Developmental Patterns in Histones and Histone Synthesis During Early Embryogenesis of the Sea Urchin Paracentrotus lividus

Abstract. Striking developmental changes in histone and histone synthesis in sea urchin embryos were observed in three histone classes, H1, H2A and H2B. In each case there is a shift in histone synthesis from the early cleavage stage types to other types of histones at the morula stage; Two new forms appear after the blastula stage. In addition, multiple changes in histone types were found during gameto-genesis in the male and female gonads where specific histones, different from the embryonic histones, were observed.     

Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos

The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages.     

The mobilization of maternal histone messenger RNA after fertilization of the sea urchin egg

The extent of protein, RNA and DNA synthesis in early cleavage stages of the sea urchin embryo (Parechinus angulosus) was determined. A histone mRNA specific cDNA was used in hybridization experiments to investigate the cytoplasmic localization of maternal histone mRNA in the unfertilized sea urchin egg and first cleavage stage embryo. In the unfertilized egg histone mRNA was localized exclusively in ribonucleoprotein particles with none in ribosomes or polyribosomes. This distribution changed after fertilization, in particular, coupled with the first cleavage telophase there was a significant transfer of histone mRNA from the ribonucleoprotein fraction to the polyribosomes. The results indicate mRNA specific translational control mechanisms.     

The early and late sea urchin histone H4 mRNAs respond differently to inhibitors of DNA synthesis

The effect of inhibiting DNA synthesis on the concentration of the alpha-histone mRNA and the late histone mRNAs in sea urchin embryos was measured. The alpha-histone mRNA concentrations did not change, while the late histone mRNA concentrations were rapidly reduced at the three developmental stages (morula, blastula, and mesenchyme blastula) tested. The rapid degradation of the late histone mRNAs was prevented when protein synthesis was inhibited.     

DNA polymerase activity in preimplantation mouse embryos.

DNA polymerase activity was measured in mouse embryos at stages before implantation to determine whether it increases in proportion to the amount of DNA synthesis, as it does in populations of differentiated mammalian cells, or remains constant, as it does in early sea urchin embryos. Total enzyme activity was found to be relatively unchanged following fertilization and in the first few cleavage stages. However, between the 12- and 120-cell (blastocyst) stage, the amount of activity increased by several-fold. These results indicate that the relationship between amount of DNA polymerase activity and DNA synthesis in mouse embryos exhibits two phases: in the early cleavage phase it is similar to that in sea urchin embryos, whereas, in the blastocyst phase, it is similar to that in differentiated mammalian cells.     

Proteins ADP-ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development

The ADP-ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine-¹⁴C]NAD or [adenylate-³²P]NAD. ADP-ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP-ribosylation of the histone fraction did not change appreciably during early development. In the TCA-insoluble protein fraction of the nuclei, the rate of ADP-ribosylation increased from fertilization to the morula stage, then decreased and again increased from the mesenchyme blastula to the late gastrula stage. After exposure of the nuclear fraction to [adenylate-³²P]NAD, a protein band with a molecular weight of 90 kDa was detected by SDS-polyacrylamide gel electrophoresis and radioautography at all stages examined. Its labeling intensity indicated that its ADP-ribosylation is higher at the morula and late gastrula stages than at other stages. In the plasma membrane fraction, proteins with molecular weights of 22 and 68 kDa were ADP-ribosylated and their rates of ADP-ribosylation hardly changed during early development.     

Translation of maternal histone mRNAs in sea urchin embryos: a test of control by 5' cap methylation

Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.     

Individual regulation of the accumulation of H1 mRNA and core histone mRNAs in sea urchin embryos.

The relative cytoplasmic accumulation of the individual histone mRNAs in sea urchins was determined by gel analysis of 3H-labeled cytoplasmic RNA isolated from embryos of the early cleavage through the mesenchyme blastula stages. A number of separate determinations showed that H1 mRNA accumulates at a molar ratio of 0.5 or less compared with each of the H2 or H3 core histone mRNAs through approximately the first 12 h of embryonic development. After this time, the accumulation of H1 mRNA increases relative to the core histone mRNAs, and approximately equimolar amounts of the histone mRNAs are produced by about the 14-h stage. The equimolar synthesis of H1 mRNA appears to be transient, returning to 0.5-molar levels several hours later. The increase in H1 mRNA accumulation, relative to the core histone RNAs, is coincident with the transition from expression of the early (alpha) sea urchin histone gene set to the late histone genes. Since all five of the early histone genes occur in a 1:1 ratio within repeating units, the data suggest that the genes within a single repeat, or their immediate products, are individually regulated. Gel analysis of the proteins synthesized in vivo by embryos demonstrates that the pattern of synthesis of the histone proteins reflects the changing ratios of the histone mRNAs.     

Evidences of two different sets of histone genes active during embryogenesis of the sea urchin Paracentrotus lividus.

Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was markedly different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74 degrees C in all cases except for the mesenchyme histone mRNAs whose Tm was 59 degrees C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.     

Effects of 5 azacytidine on DNA methylation and early development of sea urchins and ascidia

5-azacytidine (5-azaCR), an analogue of cytidine, inhibits nuclear DNA methylation in early sea urchin embryos. This inhibition is specific and dose-dependent. Exposure of sea urchin embryos at any stage between one-cell and blastula, to micromolar quantities of 5-azaCR invariably inhibits development beyond the blastula stage. In a substantial number of embryos arrested at the blastula stage, spicule formation proceeds although other morphological differentiation is lacking. No significant effect on development is seen if sea urchin embryos are exposed to 5-azaCR at post-blastula stages. 5-azaCR also inhibits the development of a mosaic egg such as the ascidian Phallusia mammilata at the blastula stage, indicating that both regulative (sea urchin) and mosaic (ascidian) embryos respond more or less similarly to 5-azaCR treatment.     

Quantitative Measurement of Rates of 5S RNA and Transfer RNA Synthesis in Sea Urchin Embryos

Embryonic differentiation is believed to be due to a programmed expression of genes, which includes their time of activation, sequence of appearance, and amount transcribed into the immediate gene product, RNA. Differential synthesis of the major RNA classes, such as the ribosomal RNAs (28S, 18S, 5S) and transfer RNA (tRNA), characterizes many animal developing systems, including the sea urchin embryological system. Previous work has shown that the genes for 5S RNA and tRNA are active during early cleavage in sea urchin embryos. The present study focused on quantitatively measuring and comparing the rate of 5S RNA and tRNA synthesis in cleavage, early blastula, and early pluteus embryos of Arbacia punctulata. At each stage, embryos were labeled for 3 h with [8-³H]-guanosine. Total cellular RNA was extracted using the cold (4°C)-phenol-sodium dodecyl sulfate method and purified (LiCl-soluble) RNA preparations were fractionated by electrophoresis on 10% polyacrylamide gels. The amount of 5S RNA and tRNA synthesized at each stage was calculated from the radioactivity coincident with the 5S RNA and with the tRNA absorbance peaks (A_{260 nm}) on each gel, from the known guanosine monophosphate (GMP) compositions of sea urchin 5S RNA and tRNA and from the average specific radioactivity of the GTP precursor pool during each 3 h labeling period. The results showed that on a per embryo basis the rates of 5S RNA and tRNA synthesis increased slightly (about 1.4-fold) from cleavage through pluteus stages, while on a per cell basis the rates declined severalfold (about 3-fold) during embryogenesis. The rates of 5S RNA and tRNA synthesis determined here parallel previously-reported levels of RNA polymerase III in sea urchin embryos, suggesting that cellular levels of RNA polymerase III may exert some positive control over 5S RNA and tRNA synthesis during sea urchin embryogenesis.     

First cell cycles of sea urchin Paracentrotus lividus are dramatically impaired by exposure to extremely low-frequency electromagnetic field

Exposure of fertilized eggs of the sea urchin Paracentrotus lividus to an electromagnetic field of 75-Hz frequency and low amplitudes (from 0.75 to 2.20 mT of magnetic component) leads to a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatids during the mitotic events. Because acetylcholinesterase (ACHE) is thought to regulate the embryonic first developmental events of the sea urchin, its enzymatic activity was assayed in embryo homogenates and decreased by 48% when the homogenates were exposed to the same pulsed field. This enzymatic inactivation had a threshold of about 0.75 +/- 0.01 mT. The same field threshold was found for the effect on the formation of anomalous embryos of P. lividus. Moreover, ACHE inhibitors seem to induce the same teratological effects as those caused by the field, while blockers of acetylcholine (ACh) receptors are able to antagonize those effects. We conclude that one of the main causes of these dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of ACh due to ACHE inactivation. The crucial role of the membrane in determining the conditions for enzyme inactivation is discussed.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.