A comparison of protein synthetic patterns in normal and animalized sea urchin embryos

Newly synthesized proteins from normal and animalized sea urchin embryos were compared by the technique of double labeling. Total embryonic protein was solubilized in SDS, urea, and 2-mercaptoethanol. The proteins were examined by coelectrophoresis on an SDS-polyacrylamide gel. The gels were sliced and the radioactivity determined. A standardized ratio of the isotopes served as the basis of comparison. A comparison of newly synthesized proteins from normal embryos 24 and 48 h old showed a shift from larger to smaller molecular weight proteins. Animalized embryos showed a similar shift. When normal and animalized embryos of the same ages were compared, differences were found. The differences were distributed over the entire range of molecular weights. These results show that although differences in protein synthesis between animalized and normal embryos are evident by 24 h, most of the changes in protein synthesis that occur in normal embryos are unaffected by animalization.     

Primary differentiation and ectoderm-specific gene expression in the animalized sea urchin embryo

Primary differentiation in sea urchin embryos, animalized by zinc, has been gauged by the formation of characteristic endodermal and mesodermal tissue derivatives and by the accumulation of the ectoderm-specific Spec 1 mRNA. Increasing the dosage of zinc diminishes the differentiation of secondary mesenchyme, primary mesenchyme, endoderm, and ectoderm, in decreasing order. Treatment is effective only during the blastula stages, involving successive periods of sensitivity for these tissues. Removal of zinc with chelator results in the resumption of differentiation to increasing degree for this series of tissues. The developmental initiation of Spec 1 gene expression, normally at the earliest blastula stage, can be delayed by zinc for at least 30 hr before being implemented by treatment of the animalized embryos with a chelator. We conclude (1) that those processes in the blastula which are required for differentiation and are suppressed by zinc are distinguishable from the determinative processes, which are not affected by the animalizing agent and occur earlier during midcleavage; (2) that animalization by zinc involves a graded failure of primary tissues to form; and (3) that animalization involves a pause in the schedule of differentiation, which can be reinstated by removal of the animalizing agent, thereby providing a survival value inherent in a flexible schedule of development.     

Ribosomal RNA Synthesis in Sea Urchin Embryos: Differential Rates of Accumulation in Chemically-induced Animalized and Vegetalized Larvae

Animalization was induced with evans blue and with Zn⁺⁺ in embryos of Arbacia punctulata and of Lytechinus variegatus , respectively. Li⁺ induced vegetalization in A. punctulata embryos. While animalization did not affect the rate of cleavage, vegetalized embryos exhibited a reduction in cell number at post-morula stages. Mid-gastrulae and corresponding experimental embryos each were labeled for 4 hr with uridine-[5-³H] and with L-[³H-methyl]-methionine. The rate of uptake of each exogenous RNA precursor was similar in control and in experimental embryos. Purified RNA preparations were fractionated by electrophoresis on 2.4% acrylamide+0.5 % agarose gels. Comparison of rates of incorporation of each RNA precursor into 26s and 18s RNAs indicated that on a per cell basis evans blue- and Zn⁺⁺-animalized embryos showed a reduction (0.53–0.56) and Li⁺-vegetalized embryos an enhancement (1.41—1.53) in the rate of accumulation of newly made 26s and 18s RNAs compared to controls (1.00). These results suggest that chemically-induced animalized and vegetalized embryos provide useful tools for studying possible differential gene expression in different embryonic germ layers of the developing sea urchin embryo.     

Chromatin proteins from normal,vegetalized, and animalized sea urchin embryos

The chemical composition of the chromatin, the fractional content of histones and nonhistone chromatin proteins (NHP), and the biosynthesis of these proteins in normal, vegetalized, and animalized embryos of the sea urchin Strongylocentrotus droebachiensis at the blastula, mesenchyme blastula, and gastrula stages have been studied. The amount of the NHP in the chromatin from normal and vegetalized embryos increases during early embryonic development while that in animalized embryos remains without change at the mesenchyme blastula stage and then decreases. During development the histone content in all three cases slightly decreases. Polyacrylamide gel electrophoresis reveals that both fractional composition of histones and their biosynthesis in normal, vegetalized, and animalized embryos display no differences. During development, however, some changes occur, so that the relative amount of histones F1 and F2a2 increases, F2b decreases, while F3 and F2a1 remains constant. Histone F1 at the blastula stage consists of two subfractions while at the gastrula stage it consists of three subfractions. The histone F2a1 consists of one and two, respectively. Histone F3 at all stages is made up of three subfractions; histone F2b is made up of two; and the histone F2a2 is electrophoretically homogeneous. Specific radioactivity of the arginine-rich histones F3 and F2a1 tends to increase during development, while that of moderately lysine-rich histones F2b and F2a2 does not change, and that of the lysine-rich histone F1 decreases. The NHP in normal, vegetalized, and animalized embryos at different developmental stages consist of 17 fractions that can be separated by isoelectrofocusing within the 4.5-8.8 pH range. Quantitative changes have been observed in the fractions focused at pH 4.5-6.1 during development and in normal and modified embryos at the gastrula stage.     

Differences in abundance of individual RNAs in normal and animalized sea urchin embryos

A library of cDNA clones was constructed representing polysomal polyadenylated RNA of mesenchyme blastulae of Strongylocentrotus purpuratus. Using this library, we determined whether or not individual RNA species are associated with animalization of embryos by zinc ions. Clones corresponding to the most actively synthesized RNAs during the period just prior to the mesenchyme blastula stage were selected by screening colonies with in vivo-labeled RNA. The most abundant of these were chosen for further study. Individual RNA abundance was measured as percent of mass of total polyadenylated RNA by hybridizing cDNA exhaustively with cloned DNA on filters. The RNAs in the selected, cloned sequences were present in abundances of 0.01 to 1% of the mass of polyadenylated RNA. Changes in abundance of individual RNA species occurred during normal development and departures from these developmental changes occurred in the zinc-animalized embryos. Two RNA species, which normally increase 10-fold in abundance, are drastically repressed and at least one RNA species increases in abundance dramatically in the animalized embryos. These departures from the normal program of presumptive gene expression may furnish insights into changes in the normal processes of development.     

Does ADP-Ribosylation of Proteins in Nuclei Contribute to Ectoderm Cell Differentiation in Sea Urchin Embryos?

In nuclei of sea urchin embryos, marked increase in ADP-ribosyltransferase activity followed by its decrease occurrs in the pre-hatching and post-hatching periods with peaks of activity at the morula and gastrula stages. Increase in its activity was blocked by cycloheximide in the pre- and post-hatching periods and by actinomycin D only in the post-hatching period. Embryo wall cells (ectoderm cells) isolated from gastrulae exhibited markedly higher activity of this enzyme than archenteron cells and mesenchyme cells. Probably, the increase in the activity of this enzyme in the post-hatching period results from expression of the gene for this enzyme mainly in ectoderm cells. In the post-hatching period, the activity increased more in animalized embryos than in normal ones, and increased little in vegetalized embryos. 3-Aminobenzamide (3-ABA), as well as luminol and nicotinamide, inhibited formation of ectoderm structures more than that of endoderm structures, such as the archenteron, in normal and animalized embryos, but had no appreciable effect on morphogenesis in vegetalized embryos. The reaction catalyzed by ADP-ribosyltransferase probably contributes to ectoderm cell differentiation. Treatment of embryos with 3-ABA in the pre-hatching period had little inhibitory effect on the morphogenesis in the post-hatching period, though it caused death of many embryos.     

Some new observations on the animalization of the unfertilized sea urchin egg

The influence of bright daylight on the animalization of the unfertilized egg of Paracentrotus lividus by exposing it to SCN-ions in Ca⁺⁺-free sea water under aerobic conditions has been studied. The processes underlying animalization were decidedly inhibited by the light (Table I and II). It was found during the summer of 1948 that eggs taken from females from two different localities, Beclem and St. Efflam in the Roscoff area of Brittany, behaved very differently against the animalizing treatment, those from Beclem being easily animalized whereas those from St. Efflam were rather resistant. The proteins of the Beclem eggs turned out to be more soluble in icecooled 0.54 M. NaI than those of the St. Efflam eggs (Table V). In the cultures obtained from eggs not very resistant to the animalizing treatment, slightly animalized larvae were more frequent than in cultures with more resistant eggs.     

Stage Specific Effects on Sea Urchin Embryogenesis of Zn2+, Li+, several Inhibitors of cAMP-Phosphodiesterase and Inhibitors of Protein Synthesis

Treatment of sea urchin embryos for 3 hr starting at the 16-64 cell stage with Li⁺ or 3-isobutyl-1-methylxanthine as well as with other inhibitors of cAMP-phosphodiesterase (PDE) and several inhibitors of protein synthesis, resulted in production of vegetalized embryos with a large exogut. However, the same treatment starting at other stages produced hardly any vegetalized embryos. The specific stage for these substances to cause vegetalization is probably the 16-64 cell stage. Treatment with Zn²⁺ between the times of fertilization and hatching, followed by culture in normal sea water produced animalized embryos with little if any archenteron, but the same treatment followed by culture with ethylenediamine-N, N'-diacetic acid (EDDA), a chelator of Zn²⁺, produced quasi-normal plutei. This chelator did not counteract the animalizing effect of Zn²⁺ when culture with EDDA was started at the post-gastrula stage. Treatment of embryos for a long period (1-3 days) starting at the blastula stage with Li⁺ and the inhibitors of PDE and protein synthesis, as well as with Zn²⁺, produced spherical embryos with little or no archenteron. The stages at which these substances produced abnormal embryos with a poor archenteron are post-hatching stages.     

香果树体细胞胚胎发生过程中4种同工酶的研究

用非变性聚丙烯凝胶电泳技术对香果树体细胞胚胎发生及形态建成过程中过氧化物酶(POD)、酯酶(EST)、淀粉酶(AMY)和超氧化物歧化酶(SOD)4种同工酶进行分析.结果表明:香果树体细胞胚胎发生及形态建成过程中,POD、EST、AMY和SOD活性变化与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织酶谱差异明显,胚性愈伤组织中EST和AMY同工酶酶带多且活性高,非胚性愈伤组织中缺乏EST和AMY同工酶表达,AMY同工酶可作为胚性细胞分化和发育的重要标志.香果树体细胞胚形态建成过程中,球形胚时期的AMY、POD、EST同_T酶活性最强,表明这一时期生理代谢旺盛,是体细胞胚形态建成的关键时期;POD、AMY和SOD 3种同工酶的酶谱及表达强弱在形态建成的不同时期呈现有规律的变化,可作为香果树体细胞胚发生发育特定时期的参考标记. 与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织酶谱差异明显,胚性愈伤组织中EST和AMY同工酶酶带多且活性高,非胚性愈伤组织中缺乏EST和AMY同工酶表达,AMY同工酶町作为胚性细胞分化和发育的重要标志.香果树体细胞胚形态建成过程 ,球形胚时期的AMY、POD、EST同_T酶活性最强,表明这一时期生理代谢旺盛,是体细胞胚形态建成的关键时期;POD、AMY和SOD 3种同工酶的酶谱及表达强弱在形态建成的不同时期呈现有规律的变化,可作为香果树体细胞胚发生发育特定时期的参考标记. 与胚性愈伤组织的诱导及体细胞胚的发生发育密切相关.非胚性愈伤组织和胚性愈伤组织     

Animalizing Effect of A23187 on Sea Urchin Embryos

Pulse treatment of sea urchin embryos with 3 μM A23187 for 2 hr starting at a stage in initial 10 hr period of development at 20°C, followed by a culture in normal sea water up to the pluteus corresponding stage (45 hr after fertilization), yielded many large exogastrulae with thin embryo walls. The pulse treatment starting at a time between 10 and 13 hr after fertilization yielded considerable number of large prisms and gastrulae having thin embryo walls. Probably, the pulse treatment exerts stimulating effects on ectodermal cell determination in whole span of pre-hatching period to produce animalized embryos. On the other hand, pulse treatment with A23187 in pre-hatching period exerts stage-specific effects on gut formation. Embryos, thus treated for 2 hr starting at stages between 3 and 5 hr after fertilization, produced quite small exoguts but those treated at stages between 7 and 8 hr formed well developed and long exoguts. In embryos treated at the other stages than above, guts or exoguts were almost the same in their size to those in normal ones. These effects of A23187 on morphogenesis were canceled by procaine, tetracaine and ruthenium red. Probably, artificial Ca²⁺signal induced by A23187 alters the determination of cell fates, programmed in pre-hatching period.     

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.     

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.     

Evans blue treatment promotes blastomere separation and twinning in Lytechinus pictus embryos

Demembraned Lytechinus pictus embryos briefly treated with the dye Evans Blue during the first cleavage division subsequently showed frequent blastomere disengagement leading to development of twinned embryos. Further development of twinned embryos was observed in hanging drops and in batch cultures. The timing of micromere production was disturbed in some twinned embryos, but this disturbance was not correlated with subsequent developmental problems. Many twinned embryos resulting from blastomere separation following Evans Blue treatment developed into small but normal-appearing plutei.     

Evaluation of anionic histological dyes as co-injectable cell markers in pre-implantation mouse embryos

The enzyme horseradish peroxidase (HRP) is a widely used microinjectable cell marker for studying cell position, lineage, and migration in many kinds of animal embryos. Marked cells are easily identified because they darken when exposed to a chromophore and an HRP substrate such as hydrogen peroxide. This assay, however, requires cytochemical fixation. Thus, when HRP-marked cells need to be identified prior to fixation, visible co-injectants such as dyes and fluorescent substances have been used with HRP. Fluorescent substances have limitations because their excitation could be harmful to the marked cells. Visible but non-fluorescent co-injectants, however, would permit visualization of HRP-marked cells without inflicting such damage. We tested the compatibility of several histological dyes and electrolytic carriers with HRP iontophoresed as a cell marker in 2-cell mouse embryos. The dyes tested were Evans Blue, Cibacron Blue F3GA, Fast Green FCF, and Patent Blue Violet; the electrolytic carriers were KCl, K2SO4, CH3CO2K, and KH2PO4. The combination found most useful was Patent Blue Violet in K2SO4. Survival of embryos incubated to the blastocyst stage following injection with HRP + Patent Blue Violet in K2SO4 at the 2-cell stage was significantly greater than that of embryos injected with any other dye. Although the proportion of embryos undergoing the 8-cell-to-morula transition was somewhat decreased by this treatment, the proportion of embryos reaching the blastocyst stage was comparable to that in the uninjected (control) group. Our results indicate that Patent Blue Violet is a useful, HRP-co-injectable dye for short-term cell marking in preimplantation mouse embryos.     

Lactate dehydrogenase isoenzymes in loach (Misgurnus fossilis) development

Isoenzyme patterns of lactatedehydrogenase (LDH, EC 1.1.1.27) were determined in the loach (Misgurnus fossilis) oocytes, unfertilized eggs, developing embryos and larvae, and the sensitivity of the isoenzymes to the effect of AgNO3, p-chlormercurybenzoate and high temperature were investigated. The presence of 4 phenotypes of LDH patterns which differed in the isozymes number, in their relative activity, in electrophoretic mobility in the polyacrylamide gel columnes and in the frequency of occurence in different populations of the investigated species is demonstrated. Before the hatching stage there appeared a new isozyme which was completely inhibited by AgNO3 and had the same electrophoretic mobility in all LDH phenotypes. During the larvae development the content of this LDH isozyme increased. Some isozymes, especially those which had low anodic mobility and decreased in their relative quantity, disappeared during the development of embryos and larvae, p-Chlormercurybenzoate in the concentration 1 mM and heating at 70 degrees C during 20 minutes did not significantly affect LDH activities in the loach eggs, embryos and larvae.     

The effects of U.V.-light, ionizing radiation and the carcinogen N-acetoxy-2-fluorenylacetamide on the development in vitro of one- and two-cell mouse embryos.

The development of one- and two-cell mouse embryos to morula-blastula stages was followed in vitro after treatment with low doses of U.V.-light, ionizing radiation or N-acetoxy-2-fluorenylacetamide. Exposure of one-cell embryos to either radiation source 18 and 24 hours after human chorionic gonadotropin injections prevented maturation, most embryos being arrested at the one-cell stage and a few at the two-cell stage. Two-cell embryos, however, were not sensitive to low doses of either U.V. or X-irradiation and developed normally. Treatment of early one-cell embryos with the carcinogen, N-acetoxy-2-fluorenyl-acetamide (0-7 muM), also arrested development, whereas exposure of late one-cell embryos did not completely prevent maturation to morula-blastula stages. Exposure of two-cell embryos to the same concentration of carcinogen had no effect on their development to blastulas. Results with all three agents showed that mouse embryos at the one-cell stage are more sensitive than those at the two-cell stage, as judged by their ability to develop in vitro.     

The role of animal-vegetal interaction with respect to the determination of dorsoventral polarity in the equal-cleaving spiralian,Lymnaea palustris

Summary Spirally cleaving embryos in which the first two cleavages generate four equal-sized blastomeres remain radially symmetrical along their animal-vegetal axis until the interval between third and fourth quartet formation. At this time animal micromeres and vegetal macromeres contact each other as they elongate and occlude the central, fluid-filled cleavage cavity. The overlying micromeres focus their contacts onto one of the four macromeres, the presumptive 3D macromere, as it elongates to a central position within the embryo. We tested the hypothesis that this animal-vegetal interaction was causally involved in the determination of the symmetry properties in both the animal and vegetal hemispheres by reversibly inhibiting animal-vegetal contact at the 24 cell stage with cytochalasin-B. Embryos remained hollow throughout the treatment period and animal-vegetal interaction did not occur. After treatment, blastomere elongation occurred but no D quadrant macromere appeared and the vegetal hemisphere remained radialized. On the basis of cleavage and ciliation patterns of first quartet derivatives, treated embryos remained fully or partially radialized, showing a strong tendancy to develop as ventral quadrants. These results show that the quadrants of this equal-cleaving spiralian are not definitively determined until after the 24 cell stage and that animal-vegetal interaction is required for D quadrant determination. The mechanisms of symmetrization in the animal and vegetal hemispheres of equal-cleaving spiralians is also discussed.     

Preliminary observations on the synthesis of glycosaminoglycans in the sea urchin embryo.

A method based on the degradation by enzymes and nitrous acid of isotopically labelled glycosaminoglycans has been employed to study the synthesis of these compounds in normal, animalized and vegetalized sea urchin embryos. According to standard criteria, these organisms synthesize dermatan sulfate, heparan sulfate, hyaluronate and keratan sulfate. The hyaluronate seems to be slightly sulfated, it may thus be mucoitin sulfate. The preliminary results obtained suggest a conspicuous difference between animalized and vegetalized embryos: the synthesis of dermatan sulfate is suppressed in the former, while proceeding normally in the latter. The synthesis of heparan sulfate is not affected by our experimental conditions, but the isotope incorporation in hyaluronate and in keratan sulfate is decreased, more in the vegetalized than in the animalized embryos.