Proc. Natl. Acad. Sci. USAVol. 92, pp. 10443-10449, November 1995

Review

Meiosis in Drosophila: Seeing is believingTerry L. Orr-WeaverWhitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142

ABSTRACT Recently many excitingadvances have been achieved in our un-derstanding of Drosophila meiosis due tocombined cytological and genetic ap-proaches. New techniques have permittedthe characterization of chromosome po-sition and spindle formation in femalemeiosis I. The proteins encoded by the nodand ncd genes, two genes known to beneeded for the proper partitioning ofchromosomes lacking exchange events,have been identified and found to be ki-nesin-like motors. The effects of muta-tions in these genes on the spindle andchromosomes, together with the localiza-tion of the proteins, have yielded a modelfor the mechanism of female meiosis I. Inmale meiosis I, the chromosomal regionsresponsible for homolog pairing havebeen resolved to the level of specific DNAsequences. This provides a foundation forelucidating the molecular basis of meioticpairing. The cytological techniques avail-able in Drosophila also have permittedinroads into the regulation of sister-chromatid segregation. The products oftwo genes (mei-S332 and ord) essential forsister-chromatid cohesion have beenidentified recently. Additional advancesin understanding Drosophila meiosis arethe delineation of a functional centromereby using minichromosome derivatives andthe identification of several regulatorygenes for the meiotic cell cycle.

The investigation of meiosis in Drosophilahas two powerful advantages. The first isthe ability of researchers to visualize themeiotic divisions in female and male mei-osis (Figs. 1 and 2). The second is avaluable collection of mutations affectingmeiosis, an inheritance accumulated overseven decades.The cytology of Drosophila meiosis has

been investigated extensively during thepast 20 years. In males, the meiotic cellsare accessible to light and electron micros-copy, so chromosome behavior as well asspindle structure and kinetochore ultra-structure have been described (1-8). Infemales, the early events of meiosis lead-ing to homolog pairing, synaptonemalcomplex formation, and recombinationwere visualized in detail by electron mi-croscopy of serial sections of oocytes (9-11). This painstaking analysis provided animage of the structure of the synaptone-mal complex and recombination nodules.

However, until recently the cytology of thechromosomes in later meiosis was not wellcharacterized. The oocyte yolk obscuresthe meiotic spindle and chromosomes inthe light microscope. One of the mostsignificant recent advances in Drosophilameiosis is the improvement of female mei-otic cytology to permit analysis of the laterstages of meiosis (12). Confocal imagingof whole mount oocytes has provided in-sights into the mechanisms that normallypartition chromosomes during the meioticdivisions and the effects of mutations onthose processes.

Frequently in biology exceptional casesprovide important insights into funda-mental mechanisms. This has certainlybeen true for Drosophila meiosis (Fig. 3).Genetic studies demonstrated that duringthe first meiotic division in Drosophilafemales homologs pair and segregate bythe common mechanism of recombina-tion, synaptonemal complex formation,and presumably chiasmata formation (13).However, there is an exception. The tinyfourth chromosome does not undergo ex-change and yet segregates faithfully.Larger chromosomes also partition prop-erly when they fail to undergo exchange. Aset of genes was identified that are neces-sary for the segregation of nonexchangechromosomes in the female, and the re-cent molecular and cytological analysis ofsome of these mutants has provided keyinformation concerning the segregation ofall chromosomes during female meiosis.

Meiosis in Drosophila males is also anexception to the general mechanism ofhomolog segregation. Recombinationdoes not occur in males, and synaptone-mal complex is not formed. Recent studiesof the molecular basis of homolog pairingin males are likely to enhance our under-standing of mechanisms of pairing inmany systems.

Inroads into some poorly understoodareas of meiosis have been opened - inDrosophila by particular mutants. Themechanisms responsible for sister-chro-matid attachments have been elusive. Mu-tations in two Drosophila genes result inpremature sister-chromatid separation inmeiosis. These genes are likely to play adirect role in controlling sister-chromatidcohesion, and their protein products havebeen identified recently. A minichromo-some derivative that is transmitted faith-fully has made the centromere amenable

to molecular analysis, and centromerefunction has been delineated to a 220-kbregion. Finally, several genes regulatingthe meiotic cell cycle have been identifiedin the past 3 years in Drosophila.

Female Meiosis

The investigation of meiosis in many or-ganisms explained the requirement forrecombination for homologous chromo-some segregation (14, 15). During meiosisI the homologs pair, and the resultingassociation between them is necessary forbipolar attachment of the bivalent on thespindle. The tension caused by the forcestoward the two poles being counteractedby the forces holding the homologs to-gether results in stable attachment of thebivalent to opposite poles (16). Whatholds the homologs together? The synap-tonemal complex, which during pachyteneforms a zipper between the homologs,cannot provide stable homolog attach-ment because it dissociates during diplo-tene. Chiasmata are cytologically definedstructures observed in many organismsthat seem to hold the homologs togetheruntil their segregation at anaphase I. Chi-asmata appear to be the visible relics ofexchange events because there is a clearcorrelation between chiasmata and theposition and number of exchange events(14, 15). However, the physical distinctionbetween a chiasma and a crossover is notclear. Chiasma could be a recombinationintermediate such as a Holliday junctionor a resolved exchange event in which thetwo DNA duplexes are held in a crossstructure. In summary, the requirementfor recombination in proper homolog seg-regation can be explained by exchangeevents becoming chiasmata that stablyhold the homologs together, ensuringtheir bipolar attachment to the spindle.

It has not been possible to observechiasmata in Drosophila; however, it isclear that exchange is important forproper chromosome segregation (13). Thelarge chromosomes have about one ex-change event per arm during meiosis. Mu-tations reducing recombination cause ab-errant segregation of the homologs inmeiosis I. The presumption is that in Dro-sophila females exchange events lead tochiasmata that serve as stable homologattachments until the metaphase I/ana-phase I transition.

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FIG. 1. Cytology of Drosophila female meiosis. The images are confocal micrographs of oocytesfixed and labeled with antibodies to visualize meiotic stages. (A) Metaphase I arrest in a mature stage14 oocyte. The chromosomes are stained with anti-histone antibodies and are shown in yellow. Thespindle is stained with anti-a-tubulin antibodies and is shown in red. The unusual female spindle hastapered, narrow poles. The nonexchange fourth chromosomes have migrated toward the poles awayfrom the chromosomal mass at the metaphase plate. (Photograph courtesy of W. Theurkauf, StateUniversity of New York, Stony Brook.) (B) Metaphase II. The chromosomes are labeled with7-amino-actinomycin D and are shown in red. The spindles are labeled with anti-a-tubulin antibodiesand are green. The two meiosis II spindles lie in a line, and the chromosomes are on the metaphaseplates. (C) Early anaphase II. Labeling as in B. The sister chromatids are separating on each spindle.InB and C mature oocytes were activated to complete meiosis in vitro, making it easier to capture theserapid stages. (Photographs in B and C are from A. Page, Whitehead Institute.)

If chiasmata serve as physical attach-ments between the homologs, how do thenonexchange fourth chromosomes segre-gate faithfully? If an additional chromo-some fails to undergo exchange (for ex-

ample, because exchange is suppressed bymultiple inversions on a balancer ho-molog) it also will segregate correctly. Thesegregation of the fourth chromosomeand other nonexchange chromosomes wasinvestigated extensively by Grell (17, 18)and termed the distributive pairing system(Fig. 3). Grell found that in Drosophilafemales chromosomes that lack homology

(heterologous chromosomes) could segre-gate from each other, and she character-ized the parameters of this segregation.She found that distributive segregation isinfluenced by the availability, size, andshape of the chromosomes. For example,the effect of free X duplication chromo-somes of increasing size on the segrega-tion of the fourth chromosomes was in-vestigated (18). These free duplicationscontain the centromeric region of the Xtogether with varying amounts of X eu-chromatic DNA and are monosomic. Ifthey pair with and segregate from another

chromosome such as the fourth, then theother fourth homolog will move randomlyto a pole. This can result in gametes thatcontain two fourth chromosomes. Thus,segregation of the free duplication fromthe fourth chromosome can be scored bythe appearance of gametes with the freeduplication but no fourth chromosomesand gametes with two fourth chromo-somes but no duplication. Grell found thatwhen theX duplication chromosome wasequivalent in size to the fourth chromo-some, the duplication segregates effi-ciently from the fourth chromosome.

In further analysis of the segregation ofnonexchange chromosomes, Hawley et al.(19) also found that heterologous chro-mosomes could segregate from each otherduring female meiosis. However, in a se-ries of experiments with free duplicationsthey observed that homology can be im-portant in the proper segregation of non-exchange chromosomes (19). This led toan alternative nomenclature, illustrated inFig. 3, in which the segregation of nonex-change homologous and heterologouschromosomes is distinguished. There isgenetic evidence for two pathways forsegregating nonexchange chromosomes inthat mutations in theAxs, ald, and mei-S51genes affect the segregation of homolo-gous nonexchange chromosomes but donot affect heterologous chromosomes (forreview, see ref. 20).There is a large collection ofDrosophila

mutants that affect homolog segregationduring the first division of female meiosis.Most of these are defective in recombina-tion (13). The reason that reduced recom-bination leads to aberrant segregation isthat it appears that female meiosis has alimited capacity for segregating nonex-change chromosomes. Thus, as more chro-mosome pairs fail to undergo exchange,the nondisjunction frequency increases.

Recent advances come from the analy-sis of two genes needed for the segrega-tion of nonexchange chromosomes. Thenod (no distributive segregation) gene isrequired only for the segregation of non-exchange chromosomes (21, 22). The genehas been cloned and found to encode amember of the kinesin family of microtu-bule motor proteins. The N-terminal do-main of the NOD protein is homologousto the motor domain of the kinesin heavychain (23). Mutations that disrupt NODfunction result in changes in conservedamino acids, demonstrating the signifi-cance of the homology (24, 25). For ex-ample, a dominant mutation, nodDTw, isthe consequence of an amino acid changein the predicted ATP binding domain.Although it has not been possible to dem-onstrate motor activity with the purifiedNOD protein, it is hypothesized to be aplus-end-directed motor based on its con-served structure with kinesin.The ncd (nonclaret disjunctional) gene

is necessary for the segregation of nonex-

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AL~~~~~~~~~ A,

AL

pi rt

rl, j ?F

FIG. 2. Cytology of Drosophila male meiosis. Shown are phase micrographs of testis squashesfixed and stained with orcein to visualize the chromosomes. (A) Prophase I in a wild-type male.The sex chromosome bivalent is at the top and has the characteristic structure resulting frompairing at a specific site. The large autosomes are paired tightly along their length, and the tinyfourth chromosomes appear as dots. (Photograph by W. Miyazaki, Whitehead Institute.) (B)Anaphase I in a wild-type male. Equal numbers of chromosomes have partitioned to the two poles,and the sister chromatids can be seen to be attached now only at their centromere regions. (C)Anaphase II in a wild-type male. The sister chromatids have segregated equally to the two poles.(D) Anaphase I in a mei-S332 mutant. Only one pole is in focus. The sister chromatids prematurelyseparated, with the exception of the pair shown by the arrow. (E) Anaphase II in a mei-S332mutant. Sister chromatids distribute unequally to the two poles and lagards are observed (arrow).(Photographs in B-E courtesy of A. Kerrebrock, Whitehead Institute.) (F) Diagram of sister-chromatid cohesion in meiosis. Early in meiosis I the sister chromatids are attached along theirentire length. In most organisms chiasma result from exchange events, as indicated by the crossedchromatid structure. These chiasmata hold the homologs together, and they may be stabilized bycohesion on the chromatid arms distal to the site of the chiasma. After the metaphase I/anaphaseI transition (arrow) cohesion is lost on the arms, and the sister chromatids remain attached at theircentromeric regions until the metaphase II/anaphase II transition.

change and exchange chromosomes (26).The ned gene also encodes a protein withkinesin homology, but in this case theconserved motor domain lies at the Cterminus of the protein (27, 28). A sur-

prising observation was that, in contrast tokinesin, the purified NCD protein has a

minus-end-directed motor activity on mi-crotubules in vitro (29, 30). NCD is alsocapable of bundling microtubules in an

ATP-dependent manner (29).A major breakthrough in our under-

standing of the mechanism of homologsegregation in female meiosis I came fromcharacterization of the cytology of thespindle in oocytes (Fig. 1) (12). The fe-male meiotic spindle differs from that ofmale meiosis or mitosis in that the micro-tubules are not organized from the polebut rather from the chromosomes them-selves. There are no apparent centro-somes. After pachytene in prophase I thechromosomes are held tightly condensedin a karyosome; individual chromosomescannot be visualized. The synaptonemalcomplex is initially present after karyo-some formation but then dissociates (7).Prior to nuclear envelope breakdownshort microtubules appear associated withthe nuclear envelope. After envelopebreakdown these appear to be captured by

the chromosomal mass. Consequently, a

bipolar spindle is formed in which thegreatest microtubule mass is at the center,with the chromosomes, rather than at thepoles (12). In several mutants individualchromosomes move away from the spindleand are isolated in the cytoplasm (seebelow). Consistent with the role of thechromosomes in organizing the spindle,these single chromosomes are able to or-

ganize bipolar spindles.At metaphase I in the stage 14 oocyte,

the spindle elongates and is very narrowand tapered at the poles (Fig. 1A). Strik-ingly, the nonexchange fourth chromo-somes move out of the chromosomal mass

at the metaphase plate and are localizedon the spindle near the poles (Fig. 1A)(12). The fourth chromosomes are ob-served near the poles in living oocytes as

well; thus, this position is not an artifact offixation (W. Theurkauf, personal commu-nication). If the dosage of the nod gene isreduced, other nonexchange chromo-somes also will move out of the chromo-somal mass toward the pole (12). Theextent of migration to the poles is a func-tion of size; the X chromosomes do notmigrate as close to the poles as do the tinyfourth chromosomes.

Mutations in the nod and ned genescause alterations in the position of chro-mosomes or spindle structure that giveinsights into the mechanism of segrega-tion of exchange and nonexchange chro-mosomes. In nod mutants the spindle isnormal but nonexchange chromosomesare frequently observed in the cytoplasmoutside the spindle (12). These wanderingchromosomes can reattach to the spindleand, when they do so, attach randomly toeither pole. Thus, the cytological obser-vations account for the nondisjunctionand chromosome loss observed in themutants.A variety of spindle and chromosomal

defects is observed in female meiosis inned mutants (31). In metaphase I thespindle is diffuse. Later in metaphaseI/anaphase I, broad, diffuse, and multi-polar spindles are observed. The chromo-somes are frequently located off the spin-dle, in the cytoplasm. The dynamics of thespindle have been characterized recentlyby studying living oocytes from ned mu-tants. In real time it is observed that in nedmutants the spindle assembles moreslowly, and the spindles that do form arenot stable (W. Theurkauf, personal com-munication). The NCD protein is local-ized along the spindle microtubules duringall of the stages of meiosis (32). Thus, theNCD protein appears to be essential forforming and stabilizing the meiotic spin-dle, possibly by virtue of its capability tobundle microtubules (29, 31, 32).NOD protein localization provides

much information concerning its function.The protein is localized all along the chro-mosomes in prometaphase- and meta-phase-arrested oocytes, the only stagesexamined to date (33). Moreover, thenonmotor domain of the NOD proteinbinds DNA with a preference for (A+T)-rich regions (33). A set of deletions span-ning a small free duplication chromosomewas used to map the sites at which NODacts (34). This minichromosome, Dp1187,is transmitted faithfully during mitosis andmeiosis in wild-type females, and it is lostat a slight but significant frequency in nodheterozygotes. However, deletions in theminichromosome result in a marked lossof transmission when the dosage of thenod gene is reduced. Consistent with theimmunolocalization of the protein alongthe length of the chromosomes, deletionsthroughout the noncentromere region ofthe minichromosome enhance loss in nodheterozygotes. In general, transmissionfrequency increases with increasing size ofthe chromosome, although some regionsinteract more strongly with NOD thanothers.The cytological and molecular observa-

tions converge to provide a model formeiosis I segregation in females that hasbeen articulated most explicitly by Hawleyand colleagues (20, 35). The general fea-tures of the model are that the chromo-

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(eg.X-*X Bal

Dp4 *--4,4DpK *--* XX)

Representative Recombination [FAxsaldMutants Deficient Mutants

nod

I nod

(eg. XX4- YA A

XX 4-*044DpX *-#-4,4)

3mei-l mei-13

Dub ord

FIG. 3. Summary of Drosophila meiosis I. The classes of homologs segregating in female andmale meiosis are indicated together with representative examples of mutations affecting segre-gation. At the bottom the types of segregation affected in the mutants are indicated by the extentof the boxes. The alternative names for the segregation of nonexchange chromosomes in femalesare shown, along with some examples of each class of segregation. Homologous chromosomes thatfail to exchange because they are in trans to a balancer (Bal) undergo achaismate homologoussegregation. Free duplications segregating from homologous normal chromosomes also areclassified as achiasmate homologous segregation. The segregation of theXchromosomes from theY, heterologous compound chromosomes, or free duplications from nonhomologous chromo-somes are termed achiasmate heterologous segregation. Note that the ord gene causes meiosis Inondisjunction of all classes of chromosomes because it results in premature separation of thesister chromatids. See the text for references for the mutants.

somes pair early in meiosis and form a

synaptonemal complex. The fact thatthere is a karyosome stage gives uniqueproperties to Drosophila female meiosis.Whereas in most organisms the homologsappear to repulse each other during dip-lotene/diakinesis, these stages do not oc-cur in Drosophila females. It is proposedthat the tight packing in the karyosomemaintains homolog associations even forthe nonexchange bivalents. When thekaryosome breaks down and the spindleforms, achiasmate chromosomes might bepredicted to dissociate and move to thepoles. The NOD motor protein, which islocalized along the chromosome arms,could act to push the achiasmate chromo-somes away from the poles, forcing themtogether into the chromosomal mass onthe metaphase plate (33). This hypothesisis consistent with the location of the NODprotein and the observation of stray non-exchange chromosomes in nod mutants. Itrequires that the direction of the NODmotor is toward the plus ends of microtu-bules, a prediction that has not yet beenverified.The model proposes that nonexchange

chromosomes that have homology pairearly in meiosis. This is certainly the casefor the fourth chromosome, as it is ob-served to have synaptonemal complex (9).Hawley et al. (20) propose that othernonexchange chromosomes can pairthrough homologous heterochromatinprior to karyosome formation. However,chromosomes that lack apparent homol-ogy are capable of accurately segregatingfrom each other in female meiosis. Hawley

advances two hypotheses to account forthe segregation of heterologous chromo-somes (20). In the first, the chromosomessomehow interact to result in the propercentromere alignment to the two poles,and this association is stabilized by thekaryosome. This explanation does notnecessarily require that the interactionbetween the heterologous chromosomesbe a physical attachment. The second hy-pothesis accounts for segregation by thenarrow female meiotic spindle. Thepremise is that the tapered poles havelimited accommodation for chromo-somes. So the orientation of the heterol-ogous chromosomes could be randomwithin the karyosome prior to spindleformation. Once the spindle is formed thenonexchange chromosomes would beginto orient and move to the pole. If oneheterolog had moved toward one pole,that pole would be crowded, thus exclud-ing its partner and resulting in its orien-tation to the other pole.

Male Meiosis

In Drosophila males recombination doesnot occur and synaptonemal complex isnot detectable (4, 5). Nevertheless, thesenonexchange homologs pair and segregatefaithfully during meiosis I. Mutations inthe genes affecting the segregation of non-exchange chromosomes in the female haveno effect on male meiosis (Fig. 3, with theexceptions of Dub and ord discussed be-low); thus, distributive segregation doesnot account for the mechanism of malemeiosis I. The distributive segregation

functions may be integrally linked to theunusual female meiotic spindle. In con-trast, the male meiotic spindle resembles amitotic spindle with a microtubule orga-nizing center at each pole, rather than atapered spindle organized by the chromo-somes as in female meiosis (3, 6). Inaddition, the chromosomes do not assumea karyosome structure in the male and arecondensed and visibly paired throughoutthe meiosis I division (Fig. 2A).

Little is known about the trans-actinggenes required for male homolog segre-gation, and some of the most interestingmutants have been lost (36). However,recently there have been exciting advancesin our understanding of the cis-acting el-ements responsible for homolog pairing.The mechanism by which homologouschromosomes find each other and pairduring meiosis is not understood. Dro-sophila males provide the advantage thatspecific sequences promote pairing, thusgiving a defined focal point from which toelucidate the molecular basis of pairing.Moreover, the ability to visualize pairingcytologically in testis squashes permitspairing to be addressed independently ofsegregation (Fig. 2A).Cooper (37) demonstrated that the X

and Y chromosomes pair at a specific site,termed the collochore. He used deletionsto map the position of this site to the baseof the long arm of theX chromosome andthe short arm of the Ychromosome. In thepast 5 years McKee and colleagues (38,39) have delineated the basis ofX-Y pair-ing to a molecular level. The repeatedrDNA genes are present on the X and Ychromosomes in the vicinity of the collo-chore defined by deletions. McKee andKarpen (38) found that a single copy ofthe rDNA repeat inserted on theX chro-mosome is capable of restoring X-Y pair-ing and segregation to an X chromosomedeleted for most of the X heterochroma-tin, including the collochore. By generat-ing a set of deletions within the rDNArepeat and measuring pairing and segre-gation, a 240-bp repeated sequence withinthe intergenic region was found to besufficient for pairing and segregation (39).Increased copies of this repeat enhanceX-Y chromosome interaction. Interest-ingly, insertion of the rDNA repeat ontoan autosome does not promote pairingwith the Y chromosome (38). Thus, thechromosomal domain in which the rDNArepeat is inserted must also influence ho-molog pairing.These experiments establish that the sex

chromosomes interact via a specific pair-ing site, a block of homology present ontheX and Ychromosomes. However, thereare unique properties of the rDNA se-quence responsible for this interaction.Other blocks of homology shared betweenthe X and Y chromosomes such as theStellate (Ste) repeats are not sufficient forproper segregation (38). One possibility

FemaleExchange Chromosomes

OtherDesignations

Female MaleNonexchange Chromosomes Nonexchange Chromosomes

Distributive Segregation

Achiasmate AchiasmateHomologous HeterologousSegregation Segregation

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for the special characteristics of the rDNArepeat is that the presence of this se-quence within a nucleolus somehow con-fers pairing ability. This explanation isunlikely, since there are deletions withinthe rDNA that prevent detectable nucle-olus formation yet still permit properX-Ysegregation (39).Chromosomal regions needed for auto-

somal pairing and segregation in Drosoph-ila males have also been investigated. Inthe most extensive study, McKee et al. (40)used a set of transpositions in which seg-ments of the second chromosome are in-serted onto the Y chromosome. They ex-amined cytologically whether these chro-mosomal segments caused the Y chromo-some to pair with the second chromosome.Using genetic assays, they also testedwhether the transposed chromosome 2 seg-ment could direct the Y chromosome car-rying it to segregate from the homologouschromosome 2The results from these experiments con-

trast with the simple, specific pairing siteused in segregation of the sex bivalent.Consistent with previous reports, McKeeet al. (40) found that the heterochromatinof the second chromosome does not pro-mote pairing and segregation. While theeuchromatin does act as a pairing region,its ability to do so is a function of the sizeof the euchromatic block. Thus, increas-ingly larger transpositions were found tobe more effective in pairing and segregat-ing with chromosome 2. These observa-tions indicate that either there are notspecific pairing sites on the autosomes orthey are distributed very frequently alongthe chromosome.Although many regions along the sec-

ond chromosome confer pairing ability,there is an exceptional region at the baseof the left arm of the second chromosomethat is particularly effective at promotingsegregation (40). By deficiency mapping,this autosomal pairing site has been shownto colocalize with the histone gene cluster(B. McKee, personal communication). InDrosophila, the histone genes are repeatedtandemly in 100-200 copies (41). Thus,this repeated gene cluster appears to playa role in autosomal pairing analogous tothat of the rDNA cluster in sex chromo-some pairing. The distinction is that this isnot the sole sequence that promotes pair-ing of the second chromosomes.

It is unclear why these two tandemlyrepeated sequences function as pairingsites whereas the highly repetitive DNAfound in heterochromatin does not. Thedifferent chromatin configuration in het-erochromatin may impede pairing. Theability of tandem repeats of specific se-

quences to promote pairing in males couldreflect the increased density of sequencehomology or, more likely, the propertiesof specific chromatin proteins bound tothese repeats.

That fact that homolog pairing in malemeiosis requires homology, even for thesex chromosomes, suggests that somefunctions promoting homolog pairingcould be shared between males and fe-males, a point emphasized by McKee (40).Such common functions have not beendescribed. However, the previous screensfor meiotic mutations were not saturating,and these genes may not have yet beenisolated.A recently identified gene Dub (Double

or nothing), is intriguing because, to myknowledge, it is the first mutation thataffects homolog segregation in meiosis Iof males and females (Fig. 3) (42). Thissuggests that the Dub gene could providea common pairing function in both sexes,or it could be involved in another aspect ofhomolog partitioning used in both sexes.The Dub mutation is a conditional domi-nant allele that leads to predominantlymeiosis I nondisjunction in males. In mu-tant females exchange chromosomes non-disjoin, but nonexchange chromosomesare affected most severely. Nonexchangechromosomes nondisjoin during meiosis Iin Dub mutant females, whether they arehomologous or heterologous. The Dubmutation also is a recessive conditionallethal, producing phenotypes consistentwith extensive cell death or mitotic abnor-malities. The possible effect of Dub onmitosis raises the potential for the Dubgene product to have a general function insegregation in all divisions. Further inter-pretation of the role of the Dub gene inmale and female meiosis requires analysisof the loss-of-function phenotypes.

Sister-Chromatid Cohesion

The cytological techniques available toDrosophila researchers have permitted in-vestigation of a critical aspect of chromo-some segregation that has remained elu-sive in most other systems: the functionsthat hold replicated sister chromatids to-gether until the metaphase/anaphasetransition (43). Mutations in genes encod-ing proteins required to hold sister chro-matids together would cause prematureseparation of the sister chromatids in mei-osis or mitosis, depending on which divi-sion they affected. Premature separationof the sister chromatids is identified mostdefinitively if it can be directly visualized(Fig. 2D). Two strong candidates for pro-teins having a primary role in maintainingsister-chromatid cohesion in meiosis arethe Drosophila ord and mei-S332 genes.Mutations in these genes cause aberrantmeiotic chromosome segregation in fe-males and males, and they have beenobserved to cause premature sister-chromatid separation in male meiosis (44-48).

Premature sister separation occurs atdifferent times in the two mutants. Cytol-ogy suggests different control of sister-

chromatid cohesion early versus later inmeiosis (43). Early in meiosis I the sisterchromatids are held together along theirentire length, as they are in mitosis. Thiscohesion along the chromatid arms hasbeen postulated to stabilize chiasmata inorganisms with chiasmate meiosis (14). Atthe metaphase I/anaphase I transitioncohesion along the arms of the sisterchromatids is lost, and they remain at-tached only at the centromere regionsuntil the metaphase II/anaphase II tran-sition (Fig. 2F). Thus, based on the cyto-logical observations, either there are dif-ferent functions controlling sister-chro-matid cohesion early and late in meiosis orthe release of cohesion is spatially con-trolled so that it persists in the centromereregion until anaphase II.Even in apparent null mutations of mei-

S332 sister-chromatid cohesion is normaluntil late in anaphase I (45, 46). Thisobservation suggests that the MEI-S332protein might act specifically to maintaincohesion in the centromere regions of thechromosomes (Fig. 2 D and E) (46). Incontrast to meiosis, sister-chromatid co-hesion is released in one step in mitosis atthe metaphase/anaphase transition. Mu-tations in the mei-S332 gene have no effecton mitosis, and the gene is transcribedstrongly during developmental stageswhen meiosis is occurring but not duringthe larval stages when mitosis occurs (49).These results argue that mei-S332 plays aspecific role in meiosis to hold sister chro-matids together at their centromere re-gions.The mei-S332 gene has been shown

recently to encode a novel 44-kDa proteinthat is highly charged (49). The MEI-S332protein was localized by fusing it to thegreen fluorescent protein from jellyfish(49). The MEI-S332-GFP fusion protein isfully functional, because it rescues mei-S332 mutants. MEI-S332-GFP localizesto the centromeric region of meiotic chro-mosomes from prophase I until meta-phase II. Strikingly, as the sister chroma-tids separate in anaphase II and cohesionis lost, MEI-S332-GFP is no longer de-tectable on the chromosomes. These re-sults indicate that MEI-S332 binds to cen-tromere regions and holds sister chroma-tids together and that it must dissociate topermit segregation.

In contrast to the specific effect ofmei-S332 on meiosis, mutations in the ordgene affect several aspects of meiosis aswell as mitosis (47-50). Premature sister-chromatid separation is observed in malemeiosis as early as prometaphase I (45,48). Null alleles give missegregation con-sistent with ratios predicted from prema-ture separation and random segregationof the four sister chromatids through bothmeiotic divisions (48). This is true for maleand female meiosis. Thus, it appears thatthe ord gene is necessary to maintainsister-chromatid cohesion beginning early

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in meiosis I when the sister chromatids areheld along their lengths. One prediction isthat the ORD protein may be localized onthe chromosome arms as well as at thecentromere regions.

In addition to loss of sister-chromatidcohesion, most mutations in the ord genecause a reduction of recombination infemale meiosis, and the exchange thatdoes occur is not sufficient to ensureproper segregation (47, 48). This is con-sistent with the prediction that prematureloss of sister-chromatid cohesion wouldresult in loss of chiasmata. ord may haveseveral independent functions in meiosis,or the effects on recombination and cohe-sion may be related. Moreover, strong ordmutations result in mitotic nondisjunction inthe male germline (48, 50). However, thesealleles show almost no effect on somaticmitotic divisions, so the requirement for thegene may be restricted to divisions in thegerm line (J. Wu, W. Miyazaki, and T.L.O.-W., unpublished results).The ord gene has been isolated recently

(S. Bickel, D. Wyman, W. Miyazaki, D.Moore, and T.L.O.-W., unpublished re-sults). There are different forms of the ordtranscript in testis and ovaries. A commonopen reading frame shared by the testistranscripts predicts a 55-kDa protein. Thesix ord mutations cause either stop codonsor missense changes within the C-terminalhalf of this open reading frame. Sincethese affect female as well as male meiosis,at least the C-terminal half of the proteinis common to both sexes. It remains pos-sible that there may be female or mitoticforms of the protein with differing Ntermini. These potentially different formsof the protein could account for the pleio-tropic effects of ord mutations.

Centromere Structure

Ultimately it will be necessary to deter-mine how segregation proteins interactwith the chromosomes to partition themcorrectly during meiosis. In particular it isessential to identify the proteins that act atthe centromere. The prerequisite forachieving this goal is to define the centro-mere at a molecular level. Drosophila hasyielded a recent important advance in thisarea. The centromeres of the chromo-somes in higher eukaryotes are complex,and they have eluded molecular charac-terization because they are embedded inheterochromatin. By studying the struc-ture and function of a minichromosome,Karpen and colleagues (51, 52) have beenable to localize a centromere.The free duplication Dp187 is a dele-

tion derivative of the X chromosome thatis only 1.3 Mb in size and is stably trans-mitted (51, 52). Deletions within the 1 Mbof heterochromatin on this minichromo-some were generated, mapped, and testedfor their effects on transmission (52, 53).The genetic assay for transmission re-

quires stability in germ-line mitotic divi-sions, meiosis, and somatic mitotic divi-sions. This analysis defined a region of 220kb essential for normal transmission of theminichromosome (52). This interval is anisland of complex DNA that is single copyor moderately repetitive DNA. Com-pletely normal transmission requires anadditional 200 kb of satellite DNA oneither side of the essential region. Inter-estingly, transmission of the minichromo-some through females is considerablymore sensitive to the effect of deletionsthan is transmission in males. The delin-eation of a centromere will permit a com-parison of cis-acting requirements for mei-osis and mitosis. It will be interesting todetermine whether different centromerefunctions are needed during the first meioticdivision. These minichromosome deletionsmay make it possible to map the sites ofMEI-S332 action and to determine whetherMEI-S332 localizes to the kinetochore orflanking centric heterochromatin.

Cell Cycle Regulation of Meiosis

A complete understanding of meiosis re-quires that we know the mechanisms bywhich chromosomes pair and segregatebut also that we elucidate the regulation ofthe steps of meiosis. Meiosis can beviewed as a variant of the normal mitoticcycle in which an extra, specialized divi-sion with homolog pairing and segregationis inserted after DNA replication. In ad-dition, in multicellular organisms, femalemeiosis is arrested during oogenesis topermit growth and differentiation of theoocyte. Developmental signals subse-quently trigger the completion of meiosis.In the case of Drosophila, the oocyte isarrested at metaphase I and the signal tocomplete meiosis involves movement intothe uterus, although the molecular basis ofthis activation is unknown. The identifi-cation of the cell cycle regulators of mei-osis is still preliminary, but recently sev-eral control genes have been isolated inDrosophila.

Several meiotic cell cycle control geneshave been identified because they alsohave a role in the mitotic cell cycle. Thetwine gene was isolated as a homolog ofstring (stg), a mitotic cell cycle regulator,and it is needed for early steps in themeiotic cell cycle in males (54, 55). twineencodes a tyrosine phosphatase. twineand string are homologs of the cdc25 geneof Schizosaccharomyces pombe, a positiveactivator of cdc2. The germ-line mitoticdivisions take place in twine mutantmales, but meiosis does not occur (54, 55).Although no spindles are formed, someaspects of meiosis such as chromosomecondensation are observed in twine mu-tants (56).The coordination of the two meiotic

divisions in males requires the properdosage of the product of the roughex (rux)

gene (57). In loss-of-function mutations ofroughex the two meiotic divisions occur,but they are followed by another inappro-priate division. Overexpression of roughexblocks the meiosis II division. Reducingthe dosage of Cyclin A (CycA) or twinecan suppress the roughex phenotype; thus,it appears that roughex acts through Cy-clin A/cdc2 to control the second meioticdivision. The effect of roughex mutationson female meiosis has not been investi-gated.The meiotic arrest of oocytes is signaled

in part by exchange events (58). In recom-bination-deficient mutants, mature oocytesare not arrested at metaphase I but areobserved in anaphase I, metaphase II, oranaphase II. An exchange event on onechromosome pair is sufficient to cause ar-rest, so it is not simply that chromosomes arephysically constrained to the metaphaseplate by chiasmata. However, it is not theexchange itself that causes arrest. Jang et aL(59) produced an "all-compound" strain inwhich the two homologs of each of themajor chromosomes were attached to thesame kinetochore. Although these com-pound chromosomes undergo exchange,there is no metaphase I arrest. Thus, itappears that the tension resulting from anexchange (and presumably chiasma) be-tween two chromosomes with separate kin-etochores sends a signal for meiosis arrest(59). In twine mutant females meiosis ini-tiates but fails to arrest at metaphase I,suggesting twine may be involved in thesignaling process (55, 56). twine mutantoocytes undergo a series of aberrant nucleardivisions and DNA replication.The Drosophila oocyte is activated to

complete meiosis by movement into theuterus, and fertilization is not required forthis process. The molecular basis of acti-vation remains a mystery, although theoocyte becomes hydrated upon oviposi-tion. Moreover, the completion of meiosiscan be efficiently activated in vitro byswelling in hypotonic buffers (Fig. 1 B andC). Eggs from mothers mutant for thegene grauzone or cortex fail to completemeiosis and arrest at metaphase II (A.Page and T.L.O.-W., unpublished results).These mutations may disrupt the signalsfor the completion of meiosis.

Future Directions

The cytological techniques and geneticsavailable in Drosophila have permittedgreat strides in our understanding of ho-molog pairing and segregation in meiosisI and mechanisms of sister-chromatid co-hesion. Other important proteins will beidentified by cloning genes known to beessential for meiosis. Insights into thefunction of these proteins will result fromanalysis of their localization on meioticchromosomes or spindles. Furthermore,antibodies to these proteins will facilitateinvestigation of the interaction and rela-

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tionships between meiotic proteins by ex-amining protein behavior in mutants. Thefoundation for the isolation of proteinsacting at the kinetochore has been laid bythe delineation of the centromere. In ad-dition to these advances in our under-standing of meiotic mechanisms, given thepace of the mitotic cell cycle field, it isanticipated that we will soon have aclearer image of the control of the meioticcell cycle.

I thank S. Endow, R. S. Hawley, and G.Karpen for providing reprints and preprints. Iam grateful to B. McKee and W. Theurkauf forpermission to cite unpublished results. A. Ker-rebrock, W. Miyazaki, A. Page, and W. Theur-kauf kindly provided cytology photographs,and H. Protzman prepared the figures. G.Karpen, B. Reed, A. Page, S. Bickel, D. Moore,and A. Kerrebrock critically read the manu-script. This work was supported by NationalScience Foundation Grant MCB 9316168.

1. Cooper, K. (1950) in Biology of Drosoph-ila, eds. Demerec, M. (Wiley, New York),pp. 1-61.

2. Goldstein, L. S. B. (1981) Cell 25, 591-602.

3. Lin, H.-P., Ault, J. G. & Church, K. (1981)Chromosoma 83, 507-521.

4. Meyer, G. F. (1960) in Proceedings of theEuropean Regional Conference on ElectronMicroscopy, eds. Houwink, A. L. & Spit,B. J. (Nederlandse Vereniging voor Elec-tronenmicroscopie, Delft, The Nether-lands), pp. 951-954.

5. Rasmussen, S. W. (1973) Z. Zellforsch.140, 125-144.

6. Cenci, G., Bonaccorsi, S., Pisano, C.,Verni, F. & Gatti, M. (1994) J. Cell Sci.107, 3521-3534.

7. Church, K. & Lin, H. P. P. (1982) J. CellBiol. 93, 365-373.

8. Church, K. & Lin, H. P. P. (1985) Chro-mosoma 92, 273-282.

9. Carpenter, A. T. C. (1975) Chromosoma51, 157-182.

10. Carpenter, A. T. C. (1975) Proc. Natl.Acad. Sci. USA 72, 3186-3189.

11. Carpenter, A. T. C. (1979) Genetics 92,511-541.

12. Theurkauf, W. & Hawley, R. S. (1992) J.Cell Biol. 116, 1167-1180.

13. Baker, B. & Hall, J. (1976) in The Geneticsand Biology ofDrosophila, eds. Ashburner,M. & Novitski, E. (Academic, New York),pp. 351-434.

14. Darlington, C. D. (1932) RecentAdvancesin Cytology (Churchill, London).

15. John, B. (1990) Meiosis (Cambridge Univ.Press, Cambridge, U.K.).

16. Nicklas, R. B. (1974) Genetics 78, 205-213.

17. Grell, R. F. (1962) Genetics 47, 1737-1754.

18. Grell, R. (1976) in The Genetics and Bi-ology ofDrosophila, eds. Ashburner, M. &Novitski, E. (Academic, New York), pp.436-486.

19. Hawley, R. S., Irick, H., Zitron, A. E.,Haddox, D. A., Lohe, A., New, C., Whit-ley, M. D., Arbel, T., Jang, J., McKim, K.& Childs, G. (1993) Dev. Genet. 13, 440-467.

20. Hawley, R. S., McKim, K. S. & Arbel, T.(1993) Annu. Rev. Genet. 27, 281-317.

21. Carpenter, A. (1973) Genetics 73, 393-428.

22. Zhang, P. & Hawley, R. S. (1990) Genetics125, 115-127.

23. Zhang, P., Knowles, B. A., Goldstein,L. S. B. & Hawley, R. S. (1990) Cell 62,1053-1062.

24. Rasooly, R. S., New, C. M., Zhang, P.,Hawley, R. S. & Baker, B. S. (1991) Ge-netics 129, 409-422.

25. Rasooly, R. S., Zhang, P., Tibolla, A. K &Hawley, R. S. (1994) Mol. Gen. Genet.242, 145-151.

26. Davis, D. G. (1969) Genetics 61, 577-594.27. Endow, S. A., Henikoff, S. & Soler-Niedz-

iela, L. (1990) Nature (London) 345,81-83.28. McDonald, H. B. & Goldstein, L. S. B.

(1990) Cell 61, 991-1000.29. McDonald, H. B., Stewart, R. J. & Gold-

stein, L. S. B. (1990) Cell 63, 1159-1165.30. Walker, R. A., Salmon, E. D. & Endow,

S. A. (1990) Nature (London) 347, 780-782.31. Hatsumi, M. & Endow, S. (1992) J. Cell

Sci. 101, 547-559.32. Hatsumi, M. & Endow, S. A. (1992)J. Cell

Sci. 103, 1013-1020.33. Afshar, K, Barton, N. R., Hawley, R. S. &

Goldstein, L. S. B. (1995) Cell 81, 129-138.34. Murphy, T. D. & Karpen, G. H. (1995)

Cell 81, 139-148.35. Hawley, R. S. & Theurkauf, W. E. (1993)

Trends Genetics 9, 310-317.

36. Lindlsey, D. & Zimm, G. (1992) The Ge-nome of Drosophila melanogaster (Aca-demic, New York).

37. Cooper, K. W. (1964) Proc. Natl. Acad.Sci. USA 52, 1248-1255.

38. McKee, B. & Karpen, G. (1990) Cell 61,61-72.

39. McKee, B. D., Habera, L. & Vrana, J. A.(1992) Genetics 132, 529-544.

40. McKee, B. D., Lumsden, S. E. & Das, S.(1993) Chromosoma 102, 180-194.

41. Moore, G. D., Sinclair, D. A. & Grigliatti,T. A. (1983) Genetics 105, 327-344.

42. Moore, D. P., Miyazaki, W. Y., Tomkiel,J. E. & Orr-Weaver, T. L. (1994) Genetics136, 953-964.

43. Miyazaki, W. Y. & Orr-Weaver, T. L.(1994) Annu. Rev. Genet. 28, 167-187.

44. Davis, B. (1971) Mol. Gen. Genet. 113,251-272.

45. Goldstein, L. S. B. (1980) Chromosoma78, 79-111.

46. Kerrebrock, A. W., Miyazaki, W. Y.,Birnby, D. & Orr-Weaver, T. L. (1992)Genetics 130, 827-841.

47. Mason, J. M. (1976) Genetics 84,545-572.48. Miyazaki, W. Y. & Orr-Weaver, T. L.

(1992) Genetics 132, 1047-1061.49. Kerrebrock, A. W., Moore, D. P., Wu,

J. S. & Orr-Weaver, T. L. (1995) Cell 83,in press.

50. Lin, H. P. & Church, K. (1982) Genetics102, 751-770.

51. Karpen, G. H. & Spradling, A. C. (1990)Cell 63, 97-107.

52. Murphy, T. D. & Karpen, G. H. (1995)Cell 82, 599-609.

53. Le, M.-H., Duricka, D. & Karpen, G. H.(1995) Genetics 141, 283-303.

54. Alphey, L., Jimenez, J., White-Cooper,H., Dawson, I., Nurse, P. & Glover, D.(1992) Cell 69, 977-988.

55. Courtot, C., Fankhauser, C., Simanis, V.& Lehner, C. F. (1992) Development(Cambridge, U.K) 116, 405-416.

56. White-Cooper, H., Alphey, L. & Glover,D. M. (1993) J. Cell Sci. 106, 1035-1044.

57. Gonczy, P., Thomas, B. J. & DiNardo, S.(1994) Cell 77, 1015-1025.

58. McKim, K. S., Jang, J. K., Theurkauf,W.E. & Hawley, R. S. (1993) Nature(London) 362, 364-366.

59. Jang, J. K, Messina, L., Erdman, M. B.,Arbel, T. & Hawley, R. S. (1995) Science268, 1917-1919.

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