Development 102. 127-134 (1988)Printed in Great Britain © T h e Company of Biologists Limited 1988

127

Polytene chromosomes in mouse trophoblast giant cells

SUSANNAH VARMUZA, VALERIE PRIDEAUX. RASHMI KOTHARY and JANET ROSSANT

Mount Sinai Hospital Research Institute and Department of Medical Genetics. University of Toronto, 600 University Avenue, Toronto.Ontario. Canada. M5G 1X5

Summary

Mouse trophoblast giant cells undergo successiverounds of DNA replication resulting in amplification ofthe genome. It has been difficult to determine whethergiant cell chromosomes are polyploid as in liver cellsor polytene as in Dipteran salivary glands because thechromosomes do not condense. We have examined thepattern of hybridization of mouse giant cells with avariety of in situ chromosome markers to address thisquestion. Hemizygous markers displayed one hybrid-ization signal per nucleus in both diploid and giantcells, while homozygous markers displayed two signalsper nucleus in both cell types. These patterns are

consistent with cytological evidence indicating thatgiant cell chromosomes are polytene rather thanpolyploid. However, in contrast to the situation inDipteran salivary glands, the two homologues do notappear to be closely associated. We conclude that themechanism of giant cell DNA amplification involvesmultiple rounds of DNA replication in the absence ofboth karyokinesis and cytokinesis, and that sisterchromatids, but not homologous chromosomes, re-main closely associated during this process.

Key words: mouse, trophoblast, giant cells, polytenechromosome, DNA replication.

Introduction

The first differentiated tissue to arise in the mouseembryo is the trophectoderm. Trophectoderm cellsform a monolayer surrounding the inner cell mass(ICM) and the blastocoelic cavity at the blastocyststage of development; those cells directly overlyingthe ICM are designated polar trophectoderm, whilethose away from the ICM are termed mural troph-ectoderm. These latter cells are believed to be in-volved in implantation of the blastocyst into theuterine epithelium (Gardner, 1972). Polar trophecto-derm continues to proliferate and forms extraembry-onic ectoderm and the ectoplacental cone, which latercontribute to the fetal placenta (Rossant & Papaioan-nou, 1977). Mural trophectoderm cells cease mitosis,but not DNA replication, beginning at 4-5 days postcoitum. These enlarged cells, called primary tropho-blast giant cells, may endoreduplicate their genomesby up to 500-fold (Zybina, 1970; Zybina & Grish-chenko, 1972; Barlow & Sherman, 1972). Secondarygiant cells develop later at the periphery of theectoplacental cone and are phenotypically indis-tinguishable from primary giant cells.

The high ploidy levels achieved by these cells makethem unique among mammalian cell types. Theincrease in C value is accomplished by DNA repli-cation in the apparent absence of mitosis and chromo-some condensation. Occasional polyploid mitoseshave been observed in trophoblast cells (Zybina &Grischenko, 1970; Ilgren, 1980), but the frequency ofsuch events is insufficient to account for the increasedploidy values. Several possible alternative modes ofreplication can be envisaged. Multiple rounds ofDNA synthesis without either karyokinesis or cyto-kinesis may result in chromosome structures in which(i) all homologous strands are closely associated, (ii)sister chromatids of individual chromosomes, but notof homologues, are closely associated, or (iii) allchromatids are unassociated. The first case closelyresembles the mode of replication of polytenechromosomes in Dipteran insects, while the last caseis similar, but not identical, to liver cell polyploidiz-ation, where karyokinesis still occurs (reviewed inBrodsky & Uryvaeva, 1985). Various cytologicalstudies have purported to show that giant cellchromosomes are polytene (Barlow & Sherman.1974; Snow & Ansell, 1974; Zybina, 1977), although

128 S. Varmuza, V. Prideaux, R. Kothary and J. Rossant

condensed polytene chromosomes are not observednormally in the interphase nuclei of trophoblast giantcells. It has also been reported that single Barr bodiesoccur in rabbit trophoblast giant cells (Zybina et al.1973).

The advent of sensitive in situ DNA-DNA hybrid-ization techniques allows resolution of the problem ofthe mode of endoreduplication in trophoblast giantcells. We have utilized in situ hybridization to severalchromosome markers to show that the number ofhybridization signals does not increase with ploidylevels in trophoblast giant cells. Moreover, the num-ber of signals per nucleus approximates the numberof marked chromosomes per diploid complement.These results suggest that sister chromatids, but nothomologous chromosomes, are closely associatedthroughout the development of trophoblast giantcells.

Materials and methods

Mouse strainsFor studies utilizing hybridization to endogenous DNAsequences, outbred CD-] mice (Charles River, St Con-stant, Quebec) were used. Two transgenic mouse strainswere also used. Cecilia Lo derived a transgenic mouse line,line 85, by DNA iontophoretic introduction of a mouse /3-globin plasmid into a hybrid C57BL/6/C3H mouse zygote(Lo, 1986). It contains 1000 tandemly repeated copies of theplasmid inserted at the telomere of chromosome 3 (Lo,1986 and C. Lo, personal communication). We obtained ahomozygous male from Lo and have crossed the marker onto the outbred CD-I strain where it is now maintained as ahomozygous outbred line, Tg-M/3G-]. A second transgenicline, Tg-HSZ-4, was produced in this laboratory andcontains 16 tandem copies of a mouse hsp68-£\ coli lacZfusion gene fragment on a CD-I background (R. Kothary,M. Perry, S. Clapoff, L. Moran & J. Rossant, unpublisheddata). The insert is located near the centromere of a largechromosome, possibly chromosome 1.

PlasmidsA plasmid containing five tandem repeats of the mousemajor satellite DNA sequence was prepared by insertingAvail fragments from the satellite peak isolated from acaesium chloride density gradient into pTZ19R (C. Davis,unpublished data). pM/3<52, a derivative of the plasmidinserted into Tg-M/3G-1 from which a mouse repetitivesequence has been removed, was a kind gift of C. Lo(University of Pennsylvania). pCH126 containing the E.coli lacZ gene was used for in situ detection of the Tg-HSZ-4insert. A cDNA clone, pH2-IIa, which cross-reacts with allClass I MHC genes, was kindly provided by L. Hood(Steinmetz et al. 1981).

Preparation of trophoblast cellsPrimary giant cells were prepared by culturing blastocysts,flushed from the uteri of 3-5-day pregnant mice, into

multichambered culture slides (Lab-Tek, Miles Scientific)in O--MEM (Gibco) supplemented with 10% fetal bovineserum, lOOi.u. ml"' penicillin, and 50/igmP1 strepto-mycin. Cultures were maintained for 4-6 days in humidi-fied air plus 5 % CO2 at 37°C, during which time giant cellsgrew out from the hatched blastocysts. Secondary giantcells were cultured from ectoplacental cones which hadbeen dissected from 7-5-day postimplantation embryos.The cells were grown as described above, except thatculture time was reduced to 2-4 days. Giant cell nuclei from10-5-day embryos were isolated by teasing apart the deci-dual cell layer and the trophoblast giant cell layer in a dishcontaining phosphate-buffered saline. The nuclei, whichfell to the bottom of the dish, were collected in a drawnPasteur pipette, applied directly to a poly-L-lysine-coatedglass microscope slide and allowed to air dry. Liver cellswere prepared by simply touching a poly-L-lysine-coatedslide with a freshly cut piece of liver. The blotted cells wereallowed to air dry.

In situ hybridization with biotinylated probesCells attached to microscope slides were fixed for 30min ormore in 3:1 ethanol: acetic acid, followed by two rinses inabsolute ethanol. After air drying, the DNA was denaturedin 70% formamide plus 2 x SSC at 70°C for 5min. Thedenatured slides were quenched by immersion in cold 70 %ethanol, dehydrated through an ethanol series and airdried.

Plasmid DNA was nick translated with biotinylateddUTP as described (Rossant et al. 1986). The hybridizationsolution, consisting of 2-4Jugml~1 denatured biotinylatedDNA, 200/igmP' sheared denatured herring sperm DNA.5 x SSC and 10% dextran sulphate, was layered over thecells under a coverslip and hybridized overnight at 60°C.Slides were washed twice at room temperature in 2 x SSC.once at room temperature in 01 x SSC and once at 50°C in0-1 x SSC.

Biotin was detected by binding of streptavidin conju-gated with horseradish peroxidase (HRP) (Detek-1-hrp,Enzo Biochem), followed by staining with 3,3'-diamino-benzidine (DAB) to detect HRP activity (Rossant et al.1986). In some cases, the DAB staining was silver enhanced(DAB enhancement kit, Amersham). Following stainingfor HRP, cells were counterstained with haematoxylin andeosin, dehydrated and mounted with Permount.

Results

In situ hybridization to satellite DNAThe first chromosome marker examined in this studywas the major satellite sequence which is associatedwith the centromeres of all mouse chromosomes.Primary and secondary giant cells displayed essen-tially identical patterns when they were hybridizedwith a cloned satellite sequence. Discrete hybridiz-ation signals of varying sizes could be seen on allinterphase nuclei (Fig. 1A,C,E). The number ofsignals per nucleus varied, but was distributed nor-mally around a mean which approximated the haploid

Polytene chromosomes in mouse trophoblast giant cells 129

14-

12-

= 10-

"5 8

I 6'3Z 4

p Blastocyst outgrowthsN = 115

Mean = 18-8a = 4-64

16

14-

I 10-

| 8

I 6'Z

4-

2

10 14 18 22 26 30 34Signals per nucleus

mi1

epc outgrowthsN = 121

Mean =17-5a = 504

IfI 4

n

10-5 d giant cell nucleiN = 53

Mean= 19-9a =3-88

10 14 18 22 26 30 34Signals per nucleus

14 IS 22 26 30 34Signals per nucleus

Fig. 1. Satellite DNA sequence distribution in giant cell nuclei. Cells were fixed and hybridized with biotinylatedpTZ19K. Photomicrographs of in situ hybridizations to giant cells from blastocyst outgrowths (A), ectoplacental coneoutgrowths (C), and 10-5-day conceptuses (E) show multiple hybridization signals in each nucleus. The bars in eachphotomicrograph represent 50fim. Histograms of the number of hybridization signals per nucleus are shown forblastocyst outgrowths (B), ectoplacental cone outgrowths (D), and isolated 10-5-day giant cell nuclei (F).Photomicrographs of blastocyst outgrowths and ectoplacental cone outgrowths which had been hybridized withbiotinylated satellite DNA were used to score the number of hybridization signals per nucleus. Only clearlydistinguishable nuclei were scored. Giant cell nuclei from 10-5-day conceptuses were scored directly under themicroscope for the number of hybridization signals per nucleus.

number of chromosomes (Fig. 1B,D,F). The signalsin 10-5-day giant cell nuclei (Fig. IE) were noticeablylarger than those on giant cells derived from ectopla-cental cone (Fig. 1C) or blastocyst outgrowths(Fig. 1A).

In situ hybridization to Tg-MfiG-1 cellsThe observation that giant cells contain a haploidnumber of centromeric hybridization signals might beconstrued as evidence of polyteny in these cells,perhaps even involving association of homologouschromosomes. However, a similar pattern could ariseby random association of centromeric sequences inthe interphase nuclei of trophoblast giant cells. In situhybridization to the major satellite sequence couldnot distinguish these possibilities because the se-quence is found on all chromosomes. Therefore, weexamined in detail the hybridization patttern of a

chromosome-specific marker, namely the mouse S-globin plasmid inserted at the telomere of chromo-some 3 in Tg-M/SG-1.

Control hybridizations of biotinylated pM/3<52 toliver cells from hemizygous or homozygous Tg-MfiG-1 mice established that Tg-M/SG-1 contained a suit-able marker with which to distinguish polyploidy andpolyteny in situ (Fig. 2A,B). Liver spreads containcells with 2C, 4C and 8C ploidy levels of DNA,derived by karyokinesis without cytokinesis. Thenumber of signals observed per cell generallyexhibited the expected variation with ploidy values.Most hemizygous cells contained one (2C), two (4C)or four (8C) hybridization signals, while homozygouscells contained two (2C), four (4C) or, less fre-quently, eight (8C) signals (Fig. 2C). Intermediatenumbers of signals were occasionally observed es-pecially in homozygous cells. This was probably due

130 S. Varmuza, V. Prideaux, R. Kotharv and J. Rossant

to overlap of signals within the nucleus. In situhybridization can thus directly determine the ploidylevel of a polyploid cell without the necessity ofdetermining total DNA content.

Primary and secondary giant cells from hemizygousor homozygous transgenic embryos were hybridizedwith pM/382 and scored for the number of hybridiz-ation signals per cell. Unlike liver cells, the number ofhybridization signals did not increase with ploidylevels in these cells. Most hemizygous Tg-M^G-1giant cells contained one discrete hybridization signal(Fig. 3A,C,E), while homozygous cells containedtwo signals (Fig. 3B,D,F). Direct microspectrophoto-metric analysis of ploidy levels was not compatiblewith the in situ hybridization technique, but previousstudies have shown that blastocyst and ectoplacentalcone outgrowths contain cells with ploidy levels up to16C (Barlow & Sherman, 1972; Rossant & Ofer,1977). Thus, polytenic association of chromatids mustoccur in the region of the Tg-M/JG-1 insert in giantcells outgrown in tissue culture.

Hybridization to 10-5-day giant cell nuclei obtaineddirectly from embryos in vivo revealed that polytenicassociation of chromatids was a consistent feature ofgiant cells even at very high ploidy levels (Fig. 3E,F).These nuclei can become as large as 100 fim indiameter and may contain DNA levels of up to 512C(Barlow & Sherman, 1972). However, only one(hemizygous) or two (homozygous) hybridizationsignals were observed per cell. The fortuitous associ-ation of the giant cell nucleus in Fig. 3F with a groupof diploid cells illustrates clearly the difference in sizeof the nuclei and the intensity of the hybridizationsignals between the diploid and giant cells. Thedifference is also clear in a sectioned 7-5-day concep-tus (Fig. 6).

Occasionally, twice the expected number of dis-crete signals were observed per giant cell (summar-ized in Fig. 4), presumably resulting from either abreakdown in the polytenic association, or a poly-ploid mitotic division. The sporadic appearance ofnondiscrete (fuzzy) hybridization signals in giant cellsgrown in vitro is less easy to explain (see arrowsFig. 3C). However, since the frequency of thesesignals varied from experiment to experiment, wesuspect it was an in vitro artefact perhaps relatingmore to the penetration of the DAB stain into thecells than the state of the DNA. Such signals werenever observed in giant cells collected in vivo or insections of intact conceptuses (Fig. 6). It should benoted that the mouse /3-globin sequences in the probedid not detect the endogenous mouse globin genes ingiant cells from nontransgenic CD-I controls, indi-cating that there may be a minimum target size forhybridization with this technique.

In situ hybridization to Tg-HSZ-4 and H-2In order to confirm that polyteny was not a propertyof the telomeric Tg-M/SG-1 insert only, we performeda limited series of hybridizations to two othermarkers. Hybridizations were performed to giantcells from hemizygous Tg-HSZ-4 and CD-I giant cellsusing an E. coli lacZ or a class I MHC proberespectively. Since the target sizes for hybridization inthese cells were smaller than in Tg-MBG-1, DAB-stained slides were silver enhanced to increase sensi-tivity. Giant cell nuclei isolated from -10-5-day em-bryos were examined since these have higher ploidylevels than cells cultured in vitro and should not besubject to any culture artefacts. Hemizygous Tg-HSZ-4 cells contained one hybridization signal as wasalso observed in diploid amnion tissue on the sameslide (Fig. 5A,B). Hybridization to the endogenousH-2 complex on chromosome 17 revealed two clearsignals per giant cell (Fig. 5C), even though the levelof sensitivity was not sufficient to detect any signal indiploid cells.

Discussion

Trophoblast giant cells in mammalian embryosendoreduplicate their genomes and become enlargedas a consequence. Until recently, the mechanism bywhich giant cells amplify their genomes could only beinferred from indirect evidence and inconclusivecytological observation. We have shown, using fourdifferent chromosome markers which are detectablein situ, that polytenic association of sister chromatidsoccurs in trophoblast giant cells despite the absenceof visible condensed polytene chromosomes. Giantcells from three different stages of embryogenesis andwith varying ploidy levels were analysed in our study.All produced essentially the same pattern of hybridiz-ation with three of the four markers used. In embryoscontaining a single marked chromosome per diploidcomplement (two hemizygous transgenjc mouselines), only one hybridization signal was observed ingiant cell nuclei; embryos containing two markedchromosomes per diploid complement (the homo-zygous transgenic Tg-M/3G-1 and endogenous H2sequences) displayed only two signals per giant cell

Fig. 2. Distribution of a telomeric marker in polyploidliver cells. Photomicrographs of liver cells fromhemizygous (A) and homozygous (B) Tg-M/SG-1 mice,which were hybridized with biotinylated pM/3<52, revealmultiple signals in polyploid cells. Bars represent 50jum.The number of hybridization signals visualized in allnuclei in several randomly chosen fields were scored andpresented in histogram form (C). The number of cellsscored was 327 for hemizygous QS/+) liver and 408 forhomozygous (/3//J) liver.

•

100-

40-

20

/?/+ liver

1

liver

1 2 3 4 1 2 3 4 5 6 7Signals per nucleus

•^^Hr mSKKs

Polytene chromosomes in mouse trophoblast giant cells 131

nucleus. In contrast, polyploid liver cells from hemi-zygous and homozygous Tg-M/JG-1 mice containedmultiple signals.

The major satellite sequence, which is located atthe centromeres of all mouse chromosomes, pro-duced a different pattern. Giant cells containedapproximately 18 hybridization signals, which isslightly fewer than the haploid number of mousechromosomes (20). Although this observation is con-sistent with polytenic association of both sister chro-matids and homologous chromosomes at the centro-meres, more convincing evidence would be providedby chromosome-specific centromeric markers. Otherinterphase nuclei display haploid numbers of 'chro-mocentres' after staining with Hoechst stain (Hilwig& Gropp, 1972), or hybridization with the satellitemarker (Manuelidis et al. 1982; Manuelidis, 1984),suggesting that association of centromeres is a generalproperty of interphase nuclei.

We have not demonstrated directly association ofsister chromatids for all regions of the genome.However, the three sequences we have used map todifferent chromosomes and to different chromosomalregions. Thus, it seems reasonable to conclude thatthe general mechanism of giant cell formation in-volves successive rounds of DNA replication in theabsence of either karyokinesis or cytokinesis, perhapsbecause of impaired mitotic functions. Sister chroma-tids remain closely associated, but homologouschromosomes do not associate with each other. Theclose association of sister chromatids may reflectsteric intertwining of strands subsequent to asynchro-nous rounds of DNA replication, or it may reflect amore concerted function, such as specific chromatidassociation mediated by a synaptonemal complex-like

Fig. 3. Distribution of a telomeric marker in giant cellsof different ploidy. Giant cells from hemizygous (A,C,E)and homozygous (B,D,F) Tg-M/3G-1 embryos werehybridized with biotinylated pM/362 DNA. In two cases(B,D), the DAB signal was enhanced as described. Thedifferent ploidy levels were represented by blastocystoutgrowths (A,B), ectoplacental cone outgrowths (C,D),and isolated 10-5-day giant cell nuclei (E,F). Barsrepresent 50^m. The arrows in C indicate examples ofdiffuse hybridization. Note that one giant cell nucleus inF lies adjacent to a cluster of diploid cells.Fig. 4. Histograms of the number of hybridization signalsin giant cells from hemizygous and homozygous Tg-M/SG-1 embryos. Hemizygous (A,C,E) and homozygous(B,D,F) giant cells from blastocyst outgrowths (A,B),ectoplacental cone outgrowths (C,D), and isolated giantcell nuclei (E,F) which had been hybridized withbiotinylated pM/3<52 DNA were scored for the number ofsignals per nucleus. All nuclei in several randomly chosenfields were scored under the microscope, and the numberof hybridization signals per nucleus were tabulated inhistogram form.

structure. The observation that occasional nucleicontained multiple signals tends to support the firstpossibility. Interphase-like association of centro-meres continues, even though the centromeres mayalso be endoreduplicated. Some evidence has beenpresented to support the notion that interphasechromosomes associate with each other in a nonran-dom fashion (Hadlaczky et al. 1986; Manuelidis,1984). It may prove useful in future to examine the

A B

f}/+ Blastocyst outgrowths fi/fi Blastocyst outgrowthsN = 334 N = 129

100

80

•3 60O

40-

20

0 1 2N.D.

100

80

= 60

^ 4 0

20

f}/+ epc outgrowthsN = 587

•

0 1 2 3 4 mixN.D.

D

fi/fi epc outgrowthsN = 483

I0 1 2N.D. 0 1 2 3 4 mixN.D.

E F

0/+ 10-5 d giant cell nuclei 0/0 10-5 d giant cell nuclei

N = 67

100

0 1 2 0 1 2 3 4

132 S. Varmuza, V. Prideaux, R. Kotharv and J. Rossant

distribution of chromosome-specific markers such asthose described here, which would unambiguouslydemonstrate whether specific chromosomes are as-sociated in interphase cells.

Other investigators have proposed a complicatedseries of endomitotic and endoreduplicative roundsof chromosome duplication to account for the ac-cumulation of DNA in rodent trophoblast giant cells(Zybina & Grischenko, 1970; Ilgren, 1980). Our datado not support the idea that initial amplification isaccomplished by endomitosis leading to polyploidcells. Indeed, the polyploid metaphases observed byIlgren (1980) are far too few to account for the degreeof DNA amplification which occurs in the giant cellpopulation. We suspect that the few polyploid meta-phases observed by Ilgren (1980) and by Zybina &Grischenko (1970) may have arisen from extraembry-onic ectoderm rather than trophoblast giant cells.

Recently, Brower (1987) reported similar findingsto ours from a limited study using in situ hybridizationof a cDNA clone of a-1 antitrypsin to giant cells. Shewas unable to detect any signal in giant cells with lessthan 8C, however, and she reported that 45 % ofgiant cell nuclei from 11-day conceptuses displayeddispersed staining. We did not observe such dispersedsignals in giant cell nuclei of similar age. Because insitu hybridization to the transgenic inserts was sensi-tive enough to detect signals in diploid cells, we areconfident that multiple signals in giant cell nucleiwould have been readily detectable with these probeshad they occurred.

Polyploidy and polyteny are widespread through-out the biological world (reviewed in Brodsky &Uryvaeva, 1985). The functional significance of poly-ploidy is unclear, but polyteny may afford a cell theopportunity to differentially amplify important se-quences. This has been demonstrated to be the casefor Drosophila chorion genes in polyploid ovarianfollicle cells (Kalfayan etal. 1986; Kafatos etal. 1986).No differential amplification of specific sequences hasyet been observed in mouse trophoblast giant cells.

Fig. 5. Distribution of a nontelomeric hemizygousmarker and of an endogenous marker in 10-5-day giantcell nuclei. Giant cell nuclei (A) and amnion epithelium(B) from 10-5-day hemizygous Tg-HSZ-4 embryos werehybridized with biotinylated pCH126 DNA. The amnionepithelium served as a diploid control. Giant cell nucleifrom 10-5-day CD-I embryos were hybridized withbiotinylated pH2-IIa DNA (C). All samples were treatedwith the DAB enhance procedure. The pH2-IIa clonerecognized endogenous H2 sequences in giant cells, butnot in diploid cells (data not shown). In Fig. 5A thearrow indicates the hybridization signal while thearrowhead is pointing at heterochromatin. These twofeatures are distinguishable in colour reproductions butappear similar in monochromatic reproduction.

Sherman etal. (1972)measured the relative amountof satellite sequence versus unique sequence DNA ingiant cells by comparing their hybridization kineticsand found no difference between giant cells anddiploid cells, indicating that satellite sequences areamplified to the same degree as unique sequences.This is contrary to the situation in Dipteran salivaryglands where satellite sequences are under-replicated(Gall et al. 1971). Further analysis of the state ofreplication of trophoblast-specific genes may shedlight in this area.

5A

Polytene chromosomes in mouse trophoblast giant cells 133

Drosophila polytene chromosomes have proved tobe invaluable tools in the mapping and cloning ofgenes. A similarly abundant source of material formapping and cloning mouse genes would be useful.Clear demonstration of polyteny in trophoblast giantcells gives new impetus to the search for a means ofcondensing these chromosomes and visualizing themmore readily. In vitro extracts of frog oocytes showingmaturation-promoting activity might be applicable(Lohka & Mailer, 1985; Miake-Lye & Kirschner,1985).

We have demonstrated here that transgenic insertscan be used as in situ chromosome markers to addressquestions related to the dynamics of the genome.

They may also be used effectively as ubiquitous cellautonomous markers in experiments involving analy-sis of cell distribution in chimaeric mice. Previousstudies employing differential in situ hybridization tosatellite sequences in two different species of mousewere limited by interspecific incompatibilities (Ros-sant & Frels, 1980; Rossant et al. 1983). Transgenicmice, which can be made congenic with the nontrans-genic partner, eliminate such barriers to experimentaldesign. We are currently using the Tg-M/JG-1 strainin a variety of chimaera studies.

Trangenic inserts may also act as closely linked insitu markers of known mutations in future chimaerastudies. At present, this approach is limited only bythe paucity of transgenic mice with large detectableinserts at interesting chromosomal locations. As thenumber of transgenic lines increases in various lab-oratories, the possibilities of this application willsurely expand.

We wish to thank Lori Regenstnef for patient collectionof giant cell nuclei and Susan Clapoff and Victor Maltby forproduction of transgenic mice. This work was supported bythe Medical Research Council of Canada, the NationalCancer Institute of Canada, and the Natural Sciences andEngineering Research Council of Canada. J.R. is an NCIResearch Associate, and S.V. and R.K. are NCI Fellows.

:•• » . •

V . f • • >.

Fig. 6. Distribution of a telomeric marker in sectionedgiant cells. Homozygous 7-5-day Tg-M^SG-1 embryoswere fixed in 3:1 ethanol:acetic acid, embedded in esterwax and sectioned to a thickness of 7^m. Sections werehybridized with biotinylated pM/S<52 DNA andcounterstained with haematoxylin and eosin. Many giantcell nuclei appear to contain only one signal, and somecontain none. This is a result of cutting the marker out ofthe plane of the section. Note that the signals in adjacentdiploid cells are smaller than those in the giant cells. Thebar represents 50jim.

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{Accepted 19 September 1987)