Chk1 is activated at the midblastula transition in Xenopus laevis embryos independently of DNA content and the cyclin E/Cdk2 developmental timer

Cell cycle checkpoints that are engaged in response to damaged and unreplicated DNA may serve additional, constitutive functions. In the developing Xenopus laevis embryo, the checkpoint kinase Chk1 is transiently activated at the midblastula transition (MBT), a period of extensive cell cycle remodeling including the acquisition of cell cycle checkpoints. The timing of many cell cycle remodeling events at the MBT, such as the lengthening of cell cycles, depends upon a critical nucleocytoplasmic (N/C) ratio. However, other events, including the degradation of maternal cyclin E, do not depend upon the N/C ratio, and are regulated by an autonomous developmental timer. To better understand what regulates Chk1 activation at the MBT, embryos were treated with aphidicolin, at different developmental times and for different lengths of time, to reduce the DNA content at the MBT. Chk1 was activated at the MBT in these embryos establishing that Chk1 activation occurs independently of the N/C ratio. Cdc25A is normally phosphorylated by Chk1 at the MBT and then degraded. The degradation of Cdc25A demonstrated partial dependence on DNA content, suggesting that factors other than Chk1 regulate its degradation. When the cyclin E developmental timer was disrupted with the Cdk2 inhibitor Δ34-Xic1, Chk1 was still activated at the MBT, indicating that activation of Chk1 at the MBT was not directly linked to the cyclin E timer. Conversely, unreplicated or damaged DNA, delayed the degradation of cyclin E at the MBT, indicating that the cyclin E/Cdk2 timer is sensitive to engagement of cell cycle checkpoints.     

斑马鱼中囊胚过渡调控机制

斑马鱼中囊胚过渡(MBT)始于受精卵的第10次卵裂,此时亦伴有细胞周期延长,分裂同步性丧失,合子型基因开始转录活化,胚胎细胞开始具备运动迁移能力等现象。斑马鱼MBT。的发生依赖于胚胎细胞的核质比,胚胎细胞周期中的G1时相则只有在合子型基因组开始被转录活化后才能出现。细胞周期检验点的激活可能也是受转录调控的,但中期检验点对DNA复制抑制状态的响应不仅在MBT前后、甚至在MBT前的不同阶段也可能有具体作用途径的差异。活化的P38蛋白在胚胎中的不对称分布是维持卵裂阶段细胞分裂同步性的关键因素。尽管大规模的合子型基因的表达发生在MBT开始后,也有少数与胚层分化有关的合子型基因是在MBT。前表达的,还有一些既有母型表达也有合子型表达的基因在MBT前后分别参与不同的信号途径来调控胚胎的发育与分化。     

Phosphorylation of Claspin is triggered by the nucleocytoplasmic ratio at the Xenopus laevis midblastula transition

At the Xenopus midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.     

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.     

The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition

BACKGROUND: Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. RESULTS: We show that embryos lacking Mei-41, a Drosophila homologue of the ATM tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homologue of the Checkpoint-1 kinase. We present biochemical, cytological and genetic data indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. This pathway is nonessential during postembryonic development, but it is required to terminate the cleavage stage at the MBT. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. These mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT. CONCLUSIONS: The Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT.     

Dissection of the XChk1 signaling pathway in Xenopus laevis embryos

Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints, xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 in Xenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.     

Chk2 Regulates Cell Cycle Progression during Mouse Oocyte Maturation and Early Embryo Development

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein γ-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.     

Maternal cyclin B levels "Chk" the onset of DNA replication checkpoint control in Drosophila

In many animals, early development of the embryo is characterized by synchronous, biphasic cell divisions. These cell divisions are controlled by maternally inherited proteins and RNAs. A critical question in developmental biology is how the embryo transitions to a later pattern of asynchronous cell divisions and transfers the prior maternal control of development to the zygotic genome. The most-common model regarding how this transition from maternal to zygotic control is regulated posits that this is a consequence of the limitation of maternal gene products, due to their titration during early cell divisions. Here we discuss a recent article by Crest et al.1 that instead proposes that the balance of Cyclin-dependent Kinase 1 and Cyclin B (Cdk1-CycB) activity relative to that of the Drosophila checkpoint kinase Chk1 determines when asynchronous divisions begin.     

The Effect of a DNA Damaging Agent on Embryonic Cell Cycles of the Cnidarian Hydractinia echinata

The onset of gastrulation at the Mid-Blastula Transition can accompany profound changes in embryonic cell cycles including the introduction of gap phases and the transition from maternal to zygotic control. Studies in Xenopus and Drosophila embryos have also found that cell cycles respond to DNA damage differently before and after MBT (or its equivalent, MZT, in Drosophila). DNA checkpoints are absent in Xenopus cleavage cycles but are acquired during MBT. Drosophila cleavage nuclei enter an abortive mitosis in the presence of DNA damage whereas post-MZT cells delay the entry into mitosis. Despite attributes that render them workhorses of embryonic cell cycle studies, Xenopus and Drosophila are hardly representative of diverse animal forms that exist. To investigate developmental changes in DNA damage responses in a distant phylum, I studied the effect of an alkylating agent, Methyl Methanesulfonate (MMS), on embryos of Hydractinia echinata. Hydractinia embryos are found to differ from Xenopus embryos in the ability to respond to a DNA damaging agent in early cleavage but are similar to Xenopus and Drosophila embryos in acquiring stronger DNA damage responses and greater resistance to killing by MMS after the onset of gastrulation. This represents the first study of DNA damage responses in the phylum Cnidaria.     

Control of cleavage cycles in Drosophila embryos by frühstart

Early metazoan development consists of cleavage stages characterized by rapid cell cycles that successively divide the fertilized egg. The cell cycle oscillator pauses when the ratio of DNA and cytoplasm (N/C) reaches a threshold characteristic for the species. This pause requires maternal factors as well as zygotic expression of as yet unknown genes. Here we isolate the zygotic gene frühstart of Drosophila and show that it is involved in pausing the cleavage cell cycle. frs is expressed immediately after the last cleavage division. It plays a role in generating a uniform pause and it can inhibit cleavage divisions when precociously expressed. Furthermore, the expression of frs is delayed in haploid embryos and requires activity of the maternal checkpoint gene grapes. We propose that zygotic frs expression is involved in linking the N/C and the pause of cleavage cycle.