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Oncogene (2005) 24, 2756–2764
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Cell-cycle control in Caenorhabditis elegans: how the worm moves from G1
to S
John Koreth1,2 and Sander van den Heuvel*,1
1
MGH Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; 2Dana Farber Cancer Institute, 44 Binney Street,
Boston, MA 02115, USA
The nematode Caenorhabditis elegans offers a powerful
model system to study cell division control during animal
development. Progress from the one-cell zygote to adult
stage follows a nearly invariant pattern of divisions. This,
combined with a transparent body and efficient genetics,
allows for sensitive identification and quantitative analysis
of cell-cycle mutants. Nearly all G1 control genes
identified in C. elegans have mammalian homologs.
Examples include a D-type cyclin and CDK4/6-related
kinase, a member of the retinoblastoma protein family and
CDK inhibitors of the Cip/Kip family. Genetic studies
have placed the currently known G1 regulators into
pathways similar to those in mammals. Together, this
validates the use of C. elegans in identifying additional
regulators of cell-cycle entry and exit. For instance, we
recently found that the CDC-14 phosphatase promotes
maintenance of the quiescent state. Here, we describe cellcycle control as an integral part of C. elegans development, summarize current knowledge of G1 control genes
in the worm, compare the results with those obtained in
other species, and discuss the possible implications of cellcycle studies in C. elegans for higher organisms, including
humans.
Oncogene (2005) 24, 2756–2764. doi:10.1038/sj.onc.1208607
Keywords: C. elegans cell cycle; G1–S; cyclin D; Cdk4/6;
lin-35 Rb; cki-1 Cip/Kip
Introduction
The nematode Caenorhabditis elegans offers an attractive genetic system to study various aspects of development (Brenner, 1974), including the developmental
regulation of cell proliferation. Mutants with altered
patterns of cell divisions during larval development have
been identified since the early days of C. elegans
genetics. Several of the mutations are now known to
affect genes that control cell-cycle progression. Cellcycle genes have also been identified in more recent
studies focused on cell-cycle entry and withdrawal.
*Correspondence: S van den Heuvel, MGH Cancer Center, Harvard
Medical School, Charlestown, MA 02129, USA;
E-mail: [email protected]
Together, a pathway for G1 control in C. elegans has
emerged, which includes positive regulators similar to
the Cyclin D and Cdk4/6 oncogenes, negative regulators
related to the retinoblastoma (Rb) tumor suppressor
and cyclin-dependent kinase (CDK) inhibitors, and
transcription factors of the E2F and DP families. Thus,
the control of G1 progression involves similar players in
C. elegans and mammals.
Studies in C. elegans benefit from a variety of
practical advantages: the animals are small (adults are
B1 mm in length) and can be grown on agar plates or in
liquid media. Its life cycle is short: in just over 3 days a
fertilized egg develops into an adult hermaphrodite or
male. Each hermaphrodite produces around 300 progeny by self-fertilization, while hermaphrodite-male
crosses can generate more than a thousand progeny.
One of its major strengths is efficient genetics. Traditionally, this meant forward genetics, which starts from
mutagenesis. However, reverse genetics by RNA interference (RNAi) is also highly efficient in the worm
(Fire et al., 1998), and genome-wide screens based on
‘feeding RNAi’ have become common (Kamath
and Ahringer, 2003; Rual et al., 2004). Despite its
simplicity, C. elegans completes an entire program
of animal development and forms a broad variety of
cell types and tissues. Its transparency, small size,
nearly invariant cell lineage, and simple body plan
have made it possible to trace the development of
every somatic cell nucleus in the adult worm back to the
single cell zygote (for accuracy we use ‘nuclei’ instead of
‘cells’, as some nuclei share a common cytoplasm)
(Sulston and Horvitz, 1977; Sulston et al., 1983). This
analysis allows for the precise prediction of the timing
and localization of each cell division in the developing
worm. Together, these characteristics offer a unique set
of tools for the identification and characterization of
cell-cycle mutants.
Throughout C. elegans development, cells decide
whether to remain quiescent or to enter another division
cycle. These decisions form part of a larger program,
which precisely coordinates cell division with cell
growth, differentiation, tissue formation, and morphogenesis. Thus, the genes that regulate cell-cycle progression form an integral part of the network that
controls formation of a well-defined organism. Major
questions include how developmental signaling cascades, mediated, for instance, by insulin-related growth
G1 progression in C. elegans
J Koreth and S van den Heuvel
2757
factors, TGF-b family members, or Wnt ligands,
impinge on the cell-cycle machinery to accomplish
proper temporal and spatial control of cell division.
As many of the regulators and pathways controlling
cell fate, cell growth, cell death, and cell proliferation are
conserved, C. elegans studies will likely help reveal how
cell division is controlled in a developmental context.
The most common model systems, yeasts and mammalian cells in tissue culture, are ill suited to address this
higher level of cell-cycle control. Therefore, developmental control of cell division is an area that remains
poorly understood. Yet, the regulatory mechanisms
involved are highly relevant to our understanding of,
and potentially interfering with, the unrestrained proliferation of cancer cells.
cycle profiles. Certain cells continue rapid divisions,
others divide after an extended interphase of 2 h or
more, yet other cells become post-mitotic or only resume
divisions during larval development, after an arrest of
over 18 h (Sulston et al., 1983). Thus, the regulation of
cell-cycle events and the introduction of G1 and G2
phases are likely determined by the cell lineage.
Maternal gene products, synthesized in the hermaphrodite and deposited into the egg, drive most embryonic
divisions. Owing to perdurance of the maternal contribution of the wild-type gene product, homozygous
cell-cycle mutants derived from a heterozygous mother
tend to complete embryogenesis. Such homozygous
mutants often display cell-cycle defects during larval
development, usually during the first larval stage.
Larval somatic cell cycles
Cell-cycle regulation and C. elegans development
As metazoans go through development, their cells
progress through various types of cell cycles. These
include the embryonic cell cycle, somatic cell cycle,
endoreduplication cycle, and meiotic cell cycle. The cellcycle machinery used is tailored towards these individual
cycles, and varies in the requirement for cell-cycle genes
and the balance between their contributions. For
instance, C. elegans cyd-1 cyclin D and cdk-4 Cdk4/6
are not required for most of embryogenesis and cdk-1 is
not needed for the endoreduplication cycles (see below;
Boxem et al., 1999). It is thus important to consider the
type of cycle and developmental context when examining the function of cell-cycle components. For these
reasons, we summarize briefly how the various cell
division cycles follow each other during C. elegans
development.
Embryonic cell cycles
As in other metazoans, early embryonic divisions in C.
elegans are fast and cycle between S and M phases,
apparently lacking the Gap phases G1 and G2 (Edgar
and McGhee, 1988). The initial mitotic division cycles
take approximately 15 min each and include cytoplasmic
cleavage, in contrast to the extremely rapid nuclear
divisions of early Drosophila embryos. Cells start to
acquire different fates right from the asymmetric first
mitotic division, and the division cycles of their
descendents are asynchronous and progressively vary
in length. Nearly all embryonic divisions occur during
the first half of embryogenesis, within the proliferation
phase that is completed approximately 7 h after fertilization. This phase is followed by a differentiation and
tissue morphogenesis phase, during which the initially
spherical embryo is stretched out into the larva that
hatches from the egg.
As C. elegans embryonic divisions are asynchronous,
it is not possible to make generalized statements about
the number of embryonic divisions that consist solely of
S and M phases, or when the G1 and G2 phase are first
introduced. Just a few hours into embryonic development, cells in different lineages diverge greatly in cell-
After hatching in the presence of adequate food, the
worm progresses through four larval stages (L1–L4) to
become an adult. In total, 55 precursor cells continue to
proliferate during larval development, thereby expanding the 558 nuclei of an early L1 larva to the 959 somatic
nuclei and extensive germline of the adult hermaphrodite (recently reviewed in Lambie, 2002). The somatic
nuclei appear to contain a 2n DNA content at the time
of hatching and go through a DNA synthesis phase
before initiating mitosis (Boxem et al., 1999; Boxem and
van den Heuvel, 2001; our unpublished results). These
divisions generally depend on the function of G1/S and
G2/M control genes. Thus, the precursor cells of the
post-embryonic lineages and their descendents follow
canonical cell cycles in which the S and M phases are
separated by Gap phases. As in embryogenesis, the
length of the interphase varies greatly between different
cell types. Divisions frequently follow each other within
1 h, but some cells remain quiescent for 20 h, before
dividing again two larval stages later (Sulston and
Horvitz, 1977).
At least some divisions incorporate developmental
information during the G1 phase. Extracellular signals
can induce cell fate decisions with specific division
patterns. For instance, heterochronic genes such as lin-4
and lin-14, which encode a micro-RNA and its target,
respectively, control the larval stage-specific patterns of
cell proliferation (Lee et al., 1993). These molecular
timekeepers exercise organism-wide control over temporal identity and patterning, a role reminiscent of Hox
gene functions in spatial control. Environmental factors
can also induce a global arrest of the cell division
program. L1 larvae that hatch in the absence of food
arrest development, growth, and cell division until the
food supply is restored. Starved larvae can also adopt an
alternative specialized developmental arrest phase
known as the ‘dauer’ (enduring) stage. Dauer larvae
do not feed, and adapt their anatomy, behavior, and
metabolism to long-term survival. Entry into the dauer
stage is determined by multiple environmental inputs
that include dauer pheromone, temperature, and food
availability. These stimuli impact a complex network of
interacting pathways that include TGF-b, insulin
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J Koreth and S van den Heuvel
2758
signaling, and cGMP coupled pathways (Kimura et al.,
1997; Riddle and Albert, 1997). Further cell divisions
are arrested until environmental conditions improve.
Upon feeding, dauer larvae complete cell divisions
characteristic of the L3 larval stage, molt, and continue
fourth-stage and adult development.
Endoreduplication cycles
Cells in intestinal and hypodermal tissues (skin) undergo
endoreduplication cycles during C. elegans larval development (Hedgecock and White, 1985). Such cycles are
characterized by a DNA synthesis phase that is not
followed by M phase, thus doubling the DNA ploidy
with each additional cycle. Of the 20 intestinal cells, 14
undergo a final nuclear division at the end of the L1
stage. Subsequently, all intestinal nuclei go through an
endoreduplication cycle during each larval stage, which
results in intestinal nuclei with a 32n DNA content in
adult animals.
A major part of the hypodermis is formed by fusion of
individual cells. Cells that are added to this syncytium
during larval development usually undergo a round of
DNA synthesis just before fusion (Hedgecock and
White, 1985). Consequently, the skin contains a mixture
of nuclei with 2n and 4n DNA contents.
The meiotic cell cycle
Late larval and adult animals contain a large number of
precursor germ cells in various stages of meiotic
progression. Hermaphrodites temporarily produce male
gametes during the third larval stage, before switching
to an oogenesis program (reviewed in Schedl, 1997). A
mitotically active ‘stem cell’ population at the distal end
of the germ line forms the precursor germ cells. These
divisions are the only mitotic divisions that continue in
adult animals. The precursor cells enter a prolonged
meiotic prophase, in which homologous chromosomes
pair, synapse and undergo recombination. Subsequently, oocytes mature and arrest during diakinesis
until fertilization. Upon fertilization, the oocyte pronucleus completes meiosis I and II, after which the female
and male pronuclei meet to form a zygote (reviewed in
Kemphues and Strome, 1997).
cell-cycle entry or withdrawal show defects in mobility
(Uncoordinated phenotype), fertility (Sterile phenotype), and formation of vulval structures (Vulvaless,
protruding-Vulva or Multi-Vulva phenotypes).
Screens for mutants with abnormal cell lineages (Lin
mutants) have defined several cell-cycle components
(Horvitz and Sulston, 1980; Sulston and Horvitz, 1981).
Examples include the Cullin LIN-19/CUL-1, the bTRCP-related F box WD protein LIN-23, the Rb family
member LIN-35, and the DP1-related transcription
factor LIN-55/DPL-1 (see below). Further, a screen
for mutants defective in vulval formation revealed cye-1
cyclin E mutations (Fay and Han, 2000), and mutations
in cyd-1 cyclin D and cdk-4 Cdk4/6 were isolated in a
screen for mutants with G1-arrested postembryonic
precursor cells (Boxem and van den Heuvel, 2001).
Finally, a cdc-14 allele was isolated in a screen for
mutants with vulval precursor cells that fail to become
quiescent (Saito et al., 2004).
The specific function of these G1 regulators in C.
elegans is discussed below. Although this list is
incomplete, strong similarities with mammalian G1
control mechanisms are apparent (Figure 1). However,
there are also striking omissions, for instance, orthologs
of the INK4 gene family remain to be identified in C.
elegans. Also, the C. elegans p53 family member (cep-1)
promotes apoptosis in response to DNA damage in the
germ line, but has not been implicated in cell-cycle arrest
(Derry et al., 2001; Schumacher et al., 2001).
C. elegans cyclin D and Cdk4/6
The C. elegans genome encodes a single D-type cyclin,
CYD-1, and single kinase of the Cdk4/6 subfamily,
CDK-4. A reverse genetic approach revealed that both
cyd-1 and cdk-4 are required for postembryonic cell
divisions (Park and Krause, 1999), and cyd-1 and cdk-4
mutants were isolated in a forward genetic screen for G1
control genes (Boxem and van den Heuvel, 2001).
Homozygous cyd-1 and cdk-4 null mutants go through
embryogenesis, complete only a few larval cell divisions
Extracellular Signals
Cyclin D1,2,3
CDK4,6
CYD-1/CDK-4
C. elegans G1 control genes
Cell-cycle genes have been identified in relatively
unbiased genetic screens and through examination of
candidate genes based on homology with other species.
Owing to the persistence of maternal product and the
nature of embryonic cell cycles, homozygous G1 control
mutants tend to complete embryogenesis and show cell
division defects during larval development. Dividing
cells contribute extensively to the nervous system (in
particular, the ventral nerve cord), the reproductive
system (both the somatic gonad and germline), and the
egg-laying system (the vulva and associated egg laying
muscles) during normal postembryonic development
(Sulston and Horvitz, 1977). Hence, mutants defective in
Oncogene
pINK family
M
G1
G2
S
pRb,p107,p130 Cip/Kip family
LIN-35
CKI-1,2
Cyclin E /CDK2
CYE-1/K03E5.3?
Figure 1 Generalized model for regulation of G1 progression in
mammalian cells. For most classes of regulators, a single family
member is expressed in C. elegans. The mammalian genes are
indicated in gray above the putative C. elegans orthologs, indicated
in bold. (-) and (–|) indicate positive regulation and negative
regulation, respectively. See text for further details
G1 progression in C. elegans
J Koreth and S van den Heuvel
2759
of the gonad precursor cells, and remain thin, uncoordinated, small, and sterile larvae. The precursor nuclei
of the postembryonic lineages arrest with a 2n DNA
content and without expression of an S-phase reporter,
the ribonucleotide reductase (rnr) promoter driving green
fluorescent protein (GFP) expression (rnrHgfp), indicating that cyd-1 and cdk-4 are required for progression
from G1 to S phase. The close similarity of the cyd-1 and
cdk-4 loss-of-function phenotypes combined with binding of the proteins in vitro strongly indicate that C.
elegans CYD-1 and CDK-4 function together in a
complex.
Based on reporter gene expression, transcription of
cyd-1 and cdk-4 initiates before the 300-cell stage (Park
and Krause, 1999). Yet, only a few very late embryonic
divisions depend on cyd-1 and cdk-4 function. This is
likely not a consequence of maternal product, as RNAi,
which usually blocks the maternal contribution efficiently, mimicked the mutant phenotype. Even when
combined with the homozygous null mutations, RNAi
of cyd-1 and cdk-4 did not cause embryonic lethality or
enhance the mutant phenotype (Boxem and van den
Heuvel, unpublished). Thus, phosphorylation of target
proteins by the CDK-4/CYD-1 kinase is not essential
during the embryonic proliferation phase (see below:
‘Comparison with other systems’).
In addition to arrest of cell divisions, homozygous
cyd-1 and cdk-4 mutants show strongly reduced larval
growth (Park and Krause, 1999; Boxem and van den
Heuvel, 2001). The growth retardation follows the arrest
of cell division, and undivided cells in the mutants are
initially larger than their divided counterparts in wildtype animals (Figure 2). Therefore, lack of cell division
does not result from reduced cell growth. Interestingly,
Drosophila Cyclin D and Cdk4 also contribute to cell
growth, while their role in G1 progression is less critical
(see below; Datar et al., 2000; Meyer et al., 2000, 2002).
The essential role of the Cyclin D/Cdk4 kinase in C.
elegans can help define in vivo substrates that regulate
G1–S progression downstream of cyd-1 and cdk-4. Loss
of function of negative regulators that act downstream
may be anticipated to rescue the cell proliferation
defects and larval arrest of cyd-1 and cdk-4 mutant
animals. Indeed, the retinoblastoma and Cip/Kip
inhibitor pathways have been found to fulfill such
functions.
The retinoblastoma and E2F protein families
The lin-35 gene was initially defined in a forward screen
for genes that redundantly regulate vulval cell fates in C.
elegans (Ferguson and Horvitz, 1989). Vulval cell fates
are controlled by at least four regulatory pathways,
including inductive signaling through an EGF-like
growth factor receptor/tyrosine kinase/Ras/MAP kinase
cascade. Mutations that increase inductive signaling
cause extra cells to adopt vulval cell fates, resulting in
animals that display a multivulva (Muv) phenotype. The
inductive pathway is antagonized by a general repressive
mechanism that involves the ‘synthetic multivulva’
(synMuv) genes. The synMuv genes form three different
Figure 2 Retardation of cell growth in cyd-1 and cdk-4 mutants
follows arrest of cell division. Cell boundaries of the hypodermal
seam cells are visualized by the junctional marker AJM-1HGFP in
a first-stage wild-type larva (Top), L3 stage wild-type larva
(Middle) and L3 stage cyd-1(he112) mutant animal (bottom).
The hypodermal seam cells in the mutants grow substantially,
indicating that the lack of division is not caused by a growth defect.
The size of the animals and individual cells are identical between
wild-type and mutants until approximately 16 h of larval development, at which point cyd-1 and cdk-4 mutant animals start to lag
behind. In contrast, cell divisions fail starting as early as late
embryogenesis (Boxem and van den Heuvel, 2001; unpublished
data)
classes, A, B, and C, that act redundantly (Ceol and
Horvitz, 2004). Only animals that contain mutations in
two different classes show an Muv phenotype.
Importantly, the synMuv class B gene lin-35 was
found to encode a member of the retinoblastoma tumorsuppressor protein family (Lu and Horvitz, 1998). The
predicted LIN-35 protein is not particularly close to a
specific mammalian family member (pRb, p107, and
p130). Although the spacer that separates the pocket A
and B domains of LIN-35 is short, like that of pRb, the
protein is somewhat more closely related to p130 and
p107 (LIN-35 shares 20, 19, and 15% overall aminoacid identity with p130, p107, and pRb, respectively,
and residues 744–839 of the LIN-35 pocket B region are
34, 34, and 30% identical to these respective proteins).
Thus, lin-35 Rb likely evolved from a single common
ancestor before it diverged into three distinct genes.
The synMuv class B genes not only include lin-35 Rb,
but also homologs of other genes known to interact with
Rb in other species. These include, efl-1 (E2F family like)
and dpl-1 (DP family like) (Ceol and Horvitz, 2001), as
well as lin-53 (RbAP46/48) and hda-1 (HDAC1), which
encode components of the Nucleosome Remodeling and
Deacetylase (NURD) complex (Solari and Ahringer,
2000). The combined genetic and molecular data suggest
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J Koreth and S van den Heuvel
2760
that class B synMuv genes inhibit vulval cell-fate
determination and antagonize Ras signaling through
transcriptional repression mediated by an E2F/pRb/
NURD complex.
These studies did not reveal a cell-cycle role for lin-35
Rb. Animals homozygous for lin-35 Rb null alleles have
a reduced brood size, but are otherwise viable and have
mostly normal cell division patterns. More direct
measurements of DNA content demonstrated that lin35 Rb is not rate limiting for S-phase entry (Boxem and
van den Heuvel, 2001). However, a role for lin-35 Rb in
G1 control became apparent in sensitized genetic
backgrounds. Firstly, the cyd-1 and cdk-4 loss-offunction phenotypes are rescued to a large extent by
concomitant inactivation of lin-35 Rb: the double
mutant animals show expression of the rnrHgfp S-phase
reporter, restore postembryonic DNA synthesis, go
through endoreduplication cycles, and show substantial
postembryonic cell division and growth. In addition, lin35 Rb inactivation greatly enhances the number of extra
cell divisions in a cki-1 Kip1 loss of function background (Boxem and van den Heuvel, 2001; see below).
These results are consistent with LIN-35 Rb functioning
as a major downstream target of the Cyclin-D/CDK-4
complex (Figure 3). Further, lin-35 Rb and cki-1 Cip/
Kip apparently provide parallel levels of control and
cooperate to inhibit G1-to-S phase progression. Both of
these conclusions agree with numerous results from
studies in mammalian systems.
The same sensitized backgrounds have been used
to examine cell-cycle regulatory functions of synMuv
cydcyd-1
Cyclin D
cdkcdk-4
CDK4/6
The CIP/KIP family of Cdk inhibitors
cdccdc-14
Cdc14
linlin-36
linlin-35
Rb
eflefl-1 dpldpl-1
E2F DP
linlin-9
ckicki-1
Cip/Kip
cyecye-1
Cyclin E ?
K03E5.3
CDK2
G1
S
Figure 3 Model for G1 regulation during post-embryonic development of C. elegans. Based on mutant phenotypes and genetic
interactions, the genes indicated have been placed in a branched
pathway. The results indicate both that G1 control is conserved
between C. elegans and mammals, and that novel control elements
can be found through genetic studies in C. elegans. (-) and (–|)
indicate positive regulation and negative regulation, respectively.
See text for further details
Oncogene
genes other than lin-35 Rb (Boxem and van den
Heuvel, 2002). While none of the class A genes examined
scored in these assays, a subset of the synMuv class B
genes was found to act in G1 control. Specifically,
efl-1 E2F4/5, dpl-1 DP, lin-9 Mip130/TWIT, lin-36, and
lin-15B contribute to inhibition of G1 progression.
In each assay, efl-1 and lin-36 behaved most similar to
lin-35 Rb: their inactivation rescued cyd-1 and cdk-4
mutants substantially and increased hyperproliferation
when combined with cki-1 Kip1 RNAi. Thus, EFL-1
E2F4/5 and the predicted Zn-finger protein LIN-36
likely act in a pathway or complex with LIN-35 Rb to
repress G1/S control genes (Figure 3). Inactivation of
dpl-1 DP and efl-1 E2F4/5 caused similar effects in
these assays; however, dpl-1 inactivation on its own
reduced cell division and expression of the rnrHgfp
S-phase reporter. Thus, DPL-1 DP acts both to promote
and inhibit G1/S progression. In accordance with
observations in other systems, an EFL-1/DPL-1 complex likely recruits LIN-35 Rb to repress transcription,
while DPL-1 may act with another E2F family member
to activate S-phase genes. As yet, a transcriptional
activating E2F member has not been identified; efl-2
E2F is a good candidate but an RNAi phenotype has
not been observed (Ceol and Horvitz, 2001; Boxem
and van den Heuvel, 2002). The role of LIN-9 and
the predicted novel protein LIN-15B remain poorly
defined. Unlike lin-35 Rb, inactivation of these genes
did not enhance the cki-1 Kip1 overproliferation
phenotype. In fact, lin-15B appeared to act in parallel
to lin-35 Rb in G1 control (Boxem and van den
Heuvel, 2002).
The C. elegans genome contains two closely linked loci
on chromosome II that are predicted to encode members
of the CIP/KIP family of Cdk inhibitors. The CKI-1
and CKI-2 proteins share approximately 30% aminoacid identity and are each about equally close to p21Cip1
and p27Kip1 (Hong et al., 1998; Feng et al., 1999).
However, a function has been described only for cki-1.
CKI-1 is considered orthologous to mammalian p27Kip1,
as it acts as a developmental regulator of cell-cycle entry
and apparently not in DNA damage response.
Overexpression of cki-1 causes cell-cycle arrest in G1,
while loss of function results in extra divisions in
multiple cell lineages, including the intestinal and
hypodermal lineages (Hong et al., 1998; Boxem and
van den Heuvel, 2001; Fukuyama et al., 2003). A
deletion that spans cki-1 and cki-2 causes embryonic
lethality, which can be rescued by cki-1 expression
(Fukuyama et al., 2003). Inactivation of cki-1 by RNAi
results in partial embryonic lethality as well as sterile
adults with hyperproliferation during larval development. Postembryonic precursor cells in cki-1(RNAi)
animals fail to arrest in G1 and ectopically express the Sphase marker rnrHgfp. Thus, cki-1 Kip1 function is rate
limiting for S-phase entry, in particular in cells that enter
a prolonged quiescent state before dividing again.
Interestingly, loss of cki-1 Kip1 also affects aspects of
G1 progression in C. elegans
J Koreth and S van den Heuvel
2761
cell differentiation, cell fate, and cell death (Fukuyama
et al., 2003; Kostic et al., 2003).
Multiple levels of control have been shown to affect
cki-1 promoter activity (Hong et al., 1998). This involves
regulators that are cell-type dependent, larval stage
dependent-such as heterochronic genes, or environmentally determined–such as L1 arrest in response of food
starvation and induction of the dauer stage. Multiple 50
upstream sequences in the cki-1 promoter region
mediate the different signals (Hong et al., 1998). Thus,
proper temporal regulation of cki-1 expression appears
to control lineage-specific and environmentally induced
arrest of cell division.
Inactivation of cki-1 significantly rescued the postembryonic arrest of cell proliferation in cyd-1 or cdk-4
loss-of-function mutants. This indicates that the worm
CDK inhibitors act downstream of or in parallel to the
Cyclin-D/CDK-4 complex. Thus, as outlined above, lin35 Rb and cki-1 Kip1 each act downstream of cyd-1/
cdk-4. In contrast to lin-35 Rb, cki-1 inactivation allows
just one round of endoreduplication in cyd-1 mutants.
As expected, CKI-1 was found to bind CYE-1 Cyclin E
in the yeast two-hybrid system (Boxem and van den
Heuvel, unpublished data). These results are consistent
with a model in which CKI-1 Kip1 inhibits the Cyclin E
kinase by direct association. As proposed for other
systems, in the absence of CKI-1 Kip1, residual Cyclin E
kinase activity may drive one more round of DNA
synthesis. Additional cycles depend on transcription of
S-phase genes, which requires inactivation of lin-35 Rb.
The C. elegans CDC-14 phosphatase
A putative null allele of cdc-14 was identified in a screen
for mutants with inappropriate divisions of the vulval
precursor cells at a time of normal quiescence (Saito
et al., 2004). Subsequent studies showed that cdc-14 is
required for developmental arrest of cell division in
multiple lineages, acting similar to cki-1 but not as
strongly. A variety of genetic experiments and transgene
expression studies indicated that cdc-14 acts upstream of
cki-1 and promotes accumulation of CKI-1 protein in
the nucleus. As mammalian p27Kip1 is degraded in a
phosphorylation- and ubiquitin-dependent fashion,
CDC-14 might counteract CKI-1 phosphorylation and
promote a stable form of CKI-1 that can accumulate to
the levels required for developmental arrest of cell
division.
Functional GFPHCDC-14 fusion proteins showed a
highly dynamic pattern of localization. In cells that
temporally arrested, GFPHCDC-14 accumulated in the
cytoplasm. In contrast, the fusion protein was recruited
into the nucleus as soon as cells entered a terminally
differentiated state. Interestingly, only cells that undergo
prolonged developmental G0/G1 phase arrest are
affected by cdc-14 loss of function, indicating that
cytoplasmic localization is critical for CDC-14 to arrest
cell division.
The lack of a mitotic Cdc-14 phenotype in these
studies was surprising. For one, cytokinesis defects
have been observed to result from cdc-14 RNAi, which
can be explained, at least in part, by the use of a
sensitized zen-4(ts) background (Gruneberg et al., 2002;
Mishima et al., 2004). Moreover, Cdc14 is required for
mitotic exit in the budding yeast S. cerevisiae (Visintin
et al., 1998). In addition, the worm GFPHCDC-14
fusion protein showed strong association with the
spindle apparatus in mitosis (Saito et al., 2004). Thus,
although cdc-14 deletion mutants do not display
defects in mitosis or cytokinesis, a nonessential mitotic
role is likely. Yeast Cdc14p dephosphorylates the
Cdk inhibitor Sic1p, which acts in mitosis as well
as G1 phase, yet Cdc14p is kept inactive for most of
the cell cycle by sequestration into the nucleolus
(Mendenhall, 1993; Schwob et al., 1994; Shou et al.,
1999; Visintin et al., 1999). In contrast, Cip/Kip family
members act predominantly in G1 phase, and Cdc14
appears to act during multiple cell-cycle phases in
animal cells. These and other differences between yeast
and animal cells could shift the balance in Cdc14
requirement.
Cyclin-E/Cdk-2
The cye-1 gene encodes the only C. elegans E-type
cyclin. Null mutations in cye-1 were found to cause a
surprisingly mild phenotype in the worm, displaying late
larval cell division defects and sterility (Seydoux et al.,
1993; Fay and Han, 2000). In contrast, cye-1 RNAi
causes embryonic arrest at the B100 cell stage, thus the
late phenotype of homozygous mutants appears to
reflect a long-lived maternal contribution of cye-1
products. CYE-1 protein is present in early embryos
and cye-1 transcription initiates as early as the 28-cell
stage (Brodigan et al., 2003). Why cye-1 RNAi does not
cause defects at an even earlier time of embryonic
development is unclear at present.
Currently, an unequivocal Cdk2 ortholog has not yet
been identified in C. elegans. A candidate gene, K03E5.3,
shares B43% amino-acid identity with human Cdk2.
Depletion of its gene product by RNAi results in highly
variable defects, ranging from sterile adults, arrest at
early and late larval stages and embryonic lethality
(Boxem et al., 1999). This range of phenotypes could
reflect incomplete inactivation by RNAi, or nonessential
gene functions. It is possible that redundancy or
developmental flexibility allows some cell divisions to
continue in the absence of Cyclin E and Cdk2 function.
This would be in accordance with the surprisingly
limited phenotypes of double knockout Cyclin E1 and
E2 mice, as well as the viability of Cdk2 knockout mice
(Berthet et al., 2003; Geng et al., 2003; Ortega et al.,
2003).
Only limited data are available to place cye-1 in a G1
control pathway, but its genetic interactions are clearly
different from cyd-1. Inactivation of lin-35 Rb does not
affect the cye-1 null-mutant phenotype and, conversely,
cye-1 RNAi is equally severe in a wild-type or lin-35 Rb
mutant background (Boxem and van den Heuvel, 2001).
However, incomplete inactivation of cye-1 by feeding
RNAi is suppressed by lin-35 Rb loss of function (Ceron
and van den Heuvel, unpublished). These results are
Oncogene
G1 progression in C. elegans
J Koreth and S van den Heuvel
2762
consistent with lin-35 Rb acting upstream and negatively
regulating cye-1, a hypothesis that is also supported by
the presence of multiple E2F binding sites in the cye-1
promoter (Brodigan et al., 2003).
SCF components
Among the first cloned C. elegans cell-cycle genes were
cul-1 (previously named lin-19). Both lin-19/cul-1 and
lin-23 mutants were identified in early screens and found
to display excessive cell divisions with accelerated G1–S
transitions in all post-embryonic lineages (Kipreos et al.,
1996, 2000). CUL-1 was the founding member of the
cullin family in metazoans, and LIN-23 is an F-box WD
repeat protein, most similar to MET30 in yeast, human
b-TRCP, and the slmb F-box protein in Drosophila
(Kipreos et al., 2000). Thus, based on similar phenotypes, both proteins are likely components of an Skp1Cul1-F box (SCF) protein complex that acts as an E3
ubiquitin ligase and targets proteins for degradation.
The critical target proteins of the CUL-1/LIN-23 SCF
are expected to be positive regulators of the G1–S
transition. In analogy to a vertebrate CUL-1 containing
SCF complex, Cyclin E is considered a candidate critical
target.
Not only SCF, but also the APCCdh1 E3 ubiquitin
ligase, contributes to G1 control. This is indicated by the
increased number of larval cell divisions in mutants that
simultaneously lack lin-35 Rb and fzr-1 Cdh1 function
(Fay et al., 2002).
Comparison with other systems
As indicated in the preceding discussion, cell-cycle
effectors and their regulatory modules tend to be
conserved between C. elegans and mammals. Consequently, many of the genetic observations made in C.
elegans support existing models of G1 control. This
includes the observed roles for a Cyclin D–Cdk4/6
related kinase in promoting G1 progression, and
cooperation between pRb and Cip/Kip family members
in inhibiting this process. Furthermore, C. elegans Rb
acts in concert with E2F/DP transcription factors, and
likely represses cyclin E transcription. Despite these
global similarities, a number of specific aspects of the
mutant phenotypes are surprising and warrant further
discussion.
CYD-1 and CDK-4 are required late in embryogenesis and throughout larval development, but why not at
earlier times? It is important to consider that S-phase
entry during embryonic divisions is controlled largely by
maternal mRNA and protein products. Our observations suggest that the most critical function of the Cyclin
D kinase is inactivation of LIN-35 Rb. If cyd-1/cdk-4
are inactive, LIN-35 Rb likely continues to act as a
transcriptional repressor. While Rb activity can prevent
S-phase entry during larval development, a transcriptional repressor cannot stop embryonic expression of
maternally provided products. Thus, cyd-1 and cdk-4
likely have limited functions during embryogenesis
Oncogene
because cell divisions can continue without zygotic
transcription of cell-cycle genes.
Mice that completely lack Cyclin D kinase activity
also proceed through development until late embryogenesis (Kozar et al., 2004; Malumbres et al., 2004).
Such embryos complete extensive proliferation as well as
cell differentiation, morphogenesis, and organogenesis.
Careful characterizations have indicated roles for
compensatory mechanisms in the triple D-type cyclin
KO mice (Kozar et al., 2004). Thus, rather than
persistent maternal gene product, developmental flexibility and genetic redundancy likely explain how more
complex animals manage to alleviate the requirement
for Cyclin D kinase activity. The fact that cell divisions
arrest abruptly in cyd-1 and cdk-4 larvae may point to a
lower degree of redundancy and flexibility during worm
development.
The situation is somewhat different again in Drosophila, as the contribution of fly Cyclin D/Cdk4 in G1
progression appears to be limited. Interestingly,
DmCycD/Cdk4 complexes do not associate with the
fly Cip/Kip inhibitor dacapo (Meyer et al., 2000).
Association of mammalian Cyclin D–Cdk4/6 complexes
with p21Cip1 and p27Kip1 has been suggested to contribute
to activation of the Cyclin E–Cdk2 kinase acting
downstream, by sequestering its inhibitor. C. elegans
cki-1 Kip1 loss of function partly suppresses the
requirement for cyd-1 and cdk-4 in vivo, and CKI-1
associates with worm Cyclins D and E in the yeast twohybrid system (Boxem and van den Heuvel, 2001). Thus,
C. elegans CYD-1/CDK-4 may contribute to CKI-1
inactivation, through sequestration or phosphorylation.
As Drosophila Cyclin D–Cdk4/6 fails to interact with
dacapo, it may have a less prominent role in G1
progression.
Vertebrate Cip/Kip inhibitors have also been suggested to act as assembly factors for Cyclin D–Cdk4/6
complexes (LaBaer et al., 1997; Sherr and Roberts,
1999). Such a role has not been detected for the C.
elegans orthologs. Inactivation of cki-1 by RNAi, or
deletion of cki-1 and cki-2 together, results in strongly
increased numbers of cell divisions. However, this
number is substantially reduced when cki-1(RNAi) is
combined with cyd-1 or cdk-4 loss of function. The
difference indicates that the CYD-1/CDK-4 kinase is
not inactivated by cki-1 loss of function, and that the
worm Cip/Kip inhibitor plays only a negative role in
cell-cycle regulation.
Another surprise is that the single Rb family member
is not essential in the worm. Hardly any extra divisions
have been observed in lin-35 Rb null mutants, although
a low number of extra intestinal nuclei are formed.
Examining the timing of DNA replication also did not
indicate premature entry into S phase in lin-35 Rb
mutants, in contrast to cki-1 (RNAi) animals in which
cells prematurely enter the division cycle (Boxem and
van den Heuvel, 2001). Thus, lin-35 Rb acts redundantly
with cki-1 Kip1 in G1 control, and cki-1 is more rate
limiting. This shift in balance compared to more
complex animals may be explained by the fact that
more than half of the somatic cells in the worm are
G1 progression in C. elegans
J Koreth and S van den Heuvel
2763
formed during embryogenesis. Given the importance of
maternal product during these embryonic divisions,
post-transcriptional levels of regulation must be more
prominent in this animal.
Conclusion
From worm to man, the genes that control G1
progression and the hierarchies or pathways in which
they act are well conserved. C. elegans can be used to
clarify the in vivo interactions of various known G1
regulators. These data form a solid base on which to
start adding additional genes and pathways involved in
the regulation of cell-cycle entry. Such genes could be
identified in forward and reverse genetic screens using,
for instance, a sensitized genetic background or specific
cell-cycle reporters.
Areas that can be addressed include: the molecular
mechanisms that couple normal development and the
cell-autonomous cell-cycle machinery and the connection between the external milieu and developmental cell-
cycle progression decisions in vivo. In these areas many
questions remain, for example, how does starvation
prevent cell proliferation in the first larval stage? How
do the pathways that induce dauer formation connect to
the cell-cycle machinery? What is the interrelationship
between cell growth, differentiation and G1 progression?
C. elegans has numerous advantages that make it an
excellent model to identify pathways regulating the
organism-wide control of cell-cycle progression during
development and environmental stress. Such pathways
will extend our knowledge of cell-cycle control, central
to both normal development and cancer.
Acknowledgements
We thank our many colleagues and collaborators for
generously sharing reagents and ideas, and apologize to those
whose valuable work could not be cited owing to space
constraints. We thank Mike Boxem, John S. Satterlee and Inge
The for their suggestions and critical review of the manuscript.
This work was funded by grants from the Claudia Adams Barr
Program (to JK) and the National Institutes of Health (to
SvdH).
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