Journal of Cell Science 102, 299-305 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

299

Distribution of a nuclear envelope antigen during the syncytial mitoses of

the early Drosophila embryo as revealed by laser scanning confocal

microscopy

MARIA GIOVANNA RIPARBELLI and GIULIANO CALLAINI

Department of Evolutionary Biology, University of Siena, via Mattioli 4, 53100 Siena, Italy

Summary

The changing distribution of a nuclear envelope antigenrecognized by a monoclonal antibody raised againsthuman fibroblast vimentin during the syncytial mitosesof the Drosophila embryo has been studied with aconfocal laser scanning microscope. The antigen appearsvery early as irregular aggregates in the peripheralcytoplasm of the preblastoderm embryo. As the firstnuclei reach the periplasm the antigen is localized on the

nuclear envelope and the cytoplasmic staining decreases.In addition to the perinuclear labeling we observedintense midzone and polar staining during the mitoticcycle. A possible relationship between polar localizationof the antigen and centrosome position is discussed.

Key words: nuclear envelope, Drosophila embryo, confocalmicroscope.

Introduction

The onset of mitosis in higher eukaryotes is ac-companied by the fragmentation of the nuclear envel-ope into large cisternae and the disappearance of porecomplexes (Robbins and Gonatos, 1964; Roos, 1973;Zatsepina et al., 1977). This general model of nuclearenvelope rearrangement, termed open mitosis (Franke,1974), shows remarkable differentiation in the syncytialembryo of Drosophila. Electron microscopy revealedthat the nuclear envelope remains essentially intactduring mitosis of the syncytial blastoderm and largeopenings appear at the poles, through which the spindlemicrotubules run. Nuclear pore complexes are lostduring mitosis and a second layer of fenestratedcisternae joins the original cisternae to form a doublefenestrated layer (Stafstrom and Staehelin, 1984).Despite this structural difference in nuclear envelopeorganization from that of most higher organisms,polypeptides homologous to vertebrate lamins havebeen described in Drosophila embryos (Risau et al.,1981; Fisher et al., 1982; Smith et al., 1987). Immuno-fluorescence observations showed that the distributionof these nuclear envelope proteins is similar to thatobserved in vertebrate cells at mitosis, but during therapid divisions in the early Drosophila embryo theseantigens are never completely dispersed throughout thecytoplasm (Fuchs et al., 1983).

To follow nuclear envelope dynamics during thesyncytial mitoses that precede cellularization, weutilized a monoclonal antibody raised against human

fibroblast vimentin that recognizes a nuclear envelopeantigen (Callaini and Riparbelli, 1991). This studydemonstrated that the antibody strongly stains thenuclear envelope at interphase and throughout themitotic cycle. However, the exact localization of theantigen on the nuclear envelope and its distributionwere still unclear.

This paper re-examines the changing distribution ofthe nuclear antigen as observed by confocal mi-croscopy, which gives more detailed information thanepifluorescence microscopy. Immunoelectron-micro-scopic techniques also allowed us to detect the spatiallocalization of this antigen.

Materials and methods

Collection of the embryosEmbryos of Drosophila melanogaster (Oregon-R strain) werecollected at 24°C on agar plates, dechorionated in a 50%commercial bleach solution, and washed with distilled water.The stage of the nuclear cycle was determined by staining thenuclei with Hoechst 33258.

lmmunofluorescenceThe dechorionated embryos were fixed according to Warnand Warn (1986), except for a final short fixation with acetonefor 5 min. The embryos were then washed in phosphate-buffered saline (PBS) and incubated for 1 h in PBS containing0.1% bovine serum albumin. After rinsing in PBS theembryos were incubated overnight at 4°C with a monoclonalantibody against vimentin (Sclavo, Siena). This antibody was

300 M. G. Riparbelli and G. Callaini

made in mouse after immunization with purified vimentinfrom human cultured fibroblasts. The samples were thenwashed with PBS and incubated at room temperature in goatanti-mouse fluorescein-conjugated IgG (Cappel, West Ches-ter, PA). For double labeling the embryos were incubated for4-5 h at room temperature with Rbl88 antiserum, whichspecifically recognizes an antigen associated with the centro-some of Drosophila embryos (Whitfield et al., 1988). Theseembryos were washed and treated with rhodamine-conju-gated goat anti-rabbit IgG (Cappel, West Chester, PA).Nuclei were stained with 1 /ig/ml of the DNA-specific dyeHoechst 33258 in PBS. After a final washing in PBS, theembryos were mounted in 90% glycerol containing 2.5% n-propyl gallate (Giloh and Sedat, 1982). Fluorescence obser-vations were carried out with a Leitz Aristoplan microscopeequipped with fluorescein, rhodamine and UV filters.

Photomicrographs were taken with Kodak Tri-X pan filmand developed in Kodak HC 110 developer for 7 min at 20°C.

Confocal microscopyThe distribution of the antigen recognized by the anti-vimentin antibody was also described using a Bio-RadMicrosciences (Cambridge, MA) model MRC-500 ConfocalImaging System. This technique provides optical thin sectionsthrough the sample. It was configured with a Nikon Optiphotmicroscope using a x63 objective.

Immunoelectron microscopyDrosophila melanogaster embryos were collected, dechorio-nated, fixed and devitellinized as described for immunofluor-escence. After washing in PBS the embryos were incubatedovernight at 4CC in anti-vimentin antibody. The embryos werethen washed in PBS and incubated for 1 h at roomtemperature with anti-mouse IgG conjugated to 10 nmcolloidal gold (BioCell, Cardiff). After washing, the sampleswere fixed in 3% glutaraldehyde in PBS for 30 min, washedagain in PBS, stained en bloc with 1% uranyl acetate in water,dehydrated in a graded ethanol series and embedded in Epon.The sections were stained with uranyl acetate and lead citrateand viewed at 60 kV in a Philips EM 400 electron microscope.Control experiments were performed by omitting the incu-bation with the first antibody.

Results

Preblastoderm embryosWhole-mount face views of fixed embryos stained withthe anti-vimentin antibody during cycles 1-8, before thesomatic nuclei reach the periplasm, show irregularassemblies of anti-vimentin-stained aggregates. Sucharrays contain compact and large aggregates of fluor-escent-stained material. These aggregates are randomlydisposed over the whole surface of the embryo in a 5 /zmlayer just below the plasma membrane (Fig. 1A). Whenthe somatic nuclei reach the embryo surface duringcycle 10 the cortical aggregates are reduced in size andnumber. Fig. ID displays a small area of the surface ofan embryo at nuclear cycle 10, the first cycle after thenuclei reach the embryo cortex. The antibody stronglystains the nuclear periphery, which appears as a brightenvelope-like structure that changes in shape during themitotic cycle. The cortical aggregates are randomlydispersed among the blastodermic nuclei, but are not

localized in cytoplasmic domains like microtubules andmicrofilaments. As nuclear division progresses theantibody staining is evident around the blastodermicnuclei, whereas the concentration of the antigen in thecytoplasm is less evident and appears as punctatefluorescence (Fig. IE). The antigen distribution in polarbodies is different from that found in somatic nuclei andno envelope-like structures are observed (Fig. IB).Double labeling for anti-vimentin and DNA revealsdiffuse staining associated with the chromosomes (Fig.1C). The fluorescence is interrupted in the centralregion of the polar body where the chromosomes,disposed in a star-like configuration, leave a small gap.

Syncytial blastodermDuring interphase the antigen becomes concentrated athigh levels around the periphery of the somatic nucleiand is localized in bright rings (Fig. 2A). Suppression ofout-of-focus fluorescence and image intensificationshow that the yolk nuclear periphery is also stained bythe antibody (Fig. 2A). The fluorescence encircling thenuclei at prophase is not uniform, but the antibodyappears to stain a series of small dots on the nuclearenvelope. The antigen is also detected in the surround-ing cytoplasm as small fluorescent aggregates. Sagittaloptical sections reveal two semiconical structures on theupper side of each somatic nucleus (Fig. 2B). Doublefluorescence images with anti-vimentin and Rbl88antibodies show that these structures coincide with thelocalization of the centrosomes (Fig. 2C). The antibodycontinues to stain the polar regions from metaphase totelophase, but the staining becomes diffuse. Fig. 2Dshows a double exposure with Rbl88 and anti-vimentinantibodies during anaphase.

The antigen binding pattern changes dramaticallyduring the transition to metaphase. The bright rings aretransformed into ovoid structures and the semiconicalformations disappear (Fig. 3A). However, stronglabeling marks the extremities of the nuclear envelope.In this region the staining is diffuse and we observe agradient of fluorescence concentration decreasing fromthe nuclear envelope to the surrounding cytoplasm(Fig. 3A). During anaphase A the staining pattern issimilar to that observed during metaphase except for afurther elongation of the bright structures and ad-ditional fluorescent staining appearing in the equatorialregion of the nuclear envelope (Fig. 3B). Anaphase Binvolves a further elongation of the envelope-likestructures, which become elliptical. At this time theantibody labeling is more complex. The extremities ofthe bright structures are smaller than during anaphaseA and the antibody binding pattern has a C-shapedappearance (Fig. 3C). The C-shaped fluorescent ex-tremities become circular during the transition totelophase. At this time we observe small rings,encircling the daughter nuclei, which are connected byinterzonal labeling (Fig. 3D). Serial optical sectionsshow that the telophase structures are U-shaped withthe nuclei in the upper position. Interzonal labeling isorganized in two longitudinal lines in the lower part,whereas fluorescence is diffuse in the upper region (Fig.

Nuclear envelope dynamic in Drosophila embryo 301

Fig. 1. Localization of the antigen recognized by the monoclonal antibody against human vimentin. (A) Surface view of anembryo during the first nuclear cycle. Antibody labeling appears as irregular fluorescent aggregates. (B,C) Surface view ofan embryo during nuclear cycle 2, double labeled with the anti-vimentin antibody and Hoechst 33258. Arrows in B andarrowheads in C indicate the antibody binding pattern and the chromosome disposition in polar bodies. (D) Surface viewof an embryo during stage 10. (E) Surface view of an embryo at prophase of stage 11. n, nuclei. Bar, 20 jun.

3D). This staining is the remnant of the anaphase Bmiddle zone staining. As telophase progresses theinterzonal fluorescence concentrates in a narrow regionbetween the daughter nuclei (Fig. 3E). At the end oftelophase the interzonal labeling reduces and diffuses inthe cytoplasm (Fig. 3F). The mitotic stages weredetermined by simultaneous staining with the DNA-specific dye Hoechst 33258 (not shown).

Immunoelectron microscopyTo determine the localization of the antigen recognizedby the anti-vimentin antibody, we performed immuno-electron-microscopic experiments on Drosophila em-bryos, which give more detailed structural information.Sagittal views of the nuclear envelope show that goldparticles are restricted to the outer side of the nucleus(Fig. 4). Controls using pre-immune serum as firstantibody failed to reveal any nuclear envelope staining.

Discussion

The antigen binding pattern as revealed by epifluor-escence and confocal microscopy may be divided intofour types of distribution: perinuclear, polar, interzonaland cytoplasmic staining.

The perinuclear staining at interphase is reminiscentof the distribution of the nuclear lamins, which areobserved in the form of ring-like structures (Fuchs etal., 1983). However, immunoelectron-microscopictechniques revealed that our antibody recognizes anantigen localized on the outer side of the nuclearenvelope. Moreover, the antibody against humanfibroblast vimentin strongly stains the nuclear boundarythroughout the mitotic cycle (Callaini and Riparbelli,1991). Instead, the antibodies against the nuclearlamina of Drosophila give a feeble staining duringmetaphase and anaphase, presumably because theantigens are partly diffuse in the cytoplasm (Fuchs et

302 M. G. Riparbelli and G. Callaini

Fig. 2. Scanning confocal microscopic view of Drosophila embryos stained with the antibody against human fibrobiastvimentin (A,B) and immunofluorescence microscopy of embryos double stained with anti-vimentin antibody and Rbl88serum (C,D). (A) Optical section through a whole mount of an embryo during interphase of nuclear cycle 11 showing theantigen binding pattern around somatic nuclei (sn) and yolk nuclei (yn). (B) Surface view of an embryo during prophase ofnuclear cycle 11; arrowheads mark the antigen binding pattern in the centrosomal region. (C) Prophase and (D) anaphaseof nuclear cycle 11. Open arrows indicate centrosomes, which are dot-shaped during prophase, and flattened at anaphase.Bars, 10 jan.

al., 1983; Frash et al., 1986; Smith et al., 1987). Thepresence of an envelope-like protein, termed otefin,that persists throughout mitosis has recently beendescribed in tissue culture cells and embryos ofDrosophila (Miller et al., 1985; Harel et al., 1989). Thepattern of distribution of otefin is very similar to thatobserved with anti-vimentin antibody. However, immu-noelectron microscopy shows that, in the case of otefin,the gold particles are restricted to the inner membraneof the nuclear envelope, but when the embryos areincubated with anti-vimentin the gold particles arelocalized on the outer side of the nucleus. The similarimmunofluorescence staining, despite the differentlocalization of the antigens, indicates that most of thenuclear envelope is conserved throughout the mitoticcycle, as suggested by the electron-microscopic findingsof Stafstrom and Staehelin (1984). When the embryosare observed with a confocal microscope, which permitshigher resolution of the fluorescence images, the

antigen distribution around the nuclear envelope seemsto be discontinuous during mitosis. This is consistentwith the observations of Stafstrom and Staehelin(1984), which showed a fenestrated nuclear envelopeduring the mitotic cycle.

Immunofluorescence observations showed that theantigenic determinant, recognized by the human fibro-biast anti-vimentin antibody, is localized at late telo-phase on the nuclear envelope and in the spindlemidzone. Confocal microscopy revealed a distinct flatstructure between the re-forming daughter nuclei.Other antigens that show a similar concentration in thespindle midzone have recently been described (Cookeet al., 1987; Kingwell et al., 1987; Sellitto andKuriyama, 1988; Andreassen et al., 1991; Earnshawand Cooke, 1991). The gradual diffusion of the midzonestaining in the cytoplasm at the end of telophasesuggests that the antigen may play a role in nuclearenvelope organization during the last phases of mitosis.

Nuclear envelope dynamic in Drosophila embryo 303

Fig. 3. Scanning confocal microscope optical section through embryos at different developmental stages incubated withanti-vimentin antiboby. (A) Metaphase. (B) Anaphase A. (C) Anaphase B. (D) Early telophase. (E) Late telophase.(F) Early interphase. Arrowheads mark midzone staining. Bar, 10 fjm.

This may be confirmed by the observation that attelophase midzone staining is suggested by strongstaining of the central region of the nuclear envelopethroughout anaphase. Fluorescence staining at ana-phase was detected in the region where microtubulesoverlap antibodies that recognize midzone antigens (seeAndreassen et al., 1991). Interzonal labeling at telo-phase, comparable to that observed with the anti-

vimentin antibody, was reported in Drosophila by usingan antibody raised against a microtubule-associatedprotein (Kellogg et al., 1989).

The antigen localization observed in somatic and yolknuclei was not found in polar bodies. In this case theanti-vimentin antibody did not demonstrate bright ringsaround the chromosomes, but the fluorescence wasdiffuse in the nuclear region. This observation may

304 M. G. Riparbelli and G. Callaini

Fig. 4. Immunogold electron-microscopic localization of thenuclear envelope antigen. Arrows indicate gold particles,n, nucleus; ne, nuclear envelope. Bar, 0.25 (w\.

indicate that polar bodies are unable to form a normalnuclear envelope or, alternatively, that the nuclearenvelope antigen has a different localization from thatobserved in somatic nuclei.

Confocal microscopy shows two semiconical struc-tures localized in the upper region of the blastodermicnuclei during prophase. As mitosis progresses thesestructures move to the spindle poles and expand,suggesting a relationship with centrosomal behavior.Double-labeling with Rbl88 and anti-vimentin anti-bodies seems to confirm this hypothesis (Callaini andRiparbelli, 1991; present data). The polar stainingobserved with anti-vimentin diffuses when the centro-somal material loses its compactness and expands.Electron-microscopic analysis failed to correlate theantigen distribution at the spindle poles with distinctsubstructures in the nuclear envelope (Stafstrom andStaehelin, 1984; Callaini and Anselmi, 1988). Only apeculiar aggregation of short cisternae near the poleswas observed. We can therefore assume that theantigen was also present in these cisternae, but onlydetailed immunoelectron-microscopic observations canclarify this point. Previous studies have shown anassociation between centrosomes and nuclear mem-branes (Bornens, 1977; Nadezhdina et al., 1979; Fais etal., 1984; Katsuma et al., 1987). If a linkage betweencentrosomes and the nuclear envelope exists, it seemsto be a feeble interaction, since there are several casesin which the centrosomes detach from the spindle poles.Drosophila embryos microinjected with anti-tubulinantibodies (Warn et al., 1987), lethal maternal mutantgnu (Freeman et al., 1986), aphidicolin-treated em-bryos (Raff and Glover, 1988) and UV-irradiatedembryos (Yasuda et al., 1991) have shown isolatedcentrosomes. To determine whether the cross-reactingDrosophila antigen remains associated with centro-

somes once they dissociate from nuclei, we followed thebehavior of the yolk nuclei. In normally developingembryos the yolk centrosomes continue their repli-cation although the yolk nuclei cease to divide.Moreover, these centrosomes separate from the yolknuclei and move into the cytoplasm, thus providing anexcellent natural system for the study of the uncouplingof the nuclear and centrosomal cycles (Callaini et al.,1990; Callaini and Dallai, 1991). In this condition polarstaining was not observed (unpublished data), sugges-ting that the labeling is closely related to the position ofthe centrosome on the nuclear envelope.

Since from fertilization to cellular blastoderm forma-tion the surface area of the nucleus increases severaltimes, a large amount of karyoskeletal proteins isrequired for the correct assembly of nuclei in rapidlydeveloping early embryos. The presence of manyfluorescent aggregates in the cortical cytoplasm of earlyembryos before the beginning of nuclear divisionsuggests that the antigen recognized by the anti-vimentin antibody might be maternally supplied to theembryo. This has already been observed for Drosophilalamins and a group of nuclear envelope proteins, whichare partly dispersed in the cytoplasm during mitosis(Frash et al., 1988). The fluorescent aggregates undergorapid disassembly after cycle 10, when the first somaticnuclei reach the cortex, and only punctate fluorescenceis visible during the last syncytial mitoses. This indicatesa direct relationship between nuclear density andquantity of fluorescent aggregates in the peripheralcytoplasm and suggests that this antigen shifts into thenuclear compartment. Other proteins such as actin(Warn et al., 1984; Karr and Alberts, 1986; Hatanakaand Okada, 1991), tubulin (Karr and Alberts, 1986;Warn and Warn, 1986; Kellog et al., 1988; Warn et al.,1990), myosin (Warn et al., 1979, 1980; Lutz andKiehart, 1987; Young et al., 1991), spectrin (Pesacretaet al., 1989), actin-binding proteins (Miller et al., 1989)and intermediate-like filaments of the vimentin type(Walter and Alberts, 1984; Biessman and Walter, 1989)have been observed in the cortex of the preblastodermembryo. These proteins, which are observed in cyto-plasmic domains, undergo drastic reorganization afternuclear migration to the periphery.

We thank to A. Rennoch for his assistance with confocalmicroscopy. We are grateful to W. Whitfield for his generousgift of the Rbl88 antibody. This research was supported by aresearch grant from MURST.

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{Received 9 January 1992 - Accepted 6 March 1992)