Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Purines inhibit the development of mouse embryos in vitro

The first cleavage of embryos derived from random-bred, inbred, and hybrid-inbred female mice was not arrested by purines at concentrations as high as 30 microM. Development after the first or second cleavage was arrested by hypoxanthine, adenosine or inosine, but not guanosine. In agreement with previous results, the purine-induced block was reversed when arrested embryos were transferred to purine-free media after 24 h in culture. The cleavage arrest was not due to elevations of cAMP as a result of inhibition of phosphodiesterase activity since similar concentrations of phosphodiesterase inhibitors or dibutyryl cAMP did not block development. Treatment with inhibitors of enzymes that convert IMP to AMP or to GMP did not reverse the hypoxanthine-induced block, thus demonstrating that mitotic arrest is mediated by a mechanism different from the hypoxanthine arrest of meiosis. Thymidine incorporation studies showed that the block did not prevent the onset of DNA synthesis. The results reveal a profound sensitivity to purine inhibition of a cell process that occurs during the first 30 h of mouse embryo development and is necessary for progession through the G2 or M phases of the second or third cleavage.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

DNA replication is required for tissue-specific enzyme development in ascidian embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stage that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Effect of actinomycin D and sibiromycin on 3H-thymidine incorporation into the early developmental stages of Nereis virens Sars

The Nereis virens embryos at the stages of 2, 8, 16 and 32 blastomeres end of cleavage and beginning of rotation were placed in the actinomycin D or sibiromycin solutions and the effect of antibiotics on 3H-thymidine incorporation during cleavage, at the beginning of rotation and in trochophore was determined by means of autoradiography after careful washing the embryos off. Under the effect of actinomycin D the intensity of 3H-thymidine incorporation during cleavage decreased insignificantly, at the gastrula stage somewhat exceeded that in the control, and at the stages of trochophore formation decreased twice. At the later stages it approached the normal level. In the experiments with sibiromycin which proved to have more distinct inhibitory effect, the stage of trochophore formation was also found to be the most sensitive to the antibiotic.     

Control of first cleavage in single-cell reconstituted mouse embryos

Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Effect of bifolar and chlofiden on DNA synthesis of sarcoma 45 cells and on mitotic activity of rat small intestine epithelium

Results of the study on the influence of new antitumor preparations chlofiden and bifolar on sarcoma 45 DNA synthesis and on mitotic activity are given. It was shown that bifolar and chlofiden led to inhibition of label incorporation into DNA (3H-thymidine). Inhibition of mitotic activity after injection of both preparations in toxic doses (DL50) was revealed. During injection of the bifolar functional character of changes was established that is mitotic activity of the preparation during injection was restored.     

Temporal and spatial pattern of differences in microtubule behaviour during Drosophila embryogenesis revealed by distribution of a tubulin isoform

Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated alpha-tubulin has revealed that acetylated and nonacetylated alpha-tubulin isoforms have different patterns of distribution during early development. Acetylated alpha-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated alpha-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated alpha-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated alpha-tubulin appeared in early embryos when micro-tubules were stabilized by taxol treatment or anoxia. Because acetylated alpha-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated alpha-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of alpha-tubulin is not merely a consequence of cellularization. After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated alpha-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated alpha-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated alpha-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated alpha-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated alpha-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4 degrees C.     

Deoxyribonucleic acid synthesis and deoxyribonucleic acid-polymerase activity during early germination of wheat embryos at high and low viability

³H-thymidine incorporation and DNA-polymerase activity during early hours of wheat embryo germination at two viability levels have been studied. The patterns of two biosynthetic activities, as well as the dependence of DNA synthesis on protein synthesis, indicated the presence of a delay in the early phase of imbibition of the aged embryos with respect to viable germs.     

DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

Isolation and characterization of a Drosophila gene essential for early embryonic development and formation of cortical cleavage furrows

We have isolated a new female sterile mutant from Drosophila melanogaster, which arrests the embryonic development during the transition from syncytial to cellular blastoderm. Cytological analysis of the mutant embryos indicates that pseudocleavage furrows in the syncytial blastoderm are abnormal but not completely disrupted. However, cleavage furrows during cellularization are totally disorganized, and no embryos can develop beyond this stage. Consistent with this observation, the expression of this gene peaks around the cellular blastoderm and not in any later developmental stages. Based on immunofluorescence experiments, the protein product of this gene is localized in both pseudocleavage furrows at the syncytial blastoderm and in the cleavage furrows during the cellularization stage. Sequence homology analysis demonstrates a modest, but statistically significant, similarity of this protein with the carboxyl-terminal domains of dystrophin and a family of proteins collectively known as apodystrophins. It is possible that this protein may play an essential role in organizing and maintaining a specialized cytoskeletal structure, a function also suggested for dystrophin and apodystrophins.     

Protection of DNA during early development: adaptations and evolutionary consequences

The rapidly dividing cleavage stages of embryos do not have the typical responses to cell damage, such as induction of the heat shock response, use of mitotic checkpoints, or use of apoptosis to eliminate severely damaged cells. This could create problems with integrity of DNA, but the solution in these embryos appears to be a "be prepared" approach, in which specific adaptations are used to minimize DNA damage during cleavage and the use of apoptosis at the mid-blastula transition to remove any cells that were nevertheless damaged. It has been assumed that this approach has evolved because of the advantage of rapid production of a motile larvae. Alternatively, this particular approach may have the selective advantage of increasing mutation rate when there are greater environmental stresses. This could provide more variants on which selective pressures could act and thus accelerate evolution during environmentally stressful periods.     

An automated procedure to measure DNA synthesis in preimplantation mouse embryos

This paper describes a sensitive, reproducible, and automated procedure to measure DNA synthesis in preimplantation mouse embryos. Conditions for the DNA synthesis assay have been optimized as follows: (1) 4 μCi/ml³H-thymidine (sp. act. 20 Ci/immole); (2) a labeling period from 2 to 7 hours; (3) a 3-hour preincubation period for blastocysts and from 0 to 7 hours preincubation for 8-cell embryos; and (4) from 1 to 64 embryos per assay. The amount of DNA synthesis per embryo was found to be directly proportional to the number of cells (nuclei) per embryo. The described assay should be useful for future studies on the effect of synthetic and natural compounds on the development of preimplantation mouse embryos, as measured by perturbations in embryonic DNA synthetic activity.     

DNA synthesis during development of adventive embryos of wild carrot

Embryo development in cultured wild carrot tissue was studied by considering the relationship between DNA biosynthesis and embryo morphogenesis. Autoradiographic data of^3H-thymirdine incorporation indicated a higherpercentage of cells undergoing DNA synthesis in adventive embryos than in proembryonic masses. A definite pattern of DNA synthesis was observed during the transition from a proembryonic mass to the torpedo stage of embryogenesis. In particular the labeling pattern for both the heart and torpedo stages was found to be coincident with specific morphogenetic events.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

Nuclear and cytoplasmic DNA synthesis in cotton embryos: a correlated light and electron microscope autoradiographic study

Summary To investigate the possibility, implied by an earlier report, that large amounts of degradable DNA are probably present in the cytoplasm of young cotton embryos, an investigation was undertaken to establish the distribution, amount and metabolic stability of DNA in cotton embryos. Several sensitive cytochemical tests failed to detect any but small amounts of extranuclear DNA. Quantitative determination of the nucleic acid content of embryos during embryogenesis showed that the amounts of DNA and RNA remained fairly constant during embryogenesis, with a ratio of RNA to DNA of about 3.5 to 1. Quantitative autoradiography at both the light and electron microscope levels of sections from embryos pulse-labeled with ³H-thymidine showed that the grain density over the nucleus and cytoplasm did not change during a seven-hour period after labeling, nor did the distribution of label in the cytoplasm. Virtually all incorporation was eliminated by the inclusion of iododeoxy-uridine in the medium. Almost all of the nuclear label and at least 90% of the cytoplasmic label after ³H-thymidine incorporation was eliminated by deoxyribonuclease. It was concluded that there are no unusual features related to DNA distribution or metabolism in cotton embryo; i.e., that only small amounts of DNA are present in the cytoplasm and that all of the DNA is metabolically stable.Approximately 40% of the cytoplasmic grains after ³H-thymidine labeling were not associated with either plastids or mitochondria (i.e., were more than 0.1 micron distant). No fully satisfactory explanation for such an apparently high figure could be given.This work was supported by a Public Health Service fellowship 5-F2-GM-22,031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-R01-Ca0356-10 and by Miller Institute for Basic Science.