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.     

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.     

Development of the Basal Lamina and Its Role in Migration and Pattern Formation of Primary Mesenchyme Cells in Sea Urchin Embryos

The development and substructure of the basal lamina and its role in migration and pattern formation of primary mesenchyme cells (PMCs) in normal as well as Li⁺- and Zn⁺⁺-treated embryos of sea urchins were investigated by electron microscopy. Major findings were as follows. 1) Network fibrils appear along the basal surface of the blastular wall by the hatching blastula stage. The area covered with fibrils is restricted to the vegetal hemisphere at earlier stages, but extends to the animal hemisphere as development proceeds. 2) Nonfibrous fuzzy material embeds the fibrils to form a basal lamina, but in places the fibrils project from the basal lamina into the blastocoel. The major components of the fuzzy material were digested by glycosidase, which failed to digest the fibrous components. 3) The fibrils can be classified into two types, one Ca⁺⁺-independent and the other Ca⁺⁺-dependent. PMCs apparently utilize the Ca⁺⁺-indepndent fibrils as a substratum for locomotion. 4) After migration, PMCs accumulate in a specific region to form the PMC pattern. This is formed in the area of greatest concentration of Ca⁺⁺-independent fibrils. 5) PMCs in embryos treated with LiCl, in contrast to normal embryos, accumulate in the animal pole region where the Ca⁺⁺-independent fibrils are markedly concentrated.     

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.     

Development of Serotonergic Neurons in Embryos of the Sea Urchin, Strongylocentrotus purpuratus

The development of the serotonergic component of the nervous system of larvae of S. purpuratus is traced using indirect immunofluorescence with a polyclonal antibody against the neurotransmitter serotonin. Initially one or two neuroblasts can be detected in the thickened epithelium of the animal plate of late gastrulae (56 hr). The number of immunoreactive cells increases to about eight during formation of the pluteus (85–90 hr). Immunoreactive axons appear simultaneously from all neuroblasts present in the 79 hr prism stage larva and form the apical ganglion. It is proposed that this component of the larval nervous system is derived from a small number of ectodermal cells associated with the apical tuft.     

The Structure and Development of the Apical Ganglion in the Sea Urchin Pluteus Larvae of Strongylocentrotus droebachiensis and Mespilia globulus

The structure of the apical ganglion is described using transmission electron microscopy and ultrastructural immunohistochemical localization with anti-serotonin, and the development of these nerve cells in animalized and vegetalized embryos, and embryos treated with Ca²⁺-deficient sea water are demonstrated with immunofluorescence microscopy. The axons within the neuropile contain clear and dense-core vesicles, but all the vesicles appear anti-serotonin immunoreactive. The principal type of neuron within the apical ganglions is anti-serotonin immunoreactive. In the animalized embryos, serotonergic neuroblasts and neurons appear in the second quarter of animal-vegetal axis. Serotonergic cells were not detected in vegetalized exogestrulae. Mespilia globulus embryos treated with Ca²⁺-deficient sea water ruptured to form a cellular sheet on the substratum. In ruptured embryos serotonergic neurons formed a ring around the area corresponding to the apical plate of normal embryos. These findings indicated that the serotonergic preoral nerve cells of echinopluteus derive from cells on the periphery of the apical plate.     

Change in the Triglyceride Level in Sea Urchin Eggs and Embryos During Early Development

Triglycerides in the embryos of the sea urchin, Anthocidaris crassispina , analyzed by gas-liquid chromatography, distributed in a range of carbon numbers between 42 and 58 in the sum of three fatty acid residues. During the development until gastrulation, the levels of triglycerides with 48, 56 and 58 carbon numbers decreased at constant rates and the levels of the others decreased at specific stages different with one another, respectively. Thereafter, the amounts of all triglycerides decreased simultaneously. The amount of oxygen consumed in the embryos is enough for the oxidation of mobilized triglycerides during post-hatching period but is not during pre-hatching period. The levels of neutral glycerides increased gradually during pre-hatching period and thereafter decreased. The fatty acid level also increased during pre-hatching and post-hatching period. These suggest that the cleavage of triglycerides and the oxidation of their cleavage-products occur during whole span of early development. During pre-hatching period, the break down of triglycerides is probably higher in its rate than the rate of their oxidation, resulting in the increase in the levels of neutral glycerides, as well as fatty acids.     

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.     

Exogut Formation by the Treatment of Sea Urchin Embryos with Ascorbate and α-Ketoglutarate

In sea urchin embryos at the stages from hatch out to the pluteus stage, [¹⁴C]proline incorporation into hot trichloroacetic acid TCA-extractable proteins occurred during an exposure to [¹⁴C]proline for 3 hrs at 20°C. The rate of [¹⁴C]proline incorporation into hot TCA-extractable proteins was higher in gastrulae and plutei than in blastulae. Percentage of [¹⁴C]hydroxyproline residue to whole radioactivity of the hot TCA-extractable proteins was quite low at the blastula stage and increased exponentially during futher development. Production of [¹⁴C]hydroxyproline residue at the blastula stage, as well as at the later stages, was stimulated by ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, and inhibited by α, α'-dipyridyl, an inhibitor of this enzyme. It is also probable that the enzyme in the embryos is not fully activated because of low amounts of activating substances. These suggest that blastulae,…, also have a potency of protocollagen hydroxylation. Blastula kept in sea water containing ascorbateand α-ketoglutarate became undeveloped embryo with large exogut. Gastrula developed normally to pluteus even in the presence of these compounds. The embryos, kept in sea water containing these compounds from fertilization to hatch out, also developed normally. Exogut formation in the embryos treated by these compounds, as well as normal archenteron formation, was inhibited by α, α'-dipyridyl.     

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.     

Evolutionary Conservation of the Larval Serotonergic Nervous System in a Direct Developing Sea Urchin

Development of the larval serotonergic nervous system is examined by indirect immunofluorescence in two congeneric species of sea urchins that exhibit divergent embryonic and larval development. Heliocidar is tuberculata undergoes indirect planktotrophic development via a pluteus larva, whereas Heliocidaris erythrogramma develops directly, passing through a brief, highly derived lecithotrophic larval stage. We have cleared the opaque embryos of H. erythrogramma and discuss internal features of its development. The serotonergic nervous system of H. tuberculata arises in the apical plate at the end of gastrulation and develops into a bilaterally symmetric ganglion lying between the anterolateral arms in the preoral hood. Putatively homologous neurons appear at the apical end of the modified larva of H. erythrogramma well after the completion of gastrulation, coincident with development of the primary podia of the adult rudiment. The neurons form a bilaterally symmetric ganglion whose orientation relative to the vestibule is conserved with respect to that found in planktotrophic larvae. This allows us to define a left and right side for this larva which lacks external points of asymmetry such as a larval mouth. The alteration in the time of nervous system development in H. erythrogramma relative to that of H. tuberculata , and other indirect developers, implicates heterochronies in cellular differentiation as an important component of the evolution of direct development.     

The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence

Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT₁ receptors, antagonists of 5-HT₂, 5-HT₃ or 5-HT₄ receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA-5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter.     

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.     

Heterotrimeric Kinesin-II Is Required for the Assembly of Motile 9+2 Ciliary Axonemes on Sea Urchin Embryos

Heterotrimeric kinesin-II is a plus end– directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti–kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti–kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti–kinesin-II–injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.     

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.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.