DNA Replication in Permeabilized Cells of Sea Urchin Embryos

For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs.
DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA: cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.     

Effects of Aphidicolin on Arylsulfatase Gene Expression in Sea Urchin Embryos

Expression of the arylsulfatase (Ars) gene in sea urchin embryos begins just before hatching and ceases at the pluteus stage. Initiation of the Ars gene expression is inhibited by aphidicolin, which inhibits DNA synthesis without arresting the total RNA synthesis. Based on these finding it is supposed that DNA replication is a prerequisite for initiation of the Ars gene expression in developing sea urchin embryos.     

The Reaggregation of Dissociated Embryonic Sea Urchin Cells

Studies carried out on reaggregating sea urchin embryonic cellsreveal that reaggregating cells can give rise to normal pluteuslarvae by three developmental pathways: (i) from clusters, (ii)from chains of beads, and (iii) from a tissue culture phase.Experiments carried out on a mixture of cells from two differentspecies, Arbacia punctulata and Lytechinus pictus, demonstratethat reaggregation is species specific and that the sortingout of cells according to species occurs. Movement of cellswithin an aggregate is non-random and unidirectional. Electronmicroscope analysis of mixed aggregates of cells of the twospecies indicates that cells of the same species make initialcontact and adhere to one another by means of numerous microvilliand with the formation of an intercellular hyaline-like material.Cells of the two different species adhere, but never by meansof microvilli and no hyaline-like material can be detected onthe cell surfaces. The results indicate a role in reaggregationof an intercellular material, possibly an intercellular cement.     

DNA Polymerase Potentials of Sea Urchin Embryos

THE possible involvement of RNA-instructed DNA polymerase in differentiation has been proposed by Temin¹. Here we present evidence that partially purified polymerase fractions prepared from 16-cell sea urchin embryos, which can undergo normal development through gastrulation, are able to support RNA-directed, as well as the expected DNA-directed, DNA synthesis. The results reported lead us to suggest that the observed RNA-instructed DNA synthesis may be mediated by polymerases other than that responsible for DNA-dependent DNA synthesis.     

Stimulation of Protein Synthesis in the Mitochondria of Sea Urchin Embryos before Gastrulation

A transient increase in protein synthesis was observed in mitochondria at the mesenchyme blastula stage of sea urchin ( Hemicentrotus pulcherrimus ) embryos. This stimulated activity was inhibited by chloramphenicol but not by cycloheximide. Reconstituting experiments in which poly U-dependent protein synthesis was carried out showed the mitochondrial peptide elongation factor to be essential for increasing the protein synthetic activity in mesenchyme blastula, but aminoacyl tRNA synthetase and ribosome fraction containing initiation factor not to be involved in this increase. These findings are discussed in relation to the differentiation of embryos at the gastrulation stage.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

Nucleic Acid and Histone Synthesis by Ethanol-Treated Cleavage-Arrested Sea Urchin Embryos

Abstract. It has been found that fertilized sea urchin eggs prevented from normal cleavage by solutions of isosmotic ethanol in sea water are able to complete some cellular and molecular aspects of the normal developmental program that are observed in control cultures. In both treated and control cultures, the type of RNA transcribed changes at 24 h (early gastrula) in favor of higher molecular weight rRNA. Ultrastructural studies reveal the presence of nucleoli in ethanol-treated as well as control embryos. The type of H1 histone synthesized also shifts at 24 h in favor of a higher molecular weight H1 in both ethanol-treated and control embryos. Replication of DNA proceeds at a slower rate in ethanol-treated embryos than in controls, resulting in DNA/embryo values in ethanol which are 20–30% of control values after 24 h. The results relate to the problem of differentiation without cleavage, and the role of normal partitioning, cell-cell interaction, and DNA synthesis in triggering the sequence of events in the developmental program.     

Timers in Early Development of Sea Urchin Embryos

To elucidate the timing mechanisms in the early development of sea urchin embryos, we measured the times of initiation of the first four cleavages, of ciliary movement, of primary mesenchyme cell ingression, and of gastrulation at four temperatures ranging from 11 to 20°C. The times of cleavage and of initiation of ciliary movement showed similar temperature dependency, indicating that these events may be controlled by a common timer (the first timer). Although batches of eggs often showed variation in the period between fertilization and the first cleavage, their subsequent cleavages were more regular. This indicates that the first timer may not start at fertilization. The ingression of mesenchyme cells and the onset of gastrulation showed similar temperature dependency that was higher than that of other events, suggesting the existence of a second timer. Temperature shift experiments indicate that the second timer starts at the mid-blastula (the 8–9th cleavage) stage when divisions of blastomeres become asynchronous.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

Localization and Characterization of an Integral Membrane Protein Antigen Expressed by Pigment Cells in Embryos of the Sea Urchin Strongylocentrotus purpuratus

Chromogenic mesenchymal cells in plutei of the sea urchin Strongylocentrotus purpuratus express a tissue specific epitope recognized by the monoclonal antibody Sp–1. In transmission electron micrographs of pre-embed immunoperoxidase labelled plutei, the epitope is localized to pigment cell surfaces. Cell membranes of epidermal cells apposed to pigment cells are also immunoreactive, but endodermal cells apposed to pigment cells are not. Separation of Sp–1 immunoreactive material into the aqueous phase after embryo extraction in butanol indicates that the antigen is a protein or glycoprotein, and other solubilization characteristics suggest that it is an integral membrane constituent. The epitope is destroyed by general proteases and treatment with guanidine hydrochloride, and is resistant to oxidation by periodate and glycosyloxidases, suggesting that it is peptide rather than carbohydrate. On immunoblots of whole embryo extracts, or after SDS-PAGE analysis of ³⁵S-methionine-labelled embryos immunoprecipitated with Sp–1, a band showing an apparent molecular mass of 110 kD is seen at all stages from mid-gastrula to 26-day pluteus. We conclude that the Sp–1 antigen is a 110 kD integral membrane protein which may interact with the epidermal cells over which the pigment cells migrate.     

A Protein That Binds an Exogastrula-Inducing Peptide, EGIP-D, in the Hyaline Layer of Sea Urchin Embryos

Exogastrula-inducing peptides are present in eggs and embryos of the sea urchin Anthocidaris crassispina . They induce exogastrulation when added exogenously to the embryos. In the present study, we investigated an EGIP-D-binding protein in the embryos. EGIP-D was incubated with homogenates of embryos. EGIP-D was then cross-linked to the binding protein by use of disuccinimidyl suberate (DSS) and the complex was analyzed by western blotting with an EGIP-D-specific antibody. A 30-kDa protein was detected in both eggs and embryos. To examine the localization of this protein, EGIP-D was added to intact embryos, cross-linked to proteins by use of DSS, and the complexes were again analyzed by western blotting. The EGIP-D-binding protein was detected in intact embryos but not in embryos treated with Ca²⁺- and Mg²⁺-free seawater (CMF-SW) that removes the hyaline layer (HL). It appeared, therefore, that this protein was present on the outer surface of the embryo, being a constituent of the HL. The CMF-SW extract that contained EGIP-D-binding protein, inhibited the induction of exogastrulation by EGIP-D. Furthermore, the treatment of embryos with CMF-SW prevented EGIP-D from inducing exogastrulation. Our observations indicate that the interaction between EGIP-D and the binding protein is a prerequisite for induction of exogastrulation by EGIP-D.     

Immunochemical Analysis of Arylsulfatase Accumulation in Sea Urchin Embryos

We have determined the expression pattern of arylsulfatase in embryos of the sea urchin Strongylocentrotus purpuratus . Polyclonal antibodies raised against a fusion protein containing sequences encoded by SpARSI (Yang et al. , 1989, Dev. Biol. 135: 53–61, 1989) detect several peptides of 65–70 kD on immunoblots. Treatment with glycopeptidase F shows that at least one of these peptides is modified by N-linked glycosylation, which accounts for some of the peptide diversity. We have also identified a second arylsulfatase gene (SpARSII) whose sequence is highly similar to ARS, a gene expressed in the Hemicentrotus pulcherrimus embryo. Arylsulfatase activity is detectable in unfertilized eggs, in which only SpARSII mRNA can be detected. Both SpARSI and SpARSII mRNAs increase greatly in abundance during embryogenesis accompanied by parallel changes in arylsulfatase activity and immunoreactivity. Immunohistochemistry with the anti-SpARSI antibody shows that arylsulfatase accumulates primarily along the apical surface of the aboral ectoderm of pluteus larvae, and to a lesser extent along portions of oral ectoderm. At earlier stages, the protein is more uniformly distributed along all presumptive ectoderm, reflecting a more uniform mRNA distribution. Treatment of embryos with glycine-EDTA, which dissociates but does not lyse cells of the embryo, releases virtually all enzymatic activity and all immunoreactive protein. Embryos cultured in sulfate-free sea water, which arrest at gastrula stage, show normal accumulation and secretion of peptide detected with the SpARSI antibody.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.