Wolbachia-induced delay of paternal chromatin condensation ... · Cytoplasmic incompatibility is an unusual form of intrapop-ulation sterility that is associated in insects with inherited

271Journal of Cell Science 110, 271-280 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS3473

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

The behavior of parental chromosomes during the firstmitosis of Drosophila simulans zygotes obtained from uni-directional incompatible crosses is described and it isdemonstrated that the condensation of parental chromatincomplements was asynchronous. The timing of paternalchromatin condensation appeared to be delayed in theseembryos, so that condensed maternal chromosomes andentangled prophase-like paternal fibers congressed in theequatorial plane of the first metaphase spindle. Atanaphase the maternal chromosomes migrated to oppositepoles of the spindle, whereas the paternal chromatin laggedin the midzone of the spindle. This resulted in dramaticerrors in paternal chromatin inheritance leading to theformation of embryos with aneuploid or haploid nuclei.These observations suggest that the anaphase onset of

maternal chromosomes is unaffected by the improperalignment of the paternal complement. Since the firstmetaphase spindle of the Drosophila zygote consists of twinbundles of microtubules each holding one parental com-plement, we suspect that each half spindle regulates thetiming of anaphase onset of its own chromosome set. Innormal developing embryos, the fidelity of chromosometransmission is presumably ensured by the relative timingrequired to prepare parental complements for the orderlysegregation that occurs during the metaphase-anaphasetransition.

Key words: Drosophila simulans, Cytoplasmic incompatibility,Fertilization, Parental chromatin condensation

SUMMARY

INTRODUCTION

During the first mitotic division of the Drosophila zygote thematernal and paternal chromosomes congress at the metaphaseplate of the spindle, but instead of mingling at the equatorialplane, as in most animal cells, they initiate anaphase as twowidely separated sets, and mingle during telophase (Huettner,1924; Sonnenblick, 1950; Callaini and Riparbelli, 1996).Parental chromosomes enter anaphase at the same time, despitethe fact that maternal chromatin condenses first. The highfidelity of chromosome transmission during the followinganaphase raises the question of how the timing of themetaphase/anaphase transition is controlled during the firstmitosis in fly embryos. Somatic cells ensure the fidelity ofchromosome transmission to their daughters by extrinsiccontrol mechanisms (cell-cycle checkpoints) that allow theinitiation of an event only when the previous event is success-fully completed (Hartwell and Weinert, 1989; Murray, 1994).Errors in DNA replication and improper chromosomealignment generate signals triggering specific mechanisms thatdelay progression through the cell cycle. Most eukaryotic cellswith damaged or unreplicated DNA are kept from enteringmitosis until DNA damage is repaired (Murray, 1992; Murrayand Hunt, 1993; Kaufmann and Paules, 1996) and the onset of

anaphase is delayed in vertebrate cells once kinetochores arenot under bipolar tension or unattached (reviewed by Wells,1996). Abnormal inheritance of the genetic material occurswhen 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 treatmentinduces checkpoint override and exit from mitosis (Andreassenand Margolis, 1991; Larsen and Wolniak, 1993; Nicklas et al.,1993). The rigid cell-cycle checkpoints that ensure propertransmission of genetic material in somatic cells appear lessaccurate in some embryonic cells. Inhibitors of DNA andprotein synthesis do not affect some aspects of cell division(Kimelman et al., 1987) and centrosome duplication (Gard etal., 1990) in Xenopus eggs. Centrosome replication may alsobe uncoupled from DNA synthesis in echinoderm embryostreated with aphidicolin (Nagano et al., 1981; Sluder andLewis, 1987). The presence of unattached maternal chromo-somes has no effect on the timing of anaphase onset of thepaternal complement in sea urchin zygotes (Sluder et al.,1994). Since it seems likely that embryonic systems wouldincur developmental alterations due to chromosome loss andaneuploidy, they might have evolved compensatory mecha-nisms 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 thenuclei that failed to complete chromosome segregation(Minden et al., 1989; Sullivan et al., 1990, 1993). However,this mechanism working in late syncytial embryos does notexplain the fidelity of chromosome transmission observedduring the first mitotic division of the zygotic nucleus. In theearly Drosophila embryo poor cell-cycle checkpoint controlsare to be expected since in aphidicolin treated embryos cen-trosome replication continues (Raff and Glover, 1988) and gnu,plu, and png mutants replicate their DNA and centrosomeswithout undergoing nuclear divisions (Freeman et al., 1986;Shamansky and Orr-Weaver, 1991). While studying cytoplas-mic incompatibility in Drosophila simulans, we found thatduring the first mitosis of zygotes obtained from incompatiblecrosses, the paternal chromosomes failed to condense properlyand lagged behind on the metaphase plate, whereas thematernal set entered anaphase. A model such as this, in whichthe anaphase onset of maternal chromosomes is not affected bythe presence of maloriented paternal chromosomes, offers theunique opportunity of clarifying whether the Drosophilazygote has feedback controls for the metaphase-anaphase tran-sition based on chromosome misorientation.

Cytoplasmic incompatibility is an unusual form of intrapop-ulation sterility that is associated in insects with inheritedbacteria of the genus Wolbachia, and is commonly found wheninfected males are crossed with uninfected females (reviewedby Rousset and Raymond, 1991; Breeuwer and Werren, 1990;Boyle et al., 1993; Giordano et al., 1995). In incompatiblecrosses of Drosophila simulans, fertilized eggs fail to hatch andless than 5% of embryos are viable (Hoffmann et al., 1986).Defective early fertilization events have been associated withthe high rate of embryo mortality in intraspecific crosses ofDrosophila simulans (O’Neill and Karr, 1990; Lassy and Karr,1996), but the cytological basis of this phenomenon is still notunderstood. We therefore conducted a detailed study todetermine what happens during the fertilization of eggs derivedfrom incompatible crosses in Drosophila simulans.

MATERIALS AND METHODS

StocksThe Watsonville and Riverside strains of Drosophila simulans (abbre-viated as DSW and DSR, respectively) used in this study were kindlyprovided by Rosanna Giordano (University of Urbana, Illinois). Thecrosses were performed by taking newly hatched DSW females andDSR males and leaving them on standard cornmeal, agar and yeastmedium in 100 ml plastic containers. Eggs from 5- to 6-day-old flieswere collected three times at 24°C on small agar plates for 20 minuteseach. Repeated egg collection was needed to avoid problems causedby the fact that females retain fertilized oocytes for different periodsof time. After discarding the first eggs, fertilized eggs were againcollected three times for 20 minutes. Three sets of collections wereperformed from two groups of rapidly laying females.

Fluorescence microscopyEggs were dechorionated in a 50% bleach solution for 2-3 minutes andwashed in distilled water. They were dried on filter paper and thevitelline envelope was removed by transfer to a vial containing 3 ml n-heptane and 3 ml of cold 90% methanol solution in water. After shakingfor 3 minutes the embryos without vitelline envelope were transferredto 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 serumalbumin (BSA). The eggs were then incubated overnight at 4°C with amonoclonal antibody against β-tubulin (Boehringer Mannheim;dilution 1:200 in PBS/BSA). The eggs were rinsed in PBS/BSA andthen incubated for one hour in goat anti-mouse-conjugated IgG coupledto fluorescein (Cappel, West Chester, PA; dilution 1:600 in PBS/BSA).After rinsing in PBS the nuclei were stained with 1 µg/ml Hoechst33258 for 3 minutes. The eggs were rinsed again in PBS and mountedin 90% glycerol containing 2.5% n-propyl gallate (Giloh and Sedat,1982). Fluorescence observations were carried out with a Leitz Aristo-plan microscope equipped with fluorescein and UV filters. Micrographswere taken with Kodak Tri-X 400 pro film and developed in KodakHC110 developer for 7 minutes at 20°C.

Fuchsin stainingThe 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) in2.5% acetic acid for 20-30 minutes. The embryos were then destainedin 5% acetic acid and mounted in glycerol.

ControlsThe process of fertilization was also examined in embryos obtainedfrom three further crosses of Drosophila simulans: (a) uninfectedembryos obtained from crosses between naturally uninfected flies(DSW strain); (b) infected embryos obtained from crosses betweennaturally infected flies (DSR strain); (c) uninfected embryos obtainedfrom crosses between naturally uninfected females (DSW) and tetra-cycline-treated DSR males. These males were obtained by culturingthe DSR stock for two generations on standard Drosophila mediumsupplemented with 0.05% tetracycline as described by Hoffmann etal. (1986).

The embryos of all these crosses were viable and the first cleavagemitosis proceeded as described in Drosophila melanogaster (Callainiand Riparbelli, 1996). However, we only show pictures of embryosobtained from crosses between naturally uninfected flies (DSWstrain).

RESULTS

In crosses of Drosophila simulans between males harboringbacteria of the genus Wolbachia (DSR) and uninfected females(DSW), incorporation of sperm by the oocyte was followed bythe formation of a prominent aster at the site of the sperm head(Fig. 1a,b), as in normal developing embryos. After the com-pletion of the female meiosis, the distance between the parentalpronuclei gradually decreased and the astral microtubules asso-ciated with the male nucleus radiated from two distinct foci.These observations suggest that in incompatible crosses thesperm centrosome retains its nucleating properties and repli-cates in a normal fashion. Once the pronuclei were side-by-side, their chromatin condensed (Fig. 1d) as in control embryos(Fig. 1e) and a bipolar array of microtubules organized at thejunction between the pronuclei (Fig. 1c). At prophase theparental pronuclei showed clearly asymmetric chromatin con-densation (Fig. 1f,g). This asymmetry was also observed incontrol embryos obtained from compatible crosses betweenmales and females of the DSW strain (Fig. 1h). However, incontrol embryos the synchrony of chromatin condensation wassoon regained and at the end of prophase two groups ofstretched chromosomes were found on either side of thespindle plane (Fig. 1k). In embryos obtained from incompati-ble crosses only one set of chromosomes was clearly visible at

273Delay of paternal chromatin condensation

Fig. 1. Microtubules and DNA in early Drosophila simulans embryos obtained from incompatible crosses between DSW females and DSRmales (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 meioticapparatus (arrow), containing two chromosome sets (arrowheads) and one irregular aster (small arrow), associated with the sperm head (smallarrowheads). (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 andin incompatible embryos, while the condensation of the male chromatin is delayed in incompatible embryos. f, female pronucleus; m, malepronucleus. Bars: a,b (15 µm); c-h (5 µm).

the end of prophase, the other set being entangled chromatinfibers (Fig. 1i,j).

At prometaphase the growth of the spindle microtubulesresulted in a mitotic apparatus with two separate parental com-plements that still showed different degrees of chromatin con-densation. One parental set consisted of highly condensedchromosomes, whereas the other was a tangle of prophase-likefibers (Fig. 2a,b). In control embryos, two distinct sets ofcondensed chromosomes were seen at this stage (Fig. 2c). Theasynchronous parental chromatin condensation persistedthrough metaphase (Fig. 2d,e), at the end of which the tangledchromatin fibers and the condensed chromosome set becameclosely apposed in the midzone of the spindle (Fig. 2g,h). In

control embryos two separate groups of coiled chromosomeswere closely apposed in the equatorial region of the first mitoticspindle at the beginning of metaphase (Fig. 2f) and fullycondensed chromosomes were found in the midzone of thespindle at the end of metaphase (Fig. 2i).

At the onset of anaphase, the sister chromatids of oneparental set left the metaphase plate, whereas the other parentalset lagged behind in the equatorial region of the spindle (Fig.2j,k). At the end of anaphase the astral microtubules grewfurther (Fig. 3a) and half of the chromatids reached theopposite poles of the spindle, whereas the other parental setstill lagged in the midzone of the spindle (Fig. 3b) or shiftedslightly toward one pole (Fig. 3c). The lagging tangled


chromatin mass condensed further during the anaphase pro-gression and distinct chromosomes were often visible at theend of anaphase in suitable preparations. In control embryos,both the parental sets of chromatids moved synchronously tothe poles of the spindle (Figs 2l, 3d).

Telophase spindles showed prominent midbodies and largeasters in control and incompatible crosses (Fig. 3e). In crossesbetween DSW females and DSR males, two classes of abnormaltelophase figures were observed: the delayed parental chromatincomplement appeared to be stretched between the two poles(Fig. 3f) or split in two portions that left the midzone of thespindle and moved toward opposite poles (Fig. 3g). In embryosobtained by crossing females and males belonging to the DSWstrain the chromosomes decondensed and mingled at telophase,and two opposite zygotic nuclei formed (Fig. 3h). In embryosobtained from incompatible crosses two nuclei of unequal sizecould be observed at each spindle pole at the end of telophase.

Fig. 2. Spindle and chromatin morphology in embryos from crosses of Dpanel show embryos from crosses between DSW females and DSR maleobtained by DSW females and DSW males. (a,b,c) Prometaphase. (d,e,f)the mitotic spindle. (g,h,i) End of metaphase; the parental complements the parental complements (arrowheads) move as distinct entities to oppoincompatible crosses. Bar, 5 µm.

Parental chromosomes in incompatible crosses thereforeentered the second mitotic division without mingling.

To verify the possibility that the first mitotic apparatus of theDrosophila zygote was composed of distinct half spindles inde-pendently acting, we gently squashed newly laid eggs under glasscoverslips. Under pressure the mitotic apparatus flattened and thehalf spindles became easily visible. From prometaphase toanaphase (Fig. 4a-f) the half spindles held parental complementswith different degree of chromatin condensation. Each comple-ment was also closely associated with its own half spindle whenthe mitotic apparatus was damaged by squashing (Fig. 4e,f). Wehave also found during metaphase-anaphase transition of the firstmitosis an asymmetric organization of the spindle microtubules(Fig. 4g,h). Microtubules corresponding to the female chromo-somes were longer and continuous while microtubules corre-sponding to the lagging male chromatin were shorter and had agap in the equatorial plane of the spindle. This suggests that two

rosophila simulans. Microtubules and upper pictures of each DNAs; lower pictures of each DNA panel are from control embryos Metaphase; note the twin microtubule bundles (open arrows) forming

are close together. (j,k,l) Anaphase A; in normally developing embryossite spindle poles. Arrows indicate paternal chromatin in embryos from


Fig. 3. Microtubules and DNA in embryos from crosses of Drosophila simulans. Pictures in the right column are from compatible crosses, theothers are from incompatible crosses. (a,b,c,d) Anaphase B; note the tangled chromatin mass lagging in the midzone of the spindle or shiftedtoward one pole (arrows). (e,f,g,h) Late telophase; mitotic figures are characterized by chromatin bridges (arrow) or asymmetrically distributedchromatin complements (arrowhead). Bar, 5 µm.

groups of spindle microtubules were independently interactingwith male and female complements during the first mitosis of theDrosophila embryo.

The abnormal position of half complements presumablyaffected the regular progression of the second mitosis and ledto the formation of aberrant figures. At higher frequency weobserved twin opposite spindles holding haploid complementsthat were joined by microtubule bridges enveloping stretchedchromosomes (Fig. 5a,d). In some embryos we also found twoseparate figures (Fig. 5b,c,e,f): one with a microtubular shellthat did not differentiate distinct spindle poles and surroundeda haploid complement close to an irregular chromatin mass; theother was a normal-looking mitotic spindle. This spindle helda haploid complement of chromosomes (Fig. 5e), butsometimes one or more supernumerary chromosomes laggedin its midzone (Fig. 5f). Indirect immunofluorescence ofhaploid embryos indicated that spindle configuration and cen-trosomal cycle were normal (Fig. 6a), whereas aneuploid

nuclei were associated with broad spindles that lost their cen-trosomes at an early stage (Callaini et al., 1996). Haploidembryos survived longer and typically died shortly beforehatching, whereas the development of embryos with aneuploidnuclei arrested after a few intravitelline mitoses.

It was not possible to determine from this data whethermaternal or paternal chromosomes lagged on the equatorial planeof the spindle and were lost in early embryos obtained fromincompatible crosses. However, indirect observations providedinsight into the parental origin of the tangled chromatin masslagging in the midzone of the spindle during first anaphase. Bothchromosome sets that left the equatorial plane of the spindle atthe onset of the first anaphase showed a stick-like chromatid witha subterminal fluorescent dot. According to Gatti et al. (1976) thisstaining pattern characterizes the X chromosome of Drosophilasimulans. Since we found this staining pattern in all the anaphasefigures with distinguishable chromosomes scored during the firstmitosis (Fig. 6b; n=79), in all the haploid complements that pro-


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. Theflattening 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). Thefirst anaphase spindle (g,h) is formed by continuous microtubules (arrowhead) corresponding to the female chromosomes (f), and by shortermicrotubules (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), andduring later mitoses of haploid syncytial embryos (Fig. 6d; n=87),we suspect that only the female chromatin complement wasproperly condensed at the time of fertilization. This was alsoconfirmed by staining the embryos with fuchsin. All 113 haploidembryos scored at different times of development (25 during theintravitelline mitoses, Fig. 6e; 47 during the syncytial blastodermstage, Fig. 6f; 41 during gastrulation, Fig. 6g) had only thematernal complement.

DISCUSSION

Wolbachia induces delay of paternal chromatincondensationAfter incorporation of the sperm into the egg, maternal andpaternal complements meet and intermix. The coordination of

these events was severely impaired in embryos obtained fromcytoplasmically incompatible crosses between males ofDrosophila simulans harboring bacteria of the genusWolbachia and uninfected females. Our observations showedthat chromatin condensation of the parental sets was extremelyasynchronous. During the first mitosis, one set of fullycondensed chromosomes congressed in the equatorial plane ofthe spindle together with a set of chromatin fibers in prophase-like configuration. During anaphase the sister chromatids ofone parental complement moved to opposite spindle poles,whereas the other parental complement lagged in the midzoneof the spindle. This irregular process led to abnormal embryoswith haploid or aneuploid nuclear complements.

In looking for sex chromosomes in surviving haploidembryos (n=113), we never found the Y chromosome, whereasall suitable anaphase figures scored during the first mitosis(n=79) showed a sister chromatid with a brightly fluorescent


Fig. 5. Microtubule (upperpanels) and DNA (lowerpanels) configurations duringthe second mitosis of embryosfrom incompatible crosses.Arrows and arrowheadsindicate maternal and paternalcomplements, respectively.Bar, 5 µm.

subterminal dot. According to Gatti et al. (1976) this stainingpattern characterizes the X chromosome of Drosophilasimulans. Chromatids with this staining pattern were alsofound in haploid complements that progressed through thesecond and further mitoses. These findings together indicatethat the paternal set of chromosomes is lost in incompatiblecrosses. This observation extends previous reports on Nasoniavitripennis (Ryan and Saul, 1968), Culex pipiens (Jost, 1970)and Drosophila simulans (O’Neill and Karr, 1990) whichsuggested that cytoplasmic incompatibility bacteria preventsyngamy in incompatible crosses. Preliminary cytologicalexaminations in Drosophila simulans indicate that the highembryonic mortality is a consequence of defects which occuras early as the first cleavage division (O’Neill and Karr, 1990).This suggestion is supported by the finding that Wolbachia hasbeen shown to disrupt the first mitosis in the wasp Nasonia byimpairing paternal chromatin condensation in crosses betweencytoplasmically incompatible strains (Ryan and Saul, 1968;Breeuwer and Werren, 1990; Reed and Werren, 1995). HowWolbachia infection selectively delays condensation ofpaternal chromatin is an intriguing question. It is widelyaccepted that the condensation of DNA involves structuralrearrangement of chromatin and scaffold proteins, but little is

known about the molecular mechanism that regulates thisprocess. There is evidence that chromatin packaging in eukary-otic cells is accompanied by phosphorylation of histones H1and H3 (reviewed by Reeves, 1992) and that topoisomerase IIis required for proper mitotic chromosome condensation(reviewed by Ernshaw and MacKay, 1994). Histone H1 doesnot seem primarly involved in chromatin condensation in theearliest phases of Drosophila development, since H1 accumu-lates in the embryo from nuclear cycle 7. However, the HMG-D protein is associated with condensed chromosomes in theabsence of the histone H1 suggesting that it might perform afunction similar to that of histone H1 (Ner and Travers, 1994).Topoisomerase II is clearly detectable on condensing chromo-somes of the early Drosophila embryo (Swedlow et al., 1993)and injection of anti-topo II antibodies impairs chromosomecondensation in Drosophila syncytial embryos (Buchenau etal., 1993). Since Wolbachia has never been detected in fertil-ized oocytes from incompatible crosses, we suppose that aWolbachia-related factor is associated with the male chromatinduring spermatogenesis. This is supported by the observationthat the penetrance of the incompatible phenotype seems dosedependent, because aged males having a lower concentrationof Wolbachia in their germinal tissues (Bressac and Rousset,


Fig. 6. Crosses between DSW females and DSR males. (a) Spindlesin 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); arrowsindicate the bright dot on the X chromosome. Details ofchromosomes found in embryos stained with fuchsin duringmetaphase of the fifth nuclear division (e), anaphase of the tenthmitosis (f), metaphase of the first postblastodermic mitosis (g). Notethat the Y chromosome is absent in haploid complements; arrowsindicate the X chromosome. The fourth dot-like chromosome is notvisible 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 ofthis kind may impair the function of the HMG-D protein,

Table 1. ClassificPronuclear Pronuclear

Total migration apposition Prophase Metaphasfigures (% ) (%) (%) (%)

(a) 927 22 63 207 224(2.4) (6.8) (22.3) (24.2)

(b) 621 12 51 122 135(1.9) (8.3) (19.6) (21.8)

Eggs were obtained from incompatible crosses between DSR males and DSW femFigures scored were defined as follows. Pronuclear migration: approach of mal

juxtapposed; Prophase: chromatin condensation; Metaphase: parental complemenincompatible crosses the condensation of the male chromatin is delayed); Anaphacrosses the paternal complement lags in the midzone of the mitotic apparatus); Aincompatible crosses paternal chromosomes segregate abnormally); Telophase: fomitosis; Irregular: abnormal chromatin configurations before exit from mitosis.

topoisomerase II, or other unknown chromosomal scaffoldproteins, delaying chromatin compaction. This workinghypothesis may explain why crosses between infected malesand females produce viable embryos. The compaction ofmaternal chromatin is presumably also delayed in theseembryos, so that the synchrony of chromosome condensationis restored. The fact that infected eggs develop into viableembryos after fertilization with uninfected sperm is a problem.Presumably the male chromatin recruits the Wolbachia-derivedfactor from the oocyte cytoplasm during replication of DNA.Maternal and paternal chromatin condensation are thereforecoupled and the first mitotic division takes place successfully.

Does a metaphase-checkpoint exist in theDrosophila zygote?Our results show that in zygotes obtained by crossing DSRmales and DSW females the metaphase spindle assembled butpaternal chromosomes condensed improperly and weredelayed at the metaphase plate, while female chromatidsattained anaphase and migrated to the opposite poles of thespindle. This delay in anaphase initiation leads to dramaticerrors in paternal chromatin inheritance. Whether paternalchromosome segregation defects arise as a consequence ofimproper chromatin condensation or defective structural organ-ization of the kinetochore regions remains to be determined. Ithas been shown that kinetochore alignment, not chromosomecondensation, is essential to overcome metaphase block andinitiate anaphase. The chromatin of mammalian cells treatedwith topoisomerase II inhibitors fails to condense properly, butthe spindle assembly checkpoint is passed as soon as the kin-etochores align at the metaphase plate (Clarke et al., 1993;Gorbsky, 1994). We are unable to directly assess whether allpaternal kinetochores captured spindle microtubules whenfemale chromatids entered anaphase, but the irregulartelophase figures, in which paternal chromatin was eitherstretched or unequally distributed at the spindle poles, point todefective kinetochore-microtubule interaction. According tothe model of metaphase checkpoint control that predicts theblock of chromosome segregation in the presence of unat-tached kinetochores (McIntosh, 1991; Gorbsky and Ricketts,1993; Rieder et al., 1994; Gorbsky, 1995), a delay in the onsetof anaphase of the maternal chromosomes is to be expected.The high background of the yolk region prevented us fromfollowing the dynamics of the parental complements in vivo,

ation of the figures scoredAnaphase Anaphase

e A B Telophase Other Irregular(%) (%) (%) (%) (%)

67 105 148 83 8(7.2) (11.3) (15.9) (8.9) (0.9)

52 78 107 64(8.3) (12.6) (17.2) (10.3)

ales (a) and from compatible crosses between DSW males and DSW females (b).e and female pronuclei; Pronuclear apposition: male and female pronucleits in the equatorial plane of two distinct half spindles (in embryos fromse A: beginning of sister chromatid separation (in embryos from incompatible

naphase B: sister chromatids near the spindle poles (in embryos fromrmation of daughter nuclei; Other: various developmental stages after the first


so we could not monitor the exact time of anaphase onset innormally developing and incompatible zygotes. However,since we found anaphase and telophase figures with the samefrequency in zygotes obtained from compatible and incompat-ible crosses, collected at short time intervals from rapidlylaying females (Table 1), we suspect that the metaphase-anaphase transition of the female complement is not affectedby maloriented chromosomes during the first mitosis.

The condition in which female chromosomes overcomemetaphase and enter anaphase despite the fact that the paternalset lags behind on the metaphase plate, phenocopies the caseof the sea urchin zygote in which the fusion of the parentalcomplements is prevented by colchicine treatment. In thiscondition, the presence of many unattached maternal chromo-somes did not affect the timing of onset of anaphase of thepaternal chromosomes (Sluder et al., 1994). Sluder and co-workers concluded that the metaphase checkpoint in sea urchinembryos does not detect maloriented chromosomes if somechromosomes are attached to the spindle in a normal fashion.Although our results also point to the absence of a feedbackcontrol mechanism monitoring chromosome assembly duringthe metaphase/anaphase transition, we must keep in mind thatthe first mitosis is not exactly the same in sea urchin andDrosophila zygotes. In the sea urchin, the parental comple-ments congress at the metaphase plate where they mingle; inDrosophila maternal and paternal sets congress at themetaphase plane, but enter anaphase as two separate groups,mingling only during telophase. The condition in which theimproper organization of half chromosomes does not affect theonset of anaphase of the other chromosome complement seemsto be exclusive to the first mitosis of the Drosophila embryo.During the subsequent syncytial mitoses, one abnormally longchromosome can delay the onset of anaphase of the wholechromosome set (Sullivan et al., 1993). Likewise, defects in theaar gene product implicated in the mechanism ensuring correctinteraction between spindle microtubules and kinetochores,have been found to result in delay of the metaphase-anaphasetransition during the syncytial mitoses (Gomes et al., 1993).These observations suggest that exit from mitosis is driven dif-ferently in early and syncytial Drosophila embryos. This dis-crepancy is presumably due to spindle architecture and/or tothe relative time interval needed for anaphase preparation. Thespindle of the Drosophila zygote consists from prophase toananphase of twin bundles of microtubules converging towardcommon poles. Half spindles seem to be acting independentlysince they hold differently condensed parental complements.In zygotes from incompatible crosses, the misaligned paternalchromosomes are delayed at the metaphase plate of one halfspindle, whereas the maternal chromatids enter anaphasecorrectly in the other half spindle. This suggests that each halfspindle independently regulates the exit from mitosis of its ownchromosome set. This regulatory process is presumably insen-sitive to the presence of misaligned chromosomes. Duringnormal development, the synchrony of the parental comple-ments at the onset of anaphase and the fidelity of chromosomesegregation may be ensured by the phase that leads to congressof the parental complements at the equatorial plane of thespindle. Preparation for anaphase presumably lasts longenough to allow proper chromosome organization in both halfspindles. This process is less subject to error because of thesmall number of chromosomes in the separate half spindles.

We are indebted to Rosanna Giordano for providing us with theDrosophila simulans stocks examined in this paper. We are gratefulto an anonymous reviewer for comments on the manuscript. This workwas supported in part by grants from Murst (40% and 60%).

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(Received 18 September 1996 – Accepted 1 November 1996)

Wolbachia-induced delay of paternal chromatin condensation ... · Cytoplasmic incompatibility is an unusual form of intrapop-ulation sterility that is associated in insects with inherited

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