J. Cell Sci. 41, 321-329 (1980) 221Printed in Great Britain © Company of Biologists Limited 1980

THE NUCLEOLTJS IN TELOPHASE, INTERPHASE

AND PROPHASE

MOHAMMAD ASHRAF AND M. B. E. GODWARDDepartment of Plant Biology and Microbiology, Queen Mary College,University of London, Mile End Road, London, El \NS, England

SUMMARYThe ultrastructure of telophase to interphase has been followed in a green alga, Spirogyra

submargaritata. A series of changes transitional between the late anaphase chromatid, thedeconden8ing chromatid of telophase, and the 'pale fibrillar material' occupying channels inthe nucleolus at interphase have been demonstrated. Early stages in the regeneration of thenucleolus are described.

It has been shown that the pale fibrillar material in the nucleolus is attached to, and con-tinuous with, the fully condensed (chromocentric) part of the nucleolar-organizing chromosomeat interphase. It is also shown that in early prophase, the channels in the nucleolonema of thenucleolus are no longer occupied by pale fibrillar material, but instead a long section of con-densed chromosome is present, traversing the nucleolonema. It is contended that these observa-tions taken together constitute evidence that the pale fibrillar material of the nucleolus is thechromatin of the nucleolar-organizing region of the chromosome, expanded for transcription.

A model of the nucleolus as it is seen in most electron-microscope sections, and as it can beinterpreted in the light of present-day knowledge about it, is presented. A brief review of therelevant literature considers the views supporting the model, and the contrary views, impli-cating the use of the term 'nucleolar organizer', that are still current at the present time.

INTRODUCTION

Despite the considerable literature on nucleolar ultrastructure (see review,Gimenez-Martin, de la Torre, Lopez-Saez & Esponda, 1977) there have as yet beenfew accounts (Chouinard, 1970, 1971, 1975) of the development of the new nucleolusat telophase, nor assessment of the relationship of the internal' channels' of the nucleo-lus with their contents, to the nucleolar organizing chromosome. There has beeninsufficient ultrastructural study of the changes in the nucleolus in prophase. Theseare the subjects of the present work.

MATERIAL AND METHODS

The species Spirogyra submargaritata (Godward, 1956) was collected from a small pondin the grounds of Royal Holloway College, Egham, Surrey, England.

The filaments were fixed in 5 % glutaraldehyde in 02 M cacodylate buffer at pH 7 for 4-5 hat room temperature, then washed and left overnight in cacodylate buffer. The material wasthen transferred to 2 % aqueous OsO4 in buffer and left in it for at least 4 h, at room tempera-ture, then again washed with cacodylate buffer 3-4 times at hourly intervals; kept in 1-5 %aqueous uranyl acetate overnight in the refrigerator at 3-4 °C, and after washing with distilledwater the material was very gradually dehydrated (24 h).

After infiltration, which was also very gradual (36 h), the material was embedded in TAABresin, placed in a 'peel-away disposable mould' 22 mm square (supplied by Schuco Inter-national Ltd) polymerized at 370 C overnight under vacuum and then transferred to an

322 M. Ashraf and M. B. E. Godward

Nucleolus in telophase, interphase and prophase 323

incubator at 6o° C for 48 h. Dividing cells were selected under the visual light microscope.The required cell was sawn carefully from the flat moulded block, trimmed and sectioned.

Sections were cut on L.K.B. and Reichert ultramicrotomes with glass knives. Silver-greyand pale-yellow sections were picked up on unsupported 200-mesh copper grids.

All the sections were stained in 1 % uranyl acetate in 5°% ethanol for 3-5 min.The Siemens Elmiskop IA and Zeiss EMo. transmission microscopes were used for the

observations; photography was with Ilford SP332 film, developed in Ilford PQ Universaldeveloper and fixed in Ilford Hypam fixer.

DEVELOPMENT OF THE NUCLEOLUS

At early anaphase or early telophase in Spirogyra submargaritata one of the chroma-tids enlarged (Fig. 1) shows a development of superficial projections, characteristic ofthis stage, while spindle microtubules are still attached at its polar end (Figs. 1, 2).The nuclear envelope has not yet fully developed, but is developing, in contact withnucleolar material, which has the appearance generally referred to as 'nucleolonema'.The chromatin of the chromatid is fibrillar (Fig. 1) and its electron density variessomewhat along its length, suggesting banding. At a later telophase large patches offibrillar material (Fig. 3) can be found, resembling that of the chromatids as they werelast seen (Figs. 1, 2). These patches of chromatin, which have clearly resulted fromexpansion of a chromatid, are separated from the granular nucleolonema by the de-velopment of an intervening layer of dark fibrillar material (Fig. 3). At a slightly laterstage, the chromatin, its dark fibrillar surround, and the granular nucleolonema, havetaken on the appearance of a new nucleolus (Fig. 4).

Turning to the interphase nucleolus, in Spirogyra submargaritata, the arrangementof its internal components, the granular nucleolonema, the fibrillar nucleolonema(dark fibrillar material) and chromatin, now greatly expanded (pale fibrillar material)are as shown in Figs. 5-9. In all cases where a part of the nucleolar-organizing chromo-some external to the nucleolus has been cut through and lies in the plane of section,it is continuous with the pale fibrillar material of the nucleolus at the point of attach-ment. Lying in line with this externally attached chromosomal element, are thenumerous ramifications of the channels occupied by pale fibrillar material, and theiraccompanying border of dark fibrillar material. Details of one such nucleolus(Figs. 5, 6) show the mingling of condensed and decondensed chromatin fibrils at the

Fig. 1. Late anaphase/telophase. One chromatid is seen with paler and lighter areasof more and less condensed chromatin, and with diffusely ragged outline, in contactwith nucleolonema channels. Microtubules are still attached at the position of thekinetochore. x 22000.Fig. 2. Late anaphase/telophase. One chromatidstill with microtubules attached at thekinetochore. It is surrounded by nucleolonema. x 18000.Fig. 3. Telophase. The chromatid chromatin has expanded and become paler exceptfor a central spot. The expanded chromatin is bordered by dark fibrillar material;outside this is granular material, x 50000.Fig. 4. A developing telophase nucleus; the chromatin mass is more extended andbecoming convoluted in its course; it is bordered by dark fibrillar material whileoutside this the granular nucleolonema begins to give the round outline typical of anucleolus. x 20000.

324 M. Ashraf and M. B. E. Godward

Fig. 5. Interphase nucleolus; there is clear continuity of the attached nucleolar-organizing chromosome with the pale fibrillar material within the nucleolus (cf. theexpanded chromatin of telophase, Fig. 3). Dark fibrillar material borders the palefibrillar zone (see Fig. 14). x 20000.Fig. 6. Enlargement of part of Fig. 5. The pale fibrillar material within the nucleolushas a central condensed spot (cf. Fig. 3) and is interpenetrated by darker fibrils fromthe condensed chromatin of the attached nucleolar-organizing chromosome. Largedark fibrils also fray out from the junction of pale (expanded) and condensed chro-matin, into the nucleoplasm. x 50000.

Nucleolus in telophase, interphase andprophase 325

attachment point of the nucleolar-organizing chromosomes to the nucleolus. Inprophase stages the recondensation region of the chromosome leads to the formationof an apparent length of chromosome within the nucleolonema of the nucleolus(Figs, io, 11). The dark fibrillar material now is seen in section as laterally attachedmasses along the length of that part of the chromosome within the nucleolonema(Fig. 11). No pale fibrillar material remains.

CONCLUSIONS

The series of stages described, illustrates a continuous process of change in theposition and appearance of the chromatin of the nucleolar-organizing chromosome,from anaphase/telophase through interphase to prophase. It offers evidence that thenucleolar-organizing region of the chromosome is contained within the nucleolusat all times in S. submargaritata. In addition, evidence that the first stage of develop-ment of the nucleolus as such in telophase, is the expansion of the chromatinof the nucleolar-organizing region and the development round it of the darkfibrillar material. Anaphase/telophase can be followed through a series of changestowards interphase, with every gradation from chromatid-chromatin to pale fibrillarmaterial of the fully formed nucleolus constituting proof that the pale fibrillarmaterial is the nucleolar-organizing chromosomal region. Interphase/prophase/metaphase can similarly be followed through, illustrating the contraction of the palefibrillar material of the nucleolus to become the nucleolar-organizing chromosomeregion. The spatial relationships of the nucleolar-organizing region of the chromosomewithin the nucleolus, are indicated in Figs. 12-14, where the chromosome is shown asa stretch of pale fibrillar material, surrounded by its dark fibrillar nucleolonema,traversing the remainder of the nucleolus - an accumulation of granular material. Forthe sake of clarity, the chromosome in Fig. 13 is shown in discontinuous pieces as itappears in sections, a result of the tortuous course followed through the nucleolus.

DISCUSSION

Presence of DNA in pale fibrillar material

The presence of DNA in the pale fibrillar material has been shown using uptake of[3H]thymidine (Revel & Hay, 1961; Lafontaine & Lord, 1973; Lord & Lafontaine,1976; Goessens, 1976). DNA under transcription was isolated from nucleolar 'cores'(Miller & Beatty, 1969).

Presence of DNA in the nucleolonema

Using [3H]thymidine, labelling was found not only in the pale fibrillar material butalso in the dark fibrillar nucleolonema (Lord & Lafontaine 1976). Similar labellinghad been found in earlier investigations (Lafontaine & Lord, 1973; Granboulan &Granboulan, 1965).

Presence of RNA in the dark fibrillar material

That the dark fibrillar material does represent regions of RNA synthesis has beenshown using uptake of [3H]uridine (Das, Micou-Eastwood, Ramamurthy & Alfert,1970; Granboulan & Granboulan, 1965).

M. Ashraf and M. B. E. Godward

14Figs. 7-9. Diagrammatic representations of individual interphase nucleoli; granularregion, regularly dotted; dark fibrillar regions, transverse lines; pale fibrillar regions,finely dotted; condensed chromatin, cross-hatched. The figures are tracings fromsections of separate nuclei.

Nucleolus in telophase, interphase and prophase 327

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Fig. 10. Prophase nucleus with dispersing nucleolus. Of the 2 nucleolar-organizingchromosomes, one is cut transversely and one longitudinally. Most of the latter iscontained within the nucleolonema; a short length projects beyond it. x 6000.Fig. 11. Enlargement of part of Fig. 10. The chromatin of the nucleolar-organizingchromosome within the nucleolus (representing contracted pale nbrillar material)resembles the chromatin projecting beyond it. The dark nbrillar material (nbrillarnucleolonema) is now seen as thick, laterally attached strands, x 24000.

Fig. 12. Simplest possible model of the nucleolus at interphase, showing the nucleolar-organizing chromosomes as pale nbrillar material, traversing the granular material(regular dots) which constitutes the bulk of the nucleolus.Fig. 13. Model of the average section through the nucleolus at interphase, showing theconvolutions of the nucleolus-organizing chromatin cut transversely in several places.Fig. 14. Model of the nucleolus with functional interpretation of the pale fibrillarmaterial as rDNA exposed for transcription in progress, and the granular material ascompleted transcription products of rDNA.

328 M. Ashraf and M. B. E. Godward

Model of nucleohis

One model which is very suitable for Spirogyra (and all other organisms exceptmaize) represents the nucleolar-organizing region of the chromosome expandinglaterally into numerous loops, like those of the lampbrush chromosome or the puffof the polytene chromosome. Since the repeated rDNA cistrons are in this region, theloops would all contain these cistrons, each loop perhaps containing many. In Fig. 14,for the obvious reason of securing clarity of the diagram, the DNA of the nucleolar-organizing region is shown with only 2 loops, but clearly the entire region wouldbecome a dense tangle of loops such as that recently illustrated for chromosomal DNAfollowing the removal of histone proteins (Paulson & Laemmli, 1977). In additionthere could be amplification and detachment of lengths (circles) of the rDNA as inthe Triturus oocyte, although to a modest extent only, resulting in the presence ofdetached lengths of rDNA in the granular nucleolonema, as well as attached loops.One circular molecule in the diagram represents this suggestion.

The loops would, when expanded but not yet being transcribed, look like the palefibrillar material, or possibly this would represent early stages of transcription. Laterstages would look like the dark fibrillar material, carrying longer and more denselypacked side-chains (the matrix of Miller & Beatty, 1969); finally the side-chainsdetached or at least rolled up if still attached, constitute the granular material, stillwith intermingled DNA strands perhaps. The nucleolus in other words is a puff,similar to any other although not polytene. It would then be understandable that thepale appearance and the increased length (resulting in excessive convolution of thechannels occupied by the chromosome in the nucleolus) would gradually develop intelophase; also that the darker central regions sometimes found in the pale fibrillarmaterial would represent as yet unexpanded chromatin. The above suggestions areconsonant with published figures of the nucleolus or of nucleolar chromatin (Miller &Beatty, 1969, fig. 3; Spring et al. 1974, figs. 6-8; Spring, Scheer, Franke &Trendelen-burg, 1975, figs. 10, 13, 15).

It seems possible that Spirogyra and other Conjugales, with their very large nucleoli,long nucleolar-organizing region, and their long-drawn-out telophase and prophase,are particularly suitable organisms for the study of the chromosome-nucleolus rela-tionship. Further work on the isolation of nucleoli and spreading of their contents inthis group could probably show further features of interest.

REFERENCES

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CHOULNARD, L. A. (1971). Behaviour of the structural components of the nucleolus duringmitosis in AUium cepa. In Advances in Cytopharmacology, vol. I (ed. F. Clementi & B.Ceccarelli), pp. 69-87. New York: Raven Press.

CHOUINARD, L. A. (1975). An electron-microscope study of the intranucleolar chromatin duringnucleogenesis in root meristematic cells of Allium cepa. J. Cell Sci. 19, 85-101.

DAS, N., MICOU-EASTWOOD, J., RAMAMURTHY, G. & ALFERT, M. (1970). Sites of synthesis andprocessing of ribosomal RNA precursors within the nucleolus of Urechis caupo eggs. Proc.natrx. Acad. Sci. U.S.A. 67, 968-970.

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GIMENEZ-MARTIN, I. F., DB LA TORRE, C, LOPEZ-SAEZ, N. F. & ESPONDA, P. (1977). Plantnucleolus: structure and physiology. Cytobiologie 14, 421-462.

GODWARD, M. B. E. (1956). Cytotaxonomy of Spirogyra. I. S. submargaritata, S. subechinata,and S. britannica. spp. novae. J. Linn. Soc. (Bot.) 55, 532-546.

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MILLER, O. L. Jr. & BEATTY, B. R. (1969). Visualization of nucleolar genes. Science, N.Y.164, 955-957-

PAULSON, J. R. & LAEMMLI, U. K. (1977). The structure of histone-depleted metaphasechromosomes. Cell 12, 817-828.

REVEL, J. P. & HAY, E. D. (1961). Autoradiographic localization of DNA synthesis in a specificultrastructural component of the interphase nucleus. Expl Cell Res. 25, 474-480.

SPRING, H., TRENDELENBURG, M. F., SCHEER, U., FRANKE, W. W. & HERTH, W. (1974).Structural and biochemical studies of the primary nucleus of two green algal species. Aceta-bvlaria mediterranea and Acetabularia major. Cytobiologie 10, 1-65.

SPRING, H., SCHEER, U., FRANKS, W. W. & TRENDELENBURG, M. F. (1975). Lampbrush-typechromosomes in the primary nucleus of the green alga Acetabularia mediterranea. Chromo-soma 50, 25-43.

(Received 2 August 1978 - Revised 16 July 1979)

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