polo, a mitotic mutant of Drosophila displaying abnormal spindle poles

CLAUDIO E. SUNKEL and DAVID M. GLOVER*

Cancer Research Campaign, liukaryolic Molecular Genetics Research Gmup, Department of Biochemistry,Imperial College of Science and Technology, London SW7 2AZ, UK

* Author for correspondence

Summary

Neuroblast cells in larvae homozygous for mu-tant alleles of the locus polo show a high fre-quency of metaphases in which the chromosomeshave a circular arrangement, and anaphase fig-ures in which chromosomes appear to be ran-domly oriented with respect to at least one of thespindle poles. These defects appear to lead to theproduction of polyploid cells. Sex chromosomedisjunction is affected in male meiosis, primarilyin the second division, and the meiotic spindles ofliving cells are abnormal. One allele is a larval

lethal, whereas another is semi-lethal with about7 % of homozygotes surviving as adults. Embryosfrom homozygous polo females have aberrantmitotic spindles that are highly branched andhave broad poles. Immunofluorescence studieswith an antibody that recognizes an antigen as-sociated with the centrosome indicate that theorganization of this organelle is disrupted in themutant embryos.

Key words: Dmsophila, mitotic mutant, spindle poles.

Introduction

Genetic approaches have been highly successful inanalysing the cell cycle in yeasts, leading to a detailedpathway of the events of mitosis (see, e.g., Hartwell etal. 1974; Pringle & Hartwell, 1981; Beach et al. 1982;Nurse, 1985). However, aspects of the mechanics ofmitosis differ in yeasts from those in the multicellulareukaryotes, and furthermore there are additional con-straints upon cell division in higher organisms becauseof the need to maintain temporal and spatial control ofcell proliferation.

Drosophila offers considerable advantages as an or-ganism in which to study cell division. Work by Bakerand his colleagues has shown that certain loci originallyidentified by meiotic mutants, and mutants showingincreased mutagen sensitivity, encode products thatfunction in mitosis (Baker et al. 1978; Baker & Smith,1979). Additional mitotic mutants have been identifiedby their effects on chromosome integrity and fidelity ofmitotic chromosome transmission (Baker et al. 1982;Smith et al. 1985). There are two major stages ofDrosophila development at which one can expect todetect mutations affecting mitotic cell division. Thefirst is during early embryogenesis, when there are 13

Journal of Cell Science 89, 25-38 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

nuclear divisions at about 10-min intervals beforeindividual nuclei undergo cellularization (Zalokar &Erk, 1976). During this time there is little or no zygoticgene expression, and so one might therefore expect aclass of maternal effect lethal mutations that disrupt thefunctions of the genes encoding these components.There are then only three to four more cell divisionsduring embryogenesis and most of larval developmentinvolves cell growth, with the endoreduplication ofDNA in the absence of mitosis. Imaginal cells, des-tined to form the adult organism and not themselvesnecessary for the survival of the larva, divide through-out larval development, as do cells of the nervoussystem. A second class of mitotic mutations can there-fore be recognized in which the imaginal cells do notproliferate within the larva and death ensues during thelate larval or early pupal stages. This late larval lethalphenotype of mutations in essential mitotic functionswas first recognized in the analysis of repair-defectivemutants (Baker et al. 1982).

We have begun to screen the embryos of flies havingmaternal effect mutations in order to identify aberrantmitoses during early embryogenesis. We expect thatsuch mutations might fall into distinct categories,corresponding to: genes expressed only during

25

oogenesis to provide a product specific for the cleavageembryo; and genes that are expressed not only atoogenesis but are also required for diploid cell prolifer-ation in the imaginal and neuroblast cells of the larvae.The mutation gnu appears to be an example of theformer category. This mutation results in uncontrolledDNA replication, leading to the formation of giantnuclei, whilst the division cycle of the centrosomesappears not to be affected (Freeman et al. 1986). Thisphenotype also develops in unfertilized eggs, indicatingthat the gene plays a role in the correct establishment ofzygotic development (Freeman & Glover, 1987). Inthis paper we describe a mutation, polo1, the wild-typefunction of which is required both in the early embryoand in the diploid cells of the larva. Using immunoflu-orescence techniques we find that the mitotic spindlesin polo embryos (from homozygous polo1 females) arehighly branched and have defective poles. Homo-zygous larvae also exhibit aberrant mitotic divisions intheir brain cells and in addition we have observedabnormal meiosis in homozygous males. A strongerallele, polo2, causes the death of homozygous larvae.

Materials and methods

Genetic variantsThe mutation polo] was isolated by Nusslein-Volhard in agenetic screen designed to select sterile females. The chromo-some used was ru st e ca mutagenized with ethylmethylsul-phonatc (EMS), polo1 is a recessive mutation that maps to theleft arm of chromosome 3 near the centromere at 3—46 on thebasis of 112 recombinants between Lyra (3-40-5) andStubble (58-2). The original polo1 mutation is recessive andsome 7 % of expected homozygotes survive to eclose.

The allele, polo1, was found among a collection of P-clement-induced lethals isolated by R. Karess. All otherstocks were decribed by Lindsley & Grell (1968). All stockswere grown at 25 °C under standard culture conditions andmedia.

Neuroblast preparalionsCytological preparations were made from late third-instarlarvae that had been grown at 25 °C, collected and thenwashed in saline. They were dissected in 0 7 % NaCl and thebrain was transferred to a drop of 45 % acetic acid for 15 s.The tissue was then placed for a further 15 s in 3 % orceindissolved in 45 % acetic acid and then cleared of excess stainin 60 % acetic acid. The brain was then placed in a small dropof 3 % orcein in 60 % acetic acid and squashed firmly.Preparations were observed under phase-contrast optics.Quantification of mitotic figures was carried out essentially asdescribed by Gonzalez (1986). The unit used was the area ofa brain-squash that can be seen under the microscope with a63X Zeiss objective and 10X eyepieces. The number ofmetaphases and the number of anaphases per field wasdetermined for at least 100 fields from 15 brains forpolo]/polol orpolo'/+.

Preparations were also made by incubating dissected brainsin 0-4% colchicine in 0'7% NaCl for l h before staining.After washing in saline the brains were incubated for 15 minin 30mM-sodium citrate and then stained and squashed asbefore.

Live testis squashes

The pharate adults or recently emerged adult males weredissected in 0 7 % NaCl on a siliconized slide. The wholetestis was placed in a large drop of 0-7 % NaCl and cut in halfacross the middle using dissecting needles. A small non-siliconized coverslip was placed on top and the wholepreparation observed under phase-contrast with a 20X objec-tive. The squash is performed by withdrawing excess liquidwith blotting paper by capillarity. Live squashes were photo-graphed within 10-20 min. Measurement of nuclear diam-eters for controls or polo[ homozygotes was done bymeasuring at least 100 nuclear diameters from photographicprints of 10 different preparations.

Fixation and staining of embryos

Embryos were fixed and stained exactly as described byFreeman et al. (1986). Taxol was used to stabilize micro-tubules exactly as described in that paper and by Karr &Alberts (1986). We are well aware of potential artefacts thatcan arise from the use of taxol and were discussed at length inthese two papers. In our hands the staining of wild-typeembryos under these conditions gives a pattern of tubulinstaining in agreement with the results of Karr & Alberts(1986).

Antibodies

The antibodies used in this study were the following: anti-centrosome antibody, Bx63 (Frash et al. 1987); anti-tubulinantibody, YLl/2 (Kilmartin et al. 1982); and anti-laminantibody, T47 (Dequin et al. 1984). Rhodamine- and fluor-escein-conjugated second antibodies were bought from Jack-son Immunoresearch Laboratories Inc. USA.

Results

The somatic phenotype

We describe the phenotypes of two mutant alleles of alocus, polo, which maps close to the centromere at 46on the third chromosome of Drosophila melanogaster.About 7% of flies homozygous for the original, EMS-induced allele eclose, and the eggs laid by homozygousfemales arrest early in development. We selected themutation for further study since these embryos showedan abnormal distribution of nuclei, leading us tosuspect a mitotic abnormality. The second, moreextreme, allele (polo2) is a P-element-induced recessivelarval lethal, which is, however, viable as a trans-heterozygote with polo1.

We have studied the effect of polo1 or polo2 onchromosome integrity and segregation in the diploidneuroblasts of third-instar larvae. Our analysis of thesecytological preparations shows that the frequency of

26 C. E. Sunkel and D. M. Glover

metaphases per field is lower in homozygous polo1

larvae than in wild-type larvae (see Materials andmethods and legend to Fig. 1). The frequency ofanaphases per field is also lower, but the relativeproportion of metaphases to anaphases is roughly thesame. About 70% of the metaphase figures showabnormalities (Fig. 1), which can be classified intothree classes. Two classes show circular arrangementsof the chromosomes. In one case there is a normalchromosomal complement (Fig. 1A). Homozygouspolo larvae have about half of their mitotic figures inthis configuration, compared to approximately 4% ofthe mitotic figures in wild-type larvae. In the secondclass, the nucleus is polyploid (Fig. 1B,C). Suchpolyploid figures are never seen in wild-type brains. Inboth circular configurations, the dot-like fourthchromosomes are seen in the centre of the mitoticfigure with the major autosomes and sex chromosomesbeing arranged around them. We are not able to saycategorically whether these figures are in metaphase orif they have proceeded to anaphase. We have, however,counted them as metaphases in our numerical analyses(see legend to Fig. 1). We do not observe circularmitotic figures, polyploid or otherwise, when thesquashes are made in the presence of colchicine,indicating the requirement for functional microtubulesfor their formation (data not shown). The third class,which is much less frequent, shows aneuploid figures.Fig. ID shows a cell that contains two extra chromo-somes, a Y and a fourth. We have seen no otherchromosomal defects; chromosomes are never frag-mented and they appear to undergo normal conden-sation.

About 90% of anaphase figures seen in polo1 /polo1

larval brains are clearly abnormal. In a typical example,shown in Fig. IE, the chromosomes attached to one ofthe spindle poles appear to be randomly oriented andare not all lying with their telomeres pointing towardsthe middle of the spindle. This arrangement is some-times observed in wild-type larvae but at a very lowfrequency. The majority of wild-type anaphases re-semble the one shown in Fig. IF, in which the chromo-some arms are all oriented towards the opposite spindlepole. Occasionally we have been able to observeanaphases that are trying to separate polyploid nuclei.

These cytological abnormalities are reflected in theviability of homozygous polo1 adults. The progeny thateclose do so on average 1-5 days later than theirheterozygous siblings. The majority die either as latethird instars or during pupariation. The polo alleleshows a more extreme phenotype. None of theexpected polo1 homozygotes survives, but they die aslarvae during the early stages of the third instar. Thecytological phenotypes of their larval neuroblasts aresimilar to polo[ homozygotes. The heterozygotes

polol/poloz do eclose, but with a slightly lower fre-quency compared to polo* homozygotes. However,these trans-heterozygotes show very abnormal cuticleformation of the abdomen affecting both tergites andsternites.

The meiotic phenotvpe

We were interested to know whether or not themutations also affected meiotic divisions and we choseto analyse this process in males homozygous for theoriginal allele, which in contrast to males homozygousfor polo2 are fertile. Not only the chromosomes, butalso mitochondria, segregate to daughter nuclei uponthe meiotic spindle in male germ cells. This associationof the mitochondria with the meiotic spindle makes itpossible to observe the spindle in living cells usingphase-contrast optics. Meiotic spindles from wild-typeand polo /'polox males are shown in Fig. 2. The wild-type cells undergoing meiotic division have verycharacteristic spindles with two clearly defined poles(Fig. 2A). However, the meiotic spindles found inpolo /polo testes are generally more irregular in shapeand structure. These range from almost normal totetrapolar spindles (Fig. 2B) or even multipolar ones(Fig. 2C).

A consequence of these abnormal spindle structureswould be irregular chromosome distribution amongstthe resulting gametes. Perhaps the easiest stage atwhich abnormal chromosomal segregation can be ana-lysed is within the cyst of spermatids referred to as the'onion stage'. Any differences in the DNA content ofthe post-meiotic products found at the onion stagewould be reflected by the nuclear diameters of thesecells (Ripoll et al. 1985; Gonzalez, personal communi-cation). In wild-type spermatids two round organellesare clearly visible at this stage (Fig. 3A). The clearorganelle is the post-meiotic nucleus, the diameter ofwhich is remarkably constant amongst differentgametes, reflecting their equal DNA content (Fig. 3B).Our measurements of the diameters of these nuclei arein good agreement with those of Gonzalez et al. (1987)and reflect a haploid DNA content. The dark organ-elle, the Nebenkern, consists of mitochondria that havebeen distributed between daughter cells on the meioticspindle, and which are associated with tubulin. Thiscomplex will later elongate to form the sperm tail.Before elongation starts this organelle is also veryregular in size and shape in the wild type (Fig. 3A). Inthe post-meiotic products of polo1 /polo1 testes, mostnuclei and associated Nebenkern appear abnormal(Fig. 3C). The histogram of nuclear diameters(Fig. 3D) indicates that there is a wide range of nuclearsizes. Gonzalez et al. (1987) have demonstrated thatthe nuclear diameter is a function of the DNA content.Comparison of the histogram in Fig. 3D withtheir standardized data indicates that the polo

Abnormal spindle poles in Drosophila 27

spermatids can contain from less than a quarter of thehaploid DNA complement up to as much as a tetra-ploid complement. Furthermore, whilst in wild-typepreparations there is always a single nucleus associatedwith one Nebenkern, there is no such correspondencein the mutant. The irregular size of the Nebenkern inpolo* homozygotes indicates unequal partitioning of

mitochondria into daughter cells on the meioticspindles.

We have analysed male meiosis further by testinghomozygote polo1 males for genetic non-disjunction ofthe sex chromosomes. This was carried out by crossingfemales carrying a Y chromosome and two A* chromo-somes sharing the same centromere (C(l)yf/Yand also

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28 C. E. Sunkel and D. M. Glover

referred to asXX,yf/Y) to males with marked A' and Ychromosomes (Xiv/YBS). Meiosis comprises two div-isions, during the first of which homologous chromo-somes are segregated to the spindle poles, the sisterchromatids not being split. In the second division, thesister chromatids are separated and the gametes receivea haploid chromosome complement. If therefore dis-junction occurs correctly, the viable progeny expectedfrom this cross would be tv males in which the A*chromosome is derived from the father and the V* fromthe mother (Xw/Y), and yfBs females, which have theattached A of the mother and the marked Y from thefather (XXyf/YBs). Non-disjunction in the maleparent during the first division results in gametescarrying both Xzv and YBS chromosomes (see thediagram of Table 1). Gametes carrying no sex chromo-somes can result from non-disjunction in the first orsecond divisions. The other products of non-disjunc-tion in the second division are gametes carrying twofree Xiv chromosomes or two free YBS chromosomes(Table 1). Thus non-disjunction in the first meioticdivision can be detected by the presence of w Bs males(Xiv/YBS/Y), and in the second division by w females(Xw/Xw/Y), whereasy/females (XXyf/O) are indica-tive of non-disjunction at either division.

In heterozygous polo[/+ males non-disjunction wasobserved at a frequency of about 007 %, a levelcomparable to that in wild type (Sandier et al. 1968).Males homozygous for polo produce two major classesof abnormal diplo-gametes (Table 1). The predomi-nance of XX gametes (1-7%) relative to AT gametes(0-23 %) suggests that most of the aberrant segregationis occurring in the second meiotic division. The low

Fig. 1. Mitotic figures in brain ncuroblasts of homozygous/>ofo' larvae. We scored 871 metaphase figures (using thecriteria described in Results) from the neuroblasts ofpolol/polox larvae of which 25-6% appeared 'normal'. Thiscontrasts with the proportion of normal figures in wild-typebrains for which we scored 962% in a sample of 1249.Homozygous polo' larvae had 47-6% of the metaphasefigures in circular configurations of the type shown in A.A significantly smaller proportion of wild-type cells (3'8%)showed similar figures. Circular polyploid figures (B,C)and aneuploid figures (D) were found in homozygous poloanimals at frequencies of 20-1 % and 6-9%, respectively,and not at all in wild types. The arrowheads in D markadditional V and fourth chromosomes in this aneuploidcell. Of 230 anaphase figures in homozygous polo1 larvae,14% were normal (F) compared with 89-6% in the 308wild-type anaphases scored. 'Abnormal' anaphases, like theone shown in E, were scored at a frequency of 86% in themutant and 10'4% in wild-type neuroblast squashes. Notethe random orientations of the chromosomes at the upperpole of the anaphase in E. Bar in A, 5 f.im (A,B,C); Bar inD, 5(im (D,E,F). These are aceto-orcein-stained squashesprepared and photographed as described in Materials andmethods.

frequency of nullo (O) gametes within this sample isstriking and contrasts with observations that have beenmade with most other mutants that affect meiosis inDrosophila (see review by Baker & Mall, 1976; and theDiscussion). It could be explained by the selective lossof these gametes. An alternative explanation would bethat directed non-disjunction is occurring, so that, inaddition to having no sex chromosomes, these spermare also either missing autosomes or have additionalautosomes and so would produce inviable zygotes.

In order to test this latter idea, we have looked forthe simultaneous non-disjunction of the sex chromo-somes and the second chromosome by crossingXw/YB* males to females having wild-type A' chromo-somes and a marked pair of second chromosomesattached through a common centromere (C(2)KN).These females will give rise to either nullo-2 orattached diplo-2 gametes and so progeny will surviveonly if non-disjunction has occurred in the male inorder that the eggs are fertilized by diplo-2 or nullo-2sperm, respectively. As the second chromosomes of themale are unmarked, we cannot distinguish betweennon-disjunction in the first or second meiotic divisions.The cross does, however, allow one to determinewhether or not non-disjunction of the second chromo-some is occurring and, if so, whether or not it isindependent of non-disjunction of the sex chromo-somes. Simultaneous non-disjunction of the sexchromosomes in the males should give the same set ofgametes as described above, and would be recognizablebyX/O male progeny (from nullo-gametes produced ineither the first or second meiotic divisions); andX/Xw/YBS females (indicative of AT gametes arisingin the first meiotic division).Xiv/Xzv gametes from themale would give tnplo-A zygotes, which are notconsidered because of their poor and irreproducibleviability.

As expected, non-disjunction does not occur inheterozygous polo/ + males, which consequently giveno progeny when mated to C(2)EN females (Table 2).The results with homozygous polo1/polo1 males indi-cate that non-disjunction of the second chromosomedoes occur (Table 2). In those cases in which secondchromosome non-disjunction is independent of thesex chomosomes (to give gametes having only one sexchromosome and being either diplo-2 or nullo-2), onesees a higher recovery of diplo-2 gametes at the expenseof nullo-2 gametes, indicating either their selective lossor their production at a lower frequency. Simultaneousnon-disjunction of the second chromosome and the sexchromosomes occurs at a high frequency and in thiscross is detected predominantly by nullo-A'gametes. Inthose cases exhibiting simultaneous non-disjunction ofboth the second and sex chromosomes, we recoveredonly one Xiv/YBS gamete (0-3% of total gametesrecovered) from the males, indicating as with the first

Abnormal spindle poles in Drosophila 29

B

Fig. 2. Meiotic spindles in live cells during spermiogenesis. A,B. A cyst of wild-type cells undergoing the second meioticdivision. C,D. A tetrapolar spindle in a homozygous polo[ cell undergoing meiosis. E,F. A multipolar meiotic spindle in ahomozygouspolo' cell. Details of the preparation are given in Materials and methods. Bars; A, lOjum; C,E, lOjitm.

C. E. Sunkel and D. M. Glover

lOOiB

2 4 6

100

2 4 6 8D(jum)

Fig. 3. Spermatids from polo* males. Spermatids from heterozygous (A) or homozygous (C) animals at the 'onion stage'.The Nebenkcrn appear as black circles, the nuclei as white circles. The histograms display the nuclear diameters (D) fromthe heterozygous (B) and homozygous (D) animals. Bar, 5jUm. The occurrence of the cytological abnormalities depicted inboth this and Fig. 2 correlates well with the decrease in fertility of polo1 /polo1 males with age. Dissection of virgin maleseither at eclosion, or when they were S or 10 days old, revealed that the proportion bearing degenerated gonads increasesfrom 18% to 23 % and up to 50% after these respective intervals. polol/poloz males show the same cytological phenotypesdescribed for polo1 homozygotes. They were, however, unable to fertilize virgin Oregon R females. This could be a directconsequence of aberrant meiosis leading to the production of abnormal sperm, but it might also be a result of the cuticularabnormalities that affect the genitalia, since some sperm are found that have a normal appearance.

cross that the frequency of sex chromosome non-disjunction in the first meiotic division is low. Of thenullo-A gametes indicating simultaneous non-disjunc-tion, 95 % are diplo-2 rather than nullo-2. These datacould be explained if the defect in polo males directedthe preferential (but not absolute) disjunction

of non-homologous chromosomes. In such a case,nullo-A" gametes would contain an extra complement ofone of the autosomes, and so would not be detected inthe first cross (Table 1), whereas the second crossallows one specifically to recover gametes that arenullo-A" and diplo-2. The dearth of nullo-A';nullo-2

Abnormal spindle poles in Drosophila 31

Table 1. Genetic non-disjunction test for the sex chromosomes in polo'/polo1 and control males

cf 9

Gross: Xui/YB>; polo'/polo' X XXyf; +/ +

Gametespolo'/polo' polo'/ +

Normal meiosis

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Non-disjunctionDivision I

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Mon-disjunctionDivision II

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1472

1822

1334

1249

12 (0-35)

7 (0-2) 2 (0-07)

71 (2-0)

* These gametes were counted together.D nullo gametes cannot be differentiated and therefore are counted together. The numbers in parenthesis

refer to percentages of total gametes recovered.

gametes (which ought to be recoverable) could then beexplained if the sex chromosome and second chromo-somes were segregating into separate gametes. Thesepossibilities are currently under investigation.

The maternal effect

The small proportion of homozygouspo/o1 females thatsurvive to become adults lay a large number of eggswhen mated with either Oregon R orpolo[/polol males.However, none of these 'polo' embryos hatch. Fig. 4shows representative polo embryos that have beenallowed to develop for increasing amounts of timebefore fixing and staining with the fluorescent dye,

Hoechst 33258. DNA replication and nuclear multipli-cation seem to proceed in a disorganized way, resultingin polyploid nuclei. Some polyploid nuclei are alreadyevident after about 30min of development (Fig. 4A),and these increase in number between 1 and 2-5 h(Fig. 4B,C). The appearance of these mutant embryoscontrasts with the wild-type embryo shown in Fig. 4F.This embryo, which is from roughly mid-way throughthis period (cell cycle 11), has regularly spaced nucleiof equal sizes. The wild-type embryo is syncytial untilthe 14th nuclear division cycle, at which point theindividual nuclei become cellularized. The nuclei ofpolo embryos, on the other hand, never cellularize, andthe nuclei continue both to increase in number and

32 C. E. Sunkel and D. M. Glover

Table 2. Genetic non-disjunction test for the sex andsecond chromosomes in polo'/polo1 males

Male FemaleCross: Xiu/Yff ;2/2; polo1/polo1 X X/X ;C(2)EN,bw

Sex chromosomes inmale gametes

Second chromosomes in male gametes0 2/2

37 (14)45 (17)

-NR

2 (0-7)

74 (29)62 (24)

1 (0-3)NR

39(15)

A"

X"X"o

The genotypes of gametes are shown that result from a crosswith polo'/polo' fathers. An analogous experiment with polo'/polo*fathers gave no progeny as expected (see the text). The numbersin parenthesis refer to the percentages of total gametes recovered.NR refers to gametes that were not expected to be recoveredbecause the progeny have low viability. Gametes of this classmarked with an asterisk were scored together.

to attain high levels of polyploidy up to 6-9 h(Fig. 4D,E). Thereafter, there seems to be generaldeterioration of the embryo and breakdown of thenuclei.

A more detailed examination of polo embryos in theearly stages of their development shows that theirnuclei appear to be at comparable stages of the divisioncycle. Thus the majority of nuclei in any given embryoappear to have chromatin that is either undergoingcondensation, or that appears in anaphase, or that isdecondensing into telophase and subsequent inter-phase (data not shown). Furthermore, the nuclearenvelope, as revealed by staining with an anti-laminantibody, also seems to undergo synchronous changesin parallel with the chromatin condensation cycle as isobserved of the nuclear lamin in wild-type embryos( F u c h s i a / . 1983). This suggests that, although nucleiare polyploid and look very abnormal, they stillundergo many of the normal cyclical aspects of mitosis.

The condensed chromatin in these early poloembryos was, however, highly disorganized and ap-peared as if it were not segregating correctly uponmitotic spindles. We therefore chose to examine themitotic apparatus more closely by immuno-stainingusing antibodies that recognize tubulin or the centro-some. In the wild-type embryo, microtubules arenucleated by centrosomes to give asters during inter-phase. These split and migrate to the positions of thespindle poles during prometaphase. In metaphase, themicrotubules of the spindle pole become attached tothe condensed chromosomes at their kinetochores. Thecentrosome remains a 'focal point' for the spindle pole,and some astral microtubules still persist (see Karr &Alberts, 1986). An example of a field of prometaphasenuclei from a wild-type embryo is shown in Fig. 5G-I .

These nuclei are regularly spaced and contain con-densed chromatin (G); the mitotic spindle has formed(H) and distinct centrosome staining can be seen withBx63 antibody at the spindle poles (I). Examples of themicrotubules of polo embryos are shown in panels Band E. In the first field, condensed chromatin (A) isassociated with spindle-like microtubular structuresthat seem to be undergoing anaphase (B). Many ofthese spindle structures share one or both of theirpoles. Furthermore, many have very broad poles incontrast to wild-type spindles in which the polarmicrotubules converge on the dot-like centrosome.Surprisingly, there is no evidence of centrosomesassociated with these highly branched structures andBx63 staining is not apparent (C). At no stage ofdevelopment were we able to observe asters of micro-tubules in the polo embryos in association with inter-phase-like nuclei, as in wild-type embryos (data notshown). Furthermore, there was no evidence of freeasters in the cytoplasm as is observed with gnu embryos(Freeman et al. 1986) and with several other mitoticmutants (Leibowitz & Glover, unpublished results). Insome embryos, however, we observed complex micro-tubular structures mainly associated with condensedchromatin, but resembling complex astral structuresmore than spindles (E). In this field, the Bx63 antibodyilluminates scattered participate matter in the cyto-plasm (F). The absence of centrosome staining byBx63 caused us to wonder whether the polo1 mutationmight eliminate or cause a structural change to theantigen recognized by this antibody. We thereforecarried out Western blotting experiments to look forthis antigen within polo embryos. We were, however,unable to detect any difference in either molecularweight or abundance of the Bx63 antigen between wild-type or polo embryos (data not shown). We suspecttherefore that centrosomal components are not organ-ized into discrete structures in early polo embryos.When we examine later polo embryos using the Bx63antibody, however, we are able to detect several well-defined punctate bodies that are associated with poly-ploid nuclei containing decondensed chromatin(Fig. 6), but never appear to be associated with micro-tubules.

Discussion

We have described a locus, polo, that is essential formitosis in Drosophila, and have examined the pheno-types of polo mutants in both larval and embryonicstages of development. A larva homozygous for polo1

can survive through embryogenesis because its hetero-zygous mother has provided sufficient polo+ geneproduct for embryonic development. Most of theensuing larval development involves cell growth, withthe endoreduplication of DNA in the cells of many

Abnormal spindle poles in Drosophila 33

Fig. 4. The development of polo embryos. Embryos were collected for 30min (A) and allowed to develop at 25°C for afurther 30min (B), 120 min (C), Sh (D) and 9h (E). They were then fixed and stained with Hoechst. A representativeembryo is shown in each panel. A wild-type embryo at cycle 11 is shown in F. Bar, 30fjm. The insets in A-C show detailof the chromatin structure and are at a fourfold higher magnification.

tissues in the absence of mitosis. The diploid imaginalcells of the larva are not themselves necessary for itssurvival. In homozygous polo larvae, these cells areeventually blocked in their mitotic division, and sobecome unable to proliferate to form adult structuresand the organism dies in the larval stage of develop-ment. In this respect, polo falls into a class of mitoticmutants that have a late larval lethal phenotype, first

recognized in the analysis of repair-defective mutants(Baker et al. 1982). In the case of the polo1 allele,however, a few homozygous animals survive to adult-hood and the females can produce embryos, whichbegin to develop but show abnormal mitoses at a veryearly stage of embryogenesis. The embryo develops ofthe order of a hundred or so polyploid nuclei and neverundergoes cellularization.

'If C. E. Sunkel and D. M. Glover

Hoechst Tubulin Bx63

Fig. 5. Double immunostaining of polo embryos with antibodies against tubulin and centrosomes. Embryos wereincubated sequentially with B.\63 antibody (mouse anti-centrosome), rhodamine-conjugated anti-mouse IgG, YLl/2 (ratanti-tubulin), fluorescein-conjugated anti-rat IgG, and Hoechst (see Materials and methods). A-F. Stainings of twodifferent polol/polo[ embryos; G-I , staining of a wild-type control. Bars: A, 10/um, D, 5/.im, G, 10/mi.

In very early embryos, the nuclei are evidentlyundergoing cyclical changes to their nuclear mem-branes reflected by the patterns of lamin staining, andthese appear to be synchronized with chromosomecondensation, metaphase and anaphase. Whilst thesecyclical aspects of mitosis are retained, at least for theearly nuclear cycles, the morphology of the mitoticspindles is grossly abnormal. Virtually all the spindlesare branched and multipolar and, rather than havingtightly focused poles, many are broad and barrel-

shaped. This could be explained by a change in thestructure of the centrosome. Barrel-shaped spindles arefound in plant cells, for example, which differ frommost animal cells in lacking centrioles and a tightlylocalized spindle microtubule-nucleating centre.Another indication of defective centrosomes is theabsence of asters associated with interphase nuclei inpolo embryos, unlike wild-type embryos. In severalother mitotic mutants, we have observed free centro-somes that are dissociated from nuclei or condensed

Abnormal spindle poles in Drosophila 35

Fig. 6. Nuclear staining of a late polo embryo with B.\63 antibody. Polyploid nuclei from a late polo embryo from an 8-hcollection, stained with Hoechst (A) and with the anti-centrosome antibody Bx63 (B). Bar, 5jUm.

chromatin. The most extreme example is gnu, in whichthere is no mitosis and yet the centrosomes replicateand nucleate asters of microtubules (Freeman et al.1986). We can also see free asters dissociated fromabnormal spindles that are found in other mutants, forexample, lodestar, aurora and thule (Leibowitz &Glover, unpublished data). These mutant embryoscontrast with polo embryos, in which we are unable todetect any free cytoplasmic asters.

Immunostaining of POLO embryos with the mono-clonal antibody, Bx63, against an antigen associatedwith the centrosome indicates that POLO centrosomesare abnormal. In the wild-type embryo, the Bx63antibody stains a large dot either at the centre of astersor at the poles of the spindle (Frasch, 1985; Frasch etal. 1986; Freeman et al. 1986). When we stain poloembryos with Bx63, we see either no distinct staining,or dispersed, very fine punctate staining with noobvious association with the spindle poles. Later indevelopment, as the polo nuclei become obviouslypolyploid, the antigen appears to coalesce in largepunctate aggregates apparently associated with thenuclear membranes of interphase nuclei. The Bx63antigen is present throughout these stages, however, inlevels comparable to wild-type embryos, as indicatedby Western blotting experiments. Together theseobservations suggest that the polo mutation mightaffect a protein required for the correct structure andfunction of the centrosome, although such a proteinneed not necessarily be a centrosome componentper se.

The abundance of tubulin within the larval brainhinders the immunocytological examination of spindlesin neuroblasts, and so our observations have been madeonly upon stained chromosome preparations. Thephenotype that we observe in larval neuroblasts couldbe explained by a lesion affecting the centrosome. Themost common mitotic aberrations that we observe inthese cells are polyploid circular mitotic figures, andanaphase figures in which the chromosomes progress-ing towards one of the poles are randomly oriented.Circular mitotic figures have also been observed in themutant merry-go-round (mgr) (Gonzalez, 1986; Gon-zalez et al. 1987), and the mutant lodestar (Leibowitz& Glover, unpublished data), both of which fullycomplement polo. They could arise if one of the spindlepoles was defective with the consequent distortion ofthe mitotic plate as all the chromosomes are pulled tothe single remaining pole. This would also explain thepolyploidy that we frequently observe in these cells andin mitotic figures of this type. The abnormal anaphasestructures could be a weaker manifestation of thisphenotype in which one spindle pole behaves normally,and the other is only weakly affected. In the wild type,the spindle elongates during anaphase and the polesmove further apart, suggesting that motive forcesacting on the centrosome play an important role. Onecan speculate that the random orientation of thechromosomes around one of the spindle poles could bea consequence of inadequate transfer of motive forcethrough a defective centrosome. We are unable todetect circular mitotic figures in brains from polohomozygotes treated with colchicine. Gonzalez et al.

36 C. /:'. Sunkel and D. M. Glover

(1987) reported a similar finding for mgr, and further-more have shown that circular figures do not formwhen the mutation abnormal spindle (Ripoll et al.1985) is present together with mgr. It seems thereforethat a functional spindle is required to generate circularfigures. Other examples of circular mitotic figures inassociation with functional monopolar spindles havebeen observed in primary cultures of newt epithelialcells (Bajer, 1982); in a mutant line of Syrian hamsterovary cells (Wang et al. 1983); and following theinactivation of centrosome replication with /3-mercap-toethanol (Mazia, 1960).

polo differs from mgr in male meiosis by showing avariety of abnormal multipolar spindles in live cellsduring spermatogenesis. Furthermore, the spermatidshave a wide range of nuclear diameters, indicating aheterogeneous DNA content resulting from chromo-some non-disjunction. Mitochondrial derivatives alsosegregate on the meiotic spindle and then aggregate asNebenkern in the spermatids. The Nebenkern are alsoheterogeneous in size in the mutant, in contrast to thewild type. This indicates that the abnormal segregationis a property associated with the spindle rather than thekinetochore of the condensed chromosomes. Our gen-etic analysis of non-disjunction during male meiosissuggests that the mutation affects mainly the seconddivision. This is not to say that there is no effect on thefirst division, and indeed gametes indicative of this arerecovered at a frequency higher than in wild-typecontrols. Aberrant segregation in the first meioticdivision, or even in the pre-meiotic divisions of sperma-togenesis, may lead to a 'meiotic catastrophe' in whichthe resulting gametes could be lost. The first meioticdivision differs from mitotic divisions in that thecentromeres do not split and instead the homologouschromosomes are segregated to the spindle poles.Therefore, a separate set of functions is requiredduring this meiotic division. The second equationaldivision is, on the other hand, comparable to mitosisand requires division of the centromeres. Over 40mutants have been described that affect meiosis inDrosophila (see Baker & Hall, 1976; Lindsley &Sandier, 1977, for reviews). The majority of theseaffect the first meiotic division, different sets of genesbeing used for this division in males and females,reflecting the restriction to females of the processes ofrecombination, distributive disjunction and the forma-tion of synaptonemal complexes. Baker et al. (1978),however, have shown that of these mutants affectingthe first division, six recombination-defective loci andfour loci required for correct meiotic segregation havean effect on mitotic chromosome behaviour. Mutantsthat preferentially affect the second meiotic division arecomparatively rare. Baker & Hall (1976) suggestedthat this might be because these genes would beexpected to play a crucial role in mitosis and

so would not have been recovered in the mutantselection schemes employed to search for meioticmutants. Only two other loci, eq and meiS332, behavesimilarly to polo in this respect. The gametes producedby mei-S332 males are indicative of a relative frequencyof non-disjunction in the first and second meioticdivisions comparable to that we observe with polo males(Goldstein, 1980). meiS322, however, tends to pro-duce an excess of nullo-gametes like most other mu-tations that affect meiosis in Divsophila and thiscontrasts with our observations on polo. The lowrecovery of nullo-gametes in polo males could reflecttheir selective loss in the tests that we have carried out.It appears, however, that simultaneous non-disjunc-tion is occurring at a high frequency. In a test designedspecifically to look for the simultaneous non-disjunc-tion of the sex and second chromosomes, we have beenable to recover a high proportion of nullo-.V gametesthat were in addition diplo-2, but there were very fewnullo-2;nullo-A' gametes. This raises the possibility,currently under further investigation, that disjunctionof non-homologous chromosomes is occurring in polomales. This incorrect segregation of chromatids couldwell reflect a defect in a component of the spindleapparatus. It could be that for correct chromatidsegregation to occur there have to be equal forcesprovided by both spindle poles. This would go someway to explaining the meiotic phenotype of polo.

Although on balance our observations point towardsthe polo defect affecting the function and/or organiz-ation of the centrosome, it is still prudent to exercisecaution in the interpretation of mutant phenotypes.This is especially true of cyclical processes such asmitosis, which involve many interacting components.A given mutation affecting one step in the cycle couldsubsequently have pleiotropic consequences. Our dis-covery of a dysgenically induced polo allele paves theway towards cloning the gene and thereby studying thegene product. We are currently in the process ofidentifying the P-element that is specifically associatedwith the lethal polo2 mutation. This P-element-taggedDNA segment will then be cloned from a library ofrecombinant DNA from the mutant, permitting a fullmolecular analysis of the protein encoded by polo.

We are most grateful to the Cancer Research Campaign forsupporting this work and for a Career Development Award toD.M.G. We thank Christianne Nusslein-Volhard for allow-ing us to screen her collection of third chromosome maternaleffect mutants from which we isolated thepolo[ allele. We areextremely grateful to Roger Karess and Alan Cheshire fortheir efforts in establishing a collection of dysgenic thirdchromosome mutants in this laboratory from which weisloated polo2. We thank Cayetano Gonzalez for teaching usabout male meiosis, for improving our brain-squashingtechniques, and together with Pedro Ripoll for communicat-ing results prior to publication. Many colleagues struggled

Abnormal spindle poles in Drosophila 37

through the draft manuscript and we thank them for theirhelpful criticisms.

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(Received 13 August 1987 -Accepted 18 September 1987)

38 C. E. Sunkel and D. M. Glover