Max-Planck Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstrasse 35, 72076 Tübingen, Germany
Author for correspondence (e-mail:
marcus.dekens{at}tuebingen.mpg.de)
Accepted 6 May 2003
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SUMMARY |
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Key words: Maternal-effect mutants, Pronuclear congression, Mitotic spindle, Cell cycle, Zebrafish
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INTRODUCTION |
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Meiosis, the process by which haploid germ cells are formed, consists of
two consecutive nuclear divisions in the absence of DNA replication
(Nebreda and Ferby, 2000).
During metazoan oogenesis, mature oocytes are arrested at specific stages of
meiosis prior to fertilization. Oocytes from fish, amphibians and most mammals
arrest at metaphase II of meiosis (Austin,
1965
; Streisinger et al.,
1981
; Selman et al.,
1993
). Activation of the oocyte is in most species induced by
sperm entry, resulting in a wave of cytosolic calcium at the moment of
fertilization (Stricker, 1999
)
thereby causing a decrease in cyclin-dependent kinase activity, and inducing
the oocyte to resume meiosis (Kline and
Kline, 1992
; Sensui and
Morisawa, 1996
; McDougall and
Levasseur, 1998
; Levasseur and
McDougall, 2000
). This leads to one set of sister chromatids being
extruded to produce a haploid oocyte nucleus.
Following the completion of meiosis the haploid maternal- and
paternal-derived genomes combine to create a diploid zygote. Studies in
Drosophila, mouse and human show that the pronuclei migrate towards
each other (congression) and subsequently enter mitosis
(Schatten et al., 1985;
Simerly et al., 1995
;
Callaini and Riparbelli, 1996
).
The mixing of the maternal and paternal chromosomes therefore occurs at the
first mitosis. In species such as sea urchin, Caenorhabditis,
Xenopus, and as demonstrated here the zebrafish, a diploid nucleus forms
through the fusion of two haploid pronuclei before the first mitosis
(Longo and Anderson, 1968
;
Ubbels et al., 1983
;
Strome and Wood, 1983
).
Entry into mitosis is induced by the activation of maturation promoting
factor (MPF), also referred to as cyclin-dependent kinase (Cdk1)
(Evans et al., 1983;
Murray and Kirschner, 1989
;
Murray et al., 1989
). Active
MPF triggers a cascade of events including nuclear envelope breakdown,
chromosome condensation, mitotic spindle assembly and cyclin destruction by
the anaphase promoting complex (APC). Cyclin destruction in turn results in
MPF inactivation, thereby terminating mitosis
(Murray and Hunt, 1993
;
Zachariae and Nasmyth,
1999
).
Early embryonic, maternally regulated vertebrate cell cycles differ
substantially from somatic cell cycles. Early embryonic cells have rapid
synchronous cell cycles with alternating M (mitosis) and S (DNA synthesis)
phases and lack G (gap) phases. The rapid repeated cleavages result in cells
with successively smaller volumes. In many organisms the cleavages of early
embryos appear to proceed irrespective of various perturbations
(Nagano et al., 1981;
Gerhart et al., 1984
; Newport
and Kirschner, 1984; Gautier,
1987
; Dabauvalle et al.,
1988
). Established techniques that interfere with meiosis or the
first mitosis can be used to generate diploid gynogenic or androgenic
zebrafish embryos (Streisinger et al.,
1981
; Corley-Smith et al.,
1996
), showing that severe disruption of these crucial processes
does not lead to cell cycle arrest at early stages of zebrafish
development.
One powerful approach to identify components that are specific for the
early cell cycles is to perform a genetic screen for maternal-effect mutants.
Such screens in Drosophila have already led to the isolation of
several mutants that are specific for the early cell cycle
(Glover, 1989;
Foe et al., 1993
). However, to
date no such screens have been done in a vertebrate system. Although early
cell cycles differ between Drosophila and vertebrates, it could be
anticipated that specialised factors are also involved in the maternally
regulated vertebrate cell cycle. Here we report the results of the first small
scale maternal-effect screen for nuclear division defects specific for the
early embryonic vertebrate cell cycle. We describe a lethal recessive
maternal-effect mutation, futile cycle (fue), that abolishes
pronuclear congression and chromosomal segregation during mitosis in all
offspring of homozygous females. This mutation does not interfere with
cytokinesis and thus permits several anucleate cleavage cycles. The homozygous
mothers exhibit no defects other than the inviability of their progeny,
suggesting the existence of a specific factor required for the early embryonic
cell cycle in a vertebrate.
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MATERIALS AND METHODS |
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In vitro fertilization
Males were anaesthetised in 0.2% ethyl-m-aminobenzoate metanesulphonate
solution pH 7 (MESAB; Sigma) and then decapitated. The testes were dissected,
and sheared in Hanks buffer (Westerfield, 1994;
Pelegri and Schulte-Merker,
1999) to release sperm (6 testes per ml buffer). The sperm
suspension was kept on ice allowing debris to settle prior to use. Oocytes
were obtained from females by dry stripping. About 50 µl of sperm
suspension was added to each clutch of eggs. After 1 minute, 1 ml E3 water (5
mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4)
was added, followed shortly thereafter by another 20 ml. All in vitro
fertilizations and subsequent development occurred at 28°C.
Fluorescent labelling
Actin labelling was carried out on formaldehyde-fixed embryos that had been
washed and permeabilised by incubation for 3 hours in phosphate-buffered
saline (PBS) with 1% Triton X-100 (toctylphenoxypolyethoxyethanol; Sigma).
Actin filaments were labelled by incubation overnight at room temperature (RT)
with 1:250 dilution of fluorescein phalloidin (Molecular Probes) in PBS
containing 0.4% Triton X-100 (PBST). Nuclei were labelled using
4',6-diamidino-2-phenyl indole (DAPI; 0.5 µg/ml) in PBST (Sigma).
Embryos were incubated in DAPI solution for 10 minutes at RT followed by three
washes in PBST of 5 minutes each.
Immunocytochemistry
In order to label microtubules, embryos were mechanically dechorionated and
subsequently fixed in 4% formaldehyde, 0.05% glutaraldehyde, 5 µM EGTA, 5
µM MgSO4 and 0.1% Triton X-100 in PBS for 1 hour at RT. One- and
two-cell stage embryos were additionally treated with pronase (1 mg/ml for 2
minutes; Sigma) prior to fixation. For all other procedures embryos were fixed
in 4% formaldehyde in PBS at 4°C overnight. The embryos were washed in
PBST, mechanically dechorionated and then dehydrated through a dilution series
into pure methanol at -20°C for at least 30 minutes. After rehydration the
yolk was removed and the embryos were washed in PBST. Subsequently the embryos
were blocked for at least 1 hour at RT in PBST with 1% bovine serum albumin
(BSA; Sigma) (blocking buffer). Incubation with the primary antibody; mouse
IgG anti--tubulin (Sigma; clone DM1
), mouse IgG
anti-
-tubulin (Sigma; clone GTU-88), mouse IgG anti-ß-catenin
(Sigma; clone 15B8), or mouse IgM anti-H5 (Hiss Diagnostics), diluted 1:1000
in blocking buffer took place overnight at 4°C. Then the embryos were
washed in blocking buffer. Bound primary antibodies were detected using Cy5 or
FITC-conjugated goat anti-mouse antibodies (both 1:300; Dianova), incubated
overnight at 4°C. Following this incubation, the embryos were washed in
PBST and transferred through a glycerol (Gerbu) dilution series into 70%
glycerol. Polyvinyl alcohol (Mowiol 4-88; Hoechst) was used for mounting
embryos and 1,4-diazobicyclooctane (DABCO; Merck) was added to the mounting
medium to reduce fading of the samples. Images were obtained using a Leica
confocal microscope, and digitally processed using Adobe PhotoShop software
(Adobe Systems Inc.).
BrdU labelling
Bromodeoxyuridine (BrdU; ICN), dissolved in PBS at a concentration of 10
mM, was injected into embryos (5 nl each). After one cell cycle the embryos
were fixed in 4% formaldehyde, 0.25% glutaraldehyde, and 0.1% Triton X-100 in
PBS to preserve the structure and they were permeabilised as described. The
embryos were then incubated in 4 M HCl for 20 minutes. Subsequently embryos
were washed in PBST several times. Immunocytochemistry was performed according
to standard protocols. After labelling with the primary mouse -BrdU
(ICN; clone II5B) antibody (1:50) followed by the secondary horse
-mouse alkaline phosphatase-conjugated (AP) antibody (Vector; 1:500)
and washing in PBST, the embryos were rinsed three times in phosphatase buffer
(100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl and 0.1% Tween 20).
The enzymatic reaction was performed in phosphatase buffer with the substrates
4-nitro blue tetrazolium chloride (NBT; Sigma) and 5-bromo-4-chloro-3-indolyl
phosphate toluidine (BCIP; Gerbu). The embryos were rinsed in PBST and fixed
after a visible precipitate had formed. Images were taken using a Zeiss
Axiophot microscope.
For in vivo sperm labelling BrdU was used as a marker. To avoid diffusion of BrdU before the fish consumed the food and to be able to give a steady dose of label, we injected bud stage embryos with BrdU dissolved in PBS in order to feed these to the fish. Young males were fed 150 µg BrdU per gram body weight, on alternate days during a period of 50 days. The males were regularly mated during this period and fertilized eggs were collected and subsequently fixed at the two-cell stage in order to test for BrdU incorporation as described.
Drug treatment
The mycotoxin aphidicolin from Nigrospora sphaerica (Sigma) was
dissolved in 50% dimethylsulfoxide (DMSO; Merck) at a concentration of 2.5
mg/ml, and diluted fivefold with PBS prior to use. A volume of 35 nl was
injected per embryo at approximately 20 minutes post-fertilization.
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RESULTS |
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Meiosis is completed normally in fue zygotes
At the end of oogenesis, the mature oocyte is arrested at metaphase of the
second meiotic division in the zebrafish
(Streisinger et al., 1981;
Selman et al., 1993
).
Fertilization triggers the resumption of meiosis with the extrusion of a
haploid set of chromosomes into a vesicle on the outside of the cell membrane,
termed the second polar body. This event, which marks the completion of
meiosis, occurs at about 7 minutes post-fertilization in wild-type zygotes
(Streisinger et al., 1981
).
Since meiosis is the earliest process that could be affected in this mutant,
we tested if the extrusion of the second polar body occurs normally in
fue zygotes. In order to visualise the polar body, both DNA and actin
were labelled. Extrusion of the second polar body in fue zygotes was
indistinguishable from that of wild type, as shown in
Fig. 2, indicating a normal
completion of meiosis.
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fue cells pass through consecutive S phases
Early embryonic cells progress through alternating cycles of S (DNA
synthesis) and M (mitosis) phases and lack the G (gap) phases characteristic
of somatic cell cycles (Murray and Hunt,
1993). We addressed whether both pronuclei in fue embryos
undergo aspects of normal embryonic cell cycle, such as DNA synthesis. To
investigate whether DNA replication occurs in fue, the embryos were
assayed for the incorporation of BrdU into the DNA during S phase. BrdU was
injected directly after the eggs were laid, and these embryos were fixed at
the beginning of the two-cell stage. BrdU labelling of DNA was detected in
both nuclei of fue embryos, indicating that DNA replication does
indeed take place (Fig. 4A,B).
To determine if replication continued during subsequent cell cycles we
injected BrdU into embryos at the onset of the formation of the second furrow,
when the DNA replication phase of the first two cycles has been completed, and
fixed these embryos at the eight-cell stage. These fue embryos also
showed nuclear BrdU labelling (Fig.
4C,D). We therefore conclude that DNA replication takes place in
fue embryos at the one-cell stage and also during subsequent cell
cycles.
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We also tested the integrity of the centrosomes in fue cells by
comparing these organelles in aphidicolin-injected embryos. Centrosomes were
detected using an antibody directed against -tubulin, a highly
conserved component of this organelle
(Oakley et al., 1990
;
Stearns et al., 1991
;
Stearns and Kirschner, 1994
).
A pair of centrosomes is present in all cells at all stages of both
fue and aphidicolin-injected embryos
(Fig. 6A-C), showing that
centrosome duplication is not affected. In addition, the presence of a pair of
asters at the first mitosis in fue embryos
(Fig. 5B,C) indicates normal
centrosomal duplication prior to this event. Centrosomes of fue and
aphidicolin-treated embryos are indistinguishable from those of wild type
(Fig. 6D-F). Since these
organelles are crucial for cytokinesis their presence and capacity to
duplicate could be expected.
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Transcription is initiated earlier in fue embryos
During the mid blastula transition (MBT) the previously silenced genome
becomes active in wild-type embryos, triggered by the decreased cytoplasm to
DNA ratio (Newport and Kirschner,
1982; Edgar et al.,
1986
; Kane and Kimmel,
1993
). As both cleavage and DNA replication proceed in
fue embryos but chromosomal segregation does not take place, the DNA
concentration increases locally more rapidly than normal as the cell size
decreases. This should lead to an earlier arrival at the critical cytoplasm to
DNA ratio in fue embryos than in wild-type embryos, and therefore
activation of the fue genome is expected to occur at an earlier stage
if transcription is not affected.
The phosphorylation of the H5 epitope of RNA polymerase II has been shown
to coincide with the transcriptional activation of the silenced genome at MBT
in several species (Seydoux and Dunn,
1997; Knaut et al.,
2000
). Therefore, we analysed fue embryos at several
early cell stages using an antibody that detects the phosphorylation of this
epitope (Bregman et al., 1995
)
as a marker for the onset of transcription.
We first observe phosphorylation of the H5 epitope of RNA polymerase II in wild-type embryos at the 512-cell stage and in fue embryos at the 32-cell stage (Fig. 7). The phosphorylation of the H5 epitope of RNA polymerase II in fue embryos suggests mRNA transcription does occur in this maternal-effect mutant. When we assume that the DNA concentration doubles with every cell cycle and cleavage proceeds normally in fue embryos one would predict transcriptional activation in these embryos should occur after half the number of cell cycles that it takes wild-type embryos to reach MBT, or five cycles (32-cell stage) instead of nine cycles (512-cell stage). Therefore the earlier initiation of transcription in fue embryos seems to provide genetic support for transcriptional initiation being regulated by a previously proposed mechanism based on the DNA to cytoplasm ratio.
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DISCUSSION |
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Cyclical oscillations in the activity of maturation promoting factor (MPF)
have been shown to constitute the core of the cell cycle oscillator,
determining when the cell enters mitosis and when it exits to enter interphase
(Evans et al., 1983;
Murray and Kirschner, 1989
;
Murray et al., 1989
). Active
MPF induces the downstream events of mitosis, while inactive, it permits
downstream events that lead to interphase. In fue embryos, cleavage
takes place as in wild-type embryos, indicating that the abolished factor is
not involved in the periodical change of MPF activity but is an event
occurring downstream of this oscillating mechanism.
Pronuclear congression is abolished in fue zygotes
Pronuclear fusion follows the completion of meiosis in several organisms
(Longo and Anderson, 1968;
Ubbels et al., 1983
;
Strome and Wood, 1983
),
however, this had not been demonstrated in zebrafish. By visualising the
nuclei of zygotes that were fixed at consecutive time points after in vitro
fertilization, we have been able to document a sequence of pronuclear events
that takes place in the zebrafish zygote. We show that pronuclear fusion
occurs in wild-type zebrafish zygotes between 14 and 20 minutes
post-fertilization. In contrast, we did not observe pronuclear fusion in
fue zygotes. Furthermore, to distinguish between the pronuclei, we
labelled sperm DNA in vivo using BrdU as a marker. Following fertilization we
determined the number of BrdU-labelled nuclei shortly after the first mitosis
was completed. Only one BrdU-labelled nucleus could be detected in two-cell
fue embryos, although two nuclei are always present at this stage,
confirming that pronuclear fusion does not occur in fue zygotes. This
phenotype suggests a role for fue in pronuclear congression as the
pronuclei are always observed at a distance from each other.
Mitotic spindle assembly is perturbed in fue zygotes
fue mutants demonstrate abnormal mitosis, suggesting that the
fue wild-type gene product may also be required for chromosome
segregation. Alternatively the mitotic defect may solely be a secondary
consequence of the perturbed pronuclear congression. However, preventing
pronuclear fusion using different approaches in several species has been shown
not to block the zygote from entering mitosis or to prevent normal chromosomal
segregation (Subtelny, 1958;
H. Schatten, 1994
;
Sluder et al., 1995
;
Sadler and Shakes, 2000
). In
addition, fue zygotes enter mitosis with the appearance of asters and
often microtubule nucleation on the chromosome clusters is observed at that
time, which is not expected if such a checkpoint exists. Therefore it would
seem unlikely that pronuclear fusion is a prerequisite for mitosis in
zebrafish zygotes, suggesting that the fue wild-type gene product may
also be required at this stage.
During the first mitosis two chromosome clusters are present in fue zygotes, which often, but not always, have a defect in microtubule nucleation. When microtubules are present on the chromosomes they do not organise into bundles or bipolar spindles, suggesting a defect in coalescence. The formation of normal asters shows that centrosome-dependent organisation of microtubules is not affected in fue zygotes, indicating that the defect is restricted to the chromosome-dependent nucleation of microtubules. At later stages chromosome clusters mostly nucleate microtubules and with a low incidence can even randomly segregate DNA. Therefore, the defect at post-zygotic mitoses is less severe than at the first mitosis, which may be due to a greater amount of DNA present in these clusters.
Since chromosome clusters are observed during mitosis, fue embryos
do not have an obvious defect in chromosomal condensation. Blow and Watson
have demonstrated a requirement of the nuclear envelope for DNA replication
(Blow and Watson, 1987),
therefore the ability of fue embryos to replicate the chromosomes
could indicate the presence of a functional nuclear envelope. In addition,
this envelope has to break down or enter a permeable state in order to allow
the subsequent round of DNA synthesis (Blow
and Laskey, 1988
; Coverley et
al., 1993
). Furthermore, the presence of microtubules on some
chromosome clusters indicates the absence of a nuclear envelope at mitosis,
therefore suggesting nuclear envelope break down does occur. The kinetochores
play a role in the stabilisation of microtubules as well as positioning of
chromosomes at the metaphase plate. Since all sperm-derived components in
fue embryos are wild type, the paternal chromosomes are expected to
posses normal kinetochores. However, a significant difference is not observed
between the paternal and maternal derived chromosome clusters at mitosis,
suggesting that the defect in fue is not related to kinetochore
function. Although, as not all kinetochore-associated proteins may be provided
by the sperm, we cannot completely exclude crucial components of kinetochores
from being affected.
Nucleation of microtubules at the chromosomes has previously been shown to
be induced by the GTPase Ran (Wilde and
Zheng, 1999; Carazo-Salas et
al., 1999
; Carazo-Salas et al.,
2001
), which is present as a gradient with the highest
concentration of the GTP-bound form on the chromosomes. Several proteins
essential for mitotic spindle assembly are inactivated by being bound to
importin ß and released around the chromosomes during mitosis by RanGTP
(Gruss et al., 2001
;
Nachury et al., 2001
), thereby
inducing spindle assembly. We have shown that fue could block the
pathway that leads to the chromatin-dependent generation of microtubules,
which is regulated by Ran, its GTP exchange factor, and its downstream
components (Karsenti and Vernos,
2001
).
fue couples karyo- to cytokinesis
Pronuclear congression is a microtubule-mediated process, in which the
centriole provided by the sperm assembles an aster that connects it to the
oocyte nucleus, thereby bringing both pronuclei together
(Lessman and Huver, 1981;
G. Schatten, 1994
). The asters
formed at mitosis in fue embryos are normal, suggesting that the
sperm aster is normal too, and that the defect may be in the following step of
bringing the pronuclei towards each other. Since the defect observed during
mitosis is due to the generation or organisation of microtubules, both defects
in fue zygotes appear to primarily affect microtubule function.
Interestingly, directly after fertilization a polar body is extruded in
fue zygotes, showing that meiosis is completed successfully and thus
indicating that the meiotic spindle is normal. Therefore, the fue
phenotype reveals a crucial difference between the processes of assembly of
meiotic and mitotic spindles. Since fue embryos initiate cleavage as
in wild-type embryos, centrosomal behaviour is not affected by the absence of
pronuclear fusion and the improper mitosis. We confirmed the presence of
centrosomes at all cell stages in fue embryos, consistent with their
ability to duplicate being an absolute requirement for cell division
(Rieder et al., 2001
).
Therefore, by specifically perturbing the contribution of the chromosomes in
mitotic spindle assembly, while not affecting centrosomal functions, we
propose a mechanism by which nuclear- and cell division are connected.
Like fue, the Drosophila maternal-effect mutant giant
nuclei (gnu) has two nuclei per embryo as well as DNA
replication in the absence of chromosomal segregation and normal centrosome
duplication (Freeman et al.,
1986). However, there are several phenotypic differences when
compared with fue mutants. gnu embryos do not show a proper
completion of meiosis as DNA replication is initiated before fertilization has
occurred (Freeman and Glover,
1987
). In addition, gnu embryos do not form pole cells.
These cells give rise to the future germ cells and are the first to be formed
in the Drosophila embryo, and therefore this maternal-effect
mutation, unlike fue, does not uncouple karyokinesis from
cytokinesis. It has been demonstrated that these processes can indeed be
uncoupled in Drosophila, since embryos injected with the DNA
replication inhibitor aphidicolin are anucleate, but still form pole cells
(Raff and Glover, 1989
). The
Drosophila embryo is initially a syncitium, and therefore pole cell
formation might be a mechanistically different process from cleavage in the
vertebrate embryo. However, the differences observed between the
Drosophila gnu and the zebrafish fue phenotype make it
unlikely that this factor could also be affected in fue embryos.
Although we believe that the function of fue is restricted to the
first embryonic cell cycles it could instead represent a hypomorphic allele
that also acts zygotically like the Drosophila mutation polo
(Sunkel and Glover, 1988;
Fenton and Glover, 1993
;
Glover et al., 1998
) which was
discovered on the basis of a maternal phenotype. Alternatively, fue
function could be redundant during the subsequent somatic cell cycles. Thus,
fue may be revealed as a factor that also acts during the later
somatic cell cycles. However, the maternal-effect mutation in fue
causes lethality in all offspring at an early stage while the homozygous
mothers appear healthy and do not exhibit either a shorter life span or
decreased fecundity. Importantly, fue is the first mutant documented
to have cell division in the absence of nuclear division, and thus represents
a unique phenotype. Therefore, the characterisation of the fue gene
product should provide important new insight into the processes of pronuclear
congression and mitotic spindle assembly at the earliest stage of vertebrate
development.
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ACKNOWLEDGMENTS |
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Footnotes |
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