Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. V. Inability of inhibitor to repress 5S RNA synthesis.

A specific inhibitor of ribosomal RNA (rRNA) synthesis was partially purified from an acid-soluble fraction of Xenopus laevis blastulae. Effects of this inhibitor on 5S rRNA synthesis of isolated neurula cells of the same species were investigated. The results show that the synthesis of both 5S rRNA and 4S RNA proceeds normally when both 18 and 28S rRNA are almost completely inhibited. Failure of the inhibitor to suppress 5S rRNA synthesis suggests that it plays an important role in the regulation of 18 and 28S rRNA synthesis during development and that the synthesis of 5S rRNA is not coordinated to that of 18 and 28S rRNA.     

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.     

Contribution of maternal mRNA for maintenance of Ca2+-dependent reaggregating activity in dissociated cells of Xenopus laevis embryos

Dissociated Xenopus laevis blastula cells, where reaggregation was inhibited in Ca2+-free medium, reaggregated immediately after the addition of Ca2+. This reaggregation was not inhibited by cordycepin or actinomycin D treatment during culture, although cycloheximide and puromycin were inhibitory. The reaggregation was not inhibited even when fertilized eggs were microinjected with cordycepin and their RNA synthesis was continuously inhibited through cleavage to blastula stages. In neurula cells, cordycepin treatment induced significant reduction in sizes of aggregates formed. These results suggest that the Ca2+-dependent reaggregating activity of blastula cells is maintained by the translation of maternal, rather than newly synthesized, mRNA.     

Non-Coordinated Synthesis of RNA's in Pre-Gastrular Embryos of Xenopus Laevis

The rates of syntheses of 18S and 28S rRNA, 5S RNA, capped mRNA and 4S RNA were determined in isolated cells from pre- and post-gastrular embryos of Xenopus laevis. The rate of rRNA synthesis per nucleolated cell Mas about 0.2 pg/hr, or about 5.5 × 10⁴ molecules/hr at the blastula stage, and this value remained constant in later stages. At the blastula stage, about 30 molecules of 5s RNA, 10 molecules of capped mRNA and 900 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA. These values were all greatly reduced during the gastrula stage, and at the neurula stage, one molecule each of 5S RNA and capped mRNA and 10 molecules of 4S RNA were synthesized per molecule of 18S or 28S rRNA.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. IV. The release of inhibitor from cultured cells.

Previous reports from our laboratory have shown that a culture medium, conditioned by the growth of isolated cells of Xenopus laevis blastulae, contains a low-molecular-weight substance which selectively inhibits 18 and 28S ribosomal RNA (rRNA) synthesis. Although the occurrence of an inhibitor in an acid-soluble fraction of blastulae has recently been demonstrated, our observation of an inhibitor in a conditioned medium has not been confirmed by other laboratories. To resolve this discrepancy, we have reexamined the effects of conditioned media and acid-soluble extracts on rRNA synthesis by neurula cells. (1) The inhibitory activity for rRNA synthesis can consistently be observed in blastula-conditioned media, provided some of the cells have been broken down during conditioning. If cell rupture is avoided, an inactive conditioned medium is obtained. (2) A homogenate of blastulae inhibits total RNA synthesis and shows no selective inhibition of rRNA synthesis. (3) Charcoal treatment of the conditioned medium and homogenate enhances their specificity for rRNA synthesis. It is then likely that cell breakdown may be involved in the release of the inhibitor into the medium and that some differences in the methods of preparation of conditioned medium may account for the above discrepancy.     

Kinetic behavior of 30S rRNA intermediate labeled in developing embryonic cells of Xenopus laevis

In Xenopus neurula cells, "30S" RNA was found to be labeled with 3H-uridine after a relatively short labeling period. Results obtained from cumulative labeling and pulse-labeling and chase experiments with cells from late gastrulae, yolk plug-stage embryos, and neurulae showed that the 30S RNA is an intermediate in rRNA processing and is derived from 40S pre-rRNA and processed to 28S rRNA. The half-life of the 30S rRNA intermediate was about 7.5 min or less at the three stages examined.     

Gene Expression from Endogenou and Exogenously-introduced DNAs in Early Embryogenesis of Xenopus laevis

In Xenopus laevis embryogenesis, gene expression from endogenous and exogenously-introduced DNAs is reported to start only after 12 rounds of cleavage, at the stage called midblastula transition (MBT). In isotopic labeling experiments, however, we found that the synthesis of heterogeneous mRNA-like RNA occurs from the early cleavage stage, and that gene expression from zygotic genomes occurs sequentially in three characteristically different phases: the pre-MBT phase, in which heterogeneous mRNA-like RNA and small amounts of small-molecular-weight RNAs are synthesized; the MBT phase, in which there is a large increase in 4S RNA synthesis, and the post-MBT phase, in which rRNA is also synthesized. Moreover in our studies on the expression of exogenously introduced DNA, we found that circular forms of bacterial chloramphenicol acetyltransferase (CAT) genes connected to viral promoters were expressed from the early cleavage stage, whereas circular forms of genes connected to Xenopus cardiac α-actin promoter were expressed only after the embryos reached the neurula stage, when the endogenous α-actin gene started to be expressed. We, therefore, conclude that in Xenopus embryogenesis, DNA-dependent RNA polymerases II, III and I are activated in this order, and that the promoter, not changes associated with the MBT, probably determine the timing of gene expression.     

Tissue-specific expression of an Ornithine decarboxylase paralogue, XODC2, in Xenopus laevis

Ornithine decarboxylase (ODC) is involved in the biosynthesis of polyamines and hence has been found in almost all types of cells studied. Therefore it is frequently used as internal standard. We isolated a cDNA, XODC2, which is a paralogue to ubiquitous ODC and expressed in a spatial and temporal manner during the early embryogenesis of Xenopus laevis. Expression of XODC2was first detected at the animal pole at stage 9. During neurula stages the signals were found both in the extreme anterior and posterior part of the dorsal body axis. In tailbud stages the expression is further shifted to both the tail and head areas and gradually restricted to distinct tissues: forebrain, inner layer of epidermis of the head area, stomodeal-hypophyseal anlage, frontal gland, ear vesicle, branchial arches, the front tip of neural tube and proctodeum. In addition, signals were also found in the inner layer of epidermis underneath the cement gland during early tailbud stages while in later tailbud stages signals were detected at the apical zone of the cement gland. Comparative studies indeed could confirm that XODC1 in contrast to XODC2 is expressed ubiquitously throughout the whole embryos during early development of Xenopus laevis.     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.     

IQGAP2 is required for the cadherin-mediated cell-to-cell adhesion in Xenopus laevis embryos

We have previously identified two Xenopus homologues of mammalian IQGAP, XIQGAP1 and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. In order to clarify function of the IQGAPs during development, we performed knock-down experiments on the XIQGAPs in Xenopus laevis embryos by microinjecting morpholino antisense oligonucleotides into blastomeres at the two-cell stage. Suppression of XIQGAP2 expression caused ectodermal lesions in the neurula stage embryos. While suppression of XIQGAP1 expression alone did not show any obvious defect in subsequent developmental processes, simultaneous knock-down of both XIQGAPs caused the ectodermal lesions during the gastrula stage. Histological studies suggested that a loss of cell adhesion in the ectodermal and mesodermal layers of the embryos caused the defect. The suppression of XIQGAP2 expression resulted in loss of actin filaments, beta-catenin, and XIQGAP1 from cell borders in the ectoderm, although it did not affect the expression levels of these proteins. Furthermore, it inhibited Ca(2+)-induced reaggregation of embryonic cells which had been dissociated in a Ca(2+)/Mg(2+)-free medium. These results strongly suggest that XIQGAP2 is crucial for cell adhesion during early development in Xenopus.     

Inhibition of proteoglycan synthesis eliminates left-right asymmetry in Xenopus laevis cardiac looping

The heart of any vertebrate is formed from an apparently symmetric cardiac tube that loops consistently in the same direction along the left-right axis of the embryo. In the amphibian Xenopus laevis, inhibition of proteoglycan synthesis by p-nitrophenyl-beta-D-xylopyranoside during a narrow period of development from late gastrula to early neurula specifically eliminated the looping of the cardiac tube. Most of the proteoglycans synthesized during this period were heparan sulfate proteoglycans. Treatment with p-nitrophenyl-alpha-D-xylopyranoside, an analogue that does not inhibit proteoglycan synthesis, did not interfere with cardiac looping. The critical period for proteoglycan synthesis was coincident with the migration of cardiac primordia to the ventral midline. The inhibition of cardiac looping was further explored in explants of cardiac primordia and anterioventral ectoderm. In recombinate embryos in which half the embryo, and thus one of the two heart primordia, was treated with p-nitrophenyl-beta-D-xylopyranoside, and the other half was untreated, cardiac looping occurred normally. It is proposed that the left-right axis in Xenopus, as reflected in cardiac looping, is established early in development, and that proteoglycan synthesis is involved in the transduction of left-right axial information to the cardiac primordia during migration.     

Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.