271
Journal of Cell Science 110, 271-280 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS3473
Wolbachia-induced delay of paternal chromatin condensation does not
prevent maternal chromosomes from entering anaphase in incompatible
crosses of Drosophila simulans
Giuliano Callaini*, Romano Dallai and Maria Giovanna Riparbelli
Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy
*Author for correspondence
SUMMARY
The behavior of parental chromosomes during the first
mitosis of Drosophila simulans zygotes obtained from unidirectional incompatible crosses is described and it is
demonstrated that the condensation of parental chromatin
complements was asynchronous. The timing of paternal
chromatin condensation appeared to be delayed in these
embryos, so that condensed maternal chromosomes and
entangled prophase-like paternal fibers congressed in the
equatorial plane of the first metaphase spindle. At
anaphase the maternal chromosomes migrated to opposite
poles of the spindle, whereas the paternal chromatin lagged
in the midzone of the spindle. This resulted in dramatic
errors in paternal chromatin inheritance leading to the
formation of embryos with aneuploid or haploid nuclei.
These observations suggest that the anaphase onset of
INTRODUCTION
During the first mitotic division of the Drosophila zygote the
maternal and paternal chromosomes congress at the metaphase
plate of the spindle, but instead of mingling at the equatorial
plane, as in most animal cells, they initiate anaphase as two
widely separated sets, and mingle during telophase (Huettner,
1924; Sonnenblick, 1950; Callaini and Riparbelli, 1996).
Parental chromosomes enter anaphase at the same time, despite
the fact that maternal chromatin condenses first. The high
fidelity of chromosome transmission during the following
anaphase raises the question of how the timing of the
metaphase/anaphase transition is controlled during the first
mitosis in fly embryos. Somatic cells ensure the fidelity of
chromosome transmission to their daughters by extrinsic
control mechanisms (cell-cycle checkpoints) that allow the
initiation of an event only when the previous event is successfully completed (Hartwell and Weinert, 1989; Murray, 1994).
Errors in DNA replication and improper chromosome
alignment generate signals triggering specific mechanisms that
delay progression through the cell cycle. Most eukaryotic cells
with damaged or unreplicated DNA are kept from entering
mitosis until DNA damage is repaired (Murray, 1992; Murray
and Hunt, 1993; Kaufmann and Paules, 1996) and the onset of
maternal chromosomes is unaffected by the improper
alignment of the paternal complement. Since the first
metaphase spindle of the Drosophila zygote consists of twin
bundles of microtubules each holding one parental complement, we suspect that each half spindle regulates the
timing of anaphase onset of its own chromosome set. In
normal developing embryos, the fidelity of chromosome
transmission is presumably ensured by the relative timing
required to prepare parental complements for the orderly
segregation that occurs during the metaphase-anaphase
transition.
Key words: Drosophila simulans, Cytoplasmic incompatibility,
Fertilization, Parental chromatin condensation
anaphase is delayed in vertebrate cells once kinetochores are
not under bipolar tension or unattached (reviewed by Wells,
1996). Abnormal inheritance of the genetic material occurs
when mutations inactivate specific cell-cycle checkpoints
(Weinert and Hartwell, 1988; Hoyt et al., 1991; Li and Murray,
1991; Humphrey and Enoch, 1995) or when drug treatment
induces checkpoint override and exit from mitosis (Andreassen
and Margolis, 1991; Larsen and Wolniak, 1993; Nicklas et al.,
1993). The rigid cell-cycle checkpoints that ensure proper
transmission of genetic material in somatic cells appear less
accurate in some embryonic cells. Inhibitors of DNA and
protein synthesis do not affect some aspects of cell division
(Kimelman et al., 1987) and centrosome duplication (Gard et
al., 1990) in Xenopus eggs. Centrosome replication may also
be uncoupled from DNA synthesis in echinoderm embryos
treated with aphidicolin (Nagano et al., 1981; Sluder and
Lewis, 1987). The presence of unattached maternal chromosomes has no effect on the timing of anaphase onset of the
paternal complement in sea urchin zygotes (Sluder et al.,
1994). Since it seems likely that embryonic systems would
incur developmental alterations due to chromosome loss and
aneuploidy, they might have evolved compensatory mechanisms to avoid defective transmission of the genetic material
(Hartwell and Weinert, 1989). For example, genetic damage is
272
G. Callaini, R. Dallai and M. G. Riparbelli
avoided in syncytial Drosophila embryos by discarding the
nuclei that failed to complete chromosome segregation
(Minden et al., 1989; Sullivan et al., 1990, 1993). However,
this mechanism working in late syncytial embryos does not
explain the fidelity of chromosome transmission observed
during the first mitotic division of the zygotic nucleus. In the
early Drosophila embryo poor cell-cycle checkpoint controls
are to be expected since in aphidicolin treated embryos centrosome replication continues (Raff and Glover, 1988) and gnu,
plu, and png mutants replicate their DNA and centrosomes
without undergoing nuclear divisions (Freeman et al., 1986;
Shamansky and Orr-Weaver, 1991). While studying cytoplasmic incompatibility in Drosophila simulans, we found that
during the first mitosis of zygotes obtained from incompatible
crosses, the paternal chromosomes failed to condense properly
and lagged behind on the metaphase plate, whereas the
maternal set entered anaphase. A model such as this, in which
the anaphase onset of maternal chromosomes is not affected by
the presence of maloriented paternal chromosomes, offers the
unique opportunity of clarifying whether the Drosophila
zygote has feedback controls for the metaphase-anaphase transition based on chromosome misorientation.
Cytoplasmic incompatibility is an unusual form of intrapopulation sterility that is associated in insects with inherited
bacteria of the genus Wolbachia, and is commonly found when
infected males are crossed with uninfected females (reviewed
by Rousset and Raymond, 1991; Breeuwer and Werren, 1990;
Boyle et al., 1993; Giordano et al., 1995). In incompatible
crosses of Drosophila simulans, fertilized eggs fail to hatch and
less than 5% of embryos are viable (Hoffmann et al., 1986).
Defective early fertilization events have been associated with
the high rate of embryo mortality in intraspecific crosses of
Drosophila simulans (O’Neill and Karr, 1990; Lassy and Karr,
1996), but the cytological basis of this phenomenon is still not
understood. We therefore conducted a detailed study to
determine what happens during the fertilization of eggs derived
from incompatible crosses in Drosophila simulans.
MATERIALS AND METHODS
Stocks
The Watsonville and Riverside strains of Drosophila simulans (abbreviated as DSW and DSR, respectively) used in this study were kindly
provided by Rosanna Giordano (University of Urbana, Illinois). The
crosses were performed by taking newly hatched DSW females and
DSR males and leaving them on standard cornmeal, agar and yeast
medium in 100 ml plastic containers. Eggs from 5- to 6-day-old flies
were collected three times at 24°C on small agar plates for 20 minutes
each. Repeated egg collection was needed to avoid problems caused
by the fact that females retain fertilized oocytes for different periods
of time. After discarding the first eggs, fertilized eggs were again
collected three times for 20 minutes. Three sets of collections were
performed from two groups of rapidly laying females.
Fluorescence microscopy
Eggs were dechorionated in a 50% bleach solution for 2-3 minutes and
washed in distilled water. They were dried on filter paper and the
vitelline envelope was removed by transfer to a vial containing 3 ml nheptane and 3 ml of cold 90% methanol solution in water. After shaking
for 3 minutes the embryos without vitelline envelope were transferred
to cold methanol for 7 minutes and then to cold acetone for 5 minutes.
After fixation the embryos were washed in phosphate buffered saline
(PBS) and incubated for one hour in PBS containing 0.1% bovine serum
albumin (BSA). The eggs were then incubated overnight at 4°C with a
monoclonal antibody against β-tubulin (Boehringer Mannheim;
dilution 1:200 in PBS/BSA). The eggs were rinsed in PBS/BSA and
then incubated for one hour in goat anti-mouse-conjugated IgG coupled
to fluorescein (Cappel, West Chester, PA; dilution 1:600 in PBS/BSA).
After rinsing in PBS the nuclei were stained with 1 µg/ml Hoechst
33258 for 3 minutes. The eggs were rinsed again in PBS and mounted
in 90% glycerol containing 2.5% n-propyl gallate (Giloh and Sedat,
1982). Fluorescence observations were carried out with a Leitz Aristoplan microscope equipped with fluorescein and UV filters. Micrographs
were taken with Kodak Tri-X 400 pro film and developed in Kodak
HC110 developer for 7 minutes at 20°C.
Fuchsin staining
The embryos were fixed essentially as described by Zalokar and Erk
(1977) and incubated in 5 N HCl at room temperature for 1 hour,
washed with distilled water and stained with 1% fuchsin (Merck) in
2.5% acetic acid for 20-30 minutes. The embryos were then destained
in 5% acetic acid and mounted in glycerol.
Controls
The process of fertilization was also examined in embryos obtained
from three further crosses of Drosophila simulans: (a) uninfected
embryos obtained from crosses between naturally uninfected flies
(DSW strain); (b) infected embryos obtained from crosses between
naturally infected flies (DSR strain); (c) uninfected embryos obtained
from crosses between naturally uninfected females (DSW) and tetracycline-treated DSR males. These males were obtained by culturing
the DSR stock for two generations on standard Drosophila medium
supplemented with 0.05% tetracycline as described by Hoffmann et
al. (1986).
The embryos of all these crosses were viable and the first cleavage
mitosis proceeded as described in Drosophila melanogaster (Callaini
and Riparbelli, 1996). However, we only show pictures of embryos
obtained from crosses between naturally uninfected flies (DSW
strain).
RESULTS
In crosses of Drosophila simulans between males harboring
bacteria of the genus Wolbachia (DSR) and uninfected females
(DSW), incorporation of sperm by the oocyte was followed by
the formation of a prominent aster at the site of the sperm head
(Fig. 1a,b), as in normal developing embryos. After the completion of the female meiosis, the distance between the parental
pronuclei gradually decreased and the astral microtubules associated with the male nucleus radiated from two distinct foci.
These observations suggest that in incompatible crosses the
sperm centrosome retains its nucleating properties and replicates in a normal fashion. Once the pronuclei were side-byside, their chromatin condensed (Fig. 1d) as in control embryos
(Fig. 1e) and a bipolar array of microtubules organized at the
junction between the pronuclei (Fig. 1c). At prophase the
parental pronuclei showed clearly asymmetric chromatin condensation (Fig. 1f,g). This asymmetry was also observed in
control embryos obtained from compatible crosses between
males and females of the DSW strain (Fig. 1h). However, in
control embryos the synchrony of chromatin condensation was
soon regained and at the end of prophase two groups of
stretched chromosomes were found on either side of the
spindle plane (Fig. 1k). In embryos obtained from incompatible crosses only one set of chromosomes was clearly visible at
Delay of paternal chromatin condensation
273
Fig. 1. Microtubules and DNA in early Drosophila simulans embryos obtained from incompatible crosses between DSW females and DSR
males (a,b,c,d,f,g,i,j) and in control embryos (e,h,k). (a,b) The metaphase II oocyte shows two main microtubule arrays; the female meiotic
apparatus (arrow), containing two chromosome sets (arrowheads) and one irregular aster (small arrow), associated with the sperm head (small
arrowheads). (c,d,e) Pronuclei are closely apposed; a bipolar array of microtubules is associated with the more condensed male nucleus. (f,g,h)
Prophase; condensation of parental complements is in progress. (i,j,k) End of prophase; female chromosomes are condensed both in control and
in incompatible embryos, while the condensation of the male chromatin is delayed in incompatible embryos. f, female pronucleus; m, male
pronucleus. Bars: a,b (15 µm); c-h (5 µm).
the end of prophase, the other set being entangled chromatin
fibers (Fig. 1i,j).
At prometaphase the growth of the spindle microtubules
resulted in a mitotic apparatus with two separate parental complements that still showed different degrees of chromatin condensation. One parental set consisted of highly condensed
chromosomes, whereas the other was a tangle of prophase-like
fibers (Fig. 2a,b). In control embryos, two distinct sets of
condensed chromosomes were seen at this stage (Fig. 2c). The
asynchronous parental chromatin condensation persisted
through metaphase (Fig. 2d,e), at the end of which the tangled
chromatin fibers and the condensed chromosome set became
closely apposed in the midzone of the spindle (Fig. 2g,h). In
control embryos two separate groups of coiled chromosomes
were closely apposed in the equatorial region of the first mitotic
spindle at the beginning of metaphase (Fig. 2f) and fully
condensed chromosomes were found in the midzone of the
spindle at the end of metaphase (Fig. 2i).
At the onset of anaphase, the sister chromatids of one
parental set left the metaphase plate, whereas the other parental
set lagged behind in the equatorial region of the spindle (Fig.
2j,k). At the end of anaphase the astral microtubules grew
further (Fig. 3a) and half of the chromatids reached the
opposite poles of the spindle, whereas the other parental set
still lagged in the midzone of the spindle (Fig. 3b) or shifted
slightly toward one pole (Fig. 3c). The lagging tangled
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G. Callaini, R. Dallai and M. G. Riparbelli
chromatin mass condensed further during the anaphase progression and distinct chromosomes were often visible at the
end of anaphase in suitable preparations. In control embryos,
both the parental sets of chromatids moved synchronously to
the poles of the spindle (Figs 2l, 3d).
Telophase spindles showed prominent midbodies and large
asters in control and incompatible crosses (Fig. 3e). In crosses
between DSW females and DSR males, two classes of abnormal
telophase figures were observed: the delayed parental chromatin
complement appeared to be stretched between the two poles
(Fig. 3f) or split in two portions that left the midzone of the
spindle and moved toward opposite poles (Fig. 3g). In embryos
obtained by crossing females and males belonging to the DSW
strain the chromosomes decondensed and mingled at telophase,
and two opposite zygotic nuclei formed (Fig. 3h). In embryos
obtained from incompatible crosses two nuclei of unequal size
could be observed at each spindle pole at the end of telophase.
Parental chromosomes in incompatible crosses therefore
entered the second mitotic division without mingling.
To verify the possibility that the first mitotic apparatus of the
Drosophila zygote was composed of distinct half spindles independently acting, we gently squashed newly laid eggs under glass
coverslips. Under pressure the mitotic apparatus flattened and the
half spindles became easily visible. From prometaphase to
anaphase (Fig. 4a-f) the half spindles held parental complements
with different degree of chromatin condensation. Each complement was also closely associated with its own half spindle when
the mitotic apparatus was damaged by squashing (Fig. 4e,f). We
have also found during metaphase-anaphase transition of the first
mitosis an asymmetric organization of the spindle microtubules
(Fig. 4g,h). Microtubules corresponding to the female chromosomes were longer and continuous while microtubules corresponding to the lagging male chromatin were shorter and had a
gap in the equatorial plane of the spindle. This suggests that two
Fig. 2. Spindle and chromatin morphology in embryos from crosses of Drosophila simulans. Microtubules and upper pictures of each DNA
panel show embryos from crosses between DSW females and DSR males; lower pictures of each DNA panel are from control embryos
obtained by DSW females and DSW males. (a,b,c) Prometaphase. (d,e,f) Metaphase; note the twin microtubule bundles (open arrows) forming
the mitotic spindle. (g,h,i) End of metaphase; the parental complements are close together. (j,k,l) Anaphase A; in normally developing embryos
the parental complements (arrowheads) move as distinct entities to opposite spindle poles. Arrows indicate paternal chromatin in embryos from
incompatible crosses. Bar, 5 µm.
Delay of paternal chromatin condensation
275
Fig. 3. Microtubules and DNA in embryos from crosses of Drosophila simulans. Pictures in the right column are from compatible crosses, the
others are from incompatible crosses. (a,b,c,d) Anaphase B; note the tangled chromatin mass lagging in the midzone of the spindle or shifted
toward one pole (arrows). (e,f,g,h) Late telophase; mitotic figures are characterized by chromatin bridges (arrow) or asymmetrically distributed
chromatin complements (arrowhead). Bar, 5 µm.
groups of spindle microtubules were independently interacting
with male and female complements during the first mitosis of the
Drosophila embryo.
The abnormal position of half complements presumably
affected the regular progression of the second mitosis and led
to the formation of aberrant figures. At higher frequency we
observed twin opposite spindles holding haploid complements
that were joined by microtubule bridges enveloping stretched
chromosomes (Fig. 5a,d). In some embryos we also found two
separate figures (Fig. 5b,c,e,f): one with a microtubular shell
that did not differentiate distinct spindle poles and surrounded
a haploid complement close to an irregular chromatin mass; the
other was a normal-looking mitotic spindle. This spindle held
a haploid complement of chromosomes (Fig. 5e), but
sometimes one or more supernumerary chromosomes lagged
in its midzone (Fig. 5f). Indirect immunofluorescence of
haploid embryos indicated that spindle configuration and centrosomal cycle were normal (Fig. 6a), whereas aneuploid
nuclei were associated with broad spindles that lost their centrosomes at an early stage (Callaini et al., 1996). Haploid
embryos survived longer and typically died shortly before
hatching, whereas the development of embryos with aneuploid
nuclei arrested after a few intravitelline mitoses.
It was not possible to determine from this data whether
maternal or paternal chromosomes lagged on the equatorial plane
of the spindle and were lost in early embryos obtained from
incompatible crosses. However, indirect observations provided
insight into the parental origin of the tangled chromatin mass
lagging in the midzone of the spindle during first anaphase. Both
chromosome sets that left the equatorial plane of the spindle at
the onset of the first anaphase showed a stick-like chromatid with
a subterminal fluorescent dot. According to Gatti et al. (1976) this
staining pattern characterizes the X chromosome of Drosophila
simulans. Since we found this staining pattern in all the anaphase
figures with distinguishable chromosomes scored during the first
mitosis (Fig. 6b; n=79), in all the haploid complements that pro-
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G. Callaini, R. Dallai and M. G. Riparbelli
Fig. 4. Spindle (a,c,e,g) and chromatin (b,d,f,h) morphology in embryos from incompatible crosses between DSW females and DSR males. The
flattening of the embryo makes clear the twin half spindles that formed the first mitotic apparatus during prometaphase (a,b), metaphase (c,d)
and anaphase (e,f). Note that paternal (m) and maternal (f) complements are associated with distinct half spindles (arrows and arrowheads). The
first anaphase spindle (g,h) is formed by continuous microtubules (arrowhead) corresponding to the female chromosomes (f), and by shorter
microtubules (arrow) ending in the equatorial plane of the spindle where paternal chromosomes (m) are lagging. Bar, 5 µm.
gressed through the second and third mitoses (Fig. 6c; n=49), and
during later mitoses of haploid syncytial embryos (Fig. 6d; n=87),
we suspect that only the female chromatin complement was
properly condensed at the time of fertilization. This was also
confirmed by staining the embryos with fuchsin. All 113 haploid
embryos scored at different times of development (25 during the
intravitelline mitoses, Fig. 6e; 47 during the syncytial blastoderm
stage, Fig. 6f; 41 during gastrulation, Fig. 6g) had only the
maternal complement.
DISCUSSION
Wolbachia induces delay of paternal chromatin
condensation
After incorporation of the sperm into the egg, maternal and
paternal complements meet and intermix. The coordination of
these events was severely impaired in embryos obtained from
cytoplasmically incompatible crosses between males of
Drosophila simulans harboring bacteria of the genus
Wolbachia and uninfected females. Our observations showed
that chromatin condensation of the parental sets was extremely
asynchronous. During the first mitosis, one set of fully
condensed chromosomes congressed in the equatorial plane of
the spindle together with a set of chromatin fibers in prophaselike configuration. During anaphase the sister chromatids of
one parental complement moved to opposite spindle poles,
whereas the other parental complement lagged in the midzone
of the spindle. This irregular process led to abnormal embryos
with haploid or aneuploid nuclear complements.
In looking for sex chromosomes in surviving haploid
embryos (n=113), we never found the Y chromosome, whereas
all suitable anaphase figures scored during the first mitosis
(n=79) showed a sister chromatid with a brightly fluorescent
Delay of paternal chromatin condensation
277
Fig. 5. Microtubule (upper
panels) and DNA (lower
panels) configurations during
the second mitosis of embryos
from incompatible crosses.
Arrows and arrowheads
indicate maternal and paternal
complements, respectively.
Bar, 5 µm.
subterminal dot. According to Gatti et al. (1976) this staining
pattern characterizes the X chromosome of Drosophila
simulans. Chromatids with this staining pattern were also
found in haploid complements that progressed through the
second and further mitoses. These findings together indicate
that the paternal set of chromosomes is lost in incompatible
crosses. This observation extends previous reports on Nasonia
vitripennis (Ryan and Saul, 1968), Culex pipiens (Jost, 1970)
and Drosophila simulans (O’Neill and Karr, 1990) which
suggested that cytoplasmic incompatibility bacteria prevent
syngamy in incompatible crosses. Preliminary cytological
examinations in Drosophila simulans indicate that the high
embryonic mortality is a consequence of defects which occur
as early as the first cleavage division (O’Neill and Karr, 1990).
This suggestion is supported by the finding that Wolbachia has
been shown to disrupt the first mitosis in the wasp Nasonia by
impairing paternal chromatin condensation in crosses between
cytoplasmically incompatible strains (Ryan and Saul, 1968;
Breeuwer and Werren, 1990; Reed and Werren, 1995). How
Wolbachia infection selectively delays condensation of
paternal chromatin is an intriguing question. It is widely
accepted that the condensation of DNA involves structural
rearrangement of chromatin and scaffold proteins, but little is
known about the molecular mechanism that regulates this
process. There is evidence that chromatin packaging in eukaryotic cells is accompanied by phosphorylation of histones H1
and H3 (reviewed by Reeves, 1992) and that topoisomerase II
is required for proper mitotic chromosome condensation
(reviewed by Ernshaw and MacKay, 1994). Histone H1 does
not seem primarly involved in chromatin condensation in the
earliest phases of Drosophila development, since H1 accumulates in the embryo from nuclear cycle 7. However, the HMGD protein is associated with condensed chromosomes in the
absence of the histone H1 suggesting that it might perform a
function similar to that of histone H1 (Ner and Travers, 1994).
Topoisomerase II is clearly detectable on condensing chromosomes of the early Drosophila embryo (Swedlow et al., 1993)
and injection of anti-topo II antibodies impairs chromosome
condensation in Drosophila syncytial embryos (Buchenau et
al., 1993). Since Wolbachia has never been detected in fertilized oocytes from incompatible crosses, we suppose that a
Wolbachia-related factor is associated with the male chromatin
during spermatogenesis. This is supported by the observation
that the penetrance of the incompatible phenotype seems dose
dependent, because aged males having a lower concentration
of Wolbachia in their germinal tissues (Bressac and Rousset,
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G. Callaini, R. Dallai and M. G. Riparbelli
topoisomerase II, or other unknown chromosomal scaffold
proteins, delaying chromatin compaction. This working
hypothesis may explain why crosses between infected males
and females produce viable embryos. The compaction of
maternal chromatin is presumably also delayed in these
embryos, so that the synchrony of chromosome condensation
is restored. The fact that infected eggs develop into viable
embryos after fertilization with uninfected sperm is a problem.
Presumably the male chromatin recruits the Wolbachia-derived
factor from the oocyte cytoplasm during replication of DNA.
Maternal and paternal chromatin condensation are therefore
coupled and the first mitotic division takes place successfully.
Fig. 6. Crosses between DSW females and DSR males. (a) Spindles
in haploid embryos during anaphase of the tenth nuclear cycle.
Details of chromosomes found in embryos during first anaphase (b),
second metaphase (c), and anaphase of the tenth mitosis (d); arrows
indicate the bright dot on the X chromosome. Details of
chromosomes found in embryos stained with fuchsin during
metaphase of the fifth nuclear division (e), anaphase of the tenth
mitosis (f), metaphase of the first postblastodermic mitosis (g). Note
that the Y chromosome is absent in haploid complements; arrows
indicate the X chromosome. The fourth dot-like chromosome is not
visible in these images. Bars: a (10 µm); b-g (5 µm).
1993) produce more viable embryos in incompatible crosses
(Hoffmann et al., 1986, 1990). A Wolbachia-related factor of
this kind may impair the function of the HMG-D protein,
Does a metaphase-checkpoint exist in the
Drosophila zygote?
Our results show that in zygotes obtained by crossing DSR
males and DSW females the metaphase spindle assembled but
paternal chromosomes condensed improperly and were
delayed at the metaphase plate, while female chromatids
attained anaphase and migrated to the opposite poles of the
spindle. This delay in anaphase initiation leads to dramatic
errors in paternal chromatin inheritance. Whether paternal
chromosome segregation defects arise as a consequence of
improper chromatin condensation or defective structural organization of the kinetochore regions remains to be determined. It
has been shown that kinetochore alignment, not chromosome
condensation, is essential to overcome metaphase block and
initiate anaphase. The chromatin of mammalian cells treated
with topoisomerase II inhibitors fails to condense properly, but
the spindle assembly checkpoint is passed as soon as the kinetochores align at the metaphase plate (Clarke et al., 1993;
Gorbsky, 1994). We are unable to directly assess whether all
paternal kinetochores captured spindle microtubules when
female chromatids entered anaphase, but the irregular
telophase figures, in which paternal chromatin was either
stretched or unequally distributed at the spindle poles, point to
defective kinetochore-microtubule interaction. According to
the model of metaphase checkpoint control that predicts the
block of chromosome segregation in the presence of unattached kinetochores (McIntosh, 1991; Gorbsky and Ricketts,
1993; Rieder et al., 1994; Gorbsky, 1995), a delay in the onset
of anaphase of the maternal chromosomes is to be expected.
The high background of the yolk region prevented us from
following the dynamics of the parental complements in vivo,
Table 1. Classification of the figures scored
Total
figures
Pronuclear
migration
(% )
Pronuclear
apposition
(%)
Prophase
(%)
Metaphase
(%)
Anaphase
A
(%)
Anaphase
B
(%)
Telophase
(%)
Other
(%)
Irregular
(%)
8
(0.9)
(a)
927
22
(2.4)
63
(6.8)
207
(22.3)
224
(24.2)
67
(7.2)
105
(11.3)
148
(15.9)
83
(8.9)
(b)
621
12
(1.9)
51
(8.3)
122
(19.6)
135
(21.8)
52
(8.3)
78
(12.6)
107
(17.2)
64
(10.3)
Eggs were obtained from incompatible crosses between DSR males and DSW females (a) and from compatible crosses between DSW males and DSW females (b).
Figures scored were defined as follows. Pronuclear migration: approach of male and female pronuclei; Pronuclear apposition: male and female pronuclei
juxtapposed; Prophase: chromatin condensation; Metaphase: parental complements in the equatorial plane of two distinct half spindles (in embryos from
incompatible crosses the condensation of the male chromatin is delayed); Anaphase A: beginning of sister chromatid separation (in embryos from incompatible
crosses the paternal complement lags in the midzone of the mitotic apparatus); Anaphase B: sister chromatids near the spindle poles (in embryos from
incompatible crosses paternal chromosomes segregate abnormally); Telophase: formation of daughter nuclei; Other: various developmental stages after the first
mitosis; Irregular: abnormal chromatin configurations before exit from mitosis.
Delay of paternal chromatin condensation
so we could not monitor the exact time of anaphase onset in
normally developing and incompatible zygotes. However,
since we found anaphase and telophase figures with the same
frequency in zygotes obtained from compatible and incompatible crosses, collected at short time intervals from rapidly
laying females (Table 1), we suspect that the metaphaseanaphase transition of the female complement is not affected
by maloriented chromosomes during the first mitosis.
The condition in which female chromosomes overcome
metaphase and enter anaphase despite the fact that the paternal
set lags behind on the metaphase plate, phenocopies the case
of the sea urchin zygote in which the fusion of the parental
complements is prevented by colchicine treatment. In this
condition, the presence of many unattached maternal chromosomes did not affect the timing of onset of anaphase of the
paternal chromosomes (Sluder et al., 1994). Sluder and coworkers concluded that the metaphase checkpoint in sea urchin
embryos does not detect maloriented chromosomes if some
chromosomes are attached to the spindle in a normal fashion.
Although our results also point to the absence of a feedback
control mechanism monitoring chromosome assembly during
the metaphase/anaphase transition, we must keep in mind that
the first mitosis is not exactly the same in sea urchin and
Drosophila zygotes. In the sea urchin, the parental complements congress at the metaphase plate where they mingle; in
Drosophila maternal and paternal sets congress at the
metaphase plane, but enter anaphase as two separate groups,
mingling only during telophase. The condition in which the
improper organization of half chromosomes does not affect the
onset of anaphase of the other chromosome complement seems
to be exclusive to the first mitosis of the Drosophila embryo.
During the subsequent syncytial mitoses, one abnormally long
chromosome can delay the onset of anaphase of the whole
chromosome set (Sullivan et al., 1993). Likewise, defects in the
aar gene product implicated in the mechanism ensuring correct
interaction between spindle microtubules and kinetochores,
have been found to result in delay of the metaphase-anaphase
transition during the syncytial mitoses (Gomes et al., 1993).
These observations suggest that exit from mitosis is driven differently in early and syncytial Drosophila embryos. This discrepancy is presumably due to spindle architecture and/or to
the relative time interval needed for anaphase preparation. The
spindle of the Drosophila zygote consists from prophase to
ananphase of twin bundles of microtubules converging toward
common poles. Half spindles seem to be acting independently
since they hold differently condensed parental complements.
In zygotes from incompatible crosses, the misaligned paternal
chromosomes are delayed at the metaphase plate of one half
spindle, whereas the maternal chromatids enter anaphase
correctly in the other half spindle. This suggests that each half
spindle independently regulates the exit from mitosis of its own
chromosome set. This regulatory process is presumably insensitive to the presence of misaligned chromosomes. During
normal development, the synchrony of the parental complements at the onset of anaphase and the fidelity of chromosome
segregation may be ensured by the phase that leads to congress
of the parental complements at the equatorial plane of the
spindle. Preparation for anaphase presumably lasts long
enough to allow proper chromosome organization in both half
spindles. This process is less subject to error because of the
small number of chromosomes in the separate half spindles.
279
We are indebted to Rosanna Giordano for providing us with the
Drosophila simulans stocks examined in this paper. We are grateful
to an anonymous reviewer for comments on the manuscript. This work
was supported in part by grants from Murst (40% and 60%).
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(Received 18 September 1996 – Accepted 1 November 1996)