Electron microscopic characterization of nuclear egress in the sea urchin gastrula

Nuclear egress, also referred to as nuclear envelope (NE) budding, is a process of transport in which vesicles containing molecular complexes or viral particles leave the nucleus through budding from the inner nuclear membrane (INM) to enter the perinuclear space. Following this event, the perinuclear vesicles (PNVs) fuse with the outer nuclear membrane (ONM), where they release their contents into the cytoplasm. Nuclear egress is thought to participate in many functions such as viral replication, cellular differentiation, and synaptic development. The molecular basis for nuclear egress is now beginning to be elucidated. Here, we observe in the sea urchin gastrula, using serial section transmission electron microscopy, strikingly abundant PNVs containing as yet unidentified granules that resemble the ribonucleoprotein complexes (RNPs) previously observed in similar types of PNVs. Some PNVs were observed in the process of fusion with the ONM where they appeared to release their contents into the cytoplasm. These vesicles were abundantly observed in all three presumptive germ layers. These findings indicate that nuclear egress is likely to be an important mechanism for nucleocytoplasmic transfer during sea urchin development. The sea urchin may be a useful model to characterize further and gain a better understanding of the process of nuclear egress.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Molecular analysis of heterochronic changes in the evolution of direct developing sea urchins

The sea urchin Heliocidaris erythrogramma is a direct developer; it progresses directly from the gastrula to the juvenile adult without forming a pluteus larva. No larval skeleton is formed by mesenchyme cells, but formation of the juvenile skeleton is accelerated. We have examined two alterations in mesenchyme cell behavior that accompany this striking change in developmental pattern. 1) Rapid cell proliferation produces 1700–2200 mesenchyme cells by mid-gastrula, compared to 30–60 primary mesenchyme cells in species with typical larval development. This change may reflect the accelerated production of adult structures in H. erythrogramma. 2) B2C2 is a monoclonal antibody that recognizes primary (Anstrom et al., 1987) and adult mesenchyme cells associated with skeleton formation in typical developers. The altered pattern of B2C2 staining in H. erythrogramma (e.g., a later initial appearance of the B2C2 antigen) suggests that H. erythrogramma has deleted part of a larval program of development and accelerated its adult program of development. These results indicate that cellular and molecular heterochronies accompany the morphological changes in H. erythrogramma development.     

Electron microscopic study of the development of amphioxus,Branchiostoma belcheri tsingtauense: The gastrula

The gastrulae of amphioxus were investigated by means of scanning and transmission electron microscopy (SEM and TEM) during 7 arbitrary stages that were seen about 4 to 10 hr after fertilization. Throughout gastrulation, SEM revealed subtle differences in cells of the blastoporal lip. In fractured specimens at early and middle stages, two opposing zones different in shape, size, and connection of the component cells were found: one which consists of columnar smaller cells in close contact in animal region and the other which is composed of round or polygonal larger cells in looser association in vegetal region. The polar body was found unexpectedly on the concave vegetal surface of the early gastrula in about 25% of cases. This might be the result of migration of the polar body. A short cilium that later elongated was recognized on each cell at mid-gastrula stage. The cilia on the dorsal surface (the neural ectoderm) of the final-stage gastrula became shorter than those on the epidermal ectoderm. TEM of thin sections demonstrated that the cytoplasmic components of gastrula cells are essentially the same as those of cleavage cells. But, the homogeneous nucleus seen during cleavage changed into a heterogeneous structure in which a nucleolus and dense particles were seen. Until the late stage, regional characteristics of the gastrulae indicating definitively the anterior-posterior and dorso-ventral polarity were not detected in the present SEM and TEM study.     

3H]serotonin binding to blastula, gastrula, prism, and pluteus sea urchin embryo cells

1. The presence of serotonin binding sites in blastula, gastrula, prism, and pluteus embryos of the sea urchin, Arbacia punctulata, was investigated by the binding of radiolabelled serotonin to dissociated embryo cells. 2. [3H]serotonin binding sites were identified in prism, early pluteus, and advanced pluteus larvae, but not in blastula or gastrula embryos. 3. The ontogeny of [3H]serotonin binding activity closely parallels that of serotonin content as previously reported in Paracentrotus lividus embryos (Toneby, 1977a). 4. Results of this study support a regulatory role of serotonin in developmental processes in postgastrula sea urchin embryos.     

A fine structural study of cytodifferentiation during cleavage, blastula, and gastrula stages of Fundulus heteroclitus

The fine structure of cleavage, blastula, and gastrula stages of Fundulus heteroclitus was investigated. Cleavage blastomeres are relatively unspecialized, containing few or poorly developed organelles. Beginning in blastula stages, signs of differentiation were noted, including development of the endoplasmic reticulum and Golgi apparatus and appearance of a primary nucleolus and polyribosomes. More extensive structural specializations occur in gastrula stages, including further development of the endoplasmic reticulum and appearance of a granular component in the nucleolus. These changes are associated with cell differentiation and an increased capacity for protein synthesis, and may be preparatory to subsequent histogenesis. The periblast is a continuous syncytial cytoplasmic layer located between the blastodisc and yolk and is formed during late cleavage by incomplete division of the cytoplasm of the blastodisc. Cytoplasmic projections extend from the periblast (and from the basal region of cleavage blastomeres prior to formation of the periblast) into the yolk and function in uptake of yolk material in the absence of pinocytosis. Yolk material appears to be digested by the periblast and transferred into the segmentation cavity where it is available to the blastomeres. Protein granules, lipid droplets, glycogen, crystalline arrays, and multivesicular bodies are related to food storage and utilization by blastomeres. The yolk gel layer enclosing the yolk sphere was found to be a thin layer of cytoplasm continuous with the margin of the periblast and is renamed the yolk cytoplasmic layer.     

Electron microscopic analysis of replicating DNA of sea urchin embryos

DNA was extracted from Paracentrotus lividus embryos at the third S phase after fertilization and analyzed with the electron microscope. The most relevant structures observed in this actively replicating DNA are clusters of short, closely spaced microbubbles (about 0.1 μm long on the average), partially or entirely single-stranded molecules and few linear forks. Unexpectedly, no long eye forms were observed. The analysis of DNA purified from gastrulae and from adult somatic tissues has revealed the same structures, although at a low frequency. A quantitative analysis has been carried out to determine the size distribution and spacing of microbubbles. A number of control experiments have been performed to characterize these structures better. Various possibilities are discussed to account for the presence of the observed forms and the absence of larger eyes.     

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.     

The study of proteins during early stages of embryogenesis of sea urchins

Summary The distribution of the preexisting and of thede novo synthesized proteins among soluble, and insoluble fractions, as well as between immunoprecipitable and non-immunoprecipitable soluble proteins has been studied in sea urchin embryos at different stages of development.In the S-100 fraction, which represented about 20% of total proteins, only a minor part of radioactivity was found. The majority of newly synthesized proteins was insoluble at neutral pH. Such distribution was practically invariant for all investigated stages of development, and was not markedly affected by Dactinomycin nor by 5-azacytidine.Only a small percentage of the total radioactivity of the S-100 fraction was found in the antigen-antibody complexes of soluble proteins. No shift of newly synthesized proteins towards the type of old, preexisting antigenic proteins was detected, and the majority of soluble newly synthesized proteins was found to be related to the non-immunoprecipitable soluble proteins.The authors gratefully acknowledge a generous gift of 5-azacytidine from Drs. Doskoil and ponar. We are also indebted to Dr. Miroslav Simi for kind interest and discussion.This work was supported in part by grants No 3111/1 from Federal Research Fund of Yugoslavia and No 02-020-1 from the National Institute of Health, U. S. Department of Health, Education and Welfare (PL-480 programme).     

Evolutionary crossroads in developmental biology: sea urchins

Embryos of the echinoderms, especially those of sea urchins and sea stars, have been studied as model organisms for over 100 years. The simplicity of their early development, and the ease of experimentally perturbing this development, provides an excellent platform for mechanistic studies of cell specification and morphogenesis. As a result, echinoderms have contributed significantly to our understanding of many developmental mechanisms, including those that govern the structure and design of gene regulatory networks, those that direct cell lineage specification, and those that regulate the dynamic morphogenetic events that shape the early embryo.     

Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins

Although eukaryotic flagella and cilia all share the basic 9+2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions.     

Enzymes of DNA biosynthesis in developing sea urchins. Changes in ribonucleotide reductase, thymidine, and thymidylate kinase activities.

The pattern of ribonucleotide reductase, thymidine kinase, and thymidylate kinase activities during development of Paracentrotus lividus eggs and the effect of actinomycin on these enzymatic activities have been studied. Ribonucleotide reductase activity is detectable, though at a low level, in the unfertilized egg; the activity increases sharply soon after fertilization and reaches a peak at the morula stage. Thereafter it decreases and remains at a lower level than that of the unfertilized egg. Actinomycin, at a concentration sufficient to inhibit messenger RNA (mRNA) synthesis does not affect the level of enzymatic activity, indicating that preexisting maternal mRNA is used for the synthesis of this enzyme. Thymidine kinase is present at a low level in the egg; it increases sharply after the hatching blastula until the pluteus stage. Actinomycin does not affect the enzyme activity from fertilization until blastula but prevents the increase in enzyme activity that is observed between blastula and pluteus. Thymidylate kinase activity shows an increase after fertilization, followed by fluctuations throughout development with a considerable decrease at the blastula stage and at the end of gastrulation. Actinomycin has no effect on the activity of thymidylate kinase regardless of when the drug is added to the embryo suspension. Possible regulatory mechanisms of DNA synthesis in sea urchin embryos are discussed: The presence in the unfertilized egg of the most important enzymes controlling the cellular flow of DNA precursors and the availability of dTTP suggest that the block in DNA synthesis observed in the unfertilized egg is due to some particular mechanism that is switched on at fertilization.     

Embryonic expression of engrailed in sea urchins

Neural patterning genes that are expressed along the anterior-posterior axis of deuterostomes are expressed late in larval development in echinoderms and are thought to function in establishing the highly-derived, adult body plan. We have used genomic resources to clone an engrailed gene (SpEn) from Strongylocentrotus purpuratus, and with this we have developed an antibody specific for SpEn. SpEn is expressed late in embryogenesis in the developing larval nervous system. At the prism stage, a small number of neuroblasts in the oral ectoderm on the edge of the larval mouth begin expressing SpEn. The cells are in bilaterally symmetric positions. The expression of SpEn precedes the expression of the neural markers, synaptotagmin and serotonin in the SpEn immunoreactive cells. The SpEn cells are located on the margin of the domain of cells expressing SpNK2.1, but they do not have nuclear SpNK2.1. Expression of engrailed in a pair of bilateral neural structures in early development appears to be a shared feature of bilaterians.