A biochemical and ultrastructural study of RNA in yolk platelets of Xenopus gastrulae

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1 /. Embryol. exp. Morph. Vol. 26, 2,pp , 1971 Printed in Great Britain A biochemical and ultrastructural study of RNA in yolk platelets of Xenopus gastrulae ROBERT O. KELLEY, 1 GEORGE S. NAKAI AND MARLENE E. GUGANIG From the Departments of Anatomy and Medicine, The University of New Mexico SUMMARY Yolk platelets from Xenopus gastrulae were isolated in a sucrosepolyvinyl pyrrolidone medium, washed, centrifuged four times, and portions of each pellet were prepared for electron microscopy. Electron microscopy revealed isolated platelets to be free of cytoplasmic contamination with progressive disruption of the superficial layer after each washing. Each washing and thefinalpellet were extracted with phenol and precipitated with ethanol. Orcinol analysis indicated that 5060 fig of RNA were present in yolk platelets isolated from 1000 gastrulae. Autoradiography of yolk platelets from cells incubated in [5 3 H]uridine revealed label in superficial and main body components after treatment with DNase but not after incubation in RNase. Acrylamidegel electrophoresis suggests that yolk platelet RNA is of both high and low molecular weight. INTRODUCTION The structure, chemical composition and role of yolk platelets during amphibian embryogenesis has been a subject of active investigation and lively controversy for several years. The ultrastructure of the yolk platelet was revealed by Ward (1962) and Karasaki (1963) to consist of a main body component with a distinct crystalline matrix, a superficial layer surrounding this main body, and a limiting membrane. Gross and Gilbert (1956) reviewed the chemical composition of the platelet in amphibians; however, Lanzavecchia & Le Coultre (1958) were among the first to propose that these structures contained nucleic acids. Rounds & Flickinger (1958) suggested that nucleic acid found in the 'yolk fraction' of mesodermal cells during primary induction participated in early morphogenesis. However, in an effort to resolve and analyse yolk platelet components, Wallace (1963 a) concluded that amphibian yolk platelets in situ contained no RNA, but was uncertain of the content of DNA. Recently, Baltus, HanocqQuertier & Brachet (1968) have reported doublestranded, linear DNA in amphibian yolk platelets which they suggest plays a role in 1 Author's address: Departments of Anatomy and Medicine, The University of New Mexico, School of Medicine, Albuquerque, New Mexico 87106, U.S.A.

2 182 R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG synthesis of enzymes for breaking down platelets during development, if this hypothesis is valid, then might yolk platelets contain RNA in addition to DNA? We chose techniques of electronmicroscopic autoradiography, phenol extraction of RNA, and separation of RNA molecular species by means of sucrosegradient centrifugation and acrylamidegel electrophoresis in an attempt to answer this question. MATERIALS AND METHODS Fertilized eggs were obtained from the South African clawed frog, Xenopus laevis, after the method of Gurdon (1967). Gastrulae (stage 11; Nieuwkoop & Faber, 1956) were carefully divested of surrounding jelly and fertilization coat in 20 % cysteine neutralized to ph 78 with NaOH and containing 02 % papain. Electron microscopy Entire embryos were preserved in a variety of fixatives (veronal acetate buffered osmium tetroxide, phosphate buffered osmium tetroxide, phosphate buffered formaldehyde, phosphate buffered glutaraldehyde, cacodylate buffered glutaraldehyde, and veronal acetate buffered glutaraldehyde). Best preservation was observed in specimens fixed with 175 % glutaraldehyde in 0.1M phosphate buffer (Polysciences, Inc.) for 1 h at 4 C, postflxed with 20 % osmium tetroxide in 0.1 M phosphate buffer at 4 C for 1 h, rapidly dehydrated through an ethanol series to propylene oxide and embedded in Epon 812. Thin sections (mounted on uncoated grids) were stained for 1 h in saturated aqueous uranyl acetate at 35 C, for 5 min in alkaline lead citrate at room temperature and examined in an Hitachi HU11C electron microscope. Isolation of yolk platelets To determine the presence of RNA chemically, yolk platelets were isolated from gastrulae using modifications of the method of Wallace & Karasaki (1963). The following procedures were performed in a cold room (5 C). Embryos were washed with NiuTwitty's (1953) solution containing penicillin (1000 i.u.) and streptomycin (001 mg/ml) and were transferred to cold 025 M sucrose50 % (w/v) polyvinyl pyrrolidone (PVP). Gradients were prepared by placing 20 ml of 10 M sucrose50 % PVP in roundbottomed 35 ml Pyrex centrifuge tubes (Sorvall rotor SS34). One thousand embryos were gently homogenized in 60 ml of the 025 M sucrose50 % PVP medium, the grey homogenate then being divided into four equal aliquots and carefully layered on to the surface of each gradient. Tubes were initially spun in a Sorvall RC2B refrigerated centrifuge (0 C) for 10 min (F max = 600 g). This procedure produced a yellowish pellet of yolk platelets, a thin superficial layer of contaminating pigment on top of the pellet, and a grey supernatant. After decanting the supernatant (which was saved for analysis), walls of the tubes were rinsed with 025 M sucrose50 % PVP, the

3 RNA in yolk platelets 183 pellets resuspended by gentle manual shaking, and centrifuged again at 590 rev/ min for lomin (F max = 500 g). This procedure was repeated four times, the final pellet containing neither pigment nor contaminating particulate material (for some experiments, final resuspension was accomplished by shaking in a vortex mixer). Portions of each pellet after each spin were fixed for examination in the electron microscope. A utoradiography To determine the localization of RNA in yolk platelets, gastrulae were incubated in [5 3 H]uridine (New England Nuclear Corp.; specific activity 1312 Ci/mmole, in sterile aqueous medium) for 2 h, and were transferred into unlabeled uridine (uracil riboside, Nutritional Biochemicals Corp.; 10 mg/ml in sterile NiuTwitty's solution) for 1 h. Some embryos were prepared for electronmicroscopic autoradiography after the method of Caro (1964), whereas others were homogenized, their yolk platelets isolated as described, and the pellets prepared for autoradiographic examination in the electron microscope. Control specimens (intact embryos and platelet pellets) were fixed in 3:1 ethanol: acetic acid and embedded in paraplast. These were sectioned at 6 /*m, mounted on gelatinsubbed slides, and subjected to one of the following procedures: (1) 3 h in DNase (Worthington, x2 crystallized, 05 mg/ml in 01 M phosphate buffer with 01 % phenol as preservative); (2) 3 h in RNase (Worthington, 13 mg/ml in 01 M phosphate buffer with 01% phenol as preservative); (3) 3 h in buffer and in water, both without enzyme; and (4) 1 N HC1 at 60 C for 12 min. These slides were treated with cold 50 % trichloracetic acid for 15 min, rinsed in 80 and 95 % ethanol and allowed to dry. Random samples of silver grains over control sections were counted using a grid (one square = 04 cm 2 ) drawn on a transparent plastic sheet. All silver grains over yolk platelets within 100 adjacent squares of the grid were counted. Autoradiographs used for counting were at the same magnification. Analytical procedures Isolated yolk platelets were analysed by the following procedures: Extraction of RNA After isolation of yolk platelets, pellets were resuspended in 50 ml of saline buffer (024 M NaCl, 001 M MgCl 2, 001 M Tris, ph 50) to which was added 05 ml of 10% sodium dodecyl sulfate (SDS), 05 ml of 25% bentonite in 001 M sodium acetate, and 50 ml of watersaturated phenol containing 01 % 8hydroxyquinoline (Cline, 1966). After manual shaking for 20 min at 0 C, phases were separated by centrifuging at 800 g for 10 min at 5 C. The aqueous layer was saved whereas the phenol layer was reextracted with 50 ml saline buffer by shaking for 5 min at 50 C. The emulsion was chilled, centrifuged at 800 g for 10 min at 5 C, the aqueous layer combined with the previous aqueous portion and reextracted twice with 50 ml phenol at 50 C for 5 min. After final

4 184 R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG extraction, the aqueous fraction was centrifuged at g for 30 min to remove bentonite. Onetenth volume of 20 % sodium acetate and two volumes of 95 % ethanol were added to the supernatant and the RNA was allowed to precipitate overnight at 20 C. The precipitate was treated with DNase (Worthington, 13 mg/ml in 01 M phosphate buffer with 01 % phenol as preservative) prior to sedimentation analysis and electrophoresis. Orcinol colorimetric test Ribonucleic acid in yolk platelet pellets was determined by standard orcinol colorimetric analysis as described by Shatkin (1969). Densitygradient separations of RNA RNA was centrifuged at g for 30 min at 5 C, the ethanol was decanted and the precipitate dried by inverting the tube at 5 C for 30 min. RNA was dissolved in SDS buffer (01 M NaCl, 01 M EDTA, 001 M TrisHCl, ph 74, 02 % SDS), layered on a 1030 % linear sucrose gradient, and centrifuged in a Spinco L265 ultracentrifuge (SW 65 rotor) for 25 h at rev/min ( g) at 25 C. The centrifuged gradients were displaced with a 40% sucrose solution and continuously monitored at 254 nm through a Model D Density Gradient Fractionator (Instrumentation Specialties Co., Inc., Lincoln, Nebraska). RNA fractionation on acrylamide gel RNA was extracted with phenol, purified as described above, and subjected to electrophoresis on an acrylamide gel according to Loening (1967). RNA from Escherichia coli (23 S and 16S) were utilized as markers in control gels. RESULTS Electron microscopy Yolk platelets are present in random pattern throughout cells of Xenopus gastrulae (Fig. 1), the larger platelets (up to 50 /im in length) being present in prospective entodermal regions. Most platelets exhibit three basic components as described by Karasaki (1963): a limiting membrane, a superficial layer and a main body component. However, platelets in presumptive neural ectoderm appear to have lost superficial layers and limiting membranes by the midgastrula stage. Isolated yolk platelets Limiting membranes are generally lost during isolation procedures, whereas superficial layers and crystalline inner matrices remain (Fig. 2). The superficial layers consist of small electrondense particles (50 A in diameter), larger, more angular particles ( A in diameter) and an amorphous background substance. The main body contains a highly structured crystalline matrix (for review of fine structure in the main body component see Karasaki, 1963).

5 RNA in yolk platelets 185 Fig. 1. Portions of cells at mesodermectoderm interface (stage 11). Note random pattern of yolk platelets (yp). I, Lipid droplet; m, mitochondrion; p, cytoplasmic particles, x Fig. 2. Portion of isolated yolk platelet revealing crystalline main body component (mbc) and superficial layer (s). Note presence of A particles (arrows) in periphery of superficial layer, x

6 186 R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG

7 RNA in yolk platelets 187 Pellets examined in the electron microscope after initial centrifugation revealed presence of membranous material, lipid droplets and disrupted mitochondria in addition to yolk platelets (Fig. 3). Superficial layers of the latter were fused with neighbouring platelets, creating a continuum of yolk substance. After resuspension and a second centrifugation, platelets regained their individuality, losing contaminating materials (Fig. 4). Following a third centrifugation, superficial layers began to separate from main body components (Fig. 5), revealing particulate material in the supernatant. Main body components were disrupted following suspension with a vortex mixer and a fourth centrifugation (Fig. 6). Autoradiography Autoradiographs of gastrula cells cultured in [ 3 H]uridine reveal silver grains over both superficial and central components of yolk platelets (Fig. 7) in addition to cytoplasmic particles, mitochondria and nuclei. In addition, label remains in isolated platelets. Table 1 illustrates the effect of acid hydrolysis and RNase on grain counts over yolk platelets in situ. Thick sections of cells prepared for lightmicroscope autoradiography have fewer silver grains after incubation in RNase and treatment with 1 NHCI than when treated with DNase, phosphate buffer, and water for similar periods of time at 38 C. Table 1. Numbers of silver grains over yolk platelets in ectodermal cells treated with 1NHCI, RNase, DNase, phosphate buffer without enzyme, and water (Results represent the average of counts from at least midgastrula ectoderm.) ten light micrographs of 1 NHCI 60 C 12min RNase 38 C 3h DNase 38 C 3h Buffer 38 C 3h Water 38 C 3h Grains/grid 387 cm Fig. 3. Micrograph of yolk platelet pellet after initial centrifugation (F max = 600g). I, Lipid droplet; m, mitochondrion (contracted, because the medium used for isolating yolk platelets is hypertonic for mitochondria); cm, cytoplasmic membranes, x Fig. 4. Micrograph of yolk platelet pellet after resuspension and a second centrifugation. Note absence of cytoplasmic contamination, x Fig. 5. Micrograph of yolk platelet pellet after gentle hand resuspension and a third centrifugation revealing partial disruption of superficial layers, x Fig. 6. Micrograph of yolk platelet pellet after resuspension by vortex mixer and a fourth centrifugation revealing disruption of both main body and superficial components, x

8 188 R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG Fig. 7. Autoradiograph of ectodermal cell (stage 11) incubated in [ 3 H]uridine for 1 h. x Fig. 8. Autoradiograph of platelets isolated from midgastrula cells cultured in [ 3 H]uridine for 1 h. x

9 RNA in yolk platelets Fraction number Fig. 9. Sucrose densitygradient pattern of RNA from purified yolk platelet pellets. Peak I at top of sucrose gradient represents contaminating sediments (nucleotides, degraded DNA and protein, some lowmolecularweight RNA). E c T LO (N f\ o n _ "46S ' A49S " /S. 58 S "Top 18S 15SI20S i Bottom "Top Fraction number _ 29 S A 1 28 S hi J Bottom Fig. 10. RNA profile after acrylamidegel electrophoresis of peaks 2 and 3 present in Fig. 9. Diagram on left represents peak 2 (Fig. 9), whereas peak 3 is presented on the right. E M B 26

10 190 R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG Analytical procedures Orcinol analysis Orcinol analysis (Shatkin, 1969) suggested that 5060/tg of RNA were present in yolk platelets isolated from 1000 Xenopus embryos (stage 11). Sucrosegradient centrifugation and acrylamidegel electrophoresis Opticaldensity measures of RNA from isolated pellets in sucrosedensity gradients revealed three distinct peaks (Fig. 9). The major peak (fraction 1) at Fig. 11. Acrylamidegel profile of phenolextractable material from supernatant of first centrifugation. 15 _ Wash _ Wash 3 I 4S O Top Bottom / \ ~ Top Fraction number 30 S 27 SA y\ \ 15S I Bottom Fig. 12. Left: acrylamidegel profile of phenolextractable material from supernatant of second centrifugation step in isolation procedure. Right: acrylamidegel profile of phenol extractable material from supernatant of third centrifugation step in isolation procedure corresponding to disruption of superficial layers (Fig. 5).

11 RNA in yolk platelets 191 the top of the tubes represented contaminating sediments (nucleotides, degraded DNA, and some protein) and lowmolecularweight RNA. Fractions 2 and 3 were isolated and electrophoresed on acrylamide gels, revealing several distinct components (Fig. 10). Peaks corresponding to 23 S and 16 S RNA from E. coli in control gels were noted and sedimentation values for peaks in experimental profiles were computed from these data. RNA extracted from washings obtained during platelet isolation procedures yielded the following results. Fig. 11 represents RNA species separated by gel electrophoresis from collected supernatants after a single centrifugation of homogenate. Numerous peaks were present, representing high and lowmolecularweight RNA in both nucleus and cytoplasm of cells from stage 11 embryos. However, after resuspension and a second centrifugation of platelet pellets, phenol extractions of supernatant did not provide optically active material demonstrable in acrylamide gels (Fig. 12, left). Extractions of washings from the third centrifugation, however (Fig. 12, right), yielded electrophoretic patterns similar to those obtained from isolated yolk platelets. Lowmolecularweight RNA as well as larger species were present. A 15 S species was present in both third washings and isolated yolk platelet pellets. Analysis of washings from a fourth resuspension and centrifugation revealed electrophoretic profiles similar to those demonstrated after the third spin. DISCUSSION Questions concerning the presence and potential developmental role of nucleic acids within amphibian yolk platelets are recurrent topics in numerous papers (see Rounds & Flickinger, 1958; Yamada, 1961; Horn, 1962). Although the existence of RNA in yolk platelets has been challenged by Wallace (1963 a, b) and Ohno, Karasaki & Takata (1964), our results indicate that yolk platelets in Xenopus gastrulae (stage 11) do possess small quantities of RNA ( [i%\ embryo). Of principal concern is the problem of contamination or artifact. Bacteria would probably not contaminate an embryonic cell fraction to the extent of contributing significant optical density to the RNA population. Furthermore, bacteria can be easily removed from media. In addition, embryos were chemically dejellied, which has been shown by Brown (1967) to decrease significantly bacterial contamination. An additional potential contaminant is yolk phosphoprotein which has a molecular weight (30000) and phosphate content (8%) similar to low molecular weight (4 S) RNA (Wallace, 1963a). Furthermore, phosphoprotein can be extracted into the aqueous phase and precipitated with ethanol. Fortunately removal of most of this contaminant is effected with 001 M MgCl 2 in the initial homogenate (Brown, 1967). Are the A particles present in superficial layers of in vitro and in situ platelets ribonucleoprotein (RNP)? And if so, are they normal components of 132

12 192 R. O. KELLEY, G. S. NAKAI AND M. E. GUGANIG superficial layers or artifacts of the isolation procedure? Generally, particles that are A in diameter and either attached to membranes or free in the cytoplasm of adult tissues are designated ribosomes (Palade, 1955), whereas those larger in size ( A in diameter) and free in the cytoplasm are regarded as glycogen granules (Drochmans, 1962). It is thought then that larger particles present in superficial layers are RNP because of their appearance and affinity for aqueous uranyl acetate. In addition, the isolation procedure of Wallace & Karasaki (1963) used in this study provides the investigator with platelets which are reasonably free from contamination visible in the electron microscope (Figs. 46). Furthermore, the absence of RNA in the supernatant following a second resuspension and centrifugation (Fig. 12, left) and the associated intact appearance of isolated platelets (Fig. 4) suggest that A particles are normal components of superficial layers and not free cytoplasmic particles displaced by centrifugation. Autoradiographs presented in this study of labeled (tritiated undine) yolk platelets may provide morphological evidence for the presence of RNA in those structures (Figs. 7, 8). Although nucleosides are not thought to be precursors of nucleic acids in normal biosynthetic pathways, [ 3 H]uridine has been used in a variety of embryos and appears to be an efficient precursor of nucleic acids. Bieliavsky & Tencer (1960) have demonstrated that during cleavage in amphibian embryogenesis, labeled uridine is incorporated into DNA rather than RNA, but the converse seems to be true during gastrulation. Since thick sections of cells, prepared for lightmicroscopic autoradiography as controls, have fewer silver grains over yolk platelets after incubation in RNase and 1 NHCI than when treated with either DNase, buffer or water, it is concluded that most [ 3 H]uridine is incorporated into RNA rather than DNA during this developmental period. Failure to extract RNA from washings following a second resuspension and centrifugation (Fig. 12, left) followed by an increase in RNA content in supernatants of successive spins (Fig. 13, right) may correlate with progressive disruption of main body components (Fig. 6), superficial layers (Figs. 5 and 6), and the apparent release of RNP particles (Fig. 2) from the latter. Hence, biochemical and autoradiographical evidence presented in this study supports the hypothesis that yolk platelets in amphibian gastrulae contain RNA. Acrylamidegel profiles suggest that RNA species of high and low molecular weight are present. Furthermore, the RNA content of isolated platelets can be released to surrounding media during isolation procedures which supports observations of Rounds & Flickinger (1958). The developmental significance of yolk platelet RNA, however, remains uncertain. Grateful acknowledgment is made to Professors A. J. Ladman and Leonard Napolitano for helpful discussions and critical reading of the manuscript, to Mrs Margo G off for technical assistance, and to the United States Public Health Service for facilities through CA10694.

13 RNA in yolk platelets 193 REFERENCES BALTUS, E., HANOCQQUERTIER, J. & BRACHET, J. (1968). Isolation of deoxyribonucleic acid from the yolk platelets of Xenopus laevis oocyte. Proc. natn. Acad. Sci. U.S.A. 61, BIELIAVSKY, N. & TENCER, R. (1960). Etude de 1'incorporation de 1'uridine tritiee dans les ceufs d'amphibians. Expl Cell Res. 21, BROWN, D. D. (1967). Nucleic acid determination in embryos. In Methods in Developmental Biology, pp New York: Thomas Y. Crowell Co. CARO, L. (1964). High resolution autoradiography. In Methods in Cell Physiology, vol. r (ed. D. M. Prescott), pp New York and London: Academic Press. CLINE, M. J. (1966). Isolation and characterization of RNA from human leucocytes. /. Lab. din. Med. 62, DROCHMANS, P. (1962). Morphologie du glycogene. Etude au microscopie electronique de colorations negatives due glycogene particulare. /. Ultrastruct. Res. 6, GROSS, P. R. & GILBERT, L. I. (1956). Chemistry and ultrastructure of amphibian yolk platelets. Trans. N.Y. Acad. Sci. 19, GURDON, J. B. (1967). African clawed frogs. In Methods in Developmental Biology, pp. 7584; New York: Thomas Y. Crowell Co. HORN, E. C. (1962). Extranuclear histone in the amphibian oocyte. Proc. natn. Acad. Sci. U.S.A. 48, KARASAKI, S. (1963). Studies on amphibian yolk. I. The ultrastructure of the yolk platelet. /. Cell Biol. 18, LANZAVECCHIA, G. & LE COULTRE, A. (1958). Origine dei mitochondri durante lo sviluppo embrionale di Rana esculenta. Studio al microscopio electtronica. Archo ital. Anat. Embriol. 63, LOENING, U. E. (1967). The fractionation of highmolecularweight ribonucleic acid by polyacrylamidegel electrophoresis. Biochem. J. 102, NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/xenopus laevis (Daudin). Amsterdam: North Holland Publishing Co. Niu, M. C. & TWITTY, V. C. (1953). The differentiation of gastrula ectoderm in medium conditioned by axial mesoderm. Proc. natn. Acad. Sci. U.S.A. 39, OHNO, S., KARASAKI, S. & TAKATA, K. (1964). Histo and cytochemical studies on the superficial layer of yolk platelets in the Triturus embryo. Expl Cell Res. 33, PALADE, G. E. (1955). A small particulate component of the cytoplasm. /. biophys. biochem. Cytol. 1, ROUNDS, D. E. & FLICKINGER, R. E. (1958). Distribution of ribonucleoprotein during neural induction in the frog embryo. /. exp. Zool. 137, SHATKIN, A. J. (1969). Colorimetric reactions for DNA, RNA and protein determinations. In Fundamental Techniques in Virology (ed. K. Habel and N. P. Salzman), pp New York: Academic Press. WALLACE, R. A. & KARASAKI, S. (1963). Studies on amphibian yolk. II. The isolation of yolk platelets from the eggs of Rana pipiens. J. Cell Biol. 18, WALLACE, R. A. (1963 a). Studies on amphibian yolk. III. A resolution of yolk platelet components. Biochem. biophys. Acta 74, WALLACE, R. A. (19636). Studies on amphibian yolk. IV. An analysis of the main body component of yolk platelets. Biochim. biophys. Acta 74, WARD, R. T. (1962). The origin of protein and fatty yolk in Rana pipiens. II. Electron microscopical and cytochemical observations of young and mature oocytes. /. Cell Biol. 14, YAMADA, T. (1961). A chemical approach to the problem of the organizer. In Advances in Morphogenesis, vol. i, 155. {Manuscript received 4 January 197T)