pre-MBT Embryos Research Articles

Nuclear Size Scaling during Xenopus Early Development Contributes to Midblastula Transition Timing

Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate. Whereas the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property, it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. Whereas previous studies suggested a role for the N/C volume ratio in MBT timing, none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. Whereas the DNA-to-cytoplasmic ratio has been implicated in MBT timing, our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development.

Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis.

RNA species whose transcription is totally silent in pre-MBT stage are not mRNA but rRNA and possible involvement of weak bases (ammonium salts and/or amines) in the transcriptional silence of rRNA genes during the pre-MBT stage in <i>Xenopus</i> early embryos

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.

Gene expression in Pre-MBT embryos and activation of maternally-inherited program of apoptosis to be executed at around MBT as a fail-safe mechanism in Xenopus early embryogenesis.

S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethione (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation of caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastocoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extents of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a "fail-safe" mechanism of early development to save the embryo from accidental damages that take place during cleavage.

Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos

In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require gamma-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition.

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.

A Maternal Form of the Phosphatase Cdc25A Regulates Early Embryonic Cell Cycles in Xenopus laevis

In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. In this paper we report the cloning of Xenopus Cdc25A and demonstrate its developmental regulation and key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus in interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortened the cell cycle and accelerated the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A displayed a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform had no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 revealed a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there was a twofold increase in the level of p-tyr in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity were both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A could be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time.

Developmental Changes in RNA Polymerase I and TATA Box-Binding Protein during EarlyXenopusEmbryogenesis

Xenopusearly embryos are transcriptionally quiescent until the midblastula transition (MBT). We have examined the question of whether the absence of rRNA synthesis is related to a deficiency in the RNA polymerase I (pol I) transcription machinery. Previously we have demonstrated that the maternally provided pol I transcription factor UBF already binds to the inactive rRNA genes of pre-MBT embryos (P. Bellet al.,1997,J. Cell Sci.110, 2053–2063). Here we have analyzed the fate of pol I and the TATA box-binding protein (TBP) through immunofluorescence and immunoblotting experiments. Pol I stockpiled in the egg is taken up byin vitroassembled pronuclei and concentrated into numerous distinct nuclear domains. Comparable storage sites of template-free pol I are also seen in nuclei of blastula to neurula stage embryos. In contrast, the amount of TBP is relatively low in oocytes and eggs but increases dramatically during the cleavage stages. Most of the newly synthesized TBP colocalizes with the stored form of pol I in the extranucleolar domains of blastula/gastrula embryos. The amount of TBP per embryo reaches peak values at the blastula/gastrula stage and then rapidly declines to normal somatic levels. The positive correlation of maximal TBP levels with the timing of the MBT suggests that overproduction of TBP is required for the formation of productive transcription complexes.

DNA sequence-specific binding activity of the heat-shock transcription factor is heat-inducible before the midblastula transition of early Xenopus development

We have examined the activity of the Xenopus heat-shock transcription factor (HSF) in extracts from stressed and unstressed embryos at various stages of development using DNA mobility shift analysis. A specific interaction between HSF and a synthetic oligonucleotide corresponding to the proximal heat-shock element (HSE) of the Xenopus HSP70B gene was greatly enhanced in heat-shocked embryos compared to controls. HSF binding was inducible at all developmental stages examined including pre-midblastula transition (MBT) stages which are incapable of expressing HSP genes. In time-course experiments with both cleavage and neurula stage embryos, the activation of HSF binding was rapid and transient. Removal of cleavage and neurula stage embryos from heat stress resulted in a rapid loss of binding activity. The molecular mass of HSF, as determined by comparative gel electrophoresis of photoaffinity-labeled factor was 88 x 10(3) in both heat-shocked cleavage and neurula stage embryos. These experiments suggest that maternally derived HSF is stored in pre-MBT embryos in a heat-activatable form and may function in the regulation of heat-shock genes immediately after the MBT.