three-dimensional chromosome arrangement of crepis ... · capillaris plants from very different...

Three-dimensional chromosome arrangement of Crepis capillaris in mitotic

prophase and anaphase as studied by confocal scanning laser microscopy

J. L. OUD, A. MANS, G. J. BRAKENHOFF, H. T. M. VAN DER VOORT, E. A. VAN SPRONSEN and

N. NANNINGA*

Department of Molecular Cell Biology, Section of Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, NL-IOIS TVAmsterdam, The Netherlands

* Author for correspondence

Summary

To estimate the extent of ordering of chromosomes,confocal scanning laser microscopy -was used tomake three-dimensional images from optical sec-tions. For Crepis capillaris, which has 2n = 6 easilyrecognizable chromosomes, a statistically signifi-cant sample of 75 Feulgen-stained root tip ana-phases was analysed. A comparison of the observedchromosome ordering and the expected randomdistribution showed a significant surplus of one ofthe arrangements with a juxtaposition of the twochromosomes with a nucleolus organizer region.Two of the arrangements with these chromosomesin opposite positions were never observed in ourmaterial. Another analysis of 30 mithramycin A-stained prophases and 30 meta- and anaphasesshowed partly different patterns of non-randomchromosome distribution in the two stages of mi-tosis. A preference for an association of the homol-ogues was observed for all pairs of chromosomes inprophase cells, whereas in meta- and anaphase theassociation only persisted for the nucleolus organ-

izer chromosomes. This indicates that there may besome relocation of the chromosome positions dur-ing the transition from prophase to metaphase. Inmeta- and anaphase one of the arrangements withjuxtaposed NOR chromosomes was preferred, i.e.the ordering in which chromosomes 1 and 3 occu-pied alternate positions. Probably, the nucleolus isan important factor in producing a non-randomdistribution, but there could be other factors thatinfluence chromosome ordering as well. A compari-son of the anaphase chromosome ordering in C.capillaris plants from very different localities, indi-cated that the observed non-random distributionwas independent of the origin of the material.Existing models of chromosome disposition are notsufficient to explain the observed non-randomchromosome ordering in C. capillaris.

Key words: chromosome ordering, Crepis capillaris, confocalscanning laser microscopy.

Introduction

From the early days of cytogenetic research, the questionhas been discussed as to whether or not chromosomes arerandomly distributed in the nucleus. A well-knownexample of ordering is the so-called Rabl orientation,which refers to a relict telophase orientation of thechromosomes in the extant prophase (Rabl, 1885).Another example refers to the physical association ofhomologous chromosomes, which is a normal feature inmeiotic prophase. It has also been reported for mitoticcells of various species. Somatic pairing seems to be quitevariable, however, since it is present in some species andabsent in others (for review and references, see Avivi &Feldman, 1980). A direct study of centromere localiz-

Journal of Cell Science 92, 329-339 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

ation in rat-kangaroo and Indian muntjac interphasenuclei, using an immunofluorescence staining technique,revealed the existence of somatic pairing (Hadlaczky etal. 1986). In addition, models have been presented on thespatial arrangement of the chromosomes, in which thesize of the chromosome arms plays a prominent role,rather than homology (Shchapova, 1971; Bennett, 1982;Fussell, 1984).

Since interphase chromosomes are difficult to study,most research on their disposition has been done onmitotic cells. The arrangement of chromosomes in ametaphase plate is then assumed to reflect the interphaseordering. To analyse the relative positions of chromo-somes, squash or air-dried preparations have been com-monly used to determine chromosome localization. A

329

disadvantage of this two-dimensional technique is the riskof introducing alterations in the relative positions ofchromosomes, even if spindle fibres have been left intact.It is better, therefore, to analyse the chromosomes intheir original three-dimensional (3-D) configuration. Forexample, Bennett (1982), Heslop-Harrison (1983) andSchwarzacher-Robinson et al. (1987) made 3-D recon-structions from a large series of electron-microscopesections of root tip metaphases. Unfortunately, thismethod is extremely time-consuming and for this kind ofwork the high resolution of the electron microscope is notnecessary. As a result, the analysis of chromosomedisposition has to be carried out with a limited number ofcells, which restricts the possibility of drawing statisti-cally satisfactory conclusions (Callow, 1985). On thelight-microscopic level, Agard & Sedat (1983) achievedoptical sectioning in conventional fluorescence mi-croscopy using an iterative deconvolution algorithm.They made 3-D reconstructions of Drosophila polytenechromosomes (Agard & Sedat, 1983; Hochstrasser et al.1986, 1987a,b). Recently, Rawlins & Shaw (personalcommunication) used the same technique to study the3-D organization of chromosomes of Crepis capillaris.

Our approach to the study of the spatial distribution ofchromosomes has been to use series of optical sectionsobtained by confocal scanning laser microscopy (Braken-hoiietal. 1979, 1985; Carlsson et al. 1985; van der Voortet al. 1985; Wijnandts van Resandt et al. 1985). Thetechnique has three important features: (1) the excep-tionally short depth of field and the almost completeabsence of information from off-focus planes ensure highquality optical sectioning; (2) digital data storage permitsall kinds of image-processing techniques, for example thegeneration of stereoscopic images (van der Voort et al.1985); and (3) the method is easy and fast (a 3-D imageon the basis of 10-20 optical sections takes a fewminutes). We used the confocal scanning light micro-scope, developed in our laboratory, for 3-D karyotypingof mitotic chromosomes, after first having demonstratedthe possibility of visualizing 3-D chromatin distributionin neuroblastoma nuclei (Brakenhoff et al. 1985).

In this paper we have analysed the spatial distributionof chromosomes in prophase and anaphase. We wished toanswer the following questions: (1) to what extent doesordering occur with respect to the spatial localization ofchromosomes? (2) Are the arrangements in prophase andanaphase the same; in other words, is it permissible toextrapolate from the anaphase arrangement to the inter-phase arrangement? (3) Is chromosome ordering aphenomenon that is independent of the geographicalorigin of the plants?

As an object for model studies we decided to use theplant Crepis capillaris (Smooth Hawksbeard, Com-positae), which has a small number (In = 6) of easilyrecognizable chromosomes (Babcock, 1947).

The present paper gives a survey of the 3-D chromo-some arrangements observed in a large number of ana-phase cells from root tips of plants of one inbred line, andin samples of cells in prophase and in meta- and anaphaseof another inbred line. Chromosome positions wereanalysed in 3-D stereoscopic images, made by stacking

series of optical sections. The occurrence of differenttypes of arrangements deviates significantly from anexpected random distribution. This holds both for pro-phase and for anaphase cells, though the pattern of thenon-random distribution partly differs between thesestages. The chromosome ordering in anaphase cells doesnot differ between plants of the two inbred lines. We havealso examined whether existing models of chromosomedisposition might apply to the situation observed in C.capillaris. The results indicate that these models areinsufficient to explain the observed non-random distri-bution. Apart from unknown factors that influencechromosome ordering in our material, the nucleolusorganizer region (NOR) appeared to play an importantrole in non-random ordering.

Materials and methods

Root tips of Crepis capillaris (L.) Wallr. plants from the inbredlines Cr9 and Crl03 were used. The Cr9 plants were a kind giftfrom the Botanischer Garten Marburg, FRG; the Crl03 plantswere collected in Wageningen, The Netherlands. Primary rootswere fixed in ethanol-acetic acid (3:1, v/v) for at least 30 min atroom temperature and stored at —20°C. Pretreatment with coldwater or spindle inhibitor was omitted to avoid possiblealterations in chromosome ordering. The roots of Cr9 plantswere treated according to the Feulgen procedure to stain DNAspecifically. This included: washing in distilled water, 5 min ofhydrolysis in 1 M-HC1 at 60°C, washing in distilled water, andstaining with Feulgen for approximately 30 min. The roots ofCrl03 plants were macerated in a mixture of 0-1 % cellulase,0-1 % cytohelicase and 0-1 % pectolyase in an acetate buffer for20 min at room temperature. After washing in an aqueoussolution of 5 niM-MgCl2, they were stained overnight in100 mgml"1 mithramycin A and 5 mM-MgC^ in distilled water.The Feulgen- or mithramycin-stained roots were placed be-tween a 24 mm x 32 mm and a 18 mm x 18 mm coverslip andsealed with nail polish. Care was taken to avoid squashing. Forthe excitation of the fluorochrome, the 530-9 nm line (Feulgen)or the 476-2 nm line (mithramycin) of a krypton ion laser wereused, in combination with, respectively, a 580 nm or 520 nmdichroic mirror and a 580 nm or 520 nm longwave pass blockingfilter.

In confocal fluorescence microscopy (Brakenhoff et al. 1985,1989), the laser-illuminated pinhole is focused on precisely thesame point in the preparation as is imaged on the detectorpinhole. A property of such an optical arrangement is that thecontributions from out-of-focus layers of the preparation arealmost completely suppressed. This is an important require-ment for making 'clean' optical sections. In our system we use aX100 (NA 1-3) objective lens, and the preparation is scannedmechanically through the confocal point. Fluorescence inten-sity data are stored in a 256x256x8 bit memory array as afunction of the position of the preparation. The microprocessorthat controls the scanning device can make a series of opticalsections through the preparation. For each cell a suitablecombination of the number of sections and the distance betweenthe sections has been chosen. For root tip cells the number ofsections varied between 10 and 32, with a mutual distance of650-1500 nm. After storage of the raw data, a median filter(3x3 pixels) was applied to the data of each section. Two-dimensional images are generated by stacking the successivesections. The 3-D images, which were used to analyse thearrangement of chromosomes, were made by the generation ofstereoscopic image pairs. For this, an algorithm was used that

330 J. L. Oud et al.

Crepis capillaris

No;.

mShortarm

Longarm

17-31 15-35 9-27

Fig. 1. Schematic representation (idiogram) of the haploidchromosome set of C. capillaris arranged according to length.No., chromosome number. RL, relative length; the data arethe average of six repeated measurements of chromosomelength in four metaphase plates. NOR, nucleolus organizerregion.

assigns the maximum value (corresponding with the highestfluorescence intensity) of the pixel values encountered alongeach of the 256X256 vertical viewing lines (van der Voort et al.1985). In all stereo micrographs shown in the paper, one picturewas generated by stacking the sections precisely on top of eachother, whereas the sections of the other picture were shifted ±1pixel per section in the X direction. The original fluorescenceintensity data array was also used for the so-called simulatedfluorescence process (van der Voort et al. 1989). This methodresembles ray tracing and results in a solid modelling of thefluorescent object with an elimination of all (non-chromosomal)background information. Addition of an artificial shadow givesthe impression of depth. The photographs were made fromimages displayed on a cathode ray tube.

Besides the C. capillaris material described above, Feulgen-stained root-tip squash preparations were also made of colchi-cine-pretreated material. This material was used to measurechromosome arm length according to the computer-aidedkaryotyping procedure described by Oud et al. (1987).

Results

Possible chromosome arrangements

C. capillaris is a diploid species with three pairs ofchromosomes. Chromosomes 1, 2 and 3 can be identifiedon the basis of their length and centromere position(Fig. 1). The largest part of the short arm of chromosome2 consists of the nucleolus organizer region. In meta-phase, the centromeres of all chromosomes are circularlyarranged in the equatorial plane (Fig. 2). This figure is an

BFig. 2. Root-tip cell of C. capillaris in metaphase with aradial arrangement of the chromosomes (A). The image wasderived from a 3-D reconstruction to which various filteringprocedures, a ray tracing technique and artificial shadowingwere applied. Bar, 10^m. B. Schematic representation withchromosome numbers and indication of the centromerepositions (white dots). 1-3, chromosome numbers.

example of the so-called simulated fluorescence processtechnique, in which solid modelling and artificial sha-dowing give a 3-D impression. The circular arrangementis already visible in prophase and is prolonged through-out anaphase. Theoretically, in a species with 2>i = 6chromosomes, 5! (= 120) different arrangements of thechromosomes can be expected. However, since it is notpossible to distinguish between the maternally and pa-ternally derived chromosomes, and because of the circu-lar arrangement of the centromeres, the 120 permutationscan be reduced to 11 different circular permutations ofthe six centromeres. All other permutations are mirrorimages of these 11. Fig. 3 shows the 11 permutations,starting with one of the chromosomes 2. A schematic

Chromosome ordering in Crepis capillaris 331

: 223311

1223113

j 221331

1223131

1232311

i212331

; 232131

: 212313

j233211

1213231

j 231231

221133

221313

232113

212133

211233

231213

213213

233112 211332:

231132 !

213312

231312 213132 i

231123 211323!

233121 213321 i

213123

231321 !

Fig. 3. Permutations of 2x3 items, starting with number 2(this number is chosen because chromosome 2 proved to benon-randomly distributed). In the five boxes with brokenlines, groups of permutations are depicted with 0-5intervening numbers between the two chromosomes 2. If thepermutations are circular, the first and last number of eachpermutation occupy adjacent positions, and all combinationson the same row are mirror images of each other. The 11different permutations A-K in the left column (continuousline box) correspond to the chromosome arrangements A-Kin Fig. 4.

®

© E F

©JL®©r,®

®

Fig. 4. The 11 chromosome arrangements (A-K) that can bedistinguished in an organism with three pairs of homologouschromosomes and circular arranged centromeres (circles withcorresponding chromosome number). If the distribution ofthe arrangements is random, types A, B, D and E(underlined) are expected to occur twice as often as theothers.

representation of the circularly arranged centromeres inthe 11 configurations A—K is depicted in Fig. 4. It can beseen that four of the permutations (i.e. A, D, E and F)have twice as many mirror images as the remaining seven.For a statistical analysis using the chi-squared test, anexpected number of at least five cells for each of thearrangements is necessary. If we assume a randomdistribution, we have to analyse 75 cells, in which the

arrangements A, D, E and F are expected 10 times, andthe arrangements B, C, G, H, I, J and K five times.

3-D analysts in anaphaseMost 3-D images were made from optical sections ofFeulgen-stained root-tip cells in anaphase. In 11 root-tippreparations we have made series of scans of all the cellsthat could be recognized as anaphase, using the lowestmagnification of the confocal microscope. At the lowestmagnification it is impossible to have an impression of thetype of chromosome arrangement. This procedure waschosen to rule out the possibility of pre-selecting cells.For the statistical evaluation of chromosome ordering inanaphase, we restricted the analysis to cells in which bothgroups of chromatids showed identical arrangements.Among 76 anaphases we observed only one cell withdifferent chromosome arrangements (described in detailat the end of the paragraph). In the remaining 75 cells weused only one set of anaphase data per cell. Fig. 5A-Jshows an example of 10 optical sections of a root tip cell inanaphase, and a stereo pair of the 3-D image that resultswhen the sections are stacked (Fig. 5K,L). Often, thearm length ratios of anaphase chromatids are morepronounced compared with metaphase chromosomes.Nevertheless, in anaphase also each type of chromatid hasits own characteristics: 1 is the longest chromatid with aclearly visible short arm; 2 is almost as long as 1, but thetiny short arm and satellite are not perceptible in mostcells; 3 is about half as long as 1. A schematic represen-tation of the chromosomes depicted in Fig. 5K,L is givenin Fig. 5M,N. In this anaphase the chromosomes arearranged according to configuration E. Fig. 6 givesexamples of stereo micrographs of 3-D images of thearrangements A, B, C, D, F, G, H and K. The imagesmight give the impression that there were no other cells inthe preparation. In fact there were often one or more cellson top of and/or below the anaphase. However, theyremain invisible because of the optical sectioning prop-erty inherent in the confocal technique.

First, 75 cells have been analysed, which were derivedfrom 11 roots of three plants of the inbred line Cr9. Thenumber of cells per root varied from 1 to 11. Table 1 givesa survey of the observed arrangements, the expectednumber of cells for each class assuming a randomdistribution, and the results of a chi-squared test. A totalchi-squared value for the arrangements A-K of 26-6(d.f. = 10, P= 0-003) indicates a non-random distri-bution of the chromosome arrangements. The mostprominent deviations from the expectation are the excessof arrangement D and the absence of the arrangements Iand J.

The anaphase stage has the advantage that the spatialdistribution of the chromosomes can be analysed twice ineach cell. It is to be expected that the same arrangementwill be observed in both groups of chromatids. We testedwhether they are exact mirror images of each other. Of 76anaphases, only one (not included in the 75 of Table 1)showed different arrangements (Fig. 7). In this case fourof the six chromatids occupied corresponding positions inthe two groups (i.e. 2-1-3-1), the remaining two (adjac-ent) chromatids switched their order in one of the groups


(3-2 versus 2-3). As a result, arrangement D was found atone pole and arrangement G at the other pole.

In addition to the analysis of the inbred line Cr9, wehave also analysed the chromosome ordering in meta-phase and anaphases of CrlO3 root tips. Since there wasno indication of an essential difference in chromosomeordering in metaphase and anaphase, we decided tocombine the data from one sample of 30 cells. A samplesize of less than 75 cells does not permit a statisticalanalysis of individual arrangements. Therefore, we re-

stricted the first survey of the CrlO3 material to ananalysis of the relative position of homologous chromo-somes (see below).

Arrangement of homologues in anaphaseTo test whether there is a preference for the homologouschromosomes to occupy adjacent positions, the numbersof cells were counted with, respectively, zero, one or twointervening chromosomes between a given pair of homol-ogues. The results for the 75 anaphases of the inbred line

Arrangement E

Fig. 5. A-J. Ten optical sections of a C. capillans root-tip cellin anaphase. Each section is approx. 500 nm thick and thedistance between the sections is l-2fim. K,L. 3-D stereoimages, provided by combining the 10 sections. In the rightimage, the sections have been shifted plus one pixel persection; in the left image there was no shift. M,N. Drawingand schematic representation of the 3-D image withchromosome numbers indicated. The chromosomes are orderedaccording to arrangement E. Bar, 10 jitm.


Fig. 6. Eight pairs of stereo micrographs of 3-D images of C. capillaris root-tip anaphases. From upper left to lower right areshown arrangements A, B, C, D, F, G, H and K, respectively. Configuration E is depicted in Fig. 5K,L, whereas types I and Jare never observed. Bar, 10 fim.

Cr9 are summarized in Table 2. For both chromosome 1and 3 the deviation between the observed and expectednumbers of cells with zero, one or two interveningchromosomes was small. The chi-squared test indicatedthat the probability with which the deviation could beattributed to chance varied from 0-74 to more than0-99 %. Thus, there is no preference for chromosomes 1

and 3 to occupy adjacent or opposite positions. Only forchromosome 2 did associations of homologues occursignificantly more often than expected and, in agreementwith that, there was a striking deficit of cells withchromosomes 2 in opposite positions.

To compare chromosome ordering in prophase and inmetaphase and anaphase, two samples with 30 cells/stage

334 jf. L. Oud et al.

Table 1. Survey of anaphase arrangements in C. capillaris inbred line Cr9

Arrangement

ABCDEFGH1JK

Total

Probability:

1 2

1

iI

i

I

1 4

*P = 0-025; **P =

3

1

11i

j

5

0-003; •

4

111

i

1

5

5

1

1I

1

2

6

0 = 0-002;

Root

6

221

1

1

7

without

7

2

13

1

7

: symbol,

8

12

5

8

P>0-05 .

9

2

31|

10

10

30

111i

ii

ii

ij

i

i

ii

Obs.

1188

2067S4006

75

Exp.

105I

ifftfto•i

# •

1•X>

75

Chi2

0-11-81-8

10-0***1-60-900-25-0*5-0*0-2

26-6**

were analysed for CrlO3 material. When the chromo-somes are randomly distributed, one expects to find in asample of 30 cells: 12 cells without an interveningchromosome between a given pair of chromosomes, 12cells with one intervening chromosome and six cells withtwo of them. The results (Fig. 8) show that in metaphaseand anaphase cells of the inbred line 103, chromosomes 2are also preferentially juxtaposed. Chromosomes 1 and 3do not show a preference for an association of homolo-gous chromosomes. The results concerning prophasecells are given below.

3-D analysis in prophaseIn the root tips of Crl03 plants we analysed chromosomeordering in mid and late prophases. 3-D images ofprophase cells (Fig. 9) showed that the chromosomes

tended to occupy the peripheral zone of the nucleus.Separate experiments using the fluorochrome acriflavine,which also stains RNA, demonstrated that the centralpart of the nucleus contains one large nucleolus (data notshown). Note that in the cell depicted in Fig. 9A, thenucleolus is not visible because of the DNA-specificstaining procedure. For prophase chromosomes,chromosome length has been the main criterion forchromosome identification. An additional characteristicof the NOR chromosomes (2 in Fig. 9B) was the obser-vation that the short arms pointed towards the centre ofthe nucleus. The centromeres have been used as areference point for the location of the chromosomes. Theanalysis of the relative position of homologous chromo-somes (Fig. 8) showed that, in prophase cells, for allchromosomes there is a strong tendency towards: (1) a

Fig. 7. A C. capillans root-tip anaphase with a difference incentromere arrangement between the left- and right-handgroup of chromatids. A,B. A pair of stereo micrographs;C, schematic representation of the chromatids and the positionof the centromeres. The centromeres of chromatids 2 and 3,which have different positions in the two groups (D and G),are indicated in outline. For the remainng six chromatids, thetwo groups are mirror images of each other. Bar, 10 fim.

chrom. 1 chrom. 2 Sliilil chrom. 3


Table 2. The. position of C. capillaris inbred line Cr9 chromosomes in relation to their homologues

Number ofinterveningchromosomes Arrangements Observed Expected Chi2 Probability

Chromosome 10\t

Chromosome 20

I2

Chromosome 30f2

A, B, E, ID, F, G , JC, H, K

A, B, C, DE, F, G, HI . J . K

A, C, F, ID, E, H, JB, G, K

253218

47226

263019

303015

3030IS

303015

0-830-130-6

9430-135-4

0-5301-07

0-9340-9980-741

0-0220-9980-067

0-971>0-999

0-999

Chromosome 1 Chromosome 2 Chromosome 3

0 1 2

Intervening chromosomes

Fig. 8. Comparison of the distribution of arrangements with, respectively, zero, one or two intervening chromosomes betweenthe given pair of homologues in C. capillans inbred line Crl03. Note the different scale of the chromosome 2 ordinate.

Fig. 9. Root-tip cell of C. capillans in prophase. A. Stereomicrograph of the 3-D image; B, schematic representation ofthe 3-D image with chromosome numbers and centromerepositions indicated (white dots). The two black dots are themicrosatellites of chromosomes 2. Bar, 10f<m.

surplus of arrangements in which the centromeres occupyadjacent positions; (2) a deficit of arrangements with oneintervening chromosome; and (3) an expected number ofcells with the homologous chromosomes at oppositepositions.

Discussion

Non-random chromosome arrangementThe study of chromosome arrangements in 3-D imagesusing a confocal scanning laser microscope illustrates thepower of the optical sectioning technique. The collecteddata provide a sound' basis for the analysis of the spatialdistribution of chromosomes. Dividing root-tip cells of C.capillans show a non-random chromosome distributionin both prophase and anaphase.

In each of the two inbred lines the arrangements withjuxtaposed NOR chromosomes are preferred. However,of the four arrangements with adjacent chromosomes 2(types A, B, C and D in Fig. 4) one was favoured, withstatistical significance, i.e. arrangement D (Table 1). Asa consequence, there is a deficit of cells with chromo-somes 2 in opposite positions, caused by an absence ofarrangements I and J.

The short arms of both of the NOR chromosomes arelocated in the central part of the prophase nucleus, whichis mainly occupied by one large nucleolus. These obser-


vations indicate that the nucleolus might be important inexplaining the frequent juxtaposition of these chromo-somes.

Several other publications have dealt with chromosomeordering in C. capillaris (Kitani, 1963; Wagenaar, 1969;Ferrer & Lacadena, 1977). All the reports involved 2-Dstudies of unsquashed root tip cells. Wagenaar (1969)described the formation of two attached chains of pro-phase chromosomes: one consisting of the two NORchromosomes, the other consisting of chromosomes 1-3-3-1. A figure in his article, which illustrates how theassociated chromosomes are thought to be arranged ininterphase, shows two attached NOR chromosomes andone common nucleolus. So far, Wagenaar's interpretationis in agreement with ours. However, two attached chainsof chromosomes were never observed in our CrlO3prophase cells. In this material the chromosomes showeda Rabl-like orientation, i.e. centromeres and long-armtelomeres were located in opposite hemispheres of thenucleus. Ferrer & Lacadena (1977) found in metaphasesa preference for somatic associations of chromosomes 1and 3, but not for the NOR chromosome. These resultsare the opposite to our observations in metaphase andanaphase. Kitani (1963) reported a side-by-side arrange-ment of all the pairs of homologous chromosomes in pro-,meta- and anaphase. We can confirm these observationsfor the prophase stage only; in the other stages there is nolonger a preference for an association of chromosomes 1and 3. It is difficult to compare these results with ours,since a reconstruction of the spatial distribution ofchromosomes from 2-D preparations is much less reliablethan using a 3-D technique.

In other species, somatic associations of NOR chromo-somes have been reported almost exclusively for thosecases in which the association of homologues is a generalfeature of all the chromosomes. A well-known exceptionis the frequently observed association of satellites inhuman metaphases (Sele et al. 1977), in which the NORsbetween centromere and satellite are held responsible forthe associations. Recently, Heslop-Harrison et al. (1988)reported that an association of the NOR chromosomeswas observed in metaphase cells of Zea mays, but not inHordeum vulgae, H. marinum and Aegilops umbellulata.In A. umbellulata one of the two pairs of NOR chromo-somes showed a significantly larger distance between thehomologues than expected assuming a random distri-bution. All other chromosomes in these species showed arandom distribution. These results indicate that theremight be considerable differences with respect to therelation of the position of the NOR chromosomes to thenucleolus. As long as we have only a limited knowledgeabout the factors that influence chromosome ordering, itis difficult to compare the data of various species. If weassume that the relative importance of such factors canvary, it is conceivable that under certain conditions therole of the nucleolus is over-ruled by other factors.

Chromosome ordering in different stages of mitosisThe bar diagrams of Fig. 8 show that for the NORchromosomes almost the same distribution of chromo-some arrangements was observed in prophase cells as in

meta- and anaphases. In both stages there is a preferencefor an association of homologous chromosomes 2. Withrespect to chromosomes 1 and 3, the results indicate thatin prophase cells there is a preference for the arrange-ments with juxtaposed homologous chromosomes,whereas in meta- and anaphases the arrangements withone intervening chromosome are most commonly ob-served. The number of arrangements of type D, illus-trates clearly the difference between the stages of mitosis.In this arrangement the NOR chromosomes occupyadjacent positions and the remaining chromosomes showan alternating order (i.e. 1-3-1-3). In prophase cells weobserved 3x'D' , and in meta- and anaphase 9x 'D ' in asample of 30 cells/stage.

Obviously, some chromosome relocations must takeplace during the transition from prophase to metaphase.Another argument in favour of this is the repeatedobservation of prometaphase cells in which the centro-mere of one of the chromosomes was outside the ring ofthe circularly arranged centromeres of the remainingchromosomes. This gives the impression of a chromo-some whose position is changing.

The observed difference in the preferential chromo-some localization between prophase and anaphase indi-cates that chromosome ordering is a dynamic process. Inconsequence, there must also be changes in chromosomeordering between anaphase and the subsequent prophasein the daughter cells. Since the timing and extent of suchrearrangements are uncertain, the predictive value ofmeta- and anaphase ordering for interphase nuclei islimited.

Comparison with recent models of chromosome orderingTo find an explanation for the preference of arrangementD and the absence of I and J, we considered whether thechromosome disposition models of Shchapova (1971),Bennett (1982), Fussell (1984), Ashley & Pocock (1981)and Lavania & Sharma (1984) might apply to ourmaterial.

In the model of Shchapova it is assumed that telomeresare attached to each other in pairs. In consequence thechromosome complement is arranged in one large, end-less chain. In interphase, a zig-zag folding of the chainwould result in a Rabl-like orientation, in which thecentromeres and telomeres are localized in oppositehemispheres. In this model the distance from centromereto telomere and back to the centromere of the adjacentchromosome is thought to be as constant as possible. Thebest approximation of a constant inter-centromere dis-tance is obtained when the longest long arm is attached tothe shortest short arm etc. In this model it is assumedthat the diploid chromosome complement is segregated intwo haploid groups. Each group is ordered according tothe above description. In the chromosome dispositionmodel for haploid genomes proposed by Bennett (1982),the difference in length of adjacent chromosome arms iskept to a minimum to ensure the maintenance of a Rablorientation, instead of a constant inter-centromere dis-tance. This is achieved by an alternation of associatedsimilar-sized long arms, followed by short arms etc.

To predict preferences in chromosome ordering in our


C. capillaris material, the chromosome arm length datashown in Fig. 1 are used instead of arm volumes (asproposed by Bennett). Moreover, it is assumed that themicrosatellite has a negligible size. On this basis, theprime prediction according to Bennett's model is forthe order 2-1-3 or the mirror image 3-1-2, the secondmost likely is 1-2-3 or 3-2-1, while the least likely orderingis 1-3-2 or 2-3-1 (Bennett, personal communication). InShchapova's model, the first and second most likelyordering are the reverse of Bennett's prediction. Exceptfor arrangement A, all circular chromosome arrange-ments (Fig. 4) can be divided into two halves, eachcontaining a haploid set of chromosomes. Whether thisside-by-side division corresponds to the separation intomaternally and paternally derived chromosomes is un-known. For example, arrangement D shows a group ofchromosomes with a 2-1-3 ordering, and the other groupwith a 1-3-2 ordering. The most likely ordering accordingto Bennett's prediction can be seen twice in the arrange-ments C and K. A comparison of the occurrence of thedifferent orders in arrangements B-K and the observednumber of arrangements does not show a clear preferencefor one of the predicted chromosome orders. However,we have to realize that the centromeres are not arrangedexactly on the points of a hexagon with constant separ-ation distances between neighbours (as depicted inFig. 4). A deviation in the position in the X, Y and/or Zdirection might result in a different choice of chromo-somes, which are thought to belong to a haploid set.Possibly, C. capillaris with a small number of radiallyarranged chromosomes is not the most suitable organismto test models for predicting preferences in chromosomeordering in haploid genomes. In this respect, it should benoted that we have chosen an organism with a smallnumber of chromosomes for statistical reasons. Theanalysis of a species with one more chromosome pairwould require more than 1000 3-D reconstructions togive a reliable statistically significant answer.

Fussell (1984) has adapted the Bennett model to thediploid number of chromosomes, organized in one chain.In the chain, chromosome order is flexible and thenumber of possibilities is a function of the number ofarms of similar length. For each of the chromosomearrangements A-K, we calculated the sum of differencesin length of the adjacent chromosome arms. The order ofexpected chromosome arrangements in C. capillaris ac-cording to Fussell's model, using the diploid set ofchromosomes, is: C and I (equally most likely),A - F - B - D and E-G, H, J and K (equally least likely). Ascreening of the position in this rank of those arrange-ments that are observed significantly more often (D) orless often (I and J) than expected indicated that similarityin arm size is not a major factor that determines the non-random chromosome ordering in C. capillaris.

The model proposed by Ashley & Pocock (1981), onthe basis of the observed chromosome ordering in Ornith-ogalum virens, was described as having a linear order ofthe chromosomes of the haploid set in germ cells. Uponzygote formation, the exterior telomeres of the two setswould attach, with the formation of a closed chain. Insuch a chain the two parental sets of chromosomes are

Table 3. Chromosome arrangements in twoC. capillaris inbred lines

Number ofinterveningchromosomes

Chromosome 1012

Chromosome 2012

Chromosome 3012

Observed/expected inCrl03 meta-, anaphases

(30 cells)

12/1213/125/6

20/126/122/6

12/1215/123/6

Observed/expected inCr9 anaphases

(75 cells)

25/3032/3018/15

47/3022/306/15

26/3030/3019/15

mirror images of each other. The C. capillaris arrange-ments that are in accordance with this model are types B,C and I. Arrangements B and C are not observedsignificantly more often than expected on a random basisand type I has never been found in 75 anaphases of theinbred line Cr9. From these data we have to concludethat there is no preference for an ordering according tothe description of Ashley & Pocock. In a comparablemodel given by Lavania & Sharma (1984), the predictedpositions of the homologous chromosomes with respect toeach other differ slightly, to ensure a side-by-side localiz-ation of the homologues. This alternative model also doesnot fit our data.

Chromosome ordering in plants from differentgeographical originsWe have analysed chromosome positions in the anaphasestage of mitosis in root tips of cells of both the inbred lineCr9 and line Crl03. Since we found no indication ofchanges in relative chromosome positions between meta-phase and anaphase, the 30 meta- and anaphase cells ofCrlO3 and the 75 anaphases of Cr9 permit a comparisonof chromosome ordering in plants of different geographi-cal origins. It can be seen from Table 3 that the numberof arrangements with, respectively, zero, one and twointervening chromosomes for a given pair of homologuesdoes not differ significantly between the Cr9 and Crl03cell lines. In both inbred lines, the number of observedarrangements is in agreement with expectations forchromosomes 1 and 3, whereas chromosomes 2 show aclear preference for being juxtaposed.

The preliminary results of the correlative study showthat the observed non-random chromosome distributionis a fundamental property of the cell. The system that isresponsible for the ordering seems rather complex. Thereis no doubt that the nucleolus is an important factor thatdetermines chromosome ordering. The assumed re-arrangement in chromosome positioning during prometa-phase suggests that there are other unknown factorsinvolved.

We are grateful to Drs M. D. Bennett and R. S. Callow forstimulating discussions about the analysis of chromosome

338 jf. L. Oud et al.

ordering, and to Dr C. E. A. van Wijngaarden-Pater forcritically reading the manuscript.

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(Received 29 September 19SS - Accepted, in revised form,29 November I9S8)

Note added in proof. Recently a paper on the same topicappeared (D. J. Rawlins & P. J. Shaw: Three-dimensional organization of chromosomes of Crepiscapillaris by optical tomography. J . Cell Sci. 91, 401-414(1988)), in which the absence of ordering was stressed.There are some differences in sampling the material forthe statistical analysis. At present it is not clear to whatextent the different sampling procedure affected theoutcome.


three-dimensional chromosome arrangement of crepis ... · capillaris plants from very different...

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