Abstract
In Xenopus laevis embryogenesis, fertilized eggs undergo 12 cycles of synchronous divisions and reach the stage called midblastula transition (MBT). It has long been believed that during the first 12 cycles of cleavage (pre-MBT stage), transcriptional activity of the zygotic nuclei is totally absent. However, heterogeneous mRNA-like RNA is synthesized in pre-MBT stage embryos, and exogenously-injected bacterial CAT genes with SV40 promoter are expressed from the cleavage stage. Nevertheless, the synthesis of rRNA as detected by rRNA-specific2’-O-methylation does not take place in pre-MBT embryos and starts only from the latter half of the MBT stage, corroborating the fact that formation of definitive nucleoli as well as the transcription of microinjected rRNA genes starts only at and after MBT stage. Thus, while mRNA-like RNA synthesis occurs from pre-MBT stage, synthesis of rRNA is controlled in the way that transcription of rRNA genes is totally silent during pre-MBT stage and is initiated only at the latter half of MBT stage. Once initiated, the rate of the synthesis of rRNA is constant throughout later stages on a per-cell basis. We searched substances which are responsible for the transcriptional silence of rRNA genes during the pre-MBT stage. Weak bases such as ammonium ion and amines selectively inhibited rRNA synthesis at the transcriptional level in post-MBT stage embryo cells. Since we found that the level of ammonia extracted from embryos is much higher in pre-MBT embryos than in post-MBT embryos, we suggest that weak bases like ammonium ion could be responsible for the transcriptional silence of rRNA genes by slightly increasing intracellular pH during the pre-MBT.
Highlights
midblastula transition (MBT) is the important time point of transition from the phase of cleavage division to the phase of morphogenetic cell interactions
Since we found that the level of ammonia extracted from embryos is much higher in pre-MBT embryos than in post-MBT embryos, we suggest that weak bases like ammonium ion could be responsible for the transcriptional silence of rRNA genes by slightly increasing intracellular pH during the pre-MBT
In our studies based on 3H-uridine-labeled RNA labeling profiles we previously proposed our working hypothesis that Xenopus early development consists of three characteristic phases of RNA synthesis: The first one is pre-MBT stage which is characterized by the synthesis of heterogeneous mRNA-like RNA and low level of small-molecular-weight RNA probably due to the activity of DNA-dependent RNA polymerase II, and the second phase is MBT stage which is characterized by additional activation of the labeling of 4S RNA (tRNA) synthesis due to the activity of DNA-dependent RNA polymerase III, and the third phase is post-MBT stage which is characterized by additional active synthesis of rRNA due to the activity of DNA-dependent RNA polymerase I (Figure 1)
Summary
MBT is the important time point of transition from the phase of cleavage division to the phase of morphogenetic cell interactions. The results obtained showed that embryos contained only a trace amount of rRNA-specific dinucleotides in the former 2 hrs of the blastula stage but contained a very large amount of rRNA-specific dinucleotides in the latter 2 hrs, indicating that rRNA synthesis starts in the latter half of the MBT stage (Figure 4) These results are consistent with the timing of the expression of exogenously-injected ribosomal RNA genes [44]. When we first cultured blastula cells in the ammonium chloridecontaining medium for 5 hrs, and labeled them for 5 hrs in the continued presence of the ammonium chloride, much clearer inhibition of rRNA synthesis was obtained This is an indication of the inhibition of post-MBT activation of rRNA synthesis (Figures 7(D)-(F)). These results exclude the possibility of aberrant processing or rapid wastage of the mature rRNAs
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