scholarly journals Geminin Deficiency Causes a Chk1-dependent G2 Arrest inXenopus

2002 ◽  
Vol 13 (10) ◽  
pp. 3662-3671 ◽  
Author(s):  
Thomas J. McGarry
Keyword(s):  
Dna Replication ◽  
Structural Integrity ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Checkpoint Pathway ◽  
G2 Phase ◽  
Essential Function ◽  
Unusual Phenotype

Geminin is an unstable inhibitor of DNA replication that gets destroyed at the metaphase/anaphase transition. The biological function of geminin has been difficult to determine because it is not homologous to a characterized protein and has pleiotropic effects when overexpressed. Geminin is thought to prevent a second round of initiation during S or G2 phase. In some assays, geminin induces uncommitted embryonic cells to differentiate as neurons. In this study, geminin was eliminated from developing Xenopus embryos by using antisense techniques. Geminin-deficient embryos show a novel and unusual phenotype: they complete the early cleavage divisions normally but arrest in G2 phase immediately after the midblastula transition. The arrest requires Chk1, the effector kinase of the DNA replication/DNA damage checkpoint pathway. The results indicate that geminin has an essential function and that loss of this function prevents entry into mitosis by a Chk1-dependent mechanism. Geminin may be required to maintain the structural integrity of the genome or it may directly down-regulate Chk1 activity. The data also show that during the embryonic cell cycles, rereplication is almost entirely prevented by geminin-independent mechanisms.

scholarly journals Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

2012 ◽  
Vol 198 (6) ◽  
pp. 973-980 ◽  
Author(s):  
Kai Yuan ◽  
Jeffrey A. Farrell ◽  
Patrick H. O’Farrell
Keyword(s):  
Cell Cycle ◽  
Embryonic Cell ◽  
S Phase ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Mitotic Entry ◽  
G2 Phase ◽  
Embryonic Morphogenesis ◽  
Do So ◽  
S Phase Checkpoint

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. We conclude that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly.

scholarly journals Dissection of the XChk1 Signaling Pathway in Xenopus laevis Embryos

2000 ◽  
Vol 11 (9) ◽  
pp. 3101-3108 ◽  
Author(s):  
Nicholas C. Kappas ◽  
Pamela Savage ◽  
Katherine C. Chen ◽  
Allan T. Walls ◽  
Jill C. Sible
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Signaling Pathway ◽  
Embryonic Cell ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Cyclin Dependent Kinases ◽  
Xenopus Embryos ◽  
Egg Extracts ◽  
Sperm Nuclei

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 inXenopus 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.

scholarly journals Mandatory coupling of zygotic transcription to DNA replication in early Drosophila embryos

2022 ◽  
Author(s):  
Chun-Yi Cho ◽  
James P. Kemp ◽  
Robert J. Duronio ◽  
Patrick H. O'Farrell
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Cluster Formation ◽  
Embryonic Cell ◽  
Rna Polymerases ◽  
Replication Forks ◽  
Transcriptional Initiation ◽  
Cell Cycles ◽  
Zygotic Transcription

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesized that collision-avoidance mechanisms safeguard the onset of zygotic transcription in these cycles. To explore this hypothesis, we used real-time imaging of transcriptional events at the onset of each interphase. Endogenously tagged RNA polymerase II (RNAPII) abruptly formed clusters before nascent transcripts accumulated, indicating recruitment prior to transcriptional engagement. Injection of inhibitors of DNA replication prevented RNAPII clustering, blocked formation of foci of the pioneer factor Zelda, and largely prevented expression of transcription reporters. Knockdown of Zelda or the histone acetyltransferase CBP prevented RNAPII cluster formation except at the replication-dependent (RD) histone gene locus. We suggest a model in which the passage of replication forks allows Zelda and a distinct pathway at the RD histone locus to reconfigure chromatin to nucleate RNAPII clustering and promote transcriptional initiation. The replication dependency of these events defers initiation of transcription and ensures that RNA polymerases transcribe behind advancing replication forks. The resulting coordination of transcription and replication explains how early embryos circumvent collisions and promote genome stability.

Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles

1992 ◽  
Vol 102 (1) ◽  
pp. 63-69 ◽  
Author(s):  
M. Leibovici ◽  
G. Monod ◽  
J. Geraudie ◽  
R. Bravo ◽  
M. Mechali
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Xenopus Laevis ◽  
Early Development ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Antigen ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Cyclical Pattern

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

Recent advances in our understanding of the temporal control of early embryonic development in amphibians

Development ◽  
1985 ◽  
Vol 89 (Supplement) ◽  
pp. 257-270
Author(s):  
Noriyuki Satoh
Keyword(s):  
Dna Replication ◽  
Embryonic Development ◽  
Intimate Relationship ◽  
The Other ◽  
Early Embryonic Development ◽  
Embryonic Cells ◽  
Temporal Control ◽  
Midblastula Transition ◽  
Developmental Event ◽  
Cell Divisions

Recent studies on temporal control of early amphibian development are reviewed. It is becoming clear that the development of an embryo is not timed by a single clock set in motion at fertilization, instead each developmental event seems to be timed by its own clock-like mechanism. The timing of developmental events is rigidly determined within embryonic cells, and usually can not be altered experimentally. One exception, however, is the timing of midblastula transition in amphibian embryos; recent studies have shown that its timing is regulated by the nucleocytoplasmic ratio. Several developmental events, particularly those associated with transcriptional activities, require DNA replication prior to their occurrence, suggesting an intimate relationship between DNA replication cycles and their onset. On the other hand, there are many other developmental events where timing is not controlled by the number of cell divisions, DNA replication cycles, or the nucleocytoplasmic ratio. Cytoplasmic machinery with autonomous oscillatory properties is thought to be involved in the timing of these events.

scholarly journals Essential Function of the Serine Hydroxymethyl Transferase (SHMT) Gene During Rapid Syncytial Cell Cycles in Drosophila

2017 ◽  
Vol 7 (7) ◽  
pp. 2305-2314 ◽  
Author(s):  
Franziska Winkler ◽  
Maria Kriebel ◽  
Michaela Clever ◽  
Stephanie Gröning ◽  
Jörg Großhans
Keyword(s):  
Wild Type ◽  
Midblastula Transition ◽  
Central Function ◽  
Catalytic Center ◽  
Cell Cycles ◽  
C1 Metabolism ◽  
Zygotic Gene ◽  
Serine Hydroxymethyl Transferase ◽  
Essential Function ◽  
Specific Timing

Abstract Many metabolic enzymes are evolutionarily highly conserved and serve a central function in the catabolism and anabolism of cells. The serine hydroxymethyl transferase (SHMT) catalyzing the conversion of serine and glycine and vice versa feeds into tetrahydrofolate (THF)-mediated C1 metabolism. We identified a Drosophila mutation in SHMT (CG3011) in a screen for blastoderm mutants. Embryos from SHMT mutant germline clones specifically arrest the cell cycle in interphase 13 at the time of the midblastula transition (MBT) and prior to cellularization. The phenotype is due to a loss of enzymatic activity as it cannot be rescued by an allele with a point mutation in the catalytic center but by an allele based on the SHMT coding sequence from Escherichia coli. The onset of zygotic gene expression and degradation of maternal RNAs in SHMT mutant embryos are largely similar to that in wild-type embryos. The specific timing of the defects in SHMT mutants indicates that at least one of the SHMT-dependent metabolites becomes limiting in interphase 13, if it is not produced by the embryo. Our data suggest that mutant eggs contain maternally-provided and SHMT-dependent metabolites in amounts that suffice for early development until interphase 13.

scholarly journals A Maternal Smad Protein Regulates Early Embryonic Apoptosis in Xenopus laevis

2002 ◽  
Vol 22 (5) ◽  
pp. 1317-1328 ◽  
Author(s):  
Yuko Miyanaga ◽  
Ingrid Torregroza ◽  
Todd Evans
Keyword(s):  
Functional Analysis ◽  
Cell Survival ◽  
Caspase 3 ◽  
Embryonic Cell ◽  
Early Embryogenesis ◽  
Cell Cycles ◽  
Smad Proteins ◽  
Smad Protein ◽  
Smad Signaling Pathway ◽  
A Cell

ABSTRACT We identified cDNAs encoding the Xenopus Smad proteins most closely related to mammalian Smad8, and we present a functional analysis of this activity (also referred to recently as xSmad11). Misexpression experiments indicate that xSmad8(11) regulates pathways distinct from those regulated by the closely related xSmad1. Embryos that develop from eggs depleted of xSmad8(11) mRNA fail to gastrulate; instead, at the time of gastrulation, they initiate a widespread program of apoptosis, via a CPP32/caspase 3 pathway. Embryos that avoid this fate display gastrulation defects. Activation of apoptosis is rescued by expression of xSmad8(11) but not xSmad1. Our results demonstrate an embryonic requirement for Smad8(11) activity and show that a maternally derived Smad signaling pathway is required for gastrulation and for mediating a cell survival program during early embryogenesis. We suggest that xSmad8(11) functions as part of a maternally derived mechanism shown previously by others to monitor Xenopus early embryonic cell cycles.

scholarly journals Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 63-80 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
A Cell ◽  
G2 Phase ◽  
Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

scholarly journals Evolution of CDK1 paralog specializations in a lineage with fast developing planktonic embryos

2019 ◽  
Author(s):  
Xiaofei Ma ◽  
Jan Inge Øvrebø ◽  
Eric M Thompson
Keyword(s):  
General Rule ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Gene Duplications ◽  
Structural Elements ◽  
Oikopleura Dioica ◽  
Cell Cycles ◽  
Protein Levels ◽  
Binding Interface ◽  
M Phase

AbstractThe active site of the essential, eukaryotic CDK1 kinase is generated by core structural elements, among which the PSTAIRE motif in the critical αC-helix, is universally conserved in metazoans. The CDK2 kinase, sharing the PSTAIRE, arose early in metazoan evolution and permitted subdivision of tasks along the S-M-phase axis. The marine chordate, Oikopleura dioica, is the only metazoan known to possess more than a single CDK1 ortholog, and all of its 5 paralogs show sequence divergences in the PSTAIRE. Through assessing CDK1 gene duplications in the appendicularian lineage, we show that the CDK1 activation loop substrate binding platform, ATP entrance site, hinge region, and main Cyclin binding interface, have all diversified under positive selection. Three of the 5 CDK1 paralogs are required for embryonic divisions and knockdown phenotypes illustrate further subdivision of functions along the S-M-phase axis. In parallel to CDK1 gene duplications, there has also been amplification in the Cyclin B complement. Among these, the CDK1d:Cyclin Ba pairing is required for oogenic meiosis and early embryogenesis and shows evidence of coevolution of an exclusive interaction. In an intriguing twist on the general rule that Cyclin B oscillations on a background of stable CDK1 levels regulate M-phase MPF activity, it is CDK1d protein levels that oscillate, rather than Cyclin Ba levels, to drive rapid, early embryonic cell cycles. Strikingly, the modified PSTAIRE of odCDK1d shows convergence over great evolutionary distance with plant CDKB, and in both O. dioica, and plants, these variants exhibit increased specialization to M-phase.

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