The early cell cycle
of Xenopus laevis
Our laboratory investigates
the regulation of the eukaryotic cell cycle during the early development
of the South African clawed frog Xenopus laevis. In the Xenopus
embryo,
the first twelve mitotic divisions after fertilization are rapid, synchronous,
and are characterized by alternating phases of DNA synthesis (S phase)
and mitosis (M phase) with no intervening gap phases (G phases).
There is no transcription during these cycles, and thus, the cell cycle
is regulated by maternally supplied products. After the twelfth cleavage,
the embryos begin the midblastula blastula transition (MBT) during which
zygotic transcription is initiated and cells become motile, events required
for gastrulation. The simple, embryonic cell cycles gain complexity
after the MBT as cell divisions become asynchronous and slower and G phases
are acquired. This period of cell cycle remodeling during early development
of Xenopus is the focus of research in our laboratory. We
are currently investigating the acquisition of cell cycle checkpoints and
a program of apoptosis by Xenopus embryos. We also collaborate
with the Tyson Group to combine
mathematical modeling and molecular biology to investigate fundamental
properties of the eukaryotic cell cycle.
The acquisition of
cell cycle checkpoints
Checkpoints in the eukaryotic
cell cycle maintain the integrity of the genome by arresting cell cycle
progression when DNA is damaged or incompletely replicated (Novak
et al. 2002). Checkpoint pathways converge upon the activities
of cyclin-dependent kinases (Cdks), the enzymes that drive cell cycle.
We are examining the roles of XChk1 and XChk2 in regulating Cdk activity
in response to DNA damage and inhibition of DNA replication during embryonic
development of the frog Xenopus laevis.
XChk1 is the homolog of mammalian and yeast Chk1, which indirectly inactivates Cdks to maintain cell cycle arrest when checkpoints are triggered. Mutations in the Chk1 signal transduction pathway are implicated in the development of many mammalian tumors. XChk1 is also homologous to Grp1, which functions in timing the midblastula transition (MBT) during Drosophila development. In the Xenopus embryo, the MBT correlates with extensive remodeling of the cell cycle, including increased regulation of Cdk activity and acquisition of DNA damage and DNA replication checkpoints. This developmental progression mimics events of tumorigenesis in reverse, and dissection of the XChk1 pathway during a developmental window that spans the MBT should indicate ways in which signal transduction may be altered during malignant transformation and suggest targets for therapeutic intervention.
To understand why Xenopus embryos lack checkpoints, XChk1 was cloned, and its expression was examined and manipulated in Xenopus embryos (Kappas et al., 2000). Although XChk1 mRNA is degraded at the MBT, 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.
In our present studies, we
are performing analogous experiments with XChk2 to assess its role in DNA
replication and damage checkpoints in the intact, Xenopus embryo. We are
also collaborating with Felicia
Etzkorn’s and David
Kingston’s groups in the Chemistry Department to design and discover
inhibitors of XChk1 and XChk2.
A program of apoptosis
in the Xenopus embryo
A developmental program
that does not respond to a severely damaged genome would be highly detrimental
to the propagation of that organism. Although Xenopus embryos
lack checkpoints before the MBT, it appears that they have evolved mechanisms
to contend with such damage in a distinct manner after the MBT. Studies
by our laboratory and others have demonstrated that many assaults to embryos
prior to the MBT result in extensive cell death shortly after the MBT.
These assaults include inhibition of protein synthesis or DNA replication
and damage of DNA by g-irradiation. The cell death that occurs after
the MBT is apoptosis as determined by morphological and biochemical criteria.
Thus, cells in which a damaged or incomplete genome has been inherited
during the rapid cell cycle prior to the MBT may be eliminated by an apoptotic
checkpoint that is engaged after the MBT. It is clear that the apoptotic
checkpoint is regulated by maternal products since it is also engaged in
response to a-amanitin, an inhibitor of RNA polymerase a which blocks all
transcription of the zygotic genome. Thus, apoptosis may be a default
program normally inhibited by a product of zygotic gene expression at the
MBT.
We are pursuing the role of XChk1 in regulating apoptosis. Xenopus embryos do not engage cell cycle checkpoints, although overexpression of the kinase XChk1 arrests cell divisions. At the MBT, XChk1 transiently activates and promotes cell cycle lengthening. In our studies, endogenous XChk1 was inhibited by the expression of dominant-negative XChk1 (DN-XChk1) (Carter and Sible, 2003). Development appeared normal until the early gastrula stage, when cells lost attachments and chromatin condensed. TUNEL and caspase assays indicated these embryos died by apoptosis during gastrulation. Embryos with unreplicated DNA likewise died by apoptosis. Embryos expressing DN-XChk1 proceeded through additional rapid rounds of DNA replication but initiated zygotic transcription on schedule. Therefore, XChk1 is essential in the early Xenopus embryo for cell cycle remodeling and for survival after the MBT.
We are continuing these studies
to determine whether a particular cyclin-dependent kinase is responsible
for triggering apoptosis in Xenopus embryos and whether XChk1 function
to inhibit this kinase.
Mathematical modeling
of the cell cycle
Our laboratory is also collaborating
with the Tyson Research Group,
fellow members of the Biology Department at Virginia Tech. Tyson
and colleagues have developed mathematical equations based on classic biochemical
kinetics and modern dynamic systems theory to model the eukaryotic cell
cycle. These equations make specific predictions about the behavior
cycling cell-free extracts derived from Xenopus eggs.
Our laboratory has begun to test the most fundamental predictions of the Novak-Tyson mathematical model (Sha et al., 2003). Cells progressing through the cell cycle must commit irreversibly to mitosis without slipping back to interphase before properly segregating their chromosomes. The Novak-Tyson model predicts that irreversible transitions into and out of mitosis are driven by hysteresis in the molecular control system. Hysteresis refers to toggle-like switching behavior in a dynamical system. In the mathematical model, the toggle switch is created by positive feedback in the phosphorylation reactions controlling the activity of Cdc2, a protein kinase bound to its regulatory subunit, cyclin B. To determine whether hysteresis underlies entry into and exit from mitosis in cell-free egg extracts, we tested three predictions of the Novak-Tyson model. (1) The minimal concentration of cyclin B necessary to drive an interphase extract into mitosis is distinctly higher than the minimal concentration necessary to hold a mitotic extract in mitosis, evidence for hysteresis. (2) Unreplicated DNA elevates the cyclin threshold for Cdc2 activation, indication that checkpoints operate by enlarging the hysteresis loop. (3) A dramatic "slowing-down" in the rate of Cdc2 activation is detected at concentrations of cyclin B marginally above the activation threshold. All three predictions were validated. These observations confirm hysteresis as the driving force for cell cycle transitions into and out of mitosis.
We are expanding our studies
to provide a more quantitative analysis of DNA replication and DNA damage
checkpoints. Furthermore, we have begun to pair modeling and experimentation
to study the less well characterized oscillations of the cyclin E/Cdk2
developmental timer in Xenopus embryos. We will continue to
work with the Tyson Group to develop ideas from theory to realistic and
testable models. The collaboration between molecular cell biologists
and kinetic theorists is a novel approach to the study of the cell cycle,
and we look forward to a long and productive collaboration.