MEIOSIS AND EARLY CLEAVAG INE MELANOGASTER EGGS: O … · MEIOSIS AND EARLY CLEAVAG INE DROSOPHILA...

J. Cell Sci. 62, 301-318 (1983) 301Printed in Great Britain © The Company of Biologists Limited 1983

MEIOSIS AND EARLY CLEAVAGE IN DROSOPHILA

MELANOGASTER EGGS: EFFECTS OF THE CLARET-NON-DISJUNCTIONAL MUTATION

MARY KIMBLE* AND KATHLEEN CHURCHDepartment of Zoology, Arizona State University, Tempe, AZ 85287, U.SA.

SUMMARY

The claret-non-disjunctional (ca1"1) mutation is a female-specific mutation in Drosophilamelanogaster, which causes a high frequency of mortality (89-90 %) in eggs laid by the homozygousfemales. Among the progeny that survive to the adult stage, greater than 50 % are aneuploid and/or mosaic for the X and number 4 chromosomes. The genetic studies of ca'd indicate that it ishomologous to the claret mutation (ca-simulans) in Drosophila simulans.

The cytological effect of the ca1"1 mutation on meiosis I and the early cleavage divisions has beenobserved at the light microscope level in stage 14 ovarian and fertilized uterine eggs, respectively.Four classes of metaphase I figures were observed. These include those with: (1) two or morespindles; (2) spindles that were abnormally wide and the bivalents widely separated; (3) unipolarspindles; and (4) apparently normal bipolar spindles. The three abnormal classes of metaphase Ifigures included 80 % of the eggs examined at this stage. Among the cleavage stage eggs examined69 % showed highly abnormal mitotic figures, including multipolar spindles, and the nuclei in theseeggs were found in clusters, rather than dispersed throughout the ooplasm.

In addition to the cytological abnormalities observed, 17-23 % of the eggs produced by the ca1"1

females showed morphological abnormalities. These abnormalities included eggs having three orfour dorsal filaments, eggs that had a truncated shape, and abnormally small eggs. These abnor-malities may not be an aspect of the ca™1 syndrome, but they are due to recessive genes located onthe third chromosome.

Although the cand and ca-simulans mutations both affect the formation of the spindle apparatusduring meiosis and the early cleavage divisions, the effects of these two mutations differ considerablyin detail. The effect of the ca-simulans mutation appears to be more severe than the effect olcand.

A model to explain the relationship between the effect of the ca1^ mutation on meiosis I and theearly cleavage divisions is presented, and evidence to support the model is discussed.

INTRODUCTION

Claret-non-disjunctional (ca"d) is an X-ray-induced mutation that was isolated byLewis & Gencarella (1952). The mutation has been shown to be allelic to the standardclaret (ca) eye colour mutation in Drosophila melanogaster (Chan & Davis, 1970),located at map position 100-7 on the right arm of chromosome 3 (Bridges & Morgan,1923; cited by Lindsley & Grell, 1968). Flies heterozygous for ca/cand have the mutanteye colour phenotype but not the meiotic phenotype observed in cd"1 homozygotes. Theclaret eye colour has been shown to be the result of a block in the pteridine biosyntheticpathway (Chan & Davis, 1970). These authors have also shown that the levels of variousintermediates of the pteridine pathway are the same in ca/ca, ca/cand and cand/ca"d

* Author for correspondence at: Department of Biology, Indiana University, Bloomington,IN47405, U.S.A.

302 M. Kimble and K. Church

flies. This suggests that the eye colour phenotype and the meiotic phenotype areactually due to different genes. In addition, these authors (Chan & Davis, 1970) werenot able to separate the two mutant phenotypes through crossing over, suggesting thatthe co!"1 mutation is probably the result of a deletion that covers the two loci.

Eggs laid by females homozygous for the ca™* mutation show a high mortality rateand over 50% of the progeny that survive to the adult stage are either aneuploidand/or mosaic for the X and/or the fourth chromosome (Lewis & Gencarella, 1952).Among the regular gynandromorph progeny, the chromosome lost is usually thematernal X chromosome (Lewis & Gencarella, 1952; Davis, 1969), although Hinton& McEarchern (1963) reported a low frequency of gynandromorphs attributable toloss of the paternal X. Additional studies have shown that ca"4 causes a high frequencyof non-disjunction of all chromosome pairs at meiosis I, chromosome loss at meiosisII and chromosome loss during the early cleavage divisions (Hinton & McEarchern,1963; Davis, 1969). In addition, Davis observed that the frequency of flies attribut-able to diplo-X diplo-4 ova and nullo-X nullo-4 ova was greater than expected if non-disjunction or loss of one chromosome was independent of the behaviour of a secondchromosome during meiosis. Davis also noted that chromosome loss during the mitot-ic divisions was not random. This latter observation has been confirmed by Portin(1978). Despite the high frequency of non-disjunction observed during meiosis I inca"d females, cand does not appear to affect the frequency of recombination along theX chromosome (Hinton & McEarchern, 1963; Davis, 1969).

The studies discussed above indicate that ca"d is genetically homologous to theclaret mutation (ca-simulans) in D. simulans, which was isolated from a naturalpopulation of D. simulans by Plunkett in 1924 and genetically characterized bySturtevant (1929). In addition, Sturtevant & Plunkett (1926) have shown, through

Table 1. Euploid, aneuploid and mosaic offspring among the progeny of claret-non-disjunctional females

X-chromosome phenotypes

Regular females (XX)Exceptional females (XXY)Gynandromorphs (XX-XO)

& (XXY-XY)fRegular males (XY)Exceptional males (XO)

4th-chromosome phenotypes

Triplo anddiplo-4

2022(21-8)*297( 32)

242( 2-6)1778(19-1)616( 6-6)

Haplo-4

1145(12-3)10( 0-1)

122( 1-3)1406(15-1)1228(13.2)

Haplo-4mosaic

107(1-2)20(0-2)

79(0-9)119(1-3)106(1-1)

Total

3274(35-2)327( 3-5)

443( 4-7)3303(35-5)1950(21-0

Total 4955(53-3) 3911(421) 431(4-6) 9297

Data collected by C. W. Hinton, published by Davis (1969). Note: The totals for the regularfemale class, the haplo-4 mosaics and total progeny as published by Davis (1969) were not consistentwith the numbers given in the body of the table. These three numbers and the percentages werechanged so that all numbers are internally consistent.

• Numbers in parentheses are percentages of total progeny in each class.f Data from these two classes were combined to facilitate comparison with data shown in Table 2.

Meiosis and early cleavage in Drosophila eggs 303

interspecific hybridization tests, that the ca-simulans locus is homologous to thestandard claret eye colour locus in£). melanogaster. The classes of normal, aneuploidand aneuploid-mosaic progeny produced by ca-simulans females are the same as seenwith ca"d, however the frequency of progeny in 6 of the 15 classes differs (see Tables1 and 2). These differences indicate that chromosome loss at meiosis II is morecommon in ca-simulans than in cand, although some of the XO and/or haplo-4progeny could be due to loss of a chromosome from both nuclei at the first cleavagedivision. Chromosome loss during the early cleavage divisions appears to be slightlyhigher in ca"d than in ca-simulans.

The cytological effect of the ca-simulans mutation on female meiosis and earlycleavage has been studied by Wald (1936). She observed that the first meiotic spindlewas distorted, being abnormally wide, and the chromosomes became separated intoseveral widely scattered groups. Some spindles appeared to be focused at one polewhile at the other pole the spindle fibres diverged, also more than one spindle figurewas occasionally observed at this stage. During the time when second meiosisoccurred in her controls the sister chromatids in the mutant eggs separated, but onlyrarely was this accompanied by the formation of a spindle apparatus. Followingseparation of the sister chromatids, the separate groups of chromosomes becamesurrounded by membranes, forming the equivalent of polar nuclei, which Waldreferred to as vesicular nuclei. The number of vesicular nuclei varied from 4 to 12depending on how widely dispersed the chromosomes had become during the preced-ing meiotic divisions. Following formation of the nuclear membranes, one or more ofthe vesicular nuclei would move towards the centre of the egg where the malepronucleus was located. In D. simulans, as in D. melanogaster, the male and femalepronuclei form separate but closely apposed half-spindles for the first cleavagedivision. In the mutant eggs the female pronucleus failed to form a spindle unless atleast two large metacentric chromosomes were present within a single vesicularnucleus, although the spindle of the male pronucleus formed normally. In those eggs

Table 2. Euploid, aneuploid and mosaic offspring among the progeny of ca-simulansfemales

4th-chromosome phenotypes

TriploX-chromosome phenotypes

Regular females (XX)Exceptional females (XXY)Gynandromorphs (XX-XO)

& (XXY-XY)Regular males (XY)Exceptional males (XO)

Total

Combined data from Tables 2, 3 and 4 published by Sturtevant (1929).• Numbers in parentheses are percentages of total progeny in each class.

Triplo anddiplo-4

226(12-2)*22( 1-2)

26( 1-4)213(11-5)142( 7-7)

629(340)

Haplo-4

373(20-2)16( 0-9)

30( 1-6)285(15-4)423(22-9)

1127(60-9)

Haplo-4mosaic

36(1-9)4(0-2)

10(0-5)21(1-1)23(1-2)

94(51)

Total

635(34-3)42( 2-3)

66( 3-6)519(28-1)588(31-8)

1850


in which the female pronucleus failed to form a spindle, both pronuclei eventuallydegenerated. If the female pronucleus was able to form a spindle for the first cleavagedivision, then the syncytial divisions occurred as in the controls.

Because of the genetic homology of the cand and ca-simulans mutations, a cyto-logical analysis of cand was undertaken. The purpose of the study was to determine:(1) what effect ca"d has on meiosis and early cleavage; (2) if this effect is identical tothat observed in ca-simulans; and (3) if the genetic effects of ca"d can be explainedby the cytological observations.

MATERIALS AND METHODS

Drosophila stocks

All Drosophila stocks were raised at room temperature on the standard cornmeal/molasses/yeastagar medium, having buffered propionic acid as inhibitor, as used at Arizona State University. Thewild-type stock used was an isogenic Oregon-R (Ore-R) strain, obtained from Dr Winifred W.Doane at Arizona State University. The mutant stock, e',ca'"1/TM6, was obtained from theDrosophila Stock Center at the California Institute of Technology, Pasadena, CA. It washeterozygous for the third chromosome balancer TM6, marked with bithorax (bx34"), Ultrabithorax(Llbx* ) and ebony (e), and the standard third chromosome marked with ebony-sooty (e1) andclaret-non-disjunctional (ca1"1) (see Lindsley & Grell, 1968, for a description of genetic markers).

To reduce the differences in the genetic background between the wild-type and mutant stocks,mutant flies were outcrossed to the Ore-R strain and reisolated through eight generations ofoutcrossing. Males homozygous for the standard no. 3 chromosome were then mated to femalescarrying the balancer chromosome. The F| progeny from this cross, which had the Ubx phenotype,were then sib mated to obtain a mutant stock in which it is reasonable to assume that most of theresidual genetic background came from the Ore-R strain.

To ensure that the mutant stock having a predominantly Ore-R genetic background, hereaftere'jCa™1/TM6 (Ore-R), retained the cd"1 meiotic phenotype, an egg hatchability study was doneusing the technique described by King (1955). The results are shown in Table 3. As the 10-1 %hatchability obtained for the e',caad/TM6 (Ore-R) was in better agreement with the 11 % hatch-ability reported by Davis (1969) for ca1"1, than was either of the rates obtained for the es,ca"d/TM6stock, all cytological observations were made on eggs from the e',caHd/TM6 (Ore-R) stock.

Fixation and embeddmentFixation of stage 14 ovarian and uterine eggs was done according to the procedure developed by

Zalokar & Erk (1977) for fixing Drosophila embryos. The procedure involves a primary fixation inbuffered glutaraldehyde, dissolved in heptane. The heptane is able to cross the hydrophobic vitelline

Table 3. Results of egg hatchability studies

Mutantstocks

First daySecond dayThird day

Total

e'.ca^/e'.ca1"*;Ore-R background

Totaleggs

987823561

2371

Numberhatched

1067361

240

%Hatched

10-78-9

10-9

10-1

<

Totaleggs

491417290

1198

>l,ca'"'/e',cam'\original stock

series 1

Number %hatched Hatched

532717

97

10-86-55-9

8 1

t

Totaleggs

846537291

1674

•',cand/e',ca'"1\original stock

series 2

Numberhatched

31199

59

%Hatched

3-73-53-1

3-5


membrane but does not diffuse into the ooplasm. However, the glutaraldehyde is free to diffuse intothe ooplasm (Zalokar & Erk, 1977). After 3-5 min in this primary fixative the vitelline membranecan be removed and the eggs placed in 5 % buffered glutaraldehyde for 2-3 h, following which theeggs are post-fixed in 1 % OsO4 for 2 h. The eggs were then stained en bloc with 2 % aqueous uranylacetate at 60cC for 1-2h, dehydrated through an ethanol/water series (30%, 50%, 70%, 95%,100% (2 changes), 12 min each change) followed by two changes (15 min) in 100% propyleneoxide, which is a transition solvent between ethanol and Epon. The egg ends were infiltrated withEpon 812 resin (Luft, 1961) through an Epon/propylene oxide series (1:3, 1:1, 3:1, 2h each)followed by two changes of 100% Epon over 24 h, then placed in moulds with fresh 100% Eponand polymerized at 70 °C for 24-48 h.

Eggs in the first meiotic division were obtained by removing the ovaries from 3 to 4-day-oldfemales and teasing the stage 14 oocytes out of the ovaries. Eggs in the early cleavage divisions werecollected from mated females by etherizing a few (5) flies at a time and gently squeezing theabdomen. 1 f an egg is present in the uterus, the pressure caused by squeezing the abdomen will causeit to slide out. For both ovarian and uterine eggs, the chorion was removed before placing the eggsin the primary fixative. Both the chorion and the vitelline membrane were removed, at theappropriate times, by placing the eggs on double-sticky tape and gently stroking the egg with atungsten needle to break the envelope.

Sectioning and staining for light microscopyEntire eggs were serially sectioned using glass knives mounted on a Sorvall MT-2B ultramicro-

tome. Section thickness was 1 ^m. Ribbons of sections were transferred, using a wire loop, to acid-washed glass slides and dried down on a warm hot-plate. Slides were then rinsed briefly in cold1 M-HCI and hydrolysed in 1 M-HCI at 60 °C for 30 min. Following hydrolysis, the slides were againrinsed in cold 1 M-HCI, followed by a single rinsing in tap water, and then stained for 3 h in Feulgenstain. They were then rinsed in running tap water for 10 min and counterstained with 4% Giemsabuffered with O'lM-sodium phosphate buffer (pH68) . A glass coverslip was placed over thesections, using Permount as the mounting medium. Sections of interest were photographed on aZeiss phase-contrast microscope.

Sectioning and staining for electron microscopyTo obtain material for electron microscopy, eggs were first sectioned on glass knives (as above)

cutting 3 nm thick sections. The sections were transferred to a glass slide, dried down on a warmhot-plate and covered with a glass coverslip using glycerol as the mounting medium. Sections ofinterest were located and photographed under phase contrast.

The sections were then remounted on Epon pegs according to the procedure of Mogensen (1961),and thin-sectioned for electron microscopy. Ribbons of thin sections (approx. 0-1 fim in thickness)were cut using a Dupont diamond knife mounted on a Sorvall MT-2B ultramicrotome. The sectionswere transferred to Formvar-coated oval-hole grids using a carrier grid, according to the techniqueof Moens (1970). Sections were stained with 2 % aqueous uranyl acetate at 60 °C for 90 min and post-stained with bismuth subnitrate (Riva, 1974) at room temperature for 15 min. Sections were viewedon a Philips 300 electron microscope at 60keV and recorded on 35 mm film.

RESULTS

Meiosis and early cleavage divisions

Meiosis I in wild-type D. melanogaster females is characterized by a typical bipolarspindle (Fig. 1A). The average pole-to-pole distance at metaphase I is approximately11—\2pLm and the width of the spindle region is 4-5 /Jm. These dimensions are basedon measurements of the spindle region in light micrographs of nine serially sectionedstage 14 ovarian eggs from Ore-R females. Metaphase I in D. melanogaster is definedas the stage when the two large autosomal bivalents and the X bivalent are located at


Fig. 1. Longitudinal sections through the metaphase I spindle in stage 14 ovarian eggsfrom the Oregon-R wild-type (A) and the camd mutant (B) showing the typical bipolarappearance. Bar, 10 /*m.

the equatorial plane of the spindle. The small fourth chromosomes have alreadyseparated and are located on either side of the equator. In addition, it was noted thatamong the wild-type eggs examined, all those that had distinct chromosomes andspindle elements, also met the criteria for being classified as metaphase I.

Like the ca-simulans mutation, cand affects the formation of the spindle for the firstmeiotic division. However, a greater diversity of effects than that described by Wald(1936) for ca-simulans, was observed. A total of 22 ovarian eggs from ca"d femaleswere examined. In six of these the nucleus was in the condensed karyosome stageindicating that the eggs were in stage 13 rather than stage 14 of oogenesis. This is notunique to eggs produced by the ca"d females. Similar frequencies of eggs, whosenuclei were in the karyosome stage, were observed among Ore-R eggs thought to bein stage 14. In an additional six eggs no nuclear region was observed. This wasprobably due to loss of sections during transfer or staining. All 10 of the remainingovarian eggs showed distinct chromosomes and spindle elements. Among these 10eggs, three classes of abnormalities were observed: (1) two or more spindles present(n = 3); (2) abnormally wide spindles and widely separated bivalents (n — 3); and (3)unipolar spindles (n — 2). In the two remaining eggs in which spindles were observed,the spindles had the normal bipolar appearance (Fig. 1B). Fig. 2 shows one of the eggsin which two separate spindles have formed. There was one section, separating thesections in A and B, in which traces of both spindles appeared, but unfortunately thissection also had a wrinkle through the area of interest and could not be photographed.The long axes of the two spindles are at right angles to each other and each spindlehas chromatin associated with it, suggesting that both spindles function in attachingto chromosomes. An example of the second class of spindle abnormality is shown inFig. 3A. Four separate groups of chromosomes (presumably the four bivalents) areobserved but the bivalents are widely separated compared to the wild-type (Fig. 3B)and the diameter of the spindle region in the mutant egg is approximately twice the

Meiosis and early cleavage in Drosophila eggs 10?

Fig. 2. Ovarian egg from the caF* mutant showing two spindles at metaphase I. The longaxes of the two spindles, indicated by the double-headed arrows, are at right-angles andeach spindle has chromatin (c) associated with it. There was one section, separating thetwo sections shown, in which traces of both spindles appeared. Bar, 10\isn.

Fig. 3. Transverse sections through the metaphase I plane of a mutant egg (A) and a wild-type egg (B). The diameter of the spindle region in the mutant egg is approximately twicethat in the wild-type. The four bivalents in the mutant egg, indicated as a, b, c, d, arewidely separated and in two of these (b, d) the homologous chromosomes appear to beseparating. Bar, 10(im.

diameter of the spindle region observed in the wild-type. Also in two of the bivalentsin the mutant egg (Fig. 3A) the homologous chromosomes appear to be separating,indicating that the spindle, although abnormal, can initiate anaphase I. Fig. 4 showsone of the unipolar spindles observed in ovarian eggs from the cand females.

Many attempts were made to observe the effect of the ca!^ mutation on the secondmeiotic division. The attempts were unsuccessful. It is possible that the failure toobtain eggs at this stage is due to the method used for collecting eggs for fixation.Uterine eggs from rapidly laying females were collected, rather than recently laid

M. Kimble and K. Church

Fig. 4. Ovarian egg from the cd"1 mutant showing a unipolar spindle. The fourmicrographs are from consecutive sections. The long axis of the spindle is indicated by anarrow, with the arrowhead at the polar end. No evidence of spindle fibres was seen distalto the chromatin, opposite the spindle pole. Bar, 10/im.

eggs, on the assumption that uterine eggs would, on the average, be in an earlier stageof development than freshly laid eggs. Out of 33 uterine eggs collected from virginfemales all had completed meiosis. In some eggs the four haploid nuclei were present,while in others the four nuclei had begun to fuse. In all of the uterine eggs collectedfrom inseminated females, six from Ore-R females and 13 from ca"d females, develop-ment had reached the early cleavage divisions. Turner & Mahowald (1976) mentionedthat the females of the Ore-R strain of D. melanogaster they worked with showed atendency to retain a fertilized egg in their uterus prior to oviposition. Perhaps in-dividual females vary in the length of time they retain fertilized eggs, and thosefemales from which a uterine egg was obtained were the ones that tend to retain theireggs for the longer period of time.

The present observations of the effect of ca"d on the early cleavage divisions differ


Fig. 5. Cleavage-stage mitotic figures in two different eggs from the cat"1 mutant, A.Shows A nucleus («) characterized by large amounts of condensed chromatin (c), but nodistinct chromosomes, suggesting a mid to late prophase. B. Shows a nucleus in earlyprophase as indicated by the small areas of condensed chromatin. Note the abnormalshapes of both nuclei. Bar,

6A f . 7

Fig. 6. Two nuclei from a single mutant egg showing different stages of mitosis, A. Showsa nucleus (n) in mid to late prophase; B, shows a nucleus in prometaphase or metaphaseas indicated by the presence of distinct chromosomes (c). The spindle of this latter nucleusappears to be tripolar. The two nuclei were located approximately 5 /jm apart. Bar, \0fim.

significantly from the effect of ca-simulans (Wald, 1936). Thirteen fertilized eggs, inearly cleavage divisions, were sectioned. None of these 13 eggs showed signs ofdegeneration. In four of these eggs the mitotic figures appeared normal while in ninethe cleavage nuclei were highly abnormal in appearance. Examples of these abnormalnuclei are shown in Figs 5 and 6. Fig. 5A, B shows micrographs of the nuclei from twodifferent eggs. In one case (Fig. 5A) the nucleus is characterized by the presence oflarge amounts of condensed chromatin, especially at the centre of the nucleus. Dis-tinct chromosomes are not observed, suggesting a nucleus in mid to late prophase.


The second nucleus (Fig. 5B) shows very small areas of condensed chromatin indicat-ing early prophase, however the shapes of both nuclei are highly abnormal. Fig. 6A,B shows two different nuclei within a single egg. These nuclei were locatedapproximately 5 fim apart, with one nucleus located peripherally (A) and one moretowards the centre of the egg (B). The one nucleus (Fig. 6A) is in mid to late prophasewhile in the second (Fig. 6B) distinct chromosomes are present, indicating aprometaphase or metaphase stage. Also the spindle of the latter nucleus appears to betripolar. The nuclei of cleavage-stage embryos showing abnormal mitotic figures werealso found in clusters (Fig. 7A) rather than being dispersed throughout the ooplasmas observed in wild-type embryos (Fig. 7B; also see Huettner, 1933; Sonnenblick,1950). The most extreme example of this clustering, observed during this study, isshown in Fig. 8. After the 3 /im thick section shown in Fig. 8A was photographed, itwas remounted on an Epon peg and thin-sectioned for electron microscopy (Fig.8B). There are four large and at least three small regions of nuclear material each ofwhich is surrounded by several layers of membrane, and several layers of membranesurround the entire region. In none of the cleavage-stage eggs from cand that wereexamined were the nuclei either abnormal in shape but dispersed, or normal in shapebut clustered.

Abnormalities of egg morphology

During the hatchability studies and while collecting eggs for fixation, eggs wereoccasionally obtained from the homozygous mutant females that had more than twodorsal filaments (Fig. 9A, B). Other eggs were abnormal in shape, having a truncatedappearance that was due to loss of the extreme anterior end of the egg (Fig. 9B), andstill others were abnormally small (Fig. 9c). To determine what percentage of theeggs, produced by the homozygous mutant females, showed these abnormalities,mass oocyte isolations were undertaken using the procedure of Petri, Wyman &

n

n

Fig. 7. The distribution of cleavage-stage nuclei within the ooplasm. A. From a mutantegg showing nine nuclei (w) clustered together; B, shows three nuclei in a wild-type egg(each nucleus is surrounded by an island of ooplasm). Bar, O

Meiosis and early cleavage in Drosophila eggs f |1

Fig. 8. Light and electron micrographs of the nuclear region in a single mutant egg. A.Light micrograph of a 3/im thick section. After this section was photographed, it wasremounted on an Epon peg and thin-sectioned for electron microscopy, B. An electronmicrograph of one of the thin sections (orientation is reversed relative to A). There are fourlarge and at least three small nuclear regions («), each of which is surrounded by severallayers of membrane (w), and several layers of membrane surround the entire region.Unstained, phase-contrast (A), 2% aqueous uranyl acetate, post-stained with bismuthsubnitrate (B). Bar, 1 fun.

Kafatos (1976) as described by Lovett & Goldstein (1977). Those eggs that could beidentified as stage 14 eggs were scored for size, shape and number of dorsal filaments.The results are given in Table 4.

DISCUSSION

Based on the observations reported here it is clear that the effect of the cand muta-tion on the formation of the first meiotic spindle does show some similarity to theeffect reported for the ca-simulans mutation (Wald, 1936). Eggs were observed


B

Fig. 9. Morphologically abnormal eggs from ca1"1 females, A. An egg that is normal in sizeand shape but has three dorsal filaments. The egg shown in B, has four dorsal filamentsand an abnormal truncated shape, c. A normal egg and an egg that was approximately twothirds of the normal size. All four of these eggs were taken from a single batch of eggs laidby ca"d females during the egg hatchabihty studies. Bar, 0-2 mm.

Table 4. Results of egg morphology counts

Egg morphology

Normal

Abormal in shapereduced size

Excess dorsalfilaments

e'.ca"1/

No. %

152

and/or28

3 - 54 - 0

Total eggs counted 185

• Homozygous ij" Females from

females from thethe e',ca"<'/7M6

82-2

15-1

2-70

original(Ore-R)

e',e',

No.

358

86

231

468

mutantmutant

Genotype

ca"d;\

76-5

18-4

4-90-2

stock,stock.

TM6No.

580

2

00

582

;t%99-7

0-3

00

Oregon-R

No. %

687

3

00

690

99-6

0-4

00

where more than one spindle formed for the first division (Fig. 2) and nuclei wereobserved where the spindle was abnormally wide and the chromosomes were widelyseparated (Fig. 3). However, in contrast to Wald's observations, unipolar and app-arently normal bipolar spindles were also observed during the first meiotic divisionin eggs from ca"d females (Figs 4 and 1, respectively). The effect of cancl on the earlycleavage divisions appears to differ significantly from the effect of ca-simulans. In ca-simulans most of the eggs degenerated without initiating the cleavage divisions, owingto failure of the female pronucleus to form a spindle for the first cleavage division(Wald, 1936). In cand all of the fertilized eggs examined were able to initiate thecleavage divisions. Based on the number of nuclei observed, the eggs were able to


undergo several rounds of division, despite the fact that most of the eggs displayednuclei that were highly abnormal in appearance (Figs 5, 6) and did not becomedispersed throughout the ooplasm (Figs 7, 8).

The abnormalities of egg morphology described above are probably not caused bythe ca"d mutation. None of the investigators who have worked with this mutation inthe past have reported observing abnormalities in egg morphology. However, if theyare not an aspect of the cand syndrome, the abnormalities must be caused by otherrecessive genes on the third chromosome, as they are observed only in eggs from thehomozygous females of both mutant stocks, and the frequency of abnormal eggsproduced is not significantly different for the homozygous females of both the originalmutant stock and the stock in which most of the residual genetic background is of Ore-R origin. One might argue, on this basis, that perhaps the cytological abnormalitiesdescribed here (i.e. abnormal spindles and clustered nuclei) are also due to a mutationat a locus distinct from the cand locus. As will be discussed below, we believe that asingle lesion could account for the cytological abnormalities as well as for the geneticobservations.

Two models explaining the effect of ca"d have been proposed by other authors andwill be considered here. Davis (1969) suggested that the primary defect in ca!"1 is theformation of imperfect spindle fibres, which fail to separate the members of a bivalentat anaphase I, and thus the intact bivalent is drawn to one pole. Chromosome lossduring meiosis II and the cleavage divisions could also be explained by imperfectspindle fibres resulting in chromosomes that fail to move towards either pole of thespindle. To account for the correlated misbehaviour of chromosomes, Davis sugges-ted that a spindle having one defective fibre would be likely to contain additionaldefective spindle fibres. However, as pointed out by Davis, this model would predictthe production of an array of exceptional-X, exceptional-4 ova in somewhat differentproportions from those observed. To explain this inconsistency, Davis suggested thatthe deficiency of certain types of ova, such as diplo-X nullo-4 and nullo-X diplo-4, wasdue to the high rates of mitotic chromosome loss.

Baker & Hall (1976) have argued that a spindle defect would not account for thenon-random chromosome behaviour observed in ca"d, especially that occurringduring meiosis II and the cleavage divisions. These authors have suggested that ca"'1

causes the formation of chromatids having defective centromere regions, and duringthose divisions in which sister centromeres orient towards opposite spindle poles (i.e.meiosis II and mitosis) those centromeres that were made during a particular 5 phaseorient towards the same pole. Thus if all the defective centromeres oriented towardsthe same pole, chromosome loss would occur only at that pole. Like Davis, theseauthors suggest that the apparent non-randomness of non-disjunction, during meiosisI in ca"d, is an artifact created by the high rates of non-random chromosome loss atlater divisions. Baker & Hall (1976) also suggested that the spindle abnormalitiesobserved at meiosis I by P. Roberts (cited by Davis, 1969) were due to interaction ofthe defective centromeres with the spindle apparatus.

Although it is not possible, from the observations reported here, to rule out thepossibility of a centromere defect in ca!"1, it is difficult to envisage how a centromere

CEL62


defect could account for the formation of two or more spindles and unipolar spindlesduring meiosis I. Also, how a centromere defect, which occurred during the premeiot-ic S phase, would account for the abnormal mitotic figures and clustered nucleiobserved during this study, is not clear. Furthermore, it can be argued that a spindledefect, as described by Davis (1969), could account for the non-randomness ofchromosome behaviour. We suggest, however, that the defect is in the overall struc-ture of the spindle apparatus rather than in individual spindle fibres. We make thisdistinction for the following reasons. (1) To say that individual spindle fibres aredefective implies that the overall structure of the spindle is normal. Our observationssuggest that this is not the case. (2) The simplest way to obtain a defect that causesimperfect spindle fibres but does not affect the overall structure of the spindle ap-paratus is to suggest that the defective fibres have a different origin (i.e. kinetochore)than those that define the overall structure of the spindle (i.e. spindle poles). Untilrecently it was thought that many, if not all, of the microtubules associated with thekinetochore were nucleated at the kinetochore, while the microtubules that deter-mined the overall structure of the spindle were nucleated at the poles (Mclntosh,Hepler & van Wie, 1969; Fuge, 1977). More recent evidence, however, suggests thatin some species all the microtubules of the spindle apparatus are nucleated at the polesof the spindle and that the kinetochores become attached to the microtubules(LaFountain & Davidson, 1979; Nicklaseia/. 1979; Tippit, Pickett-Heaps & Leslie,1980; Rieder & Borisy, 1981; Mazia, Paweletz, Sluder & Finze, 1981). Indeed,evidence that suggests that this mode of assembly may also be used inD. melanogastermales during meiosis has been reported (Church & Lin, 1982). We suggest, therefore,that perhaps ca"d affects a component of the polar microtubule-organizing centre(MTOC), the function of which is to ensure the formation of a bipolar rather thanmultipolar or unipolar spindle. In the event of the formation of a unipolar spindle theresult would be total non-disjunction of the bivalents or total chromosome loss at onepole. A multipolar spindle on the other hand could account for those situations inwhich one or more chromosomes non-disjoin or are lost from one daughter nucleuswhile a second daughter nucleus receives greater than or equal to a full complementof the chromosomes present.

A model for cand that proposes a defect in the MTOC is attractive as it also suggestsa possible explanation for the effect of ca"d on the dispersion of the cleavage nuclei.That is, a defective MTOC that results in the assembly of an abnormal spindle mightalso cause the assembly of an abnormal cytoskeleton and thereby disrupt the normalpattern of nuclear dispersion. Thus the model being proposed is that the cand muta-tion affects a component of the MTOC of the spindle apparatus, the function of whichis to ensure the formation of a bipolar spindle, and that this component is also involvedin the MTOCs of the cytoskeleton where it functions in determining the distributionof the cytoskeletal microtubules. This model is based on four assumptions andevidence to support these assumptions will be presented. The assumptions are: (1)that some component exists within the MTOC of the spindle apparatus, the functionof which is to ensure bipolarity of the spindle apparatus but is not directly involvedin the assembly of microtubules; (2) that in the event of the formation of a multipolar


spindle, specific kinetochores may attain stable associations with microtubules fromtwo or more MTOCs, and that these associations can result in chromosomemisbehaviour; (3) that a cytoskeleton exists within the cytoplasm of the Drosophilaegg and is involved in nuclear dispersal during the cleavage divisions; and (4) that theMTOCs of the spindle are also involved in the organization of the microtubules of thecytoskeleton.

The existence of a component within the polar MTOCs that functions to ensurebipolarity is suggested by the occurrence of mutations, such as ca1"1, ca-simulans andthe divergent meiotic spindle mutation oiZeamays (Clark, 1940), which disrupt thisfunction. In addition, various chemicals and drugs, such as chloral hydrate (Mole-Bajer, 1967), isopropyl N-phenyl carbamate (Hepler & Jackson, 1969), aureofungin(Kumar, Leela, Laxminarayana & Nizam, 1978) and nocoda2ole (Heidemann, Zieve& Mclntosh, 1980), have been reported to cause the formation of multipolar spindles,suggesting that they may interact with some component that is involved in ensuringbipolarity.

Heneen (1975) has described the distribution of microtubules and kinetochores inmultipolar mitoses of cultured rat kangaroo epithelial cells. He observed that manykinetochores, especially those located near the overlap region of two half-spindles,were associated with microtubules extending towards the poles of both half-spindlesand these associations were stable through metaphase. That is, the associations do notappear to be transitory as are similar associations observed during prometaphase inwild-type Drosophila primary spermatocytes (Church & Lin, 1982). Heneen did notobserve anaphase in these multipolar mitoses; however, it seems reasonable to suggestthat such associations could disrupt normal chromosome behaviour, resulting in non-disjunction or chromosome loss.

The third assumption of the model is that a cytoskeleton exists within the cytoplasmof the Drosophila egg and is involved in nuclear dispersal during the cleavagedivisions. Although no ultrastructural studies demonstrating a cytoskeleton withinthe early cleavage stage egg have been reported, Mahowald (1963) and Fullilove &Jacobson (1971) have reported the presence of a rich array of microtubules surround-ing the blastema stage nuclei during cellularization in D. melanogaster and D. mon-tana embryos, respectively. In addition, Zalokar & Erk (1976) have exposed per-meabilized Drosophila embryos to a variety of chemicals, drugs and antibiotics, in-cluding colchicine and cytochalasin B, and observed the effect on the early embryonicstages. Their results indicate that microtubules and actin filaments (microfilaments?)are somehow involved in the dispersal of the cleavage-stage nuclei, migration of thenuclei to the periphery of the egg, maintenance of the regular spacing of the syncytialblastema nuclei and elongation of the nuclei prior to cellularization. It was noted thatmany of the effects of colchicine and cytochalasin B on the early cleavage divisions,described by these authors (Zalokar & Erk, 1976) were not unlike the effects of theca"d mutation described above.

The fourth assumption of the model is that the MTOCs of the spindle are alsoinvolved in the organization of the cytoskeleton. In many cell types the microtubulesof the cytoskeleton have been described as radiating from a single perinuclear MTOC,


which is associated with the centriole pair (e.g. see Brinkley, Fuller & Highfield,1975; Osborn & Weber, 1976; Frankel, 1976; Spiegelman, Lopata & Kirschner,1979; Schliwa, Enteneuer, Herzog & Weber, 1979; Aubin, Osborn & Weber, 1980).The most convincing evidence that the MTOCs of the spindle and the primaryMTOC of the cytoskeleton are the same is seen in the reports of Aubin et al. (1980),who followed the disassembly of the cytoskeleton and assembly of the spindle ap-paratus in rat kangaroo epithelial cells in culture, and Brinkley et al. (1975), whodescribed the reassembly of the cytoskeletal microtubules following mitosis in mouse3T3 cells, rat kangaroo epithelial cells and HeLa cells.

This research was supported by grant no. PCM-7908850 from the National Science Foundation.

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(Received 17 June 1982-Accepted, in revised form, 27 January 1983)

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