an ultrastructural and radio- autographic study of … · chromosomes or over their outermost...

J. Cell Sci. 14, 263-287 (1974) 263

Printed in Great Britain

AN ULTRASTRUCTURAL AND RADIO-

AUTOGRAPHIC STUDY OF THE EVOLUTION

OF THE INTERPHASE NUCLEUS IN PLANT

MERISTEMATIC CELLS (ALLIUM PORRUM)

J. G. LAFONTAINE AND A. LORDLaboratoire de biologie cellulaire et moUculaire, D&partement de Biologie,Facultd des Sciences, University Laval, Quebec, G1K7P4, Canada

SUMMARY

Radioautography under both light and electron microscopy was exploited to investigate thestructural changes of the chromatin reticulum which characterizes the interphase nucleus of anumber of plants. Allium porrum meristematic plant cells were used for this purpose. In thisspecies, the telophase chromosomes uncoil into dense strands which, during the Gy period,gradually give rise to a coarse reticulum. There then follows an extensive unravelling of portionsof these strands, and high-resolution radioautography reveals that labelling with tritiatedthymidine predominantly occurs over zones of the nucleus consisting of diffuse fine fibrillarmaterial. As the S-period progresses, a chromatin reticulum reappears throughout the nuclearcavity, the tortuous strands being approximately 0-25 /im in diameter. Most of the radio-autographic grains still remain over the light nucleoplasmic areas but a number of these arenow located on the outermost portion of the dense chromatin profiles. By the end of the 5-period,the chromatin strands are slightly thicker {ca. 0-3 /Jm) and form a looser reticulum. Labellinghas decreased noticeably in nuclei of that period, the radioautographic grains being groupedinto clusters resting over more or less spherical regions of the chromatin reticulum. Judgingfrom their localization at the surface of the nucleolus or close to the nuclear envelope, thesestructures correspond to chromocentres. The additional interesting finding that such nuclearstructures appear much less compactly organized strongly suggests that chromocentres undergoimportant conformational modifications during duplication of their DNA.

INTRODUCTION

The organization of the plant interphase nucleus has long been known to varygreatly with species. In certain plants (e.g. Raphanus sattvus), the nucleus exhibits anumber of densely stained masses or chromocentres closely associated with the nuclearenvelope or with the nucleolus. In other species, on the contrary, Feulgen preparationsreveal rather regular chromatin strands meandering throughout the nuclear cavityand thus giving rise to an intricate reticulum. For this reason, such nuclei have com-monly been referred to as reticulate nuclei (reviewed in Delay, 1948; Lafontaine, 1968).

In recent years, a few studies have appeared on the organization of reticulate inter-phase nuclei as revealed by light and electron microscopy. In the species examined,certain interphase nuclei show chromatin strands of quite uniform diameter whichform a regular reticulum, whereas other interphase nuclei are characterized by amuch more discontinuous distribution of chromatin (Moses & Lafontaine, 1961;Tanaka, 1965; Lafontaine & Lord, 1969; Chevalier, 1970; Nagl, 1970a, b; Kuroiwa

264 J- G. Lafontaine and A. Lord-

Si, Tanaka, 1971a, b). Since radioautographic data have also clearly established thatmost of these modifications in overall organization of the chromatin reticulum occurduring the 5-period, the possibility arose that such changes are associated with thewell known process of DNA duplication during that stage.

In the present work, the radioautographic technique has been utilized in conjunc-tion with electron microscopy to better identify nuclei of the Glt S and G2 periodsand, consequently, to follow in sufficient detail the morphological evolution of thechromatin reticulum throughout interphase. An investigation was also carried out ofthe conformational changes which the dense Gx chromatin strands undergo at thetime of DNA duplication. For this purpose a plant species (Allium porrum) with smallchromocentres and quite regular reticulum was investigated.

MATERIALS AND METHODS

Fixation, embedding and microscopy

Roots from Allium porrum seeds germinated in damp Vermiculite were used for the presentstudy. The root tips were excised and fixed for 2 h or more with 1 % osmium tetroxide bufferedto pH 7-2 in O'i M sodium cacodylate. Following dehydration in acetone these specimens wereembedded in an Araldite-Epon mixture (Mollenhauer, 1964).

For light microscopy, semi-thin sections (0-3-0-7 /4m) were deposited on glass slides, stainedby means of the Feulgen procedure and counterstained with a 1 % methylene blue solutionin 1 % borax. The ultrathin preparations were stained with 2 % uranyl acetate in water followedby lead citrate (Reynolds, 1963) and examined with a Philips EM 300 electron microscopeequipped with an anti-contamination device.

RadioautographyFor radioautography, roots were immersed for periods ranging from 10 to 20 rr.in in distilled

water containing 50 /tCi/ml of [6-'H]thymidine (specific activity: 10 Ci/mmol). This DNAprecursor was obtained from Schwarz-Mann, Orangeburg, N.Y. After labelling, the specimenswere rinsed in water and the root tips excised for fixation. Light-gold sections prepared fromspecimens embedded in an Araldite—Epon mixture were affixed to collodion-coated slides andthen covered with a uniform monolayer of Ilford L-4 bulk emulsion by means of the semi-automatic instrument described by Kopriwa (1967). After a 1- to 2-month exposure, thepreparations were developed by the gold latensification-Elon ascorbic acid method (Wisse& Tates, 1968). Staining of the sections was carried out as described above.

OBSERVATIONS

Examination of semi-thin (0-3-0-7/tm) longitudinal sections of Allium porrum rootsshows that the gross morphological organization of the chromatin interphase reticulumdiffers to a quite noticeable extent from one nucleus to another. In order to minimizethe possibility that certain of these structural variations are due to cell differentiation,observations were restricted to nuclei localized within the first millimetre of theroot tip.

Gx nuclei

Allium porrum late-anaphase chromosomes, as is also the case with many otherplant species, remain well aligned at the cell poles and persist as easily recognizable

Evolution of interphase nucleus 265

filamentous structures during telophase. In semi-thin preparations the unravellingchromosomes appear to consist of at least 2 coiled filamentous subunits or chromo-nemata approximately 0-3 /tm in diameter. At the time when small nucleolar bodiesbecome clearly visible, the chromosomes have uncoiled to a significant degree but,in most cases, are still oriented in the pole-to-pole direction (Fig. 1). By this stage thenucleus has enlarged noticeably and shows much more regular contours than through-out telophase. Such nuclei with partly uncoiled chromosomes and emerging nucleoliare taken to correspond to the early Gx interphase period. Slightly later on, chromo-somes are no longer recognizable as such, the nuclear cavity being filled by tortuouschromatin strands (Fig. 2). Except for several small heterochromatin lumps or chromo-centres close to the nuclear envelope or sitting on the nucleolar bodies, these Gxchromatin strands are quite uniform in diameter.

At the ultrastructural level (Fig. 7), due presumably to the reduced thickness of thepreparations, the chromatin strands appear much more irregular in outline thanobserved under light microscopy. Here and there throughout this chromatin reticulum,segments of strands approximately o-1 /4m in width or slightly narrower may bedetected. It is also noticed that many of the strands located within the peripheralportion of the nucleus are intimately associated with the nuclear envelope by meansof finger-like projections of roughly this same diameter. However, most chromatinmasses are much too complex in outline for any measurement of diameter to be eithermeaningful or even possible. The larger of these masses undoubtedly representsections of the chromocentres observed under light microscopy in corresponding inter-phase nuclei.

By the end of the Gx period, the nuclei have taken on more roundish contours andshow large nucleolar bodies. In Feulgen-stained preparations, the nuclear reticulumappears much less conspicuous than at early interphase; due to further unravellingof the chromatin strands there now may be observed a number of small chromocentresscattered throughout the nuclear cavity and linked by fine, hardly perceptible filaments.At higher magnification (Fig. 8), these late Gx nuclei display a most irregular chromatinreticulum consisting of tortuous strands which touch one another at many places andappear to anastomose to form masses of various sizes and shapes. Due to the irregu-larity of these strands, it is quite difficult to detect any consistency in their diameter.However, the presence of tiny light areas within the chromatin strands and thechromocentres as well as the presence of short o-i-^m-wide segments projecting fromthem strongly suggest that they are made of convoluted subunits roughly o-i /tm indiameter. This conclusion is strengthened, furthermore, by the fact that the finger-like extensions attaching peripheral chromatin to the nuclear envelope are likewiseof a similar dimension.

Apart from the loosely distributed profiles of dense chromatin, electron micro-scopy reveals that late Gx nuclei also consist of enlarged, more transparent nucleo-plasmic areas predominantly containing fine fibrillar material interspersed withclusters of granules. Certain of the latter particles are approximately 15 nm in diameterand thus resemble the granules present in the peripheral portion of the nucleolus.Other nucleoplasmic particles are 25-40 nm in size and often form clusters close to

266 J. G. Lafontaine and A. Lord

the boundaries of the chromatin strands. Sufficiently high magnification pictures alsodisclose a more finely punctate texture throughout the nucleoplasm which most likelyresults from sharp twists and kinks in the delicate background fibrillar meshwork.

S nuclei

Early replicating nuclei. Examination of nuclei which become labelled following10—20 min exposures to tritiated thymidine reveals striking variations in their grossand ultrastructural organization. In order to arrange such nuclei into a logical sequenceof stages and thus follow their structural evolution during the S-period, their volumeas well as degree and pattern of labelling were taken into account. For that purpose,extensive series of semi-thin (0-7 /im) consecutive sections of roots were prepared soas to cut through complete nuclei. Volumes of the labelled nuclei were then determinedfrom camera lucida drawings and, in accord with previous studies (Woodard, Rasch &Swift, 1961), those of smaller dimensions were assumed to correspond to the earlierpart of the 5-period.

Using criteria of nuclear volume, regularity of contours and organization of thechromatin reticulum, two main classes of S nuclei can be recognized. A first type oflabelled nuclei (Fig. 3) which will be designated as type A nuclei, are morphologicallyindistinguishable from those characterizing the late Gx period. In semi-thin prepara-tions the radioautographic grains appear to be distributed rather evenly over the non-nucleolar portion of the nuclear cavity, only a few such grains being present over thenucleolus itself. More detailed analysis of the labelling pattern in electron micro-graphs (Fig. 9) clearly shows that in incorporation periods of 10-20 min the majorityof the radioautographic grains are either lying in between the dense segments ofchromosomes or over their outermost portion. It is also evident that very few grainsare actually found over the chromatin lumps or chromocentres.

Other labelled nuclei, which for the purpose of the present rough classification willalso be referred to as type A nuclei, show a slightly more discrete distribution ofchromatin than that just described. These nuclei (Fig. 10) still exhibit important butsomewhat smaller transparent zones of diffuse fibrillar material over which most ofthe radioautographic grains are localized. The various segments of dense chromatinappear, however, thicker and their profiles possibly more complex than in the caseof the first group of type A nuclei; these chromatin masses also show more clearlythan previously an intricate pattern of small light spaces which probably account fortheir most complex appearance. The regular dimensions of these lighter intra-chromatin spaces are such as to suggest strongly that the chromosomal strands consistof narrow subunits, the organization of which cannot be analysed from thin prepara-tions. However, judging from the regular distribution of these transparent areas,especially within the chromocentres, the diameter of the subunits is estimated to bein the neighbourhood of 40 nm. As noted for other type A nuclei, the nucleoplasm isrichly provided with loose fibrillar material as well as with granules often groupedinto clusters of various sizes.

Late replicating nuclei. The second main group of interphase nuclei which becomelabelled following exposure to tritiated thymidine for 10-20 min periods stain more


intensely with the Feulgen procedure than the type A nuclei owing to the reappearanceof conspicuous chromatin strands of quite constant diameter. Judging from their sizeand similarity to the G2 nuclei, these nuclei are assumed to correspond to the late S-period; in accord with the terminology introduced in a previous work (Moses &Lafontaine, 1961), they will therefore be referred to as type B nuclei. In semi-thinpreparations, certain of these latter nuclei (Fig. 4) exhibit only a few small chromo-centres and the chromatin strands, slightly thinner than 0-25 Jim, give rise to anelaborate apparent reticulum extending throughout the nuclear cavity. As a resultof their rather close packing, the strands often appear to touch or anastomose. Hereand there, segments of these coarse filaments are also noted to be separated equi-distantly. Such twin segments are particularly common at the periphery of the nucleusand are often seen running perpendicularly to the nuclear envelope. Of the differenttypes of interphase nuclei recorded in the present study, these nuclei exhibit thesmallest fraction of interchromatin space. Under electron microscopy (Fig. 11), partof the radioautographic grains are still found over diffuse chromatin, but many ofthese are now also observed over the peripheral portion of the dense component ofthe chromosomes.

Other type B interphase nuclei are approximately of the same size as those justdescribed but show somewhat thicker (0-25—0-3 /im) convoluted strands (Fig. 5).These strands are, moreover, less tortuous and more loosely distributed throughoutthe nuclear cavity, with the result that they may be followed for longer distances insemi-thin preparations than was possible in earlier interphases. Such preparationsalso reveal that the nuclei under discussion are much less labelled than other S nuclei,the radioautographic grains occurring in small clusters throughout the nuclear cavitybut especially close to the nuclear envelope or at the periphery of the nucleolus. It isoften possible, in the light microscope, to verify that these clusters are localized overchromocentres, but in certain cases no particular structure can be recognized at thesesites. Electron microscopic observations have unambiguously confirmed such localiza-tion of grains over chromocentres in certain of the late S nuclei and, interestinglyenough, have also clearly demonstrated that, in others, labelling is found over moreor less circular chromatin structures which display a glomerular organization (Fig. 12).The ultrastructural texture, size and distribution of these loose regions of thechromatin reticulum strongly suggest that they represent partly unravelled chromo-centres.

G2 nuclei

Late interphase or G2 nuclei have been identified on the basis of their size, lack oflabelling, and from the fact that they exhibit thick chromatin strands (0-25-0-3 /on)still organized into an apparent reticulum. These chromatin strands, although stillsomewhat tortuous, have now relaxed to their maximum and longer stretches can befollowed in semi-thin preparations.

Following this extension stage, coiling of the chromosomal filaments begins (Fig. 6),with the result that points of contact between neighbouring segments disappear, hereand there, thus giving rise to large irregular interchromatin spaces. As this process

IS C E L 14


continues, the interphase reticulum gradually disappears and the coiling chromatinstrands become redistributed into elongate structures, the emerging prophase chromo-somes coursing throughout the nuclear cavity.

DISCUSSION

The great variety in gross organization which interphase nuclei show is undoubtedlymost striking in the case of plant cells. This surprising diversity in appearance isknown, from early observations on both living and fixed plant material, to reflect acorresponding variety in the structure of plant nuclei (discussed in Dangeard, 1947).A finding that has emerged from these studies is that species with long mitotic chromo-somes exhibit interphase nuclei of the reticulate type (Delay, 1948). From morerecent observations, it has also become evident that reticulate interphase nuclei arefound in plant species having large amounts of DNA (discussed in Lafontaine, 1974).

Reticulate interphase nuclei have generally been visualized as consisting of slender,highly chromatic, convoluted chromatin strands which form a more or less con-spicuous network, depending on the species. Problems related to the permanence ofthese strands throughout interphase, to their longitudinal differentiation into chromo-centres, as well as to the possibility that they consist of multiple subunits or chromo-nemata, have long attracted the attention of cytologists and have been extensivelyreviewed in the past (Dangeard, 1947; Delay, 1948; Kaufmann, 1948; Kaufmann,Gay & McDonald, i960; Ris, 1961; Bajer & Mole-Bajer, 1963; Moses, 1964; Sparvoli,Gay & Kaufmann, 1965).

The present observations on Allium porrum meristematic cells are consistent withearlier views that the uncoiling telophase chromosomes of reticulate plant speciesgradually transform into convoluted strands which give rise to an apparent reticulumwithin the early interphase or G1 nuclei. Except for the presence of numerous smallchromocentres throughout this chromatin reticulum, the strands show a rather con-stant diameter in the neighbourhood of 0-3 fim. Using the emergence of the smallnucleolar bodies as a criterion for the beginning of the Gx interphase period, it is alsoapparent that figures interpreted earlier (Lafontaine & Chouinard, 1963; Lafontaine &Lord, 1969) as mid-telophases should rather be viewed as early interphases.

The finding, in the various reticulate plant species studied so far, that early andlate interphase nuclei differ noticeably in gross organization (Moses & Lafontaine,1961; Tanaka, 1965; Lafontaine & Lord, 1969; Nagl, 1970a, b; Kuroiwa & Tanaka,1971 a, b) has shed new light on the conformational changes which chromatin undergoesduring synthesis of its deoxyribonucleohistones. In the case of Allium porrum, a specieswhich exhibits relatively small chromocentres and particularly fine strands, theevolution of the chromatin reticulum is most striking. It is observed that the regularchromatin strands characterizing early Gx nuclei (Fig. 1) transform during thelater part of this period with the result that, in semi-thin preparations stained bymeans of the Feulgen procedure, their reticulum-like organization becomes much lessevident (Fig. 2). Since early S nuclei (Figs. 3, 9) exhibit a quite similar general organi-zation, it would appear that unravelling of segments of chromatin strands is a


prerequisite for replication. This duplication process, judging from the distribution ofsilver grains in high-resolution radioautographs of early S nuclei (Fig. 9), takes placewithin the diffuse chromatin. In view of the fact that certain more advanced S nuclei(Figs, io, 11) exhibit a corresponding labelling intensity, it must be further assumedthat a portion only of the chromatin relaxes at a given time of the 5-period for thepurpose of duplication. The presence of simultaneously replicating loci scattered alonginterphase chromosomes has recently been revealed in various materials by bothbiochemical and radioautographic techniques and it is also generally agreed that groupsof such loci replicate at different times of the 5-period (Plaut, Nash & Fanning, 1966;Painter, Jermany & Rasmussen, 1966; Bernardini & Lima-de-Faria, 1967; Huberman& Riggs, 1968; Okada, 1968; Prescott, 1970; Callan, 1972).

Considering that the early G1 chromatin is organized into dense strands of ratherregular diameter, the question naturally arises as to the extent a given segment mustunravel as a prerequisite for DNA synthesis. A first possibility which comes to mindinvolves a progressive looping of microfibrillar elements from the dense chromosomesegments followed by sequential spinning back into the chromosomal axis as replica-tion progresses along these loci. Such a scheme would imply that only portions ofreplicons would be in a diffuse state at a given moment of the 5-period. The abundanceof diffuse labelled fibrillar areas observed in early and mid-5 nuclei (Figs. 9-11) ismore easily accounted for, we believe, by assuming that extensive unravelling ofcomplete chromosome segments must take place prior to their replication. This latterview also appears compatible with our rather unexpected finding that certain nuclei(Fig. 8), presumably in the late Gx or very early 5-periods, exhibit large diffusechromatin areas yet remain unlabelled.

Further evidence that, in reticulate plant interphase nuclei, DNA duplication isaccompanied by marked changes in the macromolecular organization of the chromatinstrands comes from the presence of a second major group of labelled interphasenuclei, the type B nuclei, which show a noticeably more structured reticulum thanthe early interphases. Certain of these nuclei correspond to the mid-5 period (Fig. 11),while others resembling more the G2 nuclei have unmistakably reached the late5-period (Fig. 12). The facts that the meshes of the chromatin reticulum charac-terizing these nuclei have decreased in size and, moreover, that the strands themselveshave now become thicker and more uniformly dense lead to the conclusion that diffusechromatin transforms into a compact state subsequent to duplication of DNA.

An additional interesting aspect of the ultrastructural changes of chromatin duringinterphase which emerges from the present study is the striking transformation ofchromocentres into looser masses during the later part of the 5-period. A large bodyof information has been accumulated in recent years showing that heterochromatinreplicates late during the 5-period (reviewed in Lima-de-Faria, 1969; Prescott, 1970;Comings, 1972). Only a few exceptions to this rule have so far been reported in thecase of both animals (Hsu, Schmid & Stubblefield, 1964) and plants (Tanaka, 1965;Tatuno, Tanaka & Masubuchi, 1970). The vast majority of the above observationshave, however, involved exposing various materials for different periods to tritiatedthymidine and then examining which portions of the ensuing mitotic chromosomes

18-2


first appeared labelled. Such procedures unfortunately tell us little as to the actualconformation of heterochromatin at the time it duplicated in interphase. Other obser-vations carried out on synchronized KB cells showed that DNA synthesis is moreactive in the peripheral region of the nucleus, where small patches of dense chromatinare sometimes observed, and that this activity increases during the later part of the5-period. No information has been furnished as to whether these heterochromaticmasses lose part of their compactness as DNA is replicated (Blondel, 1968). Althoughrepresenting a rather special situation, decondensation of chromatin prior to DNAsynthesis has, however, been reported in phytohaemagglutinin-activated lymphocytes(Tokuyasu, Madden & Zeldis, 1968; Milner, 1969).

In their recent electron-microscopic investigation of Crepis capillaris, a plant withreticulate nuclei, Kuroiwa & Tanaka (19716) came to the conclusion that the hetero-chromatin segments of mitotic chromosomes transform into a diffuse state whenundergoing DNA duplication at interphase, but no illustration of this process wasfurnished. The present work on Allium porrum meristematic cells has revealed thatthe well organized late-5 chromatin reticulum consists of more regular and thickerstrands than those observed earlier in interphase. These late-5 nuclei not only showvery little labelling but the radioautographic grains are also observed to be restrictedto small portions of the chromatin reticulum (Fig. 12). The size of these clusters ofgrains as well as their localization close to the nucleolar surface or at the peripheryof the nuclear cavity leave little doubt that the areas in question correspond to chromo-centres. Of particular interest is the finding that certain of these grain clusters lieover corresponding masses which have apparently decondensed to a noticeable extent.Taking into account the short labelling periods used, the presence of radioautographicgrains over such unravelled structures strongly suggests that chromocentres lose muchof their compactness when they replicate. To our knowledge, this is the first clear-cutevidence that, in plant reticulate interphase nuclei at least, heterochromatin does notreplicate while in a condensed state, as seems to have been inferred generally fromprevious radioautographic studies. The facts that labelling is very limited in late-5nuclei and that high resolution is required to detect the unravelling of the chromo-centres at that stage most likely account for Nagl's (1968 a, 1970 a, b) recent conclusionthat, in Allium carinatum, hererochromatin replicates while in a condensed state.

Considered together, our high-resolution radioautographic observations confirmearlier results from both actively dividing animal (Hay & Revel, 1963; Moses &Coleman, 1964; Blondel, 1968) and plant (Kuroiwa & Tanaka, 19716) cells to theeffect that DNA synthesis predominantly takes place within the diffuse portion ofchromatin. In accord with recent studies of various plants with reticulate interphasenuclei (Tanaka, 1965; Nagl, 1968a, b, 1970a, b; Lafontaine & Lord, 1969;Kuroiwa & Tanaka, 1970, 1971a, b), the gross and macromolecular organization ofthe chromatin reticulum characterizing Allium porrum meristematic cells has also beenshown to undergo striking modifications throughout interphase. A first importantchange takes place during the late Gx or earlier part of the S-period and involvesunravelling of various segments of the dense strands with the results that the chromatinreticulum becomes much less conspicuous (Figs. 3, 8 and 9). As interphase proceeds,


the chromatin reticulum reappears (Figs. 4, 10, 11) and, by the late S-period (Figs. 5,12), is seen to consist of strands slightly thicker than those observed during GvEither as a consequence of their close packing within the nuclear cavity or as a resultof DNA duplication which is now mostly completed, many strand segments runparallel to one another. By the time the chromocentres replicate or slightly thereafter,the chromatin reticulum becomes looser and contacts between neighbouring strandsegments gradually disappear. As also shown most elegantly by Bajer & Mole-Bajer(1963) with living Haemanthus endosperm, the late interphase chromatin strandseventually become redistributed throughout the nuclear cavity and, as they coil again,give rise to elongate structures, the early prophase chromosomes.

Although the present study has clarified certain important aspects of the structuralevolution of the reticulate plant interphase nucleus, a number of basic problemsremain unanswered. Foremost amongst these, perhaps, is the present lack of informa-tion as to whether sister chromatids are formed as distinct, physically separate entitiesduring DNA duplication or whether they first coexist as intimately associated subunitswithin the coarse o-3-/tm-wide strands observed during the late 5-period and moveapart slightly further on. Although equistantly separated segments are commonlyobserved in late S nuclei, no clue presently exists that such segments represent sisterelements.

This investigation was supported by research grants from the Ministry of Education ofQuebec and the National Research Council of Canada. The authors also gratefully acknowledgethe excellent technical assistance of Mrs Diane Michaud and Mr Siegfried Gugg.

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REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

Ris, H. (1961). Infrastructure and molecular organization of genetic systems. Can. J. Genet.Cytol. 3 95-120.

SPARVOLI, E., GAY, H. & KAUFMANN, B. P. (1965). Number and pattern of association ofchromonemata in the chromosomes of Tradescantia. Chromosoma 16, 415-435.

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{Received 11 June 1973)

274 J- G. Lafontaine and A. Lord

Figs. 1-6. The following series of phase-contrast micrographs illustrates the develop-ment of the nucleus (Alliumporrum) throughout interphase. The various nuclei all comefrom the same o-7-/tm-thick section which was stained by means of the Feulgenprocedure and counterstained with a i % solution of methylene blue in I % aqueoussodium borate. x 3800.

Fig. 1. Early G1 nucleus. The uncoiling chromosomes are still oriented in the pole-to-pole direction and appear to consist of 2 coarse subunits approximately 0-3 /«n indiameter. Although Allium porrum early interphase nuclei generally display 4nucleoli, only 2 of these organelles appear in the present semi-thin preparation. Oneof these emerging nucleoli is more clearly seen to form along a specific segment ofchromosome.

Fig. 2. By the time the nucleus has reached the late Gl period, the chromosomeshave completely unravelled and can no longer be recognized as distinct elongatestructures. The nuclear cavity is now occupied by slender convoluted strands givingrise to an apparent reticulum. Although the particular plane of sectioning revealsonly small nucleoli, these organelles are, at this stage, generally much larger thanillustrated here.

Fig. 3. In this nucleus of the early 5-period, chromatin has lost part of its strand-like organization. There may now be observed a number of densely stained chromatinlumps, or chromocentres, of various sizes and shapes which appear to be linked tofine twisty filaments filling the nuclear cavity. Following short exposure (10—20 min)to tritiated thymidine, most nuclei of this type become heavily labelled.

Fig. 4. Judging from its size, appearance of the chromatin strands and labellingpattern, this nucleus is taken to correspond to the mid-5 or first portion of the late5-period. A first most distinctive morphological characteristic of this nucleus is thereappearance of densely stained strands which are rather closely packed and thusgive rise to an elaborate reticulum. These strands are approximately 025 /tm indiameter and appear slightly narrower than those seen during the early Gy period(Fig. 1). A second important aspect of this nucleus is the fact that, as compared tothat illustrated in Fig. 3, it contains fewer chromocentres, most of which are quitesmall. This type of nucleus also displays many radioautographic grains followingshort labelling periods with tritiated thymidine.

Fig. 5. Nucleus of the late 5-period. Apart from having a volume close to that ofearly prophases, this nucleus is characterized by the presence of slightly thickerstrands (ca. 0'3 /im) than those seen in Fig. 4. The coarse chromatin filaments appearsomewhat less tortuous than those of the mid-5 period and may thus be followedover longer stretches. Due presumably to the fact that these strands are also beginningto extend, the meshes of the chromatin reticulum are larger than observed in Fig. 4.As revealed much more convincingly under electron microscopy (Fig. 12), nucleiof this type show only a few radioautographic grains, the majority of which are groupedinto small clusters close to the nucleolar surface or at the periphery of the nucleus.

Fig. 6. In this late interphase or G2 nucleus, the chromatin reticulum has taken amost complex organization. Here and there, points of contact between neighbouringsegments of the strands have disappeared, thus giving rise to extended and quiteirregular interchromatin spaces. At several places 2 tortuous strand segments are seenrunning parallel, and the impression is gained that they may represent the pairedchromatids which will soon form the early prophase chromosomes. Note the presenceof a few large knots or chromocentres.

276 J. G. Lafontaiiie and A. Lord

Fig. 7. Electron micrograph of portion of a mid-Gi nucleus. The chromosomes haveuncoiled to a degree resembling that illustrated in Fig. 2 and have given rise to acomplex reticulum extending throughout the nuclear cavity. At this stage, thechromatin strands vary considerably in diameter, certain segments appearing muchcoarser than others. Segments approximately o-i (im in width may be recognized atdifferent places (arrows) and most particularly in intimate association with the nuclearenvelope. The nucleoplasm consists of fine diffuse fibrillar material and of groupsof granules 20—40 nm in diameter. The forming nucleoli mainly consist of a centralcoarse nucleolonema surrounded by a thin irregular layer of paniculate material,x 30000.

Evolution of interphase nucleus


Fig. 8. Microphotograph of an interphase nucleus which remained unlabelledfollowing a 10-min exposure to tritiated thymidine. Judging from the degree ofunravelling of the chromatin strands and the absence of radioautographic grains, thisnucleus corresponds to the late GL period. As compared to the stage illustrated inFig. 7, it is noted that the chromatin strands have unravelled further and now exhibitan alveolar internal organization. As a result, these strands appear to consist of quitenarrow subunits in the neighbourhood of c i /tm in diameter. The enlarged inter-chromarin spaces are richly provided with fine fibrillar material and clusters ofparticles of various sizes (arrows), x 34000.


mm


Fig. 9. High-resolution radioautograph of an early prophase nucleus, the generalorganization of which corresponds to that of the nucleus illustrated in Fig. 3. In thismicrograph, the dense chromatin is represented by profiles of different sizes andshapes, the largest ones representing chromocentres (cht). Although these variouschromatin masses are generally close to one another and may touch, the overallimpression is not that of a reticulum, contrary to the situation observed in certain Gtnuclei (Fig. 7) or during the later parts of the S-period (Figs. 11, 12). Most of theseprofiles display a complex pattern of small light spaces suggesting the presence ofnarrow tortuous subunits within the chromatin strands. Following a 10-min incor-poration period, thymidine labelling is obsen'ed to occur predominantly within thelight interchromatin areas. Although a few radioautographic grains are found overdense masses of chromatin, it should be noted that hardly any of these rest over thevarious chromocentres. The nucleoplasmic areas consist mostly of loose fibrillarmaterial and of scattered granules 20—40 nm in diameter. As recently demonstrated(Lafontaine & Lord, 1973), interphasc nucleoli become labelled in the presence oftritiated thymidine, the majority of the radioautographic grains being localized overthe central nucleolonemal portion or at the surface of this organelle close to theperipheral chromatin. x 28000.


Fig. 10. Electron micrograph of portion of an early 5 nucleus which appears slightlymore advanced than that in Fig. 9. In this particular picture one also clearly recognizesthe many small chiomocentres which are revealed by Feulgen staining in corie-spondingly thicker preparations (Fig. 3). These chromocentres, like the more un-ravelled chromosome segments, exhibit numerous light spaces which confer to them amost complex appearance. At this early portion of the S-period, short labellingexperiments give rise to the appearance of radioautographic grains predominantlyover the nucleoplasmic areas or over the outermost regions of the dense chromatinprofiles. It is evident, moreover, that the larger chromatin masses or chromocentresare not labelled. As also observed in earlier interphases, the nucleoplasm consistsmostly of loose microfibrillar elements and granules 20-40 nm in diameter. Closeexamination finally reveals that the boundaries of the dense chromatin masses arerather ill-defined and merge imperceptibly with the immediately adjacent nucleo-plasmic substance. The nucleolus is similar to the organelles seen during mid andlate interphase and essentially consists of denser fibrillar zones (Jz) immersed ingranular ones (gz). x 31000.

Evolution ofinterphase nucleus 283

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Fig. 11. In this mid to late-5 interphase nucleus, the chromatin strands are slightlyless irregular in outline than in the preceding figure and begin to re-form a reticulum.The interchromatin spaces are also smaller than in earlier interphases. Although manyradioautographic grains are still resting over the lighter portions of the nuclear cavity,a certain number of these may now be seen over the chromatin strands themselves.As for the nucleolus, it still exhibits a central fibrillar zone surrounded by granularmaterial, x 26000.

Evolution of interphase nucleus 28s

19-2


Fig. 12. Portion of a late-5 nucleus depicting the thickening of chromatin strandswhich takes place at that stage. These coarse chromosomal filament9 appear lesstortuous than in earlier interphases and, as a consequence, may be followed for longerdistances in electron-microscopic preparations. A most interesting characteristic ofthis radioautograph is the localization of most of the silver grains over restricted areasof the chromatin reticulum. Two of these areas, one lying on the nucleolar surfaceand the other close to the nuclear envelope, are particularly obvious and appear tocorrespond to regions of the chromatin reticulum which have undergone extensiveunravelling. Judging from their localization, size and shape, these structures representchromocentres (cht) in the process of duplicating their DNA. The basic organizationof the nucleolus with both fibrillar (Jz) and granular zones (gz) is indistinguishablefrom that observed during the earlier periods of interphase. x 31000.

Evolution of interphase nucleus

fc*

an ultrastructural and radio- autographic study of … · chromosomes or over their outermost...

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