (multiple sperm entry) when present at fertilization. Experimental evidence suggests that anesthetic-induced polyspermy involves impairment of both the fast (electrically mediated) and slow (morphological) blocks to multiple sperm entry. These observations clearly show that relatively brief exposures to halothane at fertilization cause polyspermy and that this effect is almost certainly responsible for the ensuing abnormal development observed at the first cell division.     

Role of syndecan in the elongation of postoral arms in sea urchin larvae

Ac-SYN is the core protein of a cell surface proteoglycan of the sea urchin Anthocidaris crassispina. To examine the functions of Ac-SYN, embryos were cultured in the presence of affinity-purified antibody against Ac-SYN. At the late pluteus stage, severe inhibition of elongation of the postoral arms was seen in treated embryos compared with control embryos. Blastocoeleic microinjection of the antibody did not affect morphogenesis. The relationship between the number of cells in the postoral arms and the length of the postoral rods was investigated in normal embryos. This showed that postoral arm elongation has two phases: the first phase accompanies the increase in cell numbers while the second does not. The syndecan antibody inhibited the increase in cell numbers in the postoral arms. Furthermore, in the treated embryos, cell numbers continued to increase normally until 31 h post fertilization (hpf), while cell division stopped after 31 hpf. These results suggest that Ac-SYN participates in postoral arm formation via cell division in sea urchin embryos.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

Calcium and mitosis

Calcium is thought to be involved in regulating mitotic transitions. The basis for this view is set out. Recent data from experiments on sea urchin embryos is discussed. The relative simplicity of the embryonic cell cycle and the relative ease with which cell physiology can be done in sea urchin embryos has allowed the clear demonstration that the phosphoinositide-calcium-calmodulin signalling pathway is required for and regulates mitosis entry and anaphase onset. The relevance of the sea urchin work to mitosis in other cell types is briefly discussed.     

A sea urchin in vivo model to evaluate Epithelial‐Mesenchymal Transition

Epithelial‐mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti‐EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti‐EMT candidates.     

Effects of removing sea urchins (Strongylocentrotus droebachiensis): Stability of the barren state and succession of kelp forest recovery in the east Atlantic

Stability properties of the barren state of a kelp forest-sea urchin system were studied in northern Norway. The ability of the sea urchin Strongylocentrotus droebachiensis to maintain high population densities and recover from perturbations, and the succession of kelp forest revegetation, were studied experimentally by reducing the sea urchin density on a barren skerry. Additional information was obtained from community changes following a natural, but patchy, sea urchin mortality that varied between sites. On the barren grounds, high sea urchin densities (30 50 per m²) is maintained by annual recruitment. Severe reductions of sea urchin densities initiated luxuriant kelp growth, while more moderate reductions allowed establishment of opportunistic algae (during spring and early summer), but no kelps. Succession of algal growth, after the severe decline in sea urchin densities, followed a predictable pattern. At first the substrate was colonized by filamentous algae, but within few weeks they were outcompeted by the fast growing kelp Laminaria saccharina. After 3–4 years of the removal experiment, the slower-growing, long-lived kelp L. hyperborea became increasingly dominant. Increased food availability after reduction in sea urchin density led to increased individual growth of the remaining sea urchins. However, the population density did not increase, neither from recruitment nor immigration from adjacent areas with high sea urchin densities. Possibly, early establishment of a dense kelp stand, may represent a breakpoint in the ability of sea urchins to reestablish a barren state. The ability of L. saccharina quickly to invade and monopolize an area may have both positive and negative effects on the succession towards the climax L. hyperborea kelp forest. Competitive interactions may slow the process, but development of a dense stand of L. saccharina will also reduce grazing risk on scattered recruits of the more slowly growing L. hyperborea.     

Characterization of bep1 and bep4 antigens involved in cell interactions during Paracentrotus lividus development

We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.     

Dermatan sulfate formation in gastrulae of the sea urchin Clypeaster japonicus

Gastrullation of sea urchin embryos is arrested in sulfate-free sea water. This developmental arrest has been considered to be due to lack of sulfation of glycosaminoglycans in the extracellular matrix of the embryos. In the present study, we characterized a dermatan sulfate type component formed in gastrula-stage embryos of the sea urchin Clypeaster japonicus and examined the effects of sulfate deprivation on the formation. Glycosamino-glycans were prepared from gastrula-stage embryos incubated with [3H]acetate in normal and sulfate-free sea water. Enzymatic analyses indicated that embryos formed a glycosaminoglycan of the dermatan sulfate type which contained an N-acetylgalactosamine-6-sulfate-containing disaccharide as a major unit, plus a minor unidentified component. Under sulfate-free conditions, embryos formed an under-sulfated chondroitin/dermatan sulfate copolymer which mainly consisted of non-sulfate, glucuronic acid-containing (chondroitin) disaccharide units. These results suggest that sulfate deprivation diminishes not only the degree of sulfation but also the formation of L-iduronic acid-containing (dermatan) disaccharide units in dermatan sulfate in sea urchin embryos.     

Septate junctions mediate the barrier to paracellular permeability in sea urchin embryos

In sea urchin embryos, blastula formation occurs between the seventh and tenth cleavage and is associated with changes in the permeability properties of the epithelium although the structures responsible for mediating these changes are not known. Tight junctions regulate the barrier to paracellular permeability in chordate epithelia; however, the sea urchin blastula epithelium lacks tight junctions and instead possesses septate junctions. Septate junctions are unique to non-chordate invertebrate cell layers and have a characteristic ladder-like appearance whereby adjacent cells are connected by septa. To determine the function of septate junctions in sea urchin embryos, the permeability characteristics of the embryonic sea urchin epithelia were assessed. First, the developmental stage at which a barrier to paracellular permeability arises was examined and found to be in place after the eighth cleavage division. The mature blastula epithelium is impermeable to macromolecules; however, brief depletion of divalent cations renders the epithelium permeable. The ability of the blastula epithelium to recover from depletion of divalent cations and re-establish a barrier to paracellular permeability using fluorescently labelled lectins was also examined. Finally, septate junction structure was examined in embryos in which the permeability status of the epithelium was known. The results provide evidence that septate junctions mediate the barrier to paracellular permeability in sea urchin embryos.     

A Century of Sea Urchin Development

SYNOPSIS. In the last quarter of the nineteenth century severalinvestigators including Richard and Oskar Hertwig, Theodor Boveri,Hans Driesch, Curt Herbst, T. H. Morgan and others turned theirattention to sea urchin eggs and early embryos. This favorablecombination of outstanding investigators and the sea urchinembryo as an experimental organism contributed to a fundamentalunderstanding of the cell, fertilization and heredity. The advantagesof the sea urchin continued to be recognized as experimentalembryologists used these embryos to develop the concepts ofgradients, regulative development and inductive interactions.Then, as developmental biology arose from chemical embryology,the sea urchin embryo once again emerged as an ideal experimentalanimal, pivotal in the understanding of the molecular and developmentalbiology of eukaryotic organisms.     

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.     

Focal adhesion kinase (FAK) expression and phosphorylation in sea urchin embryos

We have cloned three cDNA isoforms of focal adhesion kinase (FAK) from the sea urchin, Lytechinus variegatus. The sea urchin FAK is more closely related to FAK from other deuterostomes than from invertebrate protostomes or to cell adhesion kinase beta (CAKbeta/Pyk2/FAK2). FAK is expressed in all cells of sea urchin embryos by the 120-cell stage and strongly in blastulae. Phospho-FAK concentrates on basal surfaces of epithelial cells in early blastulae and occurs in syncytial cables of primary mesenchyme cells (PMC). Inhibition of FAK by constructs of FAK-related non-kinase delays blastocoel expansion and early PMC ingression. These results suggest that FAK has roles in cell adhesion and in the shape and integrity of the epithelial cells in sea urchin embryos.     

The Effects of Chemical Animalizing and Vegetalizing Agents and of Cell Dissociation on the Synthesis of 5S RNA and Transfer RNA in Cleaving Sea Urchin Embryos

The effect of altering normal cell associations and interactions on the synthesis of 5S RNA and transfer RNA (tRNA) was studied in cleaving embryos of the sea urchin, Arbacia punctulata. Cell interactions were altered: (1) by culturing cleaving embryos in the animalizing agent, Evans Blue, and in the vegetalizing agent, Li⁺ as LiCl and (2) by culturing dissociated cells. Control and experimental embryos each were labeled from 3 h to 6 h post fertilization with [8-³H]-guanosine. Sixteen-cell embryos, whose GTP precursor pools had been preloaded, were dissociated, labeled and cultured under conditions which prevent reaggregation. Quantitative measurements of rates of accumulation of newly synthesized 5S RNA and tRNA showed that these rates are similar in cleaving sea urchin embryos and in corresponding embryos cultured in the presence of Evans Blue and of Li⁺. In addition, cells dissociated from cleavage embryos and maintained under conditions which prevent reaggregation retained the ability to synthesize 5S RNA and tRNA. These results suggest that normal cell associations and interactions are not necessary for the synthesis of 5S RNA and tRNA to occur in cleaving sea urchin embryos.     

Effects of 5 azacytidine on DNA methylation and early development of sea urchins and ascidia

5-azacytidine (5-azaCR), an analogue of cytidine, inhibits nuclear DNA methylation in early sea urchin embryos. This inhibition is specific and dose-dependent. Exposure of sea urchin embryos at any stage between one-cell and blastula, to micromolar quantities of 5-azaCR invariably inhibits development beyond the blastula stage. In a substantial number of embryos arrested at the blastula stage, spicule formation proceeds although other morphological differentiation is lacking. No significant effect on development is seen if sea urchin embryos are exposed to 5-azaCR at post-blastula stages. 5-azaCR also inhibits the development of a mosaic egg such as the ascidian Phallusia mammilata at the blastula stage, indicating that both regulative (sea urchin) and mosaic (ascidian) embryos respond more or less similarly to 5-azaCR treatment.     

The effect of 5-bromodeoxyuridine on the synthesis of nuclear and cytoplasmic DNA-binding proteins isolated from sea urchin embryos.

Newly synthesized DNA-binding proteins were isolated from the nuclei and, separately from, the cytoplasm of sea urchin mofula stage embryos. The presence of 5-bromodeoxyuridine during embryogenesis did not appear to alter the synthesis of either class of DNA-binding proteins. This result tends to argue that cell differentiation in early embryos is not regulated by differential synthesis of DNA-binding proteins. Sea urchin mofulae synthesize a broad range, by molecular weight, or cytoplasmic DNA-binding proteins which dissociate from sea urchin DNA-cellulose at relatively high salt concentrations (0.6-2.0 M NaCl). The most prominant of these apparently high-binding-affinity proteins has an approximate molecular weight of 33,000.