The ultrastructure and RNA metabolism of nucleoli in early sea urchin embryos

Q 1968 by

hcademic Press Inc.

Experimental

13

Cell Research 52, 13-26 (1968)

THE ULTRASTRUCTURE AND RNA METABOLISM OF NUCLEOLI IN EARLY SEA URCHIN EMBRYOS S. KARASAKI Laboratoires de Recherche, Institut da Cancer de Montrkzl, H6pital Notre-Dame et Universit6 de Montre’al, MontGal, Canada

Received November 6, 1967

THEearly

embryonic development of various animals is characterized by the absence of a nucleolus [4]. In sea urchins, nucleoli and chromocenters first become visible at the gastrula stage of development [7]. Thus, sea urchin embryos appear to be favorable for an examination of the origin of the nucleolus and of its relation to the chromosomes. Although embryos have been subjected to much investigation by electron microscopy [l, 31, relatively little attention has been paid to the fine structure of the nucleus. Recently, Comb and Silver [6] have indicated that a sequential initiation of the synthesis of different RNA species clearly takes place during early sea urchin development. RNA synthesis can be definitely detected at the blastula stage [6], although a low level of the synthesis may occur during cleavage [12, 14, 291. Up to the gastrula stage, the synthesized RNA appears to be a messenger-type [S, 12, 14, 291. Ribosomal RNA synthesis first occurs at gastrulation [S, 11, 291, thus coinciding with the appearance of a nucleolus [S, 7, 221. Radioautographic studies on RNA metabolism have been carried out with special reference to region-specific [8, 271 and stage-specific [lo] patterns in the developing embryos. Although these studies [t), 10, 271 have demonstrated synthesis of RNA in the nucleus as well as transfer of the nuclear RNA into the cytoplasm, no detailed information is available about the precise localization of the RNA-synthesizing sites in the nucleus. The present study surveying the fine structural changes of the nuclei in developing sea urchin embryos has been undertaken to determine the pattern of nucleolar appearance and its relationship to chromosomal components. It is also concerned with a submicroscopic analysis of the initial sites of nuclear RNVA synthesis and its possible correlation to nucleolar formation. For this purpose, electron microscopic radioautography has been applied to the detection of the sites of uridine incorporation in the nuclei of blastomeres at the cleavage, blastula and gastrula - stages of Arbacia punctulatn. Experimental

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MATERIAL

AND

METHODS

were obtained from the Marine Biological The sea urchins, Arbtrcirr punctulata, Laboratory, Woods Hole, Mass., during the months from June to August. The eggs were fertilized and allowed to develop at 23°C 1151. The following series of embryos were examined: the 2-cell stage (1 11 embryo), the 16K32 cell stage (3 h), the earl) blastula stage (6 h, prehatching), the late blastula stage (9 h, swimming), the earl! gastrula stage (12 h, beginning of gastrulation), the prism gastrula stage (18 h) and the pluteus stage (30 h). Some embryos were also stored in a refrigerator at 6 C for several hours. At different stages, the embryos were collected by centrifugation and suspended in filtered sea water. They were transferred in the sea water containing 3H-5-uridine (New II].

RESULTS Obserncrtions

with

the light

microscope

n’ith the light microscope, nucleoli or similar intranuclear hodies cannol be detected until the late blastula stage. \Vhen cleavage embryos are kept at 6°C for 5 h, however, a number of spherical inclusions become cltarl? visible in the nuclei. As seen in I=ig. 1, they are as large as or larger than the Experimmtnl

Crll Kesettrch 52

Nucleolus during

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Figs 1 and Z.-Light microscopic appearance of Arbacia embryos, fixed in GTA and embedded in GMA for electron microscopy. The thick sections were stained with toluidine blue. Fig. 1, Part of a 2-cell stage embryo, incubated for 5 h at 6°C. Dense bodies (arrows) in the nucleus. x 900; Fig. 2, Part of a pluteus, developed normally at 23°C. In each nucleus, one or two nucleoli (arrow) are present among numerous chromocenters. x 900.

typical nucleoli (cf. Fig. 2). These intranuclear bodies disappear when the embryos are returned to 23°C. The bodies are negative to the Feulgen reaction, but are stained with basic dyes such as toluidine blue, azure B, methylene blue and hematoxylin; this basophilic property is not significantly altered by treatment with RNase. They also give a positive reaction to mercuric bromphenol blue suggesting the presence of protein. At the late blastula and early gastrula stages, one or tjvo small nucleoli are observed in many interphase nuclei. The low temperature treatment of the embryos does not change the number and size of nucleoli. The staining characteristics closely correspond to those of the nuclear bodies observed at the cleavage stage. However, the basophilia is slightly decreased by RNase treatment. The Feulgen test reveals positively reacting chromocenters and a chromatin ring surrounding the nucleolus. At the prism gastrula and the pluteus stages the interphase nuclei contain prominent nucleoli and chromocenters (Fig. 2). The basophilia is significantly decreased by RNase treatment, indicating that this phenomenon is mainly due to RNA. Observafions

with

the electron

microscope

Regardless of the methods of preparation employed, the results obtained on the fine structure of the nucleus during successive stages of embryonic development are comparable; the GTA-GMA series are illustrated in Figs 3-7 and the O,O,-Epon series in Figs 8 and 9. However, after uranium staining, the chromatin is more intensely stained than the nucleolar material; and Experimenfal

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Figs 3--7.--Electron micrographs of small portions of interphase nuclei showing ~~ucleolar modfication. Fixed in GTA and embedded in GMA. Thin sections stained with uranyl acetate. I’ig. 3, Early blastula. Two dense bodies of amorphous material are sharply delineated from the rest of nucleus. x 35,000; Fig. 4, Late blastula. A nucleolus is surrounded by chromatin (arrorr~) and contains fibrillar and amorphous components. r 35,000; Fig. 5, Early gastrula. Nucleolus and the associated chromatin (arrow). The nuclcolar body is mainly composed of fibrillar and amorphous components. x 35,000, Pig. 6, Prism gastrula. Note particulate components in the marginal zone of nucleolus. Nucleolus-associated chromatin (arrow). * 35,000: Fig. 7, Pluteus. A large nucleolus contains particulate and fibrillar components which arc intermingled each other. x 35,000.

Figs 8 and Y.-Electron micrographs of the embryonic cells, fixed in 0~0, and embedded in Epon. The thin sections were stained with alkaline lead. Fig. 8, Part of an interphase cell from an earl? hlastula. Within chromatin clumps (CH), dense bodies can be found (arrows). The body seems to be a compact mass of fibrillar components. x 70,000; Fig. 9, Part of an interphase cell from a prism gastrula. The nucleolus consists of fibrillar and particulate components. The particulate components are clearly seen at the periphery (arrows). Chromatin components are fotmd as strands and clumps (Cff). Diffuse chromatin fibrils are closely intermingled with interchromatin granules (IG). X 70,000. Experimental

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I’$. lO.-~ Light microwopic radioautographs of embryos incubated for 1 h with YH-5-uridine. Early blastula (En), late blastula (L R) and early gaslrula (EC;). Silver grains are located over the nuclei in each embryo. Note the difference of grain density. ‘ 300.

alkaline lead stains nucleolar material more intensely than chromatin. ‘l’hr morphological distinction l)et\vecn t\vo components in the electron mic-rographs is clearly demonstrated 1)~ these staining procetlurrs, as suggcstccl 1)~ Marinozzi and Bernhard [28 I. In the cleavage and early blastula c~nh~~yos, the interphase nucleus does not show a lypical nucleolus hut contains many dense hodics. As SWII in Figs 3 anti 8, each hotly is less than 1 p in diameter and is mow or less spherical. It has a homogeneous texlurt consisting of fihrils anti amorl)hous suhstanw, and is as a rule sharply delineated from the other part of the nutleus (Fig. 3). Occasionally, they are sm-rounded by chromatin material (l’ig. 8). .\fter staining with heavy metals, these fibrous bodies appear to havr an opacity of the same order as that of the typical nucleolus which is visihlc at the later stages. Their number is countctl up to ten in sections of the inlcI.l;igs 11 -16. -Electron microscopic radioautographs of embrvos labeled in sea waler I\-itk .Xl f/C: 3H-5-uridinr. Fixed with GTA and cmhedded in GblA. Grains of Ilford I.-1 cmul?ion over Ihr thin sections were developed with Rlicrodo-S. The sections were stained with uranyl acetate. Developed grains generally appear as single filaments of metalic silver in the ciectron micrographs. Fig. II, Labeled nucleus from a late cleavage embryo. The 1 h embryo (2.cell stag!c) was Iknsc incubated for 3 h with the isotope. Silver grains are located over the dispersed chromatin. bodies (arrows) arc not labeled. Y 16,000; Fig. 12, Labeled nucleus from an early I)lastula. The .3 h embryo (16.cell stage) was incubated for 3 h with the isotope. Two dense bodie? Cctrrorus) are not laheled, while the rest of the nucleus is labeled. * 16,000; I:ig. 13, I.abeled nucleus from a late blaslula. The 6 h embryo (early blastula stage) was incuhalcd for 3 h with the isotope. IIan? silver grains occur over the nucleus except over a dense body (arrow). y 16,000; h’ig. 14, I,abrletl nucleus from an early gastrula. The 12 h embryo was incubated for 1 h with the isotope. I.abeling occurs over the nucleolus as well as over the nucleus proper. The nucleolus is associatctl with ‘I’hc 12-h a chromatin strand (arrow). \’ 16,000; I’ig. ZZ, I.abeled nucleus from a mitl-gastrula. embryo (early gastrula stage) was incubated for 3 h with the isotope. Silver grains are localctl both over the nucleoli (arrows) and the rest of nucleus. x 16,000; Fig. 16, I.aheled nucleus from a prism gastrula. The 18 h embryo was incubated for 1 h with the isotope. 1,abeling occurs throughout the nucleus proper and is concentrated over the nucleoli (arroclks). ) 16,000.

LVucleolus during

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phase nuclei. On the other hand, such bodies are not visible in the mitotit nuclei. Therefore, it seems that they may be undergoing cyclic disappearance and reappearance at each mitosis during the early stages. ;\s stated earlier, dense fibrous bodies increase in size in prolonged interphase, induced by lo\\. temperature treatment. In the late blastula (Fig. 4) anti early gastrula embryos (Fig. 31, each inlerphase nucleus contains one or t\vo nuclcolar bodies \\-ith the associated chronatin. This nucleolus is a spherical compact mass less than 2 p in diameter, ant1 composed of fibrous and amorphous material. I’arliculate components arc often found in these nucleoli, although they appear to bc smaller ant1 lrss uniform than cytoplastnic ribosomcs. 11~ the time of gastrulation, particulate components become clearly \.isible at the periphery of the fibrous nucleolar hotly (Figs 6 and 9). The particles are about 150 A in diameter and closcl~ resemble the cytoplasmic ribosonncs. In the pluteus embryos, amount of the particulate components bccomw increasingly prominent and are distributed throughout the nucleolar body (Fig. 5). The particulate anti fibrillar components of the nucleolus no\\’ aplwar intermingled \vith each other. Throughout the development, the nucleolusassociated chromatin is al\vays located at the periphery of the nucleolal body. To date no chromatin-like structure has been found inside the nucl~lus. Radiocrutog~rrl)hi~

e.xcrminniions

with

the light

microscope

Hatlioautographir studies of uridine incorporation into developing dhrcirr embryos \\-cre undertaken with the light microscope. Embryos at diiYewn1 stages of development exposed to 3H-5-uridine for 1 h ant1 fixed in glutaraltlchyde generally she\\- the silver grains over the interphase nuclei. 1)uring progressive stages of embryonic clerelol~ment, there is a significant incwaw in the incorporation of the radioisotope in the nuclei. Fig. 10 represenls an example of the labeling experiment in a mixture of embryos at three dift’crcnl

Figs 17-X0.--Electron microscopic radioautographs of embryos incubated in sea waler with .iO I’(: 3H-5-uridine. Fixed with 0~0, and embedded in Epon. Grains of Ilford L.4 emulsion over I hin sections were developed in Microdol-X. Slaincd with alkaline lead. Fig. 17, Labeled nucleus from an early blastula embryo. The 6 h embryo was incubated for 1 h with the isolopc. Only two silvcl grains are found over the dispersed chromatin area. Two dense bodies (arrotr~s) are nol lahelr~d. 16,000; Fig. 18, Labeled nucleus from a late blastula embryo. The 9 h embryo was incubated for 1 h with the isotope. Two nucleolus-like bodies are not labeled. Grains are located over lhe rest of the nucleus. * 16,000; Figs 19 and 20, J.abeled nuclei from early gastrulae. The 13 h embryos were incubated for 1 h with H3-uridine. The nucleoli are surrounded by associated chromalin. Silver grains arc found over Ihr periphery region of nucleoli (arrotrrs). ,’ 18,000.

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stages. The grain density of each nucleus appears to be undetectable at the early blastula stage, just noticeable at the late blastula stage and quite conspicuous at the early gastrula stage. Treatment of the section with RNasc removes the radioactire label from the gastrula embryos. RSase-resistant labeling occasionally occurs in some nuclei of the cleavage and blastula TABLE

1. Incorporation of’ 3H-5-uridine in to nucleoli or nucleolm-like bodies rrt the diflerent stcrges &win{/ errrly deuelopnient of Arhacia punctulata Hadioautographic

Incubation time (11) in the isotope

Embryonic stage (h aftrl fertilization) Cleavage

rxamination

(3)

Blastula

(6)

Gastrula

(12)

Prism (115) a ANucleoli or nucleolus-like

with the electron Number of nucleolia examined

microscope

Sumber of labeled nucleolia

1

6X

2

2.9

3

65

2

1 3 1 3 1

113 Xl 65 35 84

13 -1 32 26 62

3.1 13.3 $.!I 49.2 71.2 73.X

bodies.

embryos. Thus it appears that most, if not all, the grains observed over nuclei at the gastrula stage reflect the incorporation of 3H-3-uridine into RNA. These data generally support the previous radioautographic studies of Ficq et ~1. [ill on the incorporation of 3H-uridine into developing sea urchin embryos. Ho\ve\-er, the use of 3H-S-uridine in this study diminishes conversion of labeled uridine to labeled thymidine with subsequent uptake into DNA. Rodioautographic

emminntions

with

the electron microscope

The subcellular sites of 3H-3-uridine incorporation have been examined in electron microscopic radioautography of both GXlA- and Epon-embedded samples of developing Arbacirr embryos. The results obtained with the GRIAembedded material are comparable with those obtained by light microsopic radioautography, reported in the preceding section. In all derelopmcntal stages studied, silver grains are located primarily over the interphase nuclei regardless of the preparatory procedures used (GTA-GMA: Figs 1 l&l 6; OsO-Epon: Figs:1 Y-20). In general, more incorporation has been dctec*tecl after the GTA-G111,4 procedure than after the OsO,-Upon procedure. Trcat-

Nucleolus during

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ment of GMA sections in 5 per cent perchloric acid at 4°C for 5 min does not affect the labeling. However, the labeled material is completely extracted from the sections after treatment with 0.1 per cent RNase in distilled water at 37°C for 30 min. The dense bodies of the cleavage nuclei are not labeled after 1 to 3 h incubation (Figs 11 and 12, Table 1). In the majority of the cells, only a few silver grains are detected over the rest of the nucleus. In blastula embryos, after 1 to 3 h incubation, silver grains are located over the nucleoplasm except the nucleolus-like bodies (Figs 13, 17 and 18, Table 1). At the early gastrula stage, the incorporation definitely occurs within the nucleoli after 1 h incubation (Figs 14, 19 and 20, Table 1). The silver grains are found clustered over at which the chromatin is associated the peripheral region of nucleoli, (Figs 19 and 20). As the incubation is prolonged, more nucleoli become labeled (Table 1) and the number of grains per nucleolus increases (Fig. 15). The majority of nucleoli from the prism gastrula are heavily labeled even after 1 h treatment with the radioisotope (Fig. 16, Table 1). DISCUSSION

In the developing embryos of Arbacia punctulata, the interphase nucleus lacks a typical nucleolus up to the gastrula stage. This confirms the cytochemical study of Cowden and Lehman [7] who examined the nuclei of the embryos of Lytechinus virigatus. However, in Arbacia the interphase nucleus possesses a number of submicroscopic spherical bodies as early as the cleavage stage. As seen in the electron microscope, the nuclear bodies consist mainly of tightly packed fibrils which closely resemble a component within the typical nucleolus in differentiated cells of the sea urchin [20]. Morphologically, these bodies are clearly distinguishable from the typical nucleolus by the absence of a particulate component, the lack of an RNA content, and the variable numbers and sizes. Radioautography has also revealed that each nuclear body is inactive with respect to RNA synthesis and that there is 110 detectable transfer of newly synthesized RNA from chromosomal components during cleavage and blastula stages. *Judging from the cytochemical evidences, it appears likely that the body is mainly composed of protein. The small amount of proteins synthesized at this time are believed to be histones [S, 321 and structural proteins associated with the mitotic apparatus [26]. Therefore, the appearance of the nuclear body might involve the aggregation into a visible structure of protein which had been preformed during oogenesis. In fact, large spherical bodies with similar appearance as an oocyte nucleolus can be formed in the nucleus of a cleavage embryo, when it Experimenfal

Cell Research 52

is simply incubated in low tcmpcralure. Recently, Das anti AMfert -0 have reported that the lo\\- temperature induces the formation of n~1n~cro11s intranuclear bodies in unfertilized eggs of ITrcr~hi,s \vithout Ihc inc.o~I)oi‘:ltit,n of precursors of KS.4 and protein. ‘This type of a submici.osropir nuclear woody has hcen pre\-iously tl~~s~~rihetl in a vide varict? of embryonic cells in the first phase of the tlevclopmc~nl, \vhen a nutleolus is absent from the nucleus ;16, 19, 21, 22, 23, 25, Xj .I large fibrous “nuclrolus”, \vhich is already present at the heginning of’ cleavage stage of mammalian [34] anti molliisran [30/ spccics is cl?\-oitl of the particulate component. In the “nucleolus”, cytochemical methods have often failed to demonstrate the presence of I
Sucleolus during

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ribonuclease treatment, they have been referred to as ribonucleoprotein particles [13, IS]. The above stated sequence of events in the nucleolus has been reported by Karasaki [21] in amphibian embryogenesis, probably occurs also during embryogenesis of many other species [16, 19, 23, 25, 33, 341. It is now generally held that genes involved in the production of ribosomal RNA are located in the nucleolus-organizing chromosome [4, 5, 31, 3,?]. The association of the chromosome with the nucleolus may represent the actual functioning of the organelle involved in the synthesis of specific HNA molecules. In Arbncin embryos, the boundary region bet\\een the nucleolar body and the associated chromatin strand is thought to be the initial site of nucleolar RSA synthesis. In the preyious study on the amphibian embryonic cells, it has been suggested that the initial site of nucleolar RNA labeling is the central fibrous region of the nucleolus and not in the chromatin surrounding the nucleolus [21]. The apparent contradiction, holvever, seems to depend on the distribution of active chromatin in the nucleolus where the organize1 the presence of submicroscopic is located. In the amphibian nucleolus, chromatin fibrils has been found in the central fibrous region [21]. Recent ultrastructural examinations have also shown that the “nucleolus-associated chromatin” penetrates in a highly dispersed fashion throughout the region of a nucleolus [13, 17, IS]. It is thus reasonable to assume that in the nucleolus of the Arbrrcirr gastrula the active chromatin fibrils are localized in the peripheral region and participate in RNA synthesis. SUMMARY

The relationship between the structural modification of the nucleolus and the pattern of nuclear RNA synthesis has been studied by electron microscopic radioautography in the developing embryos of Arbrrcin punctrdrctrr. I)uring the cleavage and blastula stages, the interphase nuclei contain many dense bodies consisting of fibrillar components. There is no uridine incorporation into these bodies, although the rest of the nucleus becomes labeled. From the late hlastula to the early gastrula stage, when nuclear RNA synthesis is significantly enhanced, one or two fibrous nucleoli appear within the chromatin strands. Then, the nucleolus appears to initiate incorporation of 3H-3-uridine into the peripheral region at \vhich the chromatin is associated. During gastrulation, the nucleolus acquires particulate components and hecomes ljositiye to cytochemical tests for RNA.

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The initial phase of this study was performed at Marine Biological Laboratory. Woods Hole and Biology Division, Oak Ridge National Laboratory. The work was supported in part by a grant from the National Cancer Institute of Canada. I wish to thank my colleagues for their critical review of the manuscript. A part of this study was previously published in abstract [22]. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 27 2i: 25. 26. 27. 28. 29. 30. 31, 32. 33. 34. 35.

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I’ress.