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/. Embryol. exp. Morph. Vol. 19, 3, pp. 363-85, May 1968 Printed in Great Britain 363 The relative rates of synthesis of DNA, sRNA and rRNA in the endodermal region and other parts of Xenopus laevis embryos By H. R. WOODLAND 1 & J. B. GURDON 1 From the Department of Zoology, University of Oxford The onset and rates of synthesis of the major classes of nucleic acids have been extensively studied during the development of whole frog embryos (reviews by Brown, 1965; Gurdon, 1967a). Such information is of interest because nucleic acids are the immediate products of genes, and their rates of synthesis therefore provide a direct measure of changes in gene activity. To date nucleic acid synthesis in parts of frog embryos has been analysed mainly by methods which do not distinguish different classes of RNA (e.g. Bachvarova & Davidson, 1966; Flickinger, Miyagi, Moser & Rollins, 1967). Since embryos consist of many different cell types, it is important to know to what extent the pattern of nucleic acid synthesis observed in the whole embryo is true for its individual regions, and in particular for one differentiating cell type. Any differences between parts of early embryos in respect of nucleic acid synthesis are of further interest, since they are likely to be related to unequally distributed components of the egg cytoplasm. Such a relationship may eventually lead to the identification of cytoplasmic components which regulate the activity of genes during cell differentiation. In this article we compare three types of gene activity, the synthesis of soluble RNA (sRNA), ribosomal RNA (rRNA) and DNA, in the endoderm and in the rest of the embryo. These three classes of nucleic acid have been studied because they are easy to extract quantitatively, separate and characterize. sRNA (believed for reasons given below to consist primarily of transfer RNA) and rRNA are not homogeneous populations of molecules, but the different kinds of molecules within each class are functionally related. DNA is quite another kind of gene product, but like different kinds of RNA its rate of synthesis changes markedly and consistently during early development. DNA synthesis is also related to the final differentiation of a number of specialized cells (e.g. Stockdale & Topper, 1966). The endoderm region of embryos was chosen be1 24 Authors' address: Department of Zoology, Parks Road, Oxford, England. J EEM 19 364 H. R. WOODLAND & J. B. GURDON cause it can be dissected out relatively easily and cleanly at all the developmental stages studied, and also because visible signs of cytodifferentiation appear later than in most other tissues. MATERIALS AND METHODS A. Materials and culture conditions Embryos and tadpoles of Xenopus laevis (Daudin) were obtained and reared in the usual way (Gurdon, 19676), using Barth's solution (Barth & Barth, 1959) modified by the exclusion of bovine serum globulin and by addition of 0-01 g/1. each of sodium benzylpenicillin and streptomycin sulphate. This solution was used at full strength for embryos up to the late blastula stage and at one-tenth concentration for embryos beyond this stage. Anucleolate (O-nu) mutant embryos (Elsdale, Fischberg & Smith, 1958) were obtained from a mating of two heterozygous (\-nu) individuals, and were identified individually by phasecontrast examination of small portions of each embryo (Elsdale, Gurdon & Fischberg, 1960). Developmental stages are those of Nieuwkoop & Faber (1956). B. Labelling of embryos H-thymidine, H-uridine and 3H-guanosine (The Radiochemical Centre, Amersham) were evaporated to dryness and redissolved in modified Barth's saline solution to give a final concentration of 5-10 me/ml. 50-100 m/tl were injected into individual embryos (Gurdon, 1967 a) by a method similar to that used for nuclear transplantation (Elsdale et al. 1960). At the end of the labelling period unhatched embryos were de-jellied and manually removed from their vitelline membranes. After dissection they were stored at — 70 °C until processed. This procedure eliminates any detectable incorporation of labelled precursor by bacteria, as judged by sucrose density-gradient centrifugation (Brown & Littna, 1964). C. Dissection of embryos 3 3 Embryos were dissected in the incubation medium. Tadpoles were anaesthetized with MS 222 (Sandoz, Ltd.). The dissected parts were frozen in vials cooled with solid carbon dioxide. To prevent alteration of the patterns of incorporation as a result of the operation and to avoid degradation of labelled molecules, the parts of each embryo were frozen within 5 min of commencing dissection. Fig. 1 shows diagrammatically what was achieved by the dissections. In all cases some mesoderm cells are likely to have contaminated the preparation of endoderm tissue. At stages 26 and beyond, somatopleure mesoderm and neural crest pigment cells were removed, but at least part of the splanchnopleure remained with the ' endoderm' portion. At stage 39 the liver, pancreas and gall bladder rudiments were removed from the tubular part of the gut. The embryos were thus separated into two parts for subsequent analysis. One comprised the endoderm and a small but variable amount of mesoderm; Embryonic nucleic acid synthesis 365 the other included the remaining parts of the embryo. For the sake of brevity, these will now be referred to as the 'endoderm' and the 'rest of the embryo' respectively. Late blastula, stage 9 Gastrula, stage 12 Neurula, stage 18 Tadpole, stage 39 Fig. 1. Diagrams showing the parts of an embryo separated by dissection. The stippled regions, which include endoderm and some mesoderm, were frozen separately from the rest of each embryo and are described as endoderm. D. Nucleic acid extraction and fractionation The embryos were thawed and homogenized in 0-2M-NaCl, 0 0 5 M tris-HCl (pH 7-5) at 0 °C. Sodium dodecyl sulphate was added to give a concentration of 0-5% and the homogenate shaken. It was then extracted twice with watersaturated redistilled phenol at 4 °C and the aqueous phase was dialysed against 0-2M-NaCl, 0 0 5 M tris-HCl (pH 7-5) for 3 h. The samples were stored at - 70 °C. This procedure gives a good recovery of RNA and a consistent recovery of DNA at each stage (Table 1). Methylated serum albumen-Kieselguhr (MAK) columns were used to fractionate the nucleic acids, using Brown & Caston's (1962) modification of the procedure of Mandell & Hershey (1960). Nucleic acids were eluted from the column with a linear gradient of NaCl concentration buffered to pH 7-5 with 0-05M tris-HCl, followed by elution with hot l-5M-NaCl to release any radioactive material still adsorbed. Since different preparations of MAK released the nucleic acids at varying NaCl molarities, the exact range of salt gradient for 24-3 1 + 2-nu 0-nu 2-nu 2-nu 1 + 2-nu 0-nu 2-nu 2-nu 1 + 2-nu 0+l+2-nu 3 Rest Endoderm Whole Whole Whole Rest Endoderm Rest Endoderm Rest Endoderm Rest Endoderm Rest Endoderm Whole Whole Rest Endoderm Region of embryo H-thymidine enters only DNA, but only 80-90% of H-guanosine or 3H-uridine enters RNA and the rest goes into DNA. The 3 H-thymidine experiments therefore show the DNA recovery. — in- 3 Hatching tadpole, stage 33 Swimming tadpole, stages 39-40 Tail-bud, stage 28 Late gastrula, stages 12-13 0+l+2-nu Early gastrula, stages 10-11 0+l+2-nu Genotype Stage of development H-G, 54 h H-T, 6 h H-G, 4 h H-T, 4 h H-T, 9 h H-U, lOh H-U, 8 h H-T, 6 h — — 87 80 44 39 43 51 60 90 74 — — — — 90 90 87 86 91 92 67 55 30 34 34 108 90 80 85 26 33 76 68 64 66 73 80 91 81 58 53 30 31 32 76 74 60 52 25 30 74 61 63 64 69 70 dicates value not recorded. In this and subsequent tables T = thymidine, G = guanosine, U = uridine. 3 3 3 3 H-U, 10 h H-U, 5 h 3 3 3 3 3 3 Precursor and labelling duration Recovery after Recovery after Recovery after MAK 2nd phenol dialysis and chromatography extraction (%) freezing (%) Table 1. Recovery during extraction and fractionation of nucleic acids d O C; i—\ O CO o o r i-l-l OS OS AND Embryonic nucleic acid synthesis 367 elution was chosen according to the preparation used. Approximately sixty fractions were collected in a total volume of 250 ml. To each was added one drop of carrier yeast RNA solution (1 mg/ml) and trichloroacetic acid to give a 5 % solution. The precipitate was caught on Millipore filters, washed with 5 % trichloroacetic acid, and the radioactivity assayed in a liquid scintillation counter at an efficiency of 15-20% for tritium. _ \ Whole blastula 2000 - 1000 - "..., J v I A 4000 3000 2000 - Whole tadpole \ 'V ", J \ 1 1 ^k"# 1 20 10 1 30 Tube no. 3 Fig. 2. MAK profile of H-TdR-labelled nucleic acids. The peak of radioactivity is DNA. Upper figure, embryos labelled for 2 h from stages 6-8; lower figures, embryos labelled for 8 h from stages 35-39. Nucleic acids were eluted from the columns in three regions (Figs. 2-8). On the basis of our own results described below and on those of others using a variety of different organisms, we have identified these regions as follows: (i) If embryos are labelled with 3H-uridine or 14C-leucine, the 14C-radioactivity coincides with the first peak of 3H-radioactivity (Belitsina, Aytkhozhin, Gavrilova & Spirin, 1964). It may be concluded that this peak is at least partly composed of transfer RNA. -CCA end-group turn over is unlikely to have 368 H. R. WOODLAND & J. B. GURDON contributed significantly to the labelling of this peak since after incubation with 3H-uridine for 1 h only 3-5 % of tRNA counts are present in 3H-cytidine (Bachvarova, Davidson, Allfrey & Mirsky, 1966). Less than 10% of injected 3 H-guanosine is converted to 3H-adenosine during 3 h in Xenopus embryos (J. B. Gurdon, unpublished). 5S RNA is normally eluted from MAK columns in about the same region as tRNA, but is unlikely to have contributed significantly to our results, since this class of RNA appears not to be synthesized until after the neurula stage (Brown & Littna, 1966). (ii) The second peak may be identified as DNA since it is the only one labelled with 3H-thymidine in both early and late stages (Fig. 2). 3H-thymidine is known to be incorporated into DNA alone in this material (Tencer, 1961). Further, Gurdon (1967 a) has shown that this is the only RNase-resistant peak obtained from Xenopus laevis embryos labelled with 3H-guanosine. (iii) The third peak contains 28 s, 18 s and precursor ribosomal RNA (Otaka, Mitsui & Osawa, 1962). This peak is very much smaller in O-nu embryos (Figs. 6 c, d, 1b, 86), which are known from extensive evidence of various kinds to be entirely deficient in rRNA synthesis (Brown & Gurdon, 1964, 1966). However it can be seen that some RNA from O-nu embryos is eluted at high salt molarities in the region where rRNA is normally expected. Part of the peak from wild-type embryos must therefore be non-ribosomal. E. Computation of relative rates of nucleic acid synthesis The incorporation of precursor into each of the three classes of nucleic acids studied has been calculated from the MAK separations. It is assumed that sRNA and DNA counts are superimposed on a base of heterogeneously eluting RNA. A line is drawn between the points where each peak begins to spread at its base (Fig. 5), and the counts above this are summed to give a value for radioactivity in each class of nucleic acid (Gurdon, 1967a). In the rRNA region all the counts between the boundaries of the peak are summed in both the wild type and the O-nu embryos, as indicated in Fig. 5. We have regarded the difference between O-nu and 1- or 2-nu embryos in respect of radioactivity in the third peak as a measure of rRNA synthesis. The calculation was conducted in the following way. The O-nu counts per embryo are corrected to correspond to those in the wild type on the assumption that both synthesize similar amounts of sRNA. The revised values for O-nu counts in the rRNA region are then subtracted from those of the wild type to give a value for true rRNA synthesis. This correction is based on Brown & Gurdon's (1964, 1966) finding that only rRNA synthesis is deficient during early development of O-nu embryos. In this article we have also considered all labelled RNA which is not sRNA or true rRNA under a single heading. This category we have termed ' heterogeneous' RNA. Presumably it includes the messenger RNA (m-RNA) synthesized by the embryo. Embryonic nucleic acid synthesis 369 F. Determination of relative rates of synthesis Determination of absolute rates of synthesis using incorporation of labelled precursor demands a knowledge of the specific activity of the immediate precursor pool. We have not been able to estimate this in parts of embryos because it would be hard to obtain sufficient material, and because the specific activity of the pool changes rapidly during short periods of labelling. We have therefore expressed sRNA and rRNA incorporation as a ratio to DNA incorporation. Almost all our comparisons are between the endoderm region and the rest of the same embryos. If these are to represent real ratios of synthesis, three conditions must be fulfilled: (i) At each stage RNA and DNA must be extracted with the same efficiency from both parts of the embryo. Evidence that this was so at each developmental stage is presented in Table 1. It can be seen that RNA and DNA are each extracted with similar efficiency from both parts of the embryos for any given developmental stage. DNA recovery during extraction is always lower than that of RNA, but is similar in the two regions of the same embryos at any one stage. Since we are concerned with comparisons at one stage we have not corrected the cpm to allow for incomplete extraction. It is interesting that loss of DNA at the phenol interphase increases as development proceeds, possibly because of a change in its association with protein. (ii) The sizes of the relevant precursor pools and their rates of interconversion must be similar in both regions of embryos at the same developmental stage. There is no direct evidence for this assumption but the following facts provide indirect support. First, the major conclusions drawn below are supported by experiments using, either the pyrimidine, 3H-uridine, or the purine, ^-guanosine, as precursors. These substances have quite distinct synthetic pathways and, in Rana pipiens at least, their ribonucleotide derivatives have different pool sizes (Warner & Finamore, 1962), yet similar synthetic differences between the endoderm and the rest of the embryo are obtained whichever kind of precursor is used, and whatever the stage. As one might expect, however, the precise ratio of incorporation into DNA compared to RNA differs according to the kind of precursor used. Secondly, we find that the amount of radioactivity in extracted DNA closely reflects the actual rate of its synthesis as judged by increase in cell numbers. This point is demonstrated in Table 2. It strongly suggests that in early stages the DNA precursor pool entered by 3H-guanosine, presumably dGTP, has the same specific activity in all parts of the embryo. This in turn suggests that the RNA precursor pool (GTP) has a similar specific activity in both regions of the embryo. (iii) The supply of labelled precursor must not be used up by incorporation into one kind of nucleic acid much more quickly in one part of an embryo than in another. Thus, a very high rate of DNA synthesis in the rest of the embryo might rapidly reduce the specific activity of the RNA precursors, resulting in a 370 H. R. WOODLAND & J. B. GURDON false comparison of RNA synthesis in this region compared with the endoderm. This effect will be minimal if the acid-soluble pool is not greatly depleted of labelled precursor during the incubation period. In our experiments we have found that never more than half of the injected precursor becomes acid insoluble. At early stages as much as 90 % may remain acid soluble. These arguments lend some validity to the calculation of relative rates of synthesis, but our calculations must be regarded only as approximations to the actual rates. Therefore such results are meaningful only when they indicate a great difference in the rate of synthesis of two classes of nucleic acid, particularly at any one developmental stage. It is intended that the results presented should be taken only as general guides to alterations in synthetic patterns. However, they clearly show the stage at which synthesis of a particular type of nucleic acid begins. G. Estimation of cell numbers in different parts of embryos The dispersal of embryos in orcein to yield free nuclei (the method of Sze, 1953, adapted by Deuchar, 1958) was considered unsatisfactory for rapid counting, since free nuclei appeared superficially similar to some of the undissolved yolk platelets or to cell debris. The following procedure was therefore devised. The endoderm was dissected away from the rest of the embryo in Table 2. Incorporation of3H-guanosine into DNA compared to increase in cell numbers Part of embryo and stages passed during labelling Rest, stages 10-12 Endoderm, stages 10-12 Rest, stages 12|-18 Endoderm, stages 12^-18 Increase in cell no. Cell increase in part as % of increase in whole DNA cpm DNA cpm in part as % of DNA cpm in whole 14100 97 32350 97 400 3 920 3 36000 96 54500 97 1400 4 1720 3 Cell numbers were calculated from the data in Table 3. modified Barth medium, and incubated for 20-30 min in Barth solution in which Ca and Mg were replaced by Versene. The fully dissociated cells were then gently dispersed in 50 ji\ of the same medium and counted on a haemocytometer. The method proved entirely satisfactory for early stages, since the recovery of cells appeared to be virtually complete, as judged from the facts that very few were observed to be broken, and that the number of cells recorded by Embryonic nucleic acid synthesis 371 this method agrees closely with the expected number calculated from the DNA content of the embryos (see Table 3). At late stages it proved difficult to disaggregate the rest of the embryo effectively. The number of cells in the endoderm region was therefore determined as above and then subtracted from the number of cells calculated, from Dawid's (1965) estimation of DNA content, to be present in the whole embryo. RESULTS A. DNA content We relate our results on RNA and DNA synthesis to nuclear DNA content, in order to provide a measure of the rate of gene activity. In view of the limited amount of dissected endoderm material available, we have estimated nuclear DNA content from cell numbers rather than by chemical analysis. We assume that the average DNA content of each nucleus is the same in both parts of an embryo at a given developmental stage. The methods we have used to determine cell numbers are described, and their validity discussed above. The results Table 3. Cell numbers and DNA synthesis in parts of embryos Stage of development Part of embryo Early gastrula, stage 10 Late gastrula, stage 12 Late neurula, stage 19 Hatching tadpole, stage 32 Swimming tadpole, stage 39 Rest of embryo Endoderm Whole embryo Rest of embryo Endoderm Whole embryo Rest of embryo Endoderm Whole embryo Rest of embryo Endoderm Whole embryo Rest of embryo Endoderm Whole embryo Number of cells 22400 7600 30000 — — — 80000 9700 89700 182800 17200 200000 401000 27000 428000 DNA cpm in Cells in part of part as % of embryo as % DNA cpm in of total cells whole 75 25 — — — — 89 11 — 91 9 — 94 6 — • 89 11 — 97 3 — 96 4 — 97 3 — 87 13 — The values for cell numbers were obtained by direct counting, except for the rest of the embryo at stages 32 and 39, the cells of which could not be readily dispersed. These last two figures were calculated by dividing Dawid's (1965) value for DNA content of the embryo by the DNA content of a red blood cell. The same calculation from Dawid's figures would give the total number of cells at stage 10 as 33000, and at stage 19 as 70000. Deuchar (1958) found much higher values, especially at later stages, by counting orcein-stained isolated nuclei. DNA cpm (radioactivity) values were calculated from the total radioactivity in the DNA peak eluted from MAK columns, as described in the Methods section. The labelled precursors were either 3H-guanosine or 3H-uridine. 372 H. R. WOODLAND & J. B. GURDON are given in Table 3, and show that the endodermal region we have used in these experiments constitutes a progressively smaller proportion of the total cells in an embryo as development proceeds. B. DNA Synthesis Three independent kinds of experiment have shown that DNA is synthesized at a much lower rate in the endoderm than in the rest of the embryo. From Table 3 it is clear that cells increase more slowly in the endoderm than they do in the rest of the embryo. The proportion of endoderm cell nuclei labelled by 3 H-thymidine in 1 h is less than half that found in the rest of the embryo during late blastula, gastrula, and neurula stages (observations on unpublished autoradiographs kindly provided by C. F. Graham). Lastly, Table 3 shows that incorporation of 3H-uridine or 3H-guanosine into DNA is less in the endoderm than in the rest of the embryo. 1200 800 1 Blastula, re st IV 400 • t 1 \ m Blastula, endoderm 600 400 200 J L 14 28 42 56 Tube no. Fig. 3. MAK profiles of total nucleic acids extracted from two parts of the same embryos, labelled for 2 h from stage 7£-8| with 3H-guanosine. The main peak of radioactivity is DNA. C. sRNA synthesis sRNA synthesis is known to begin in embryos of X. laevis between stages 8 and 9 (Brown & Littna, 1964; Shiokawa & Yamana, 1965; Bachvarova et al. 1966; Gurdon, 1967 a). It can be seen from Fig. 3 that sRNA synthesis was not detected in either the endoderm or the rest of the embryo during stage 8. During the 2 h period between stages 9£ and 10£ intense sRNA synthesis becomes evident in both regions (Fig. 4). It therefore appears that there is no gross difference in the time at which sRNA synthesis begins in the two parts of an Embryonic nucleic acid synthesis 373 embryo, but our results do not exclude the possibility that there is a difference of the order of one cell division. At all stages of development investigated, we have found that 3H-nucleoside incorporation into sRNA compared to that into DNA is consistently higher in the endoderm than in the rest of the embryo (Figs. 4-8). The ratio of incorporation into sRNA compared to that into DNA is generally 2-4 times as great in the endoderm as in the rest of the embryo (Table 4, column 4). This difference could theoretically be attributed to different precursor pool sizes in the endoderm and in the rest of the embryo, but reasons were given above which indicate that there are no great differences in the specific activity of the RNA and DNA precursor pools following our labelling procedure. We therefore conclude that the ratio of sRNA synthesis to DNA synthesis is truly higher in the endoderm than in the rest of the embryo. sRNA DNA Early gastrula, rest 1 X Fig. 4. MAK profiles of nucleic acids extracted from parts of the same embryos labelled with 3H-uridine for 2 h from stages 9f-10i. This effect can be explained if the endoderm cells are characterized by a higher rate of sRNA synthesis, a lower rate of DNA synthesis, or both. We have shown above that the rate of DNA synthesis is lower in the endoderm, and the following reasoning indicates that sRNA synthesis per cell is higher. To obtain a measure of the rate of sRNA synthesis we have first divided the sRNA radioactivity by the total acid insoluble radioactivity in the same sample. This procedure gives the same general result as dividing by the acid-soluble radioactivity, and its purpose is to compensate for the fact that the amount of 3 H-nucleoside reaching different parts of the embryo may vary. We have then Whole Whole Rest \ Endoderm / Whole Whole Rest Endoderm 2-nu 0-nu \ 1 + 2-nu) 0-nu \ 1 + 2-nu) 1 + 2-nu 1 + 2-nu 0-nu 2-nu 0+1+2-nu 2-nu 2-nu (2) Genotype 3 3 3 3 /140 111-7 /0-98 12-60 /7-70 12-82 /208 1508 /1-48 \3-46 /0-81 11-14 /0-69 \l-04 fO-62 \2-8 /0-68 12-13 U-7 /2-8 (4) sRNA cpm DNA cpm 0 96-3 12-3 22-2 0 12-5 8-4 110 2-31 017 0 0 0-42 008 0 0 0 0 0 0 (5) rRNA cpm DNA cpm 0 8-3 12-5 8-3 0 4-3 40 2-2 1-56 005 0 0 0-62 008 0 0 0 0 0 0 (6) rRNA cpm sRNA cpm 54 140 8-2 10-5 11-9 3-7 9-4 16-2 3-3 5-8 1-7 1-5 11 20 1-7 2-8 1-3 2-9 7-8 10-6 (7) Heterogeneous RNA cpm D N A cpm justified by the finding that l-nu and 2-nu embryos show an identical pattern of RNA synthesis (Brown & Gurdon, 1964; Gurdon, 1967 a). H-U, 7 h H-U, 4 h H-U, 5 h H-U, 10£ h H-G, 6 h H-G, 3± h H-G, 5 h H-G, 4 h H-U, 2 h 3 3 3 3 3 (3) Precursor and duration of labelling Incorporation into true rRNA was calculated from the data provided by the O-nu mutant. In some experiments a mixture of 1 + 2-nu embryos was used to provide wild-type values. This is 39-40 37-38 28-30 28-30 12^-18 13-17 Rest \ Endoderm / Rest \ Endoderm/ Rest \ Endoderm/ Rest \ Endoderm/ Rest \ Endoderm / 10-12 10-13 Rest \ Endoderm / Part of embryo 9*-10± Stages passed during labelling (1) Table 4. Relative incorporation of 3H-nucleosides into sRNA, rRNA, heterogeneous RNA and DNA o 0 C W 9? D bad > • r 0 o o X OJ Embryonic nucleic acid synthesis 375 related this value to the number of cells in each part of the embryo. Since the specific activity of the precursor pools is not grossly different, the final value provides an estimate of the rate of sRNA synthesis per cell, and this is usually 5-10 times greater for the endoderm cells than for the rest of the embryo (Table 5, column 5). We conclude that sRNA synthesis proceeds at a greater rate relative to DNA synthesis in the endoderm, and that each endoderm cell appears to synthesize more sRNA molecules per unit time than do other cells in the embryo. Mid-gastrula, rest 1000 - e ex Tube no. Fig. 5. MAK profiles of nucleic acids extracted from parts of the same embryos labelled with 3H-guanosine for 4 h from stages 10-12. D. rRNA synthesis In whole X. laevis embryos rRNA synthesis is first detected during gastrulation (Brown & Littna, 1964); it increases rapidly during neurula stages and reaches a nearly constant rate relative to other kinds of nucleic acid synthesis just after hatching (Gurdon, 1967a). The amount of incorporation into rRNA has been computed by subtracting the substantial number of non-ribosomal RNA counts found in this region of the elution, by use of O-nu embryos as described above. Figs. 5-8, summarized in Table 4, columns 5 and 6, show that the pattern of rRNA synthesis characteristic of the whole embryo and the rest of the embryo does not also apply to the endoderm. Thus little if any rRNA is synthesized by endoderm neurula cells compared to cells in the rest of the embryo. This is true whether rRNA synthesis is compared to DNA or sRNA synthesis. Although the O-nu mutant provides a unique tool for determining how much RNA extracted from embryos is truly ribosomal, the accuracy of the 376 H. R. WOODLAND & J. B. GURDON Neurula Rest, 0-nu 8000 - I 30 50 Tube no. Fig. 6. MAK profiles of nucleic acids extracted from embryos labelled with 3Hguanosine for 5 h from stages 12^—18. A and B refer to the two parts of one sample of embryos and C and D to parts of another sample of embryos. All figures were obtained from embryos of the same mating. method is not sufficient to be certain whether or not the very low level of incorporation observed in the endoderm really represents rRNA synthesis. These data therefore do not distinguish an absence of rRNA synthesis from its presence at a very low rate. Using the criteria described in the preceding section, rRNA synthesis may be compared to DNA synthesis and cell number. It would appear from Table 5, column 6, that by the tail-bud stage (28-30) each endoderm cell synthesizes much more rRNA on average than other cells. The rate of synthesis per cell is maximal in the rest of the embryo by stage 16. In the endoderm, on the other hand, rRNA synthesis is ahnostundetectable at stage 16. The differences between the two regions are summarized in Fig. 10, which shows the proportion of the total acid-insoluble radioactivity constituted by sRNA or rRNA. 377 Embryonic nucleic acid synthesis E. Synthesis of heterogeneous RNA In analysing the MAK chromatograms we have also obtained data on all labelled RNA which is neither sRNA nor rRNA. This we have called 'heterogeneous ' RNA, for it is eluted throughout the salt gradient, and some of it is released only by hot l-5M-NaCl. Thus its component molecules probably differ Table 5. The distribution of cells and 3H-nucleoside incorporation in parts of embryos 10-12 10-13 13-17 12f-18 28-30 39-40 2-nu 3 2-nu 3 0+1+ 2-nu 3 2-nu 3 H-G, 3± h 1+2-nu 3 H-G, 6 h 1 + 2-nu 5 H-U, 5 h 2-nu 3 H-U, 4 h H-U, 2 h H-G, 4 h H-G, 5h no. no. 22400 7600 32000 7800 36000 7900 64000 8800 66000 8900 173000 16600 401000 27000 © S Heterogem cpm: otalNAcp X rRNA cj otalNAcp X sRNA C] © otal NAcp bel ling recursor, ai time at 21 renotype tages passe:ddu rin, 'abelling Rest Endoderm Rest Endoderm Rest Endoderm Rest Endoderm Rest Endoderm Rest Endoderm Rest Endoderm (7) < )XUI *o Z, 9f-10i (6) XUIC -2 (5) )XUI art of emb ryo 60 (4) fo. cells in part smbryo (3) (2) (1) A H H H 11 36 8-3 54 6-3 44 2-9 38 3-3 28 0 0 0 0 0 0 4-5 1-9 20 2-2 2-7 30 87 23 55 12 61 6-3 63 5-1 54 0-26 19-9 1-5 29-3 10 23 11-3 0-65 9-2 011 2-7 Cell numbers in column 4 were taken directly from Table 3, or calculated from the figures in that table. The ratios expressed in columns 5, 6 and 7 are multiplied by 106 as indicated to give values of a convenient order of magnitude. NA = acid insoluble nucleic acid. considerably in characteristics such as base composition and molecular weight. The synthesis of this class of RNA can be seen clearly at stage 8 (Fig. 3), since neither sRNA nor rRNA are synthesized at this stage. Late-eluting heterogeneous RNA can also be seen in the experiments using the O-nu mutant, which is deficient in rRNA synthesis (Fig 6 c, d, 7b, &b). The data presented in Table 4, column 7, indicate that this type of RNA is synthesized at a greater rate, relative to DNA, in the endoderm than the rest of the embryo. This is not due entirely to the reduced rate of DNA synthesis in the endoderm, since the same conclusion is reached when the rate of heterogeneous RNA synthesis is expressed per cell, and therefore independently of DNA synthesis (Table 5, column 7). 378 H. R. WOODLAND & J. B. GURDON Hatching tadpoles 1600 C Whole, 1+2-nu TVT I M Rest, 1 +2-nu 1000 " ft It 800 500 i 20 30 i 40 6 .8: D f n 800 150 - 100 400 Endoderm, 1+2-nu A IA i i i 20 30 40 Tube no. Fig. 7. MAK profiles of nucleic acids extracted from tadpoles labelled with 3Huridine. A and B, 10 h, stages 28-33; C and D, 5 h, stages 28-31. All figures were obtained from embryos of the same mating. F. Appearance of nucleoli The presence or absence of definitive nucleoli corresponds remarkably closely to the synthesis or lack of synthesis of rRNA in embryonic cells of X. laevis and other vertebrates (Brown & Gurdon, 1964; Brown, 1965; Gurdon & Brown, 1965). In view of the finding that endoderm cells show a delayed onset of rRNA synthesis compared to cells elsewhere in the embryo, it would be expected that nucleoli should appear in the ectoderm and mesoderm cells much earlier than in endoderm cells. We have looked for definitive nucleoli in embryos fixed in Bouin's fluid and stained with Mayer's haemalum. Observation with the light microscope fails to reveal definitive nucleoli in any cleavage cells, and only very rarely are they observed in gastrula cells. However at stage 20 (late neurula) at least 80 % of cells in the rest of the embryo contain nucleoli, whereas less than 20 % of endoderm cells in the same embryo did so. These observations agree with the results of an electron-microscope study of nucleoli in X. laevis embryos (Hay & Gurdon, 1967). The conclusions on rRNA synthesis derived Embryonic nucleic acid synthesis 379 from chemical data are therefore satisfactorily confirmed by cytological observations. Swimming tadpoles c Rest, 2-nu - 4000 • 400 - 2000 20 30 40 20 B 2000 - 1000 - D 30 Endoderm, Whole, 0-nu 200 L 100 - * 20 30 40 i i 20 30 40 k \ 40 Tube no. Fig. 8. MAK profiles of nucleic acids extracted from tadpoles labelled with 3Huridine. A and B, embryos of the same mating labelled for 4 h from stages 37-39. C and D, parts of the same embryos labelled for 7 h from stages 39-40. DISCUSSION (i) Conclusions and their relationship to previous work We have pointed out in the Methods section the difficulties which arise if conclusions are to be drawn about rates of synthesis after short labelling periods. These difficulties do not exist if we are concerned with determining the stage at which a certain class of nucleic acid starts to be synthesized in different parts of the embryo. Thus our conclusions are of two kinds. The first kind concerns the stage of initiation of nucleic acid synthesis, and is not affected by uncertainties regarding pool specific activities. The second kind of conclusion relates to rates of synthesis, and their security is limited by the problems of dealing with very small amounts of material, in which the pool specific activity changes considerably during the labelling period. Such conclusions are therefore tentative to the extent that we have indicated in the Methods section. The incorporation of nucleosides into DNA and cell counts have both shown that the rate of DNA synthesis is lower in the endoderm than in the rest of the embryo, and that this difference becomes most marked between stages 9 and 18. This conclusion is consistent with the finding of Graham & Morgan (1966) that 25 JEEM 19 380 H. R. WOODLAND & J. B. GURDON cells in one region of the endoderm show a marked increase in the length of the whole cell cycle between stages 8 and 10. Previous work has shown that sRNA synthesis, as opposed to terminal -CCA exchange, can first be detected in X. laevis between stages 8|- and 9\ sRNA 10 20 30 Developmental stage Fig. 9. Summary of changing rates of RNA synthesis per cell during development. Upper figure, sRNA; lower figure, heterogeneous RNA. (Brown & Littna, 1964; Shiokawa & Yamana, 1965; Bachvarova et al. 1966; Gurdon, 1967 a). The experiments reported here have shown that sRNA synthesis is first detected in both endoderm and the rest of the embryo between stages 8^ and 9%, but our labelling periods were not sufficiently short to detect a difference in the time of initiation of sRNA synthesis of the order of 1 h, or about 1 cell division. Although the accumulation, and probably the cell content, of sRNA bears a constant relationship to that of DNA after the end of cleavage (Brown & Littna, 1966), Gurdon (1967 a), using methods similar to those employed here, found that sRNA was not always synthesized at the same rate as DNA. This conclusion is reinforced by the results reported here, which indicate (i) that the relative rate of sRNA and DNA synthesis is quite different in the two regions of embryos at all stages studied, and (ii) that endoderm cells show a higher rate of sRNA synthesis as well as a lower rate of DNA synthesis per cell than other cells. These points are summarized in Fig. 9 a. When allowance is made for the fact that 3H-uridine and 3H-guanosine do not label DNA equally effectively, the results reported here are not inconsistent with the progressive Embryonic nucleic acid synthesis 381 decline after gastrulation in the relative rate of sRNA synthesis compared to DNA synthesis previously reported for whole embryos (Gurdon, 1967 a). rRNA synthesis is first detected in whole X. laevis embryos at the gastrula stage (Brown & Littna, 1964; Shiokawa & Yamana, 1965; Gurdon, 1967 a). Our results, summarized in Fig. 10, have shown that this is not true of endoderm cells, which commence rRNA synthesis, and show an acceleration in its rate of Developmental stage Fig. 10. Comparison of the stage of initiation, and changing rates, of synthesis of sRNA and rRNA in two regions of developing embryos. The graphs show what proportion of the total nucleic acids synthesized at each stage is sRNA or rRNA. They therefore show relative rates of synthesis and not the rate of synthesis per cell. synthesis, several hours later than do cells in the rest of the embryo. This result was confirmed cytologically by the time of appearance of nucleoli. The rate of rRNA synthesis per cell appears to increase relative to DNA synthesis throughout the period of development studied, in marked contrast to that of sRNA. This result is consistent with Brown's (1965) finding that the long-term accumulation of 32PO4-labelled rRNA rises in relation to that of DNA between stages 10 and 25. The only previous experiment on rRNA synthesis in parts of X. laevis embryos is that of Gurdon & Brown (1965). They found a similar specific activity of rRNA in the gut and in the rest of stage 41 X. laevis tadpoles after a sucrose gradient analysis of 14CO2-labelled RNA. The actual rate of rRNA synthesis in the two parts of the embryo was not revealed by these experiments. The results reported here, as well as previous work, strongly indicate that rRNA synthesis is regulated independently of DNA and sRNA synthesis. We have concluded that endoderm cells are more active in accumulating 'heterogeneous RNA' than are cells in the rest of the embryo. This class of RNA is defined here as that which is not included in the elution peaks of sRNA or rRNA, and is likely to include mRNA. It has indeed been demonstrated that 25-2 382 H. R. WOODLAND & J. B. GURDON fish (Belitsina et al. 1964) and sea-urchin embryos (Comb, Katz, Branda & Pinzino, 1965) synthesize molecules which elute in the rRNA region of MAK gradients and which have properties associated with mRNA. Our conclusions on heterogeneous RNA synthesis are summarized in Text-Fig. 9b. Since our experiments are not designed to distinguish different kinds of mRNA molecules, we cannot conclude in what ways mRNA synthesis varies during development. Experiments intended to provide such information have been carried out on Amphibia by Waddington & Perkowska (1965) and Flickinger, Greene, Kohl & Miyagi (1966), and on chick embryos by C. H. Waddington, E. Perkowska & K. Takata (personal communication). Mechanism of regulation of nucleic acid synthesis Our results are consistent with the view that some kind of feedback control may be important in respect of sRNA and rRNA synthesis. It has been suggested before (Brown, 1965; Gurdon, 1967 a) that the absence of sRNA and rRNA synthesis in the early development of Amphibia and other animals may be connected with the large store of these classes of molecules in egg cytoplasm. Thus the unfertilized egg of X. laevis contains 40 m/ig of sRNA and 4/*g of ribosomes (Brown, 1965). The synthesis of sRNA and rRNA might therefore be repressed until the content of them or other cell components containing them becomes limiting. This is likely to happen when the translation of new mRNA places a demand on the supply of these molecules. Bachvarova et al. (1966) have shown that there is a pronounced increase in heterogeneous RNA synthesis at stage 8, apparently in all regions of the embryo. Since these results and ours both show the initiation of sRNA synthesis at this stage, they are consistent with the hypothesis that this represents a response to a rise in cytoplasmic mRNA concentration. The fact that rRNA synthesis starts much later in development than sRNA synthesis in both regions of the embryo studied may be connected with the much greater store of ribosomes than of sRNA in the unfertilized egg. SUMMARY 1. The injection of tritiated nucleosides followed by MAK column chromatography was used to determine the relative rates of synthesis of sRNA, rRNA and DNA in the endoderm and the rest of developing embryos of X. laevis. O-nu embryos were used to determine the amount of non-ribosomal RNA in what is usually regarded as the rRNA elution peak. 2. Incorporation of nucleosides into DNA and counts of cells showed that the rate of DNA synthesis is much lower in the endoderm than in the rest of the embryo and that this difference becomes most pronounced during gastrula and neurula stages. 3. sRNA synthesis was first detected between stages 8£ and 9\ in endoderm cells as well as in the rest of the embryo. sRNA synthesis, expressed per cell or Embryonic nucleic acid synthesis 383 per DNA synthesis, always appeared greater in the endoderm than in the rest of the embryo. 4. rRNA synthesis was detected in the rest of the embryo several hours earlier than in the endoderm, a finding which was correlated with the later appearance of nucleoli in the latter region (as judged by light microscopy). After the neurula stage rRNA appears to be synthesized at a greater rate per cell and per DNA synthesis in the endoderm than in the rest of the embryo. 5. 'Heterogeneous' RNA, defined as that RNA which is neither sRNA nor rRNA, is synthesized rapidly from stage 7 onwards. It appears to accumulate at a greater rate in the endoderm than in the rest of the embryo, both per cell and per DNA synthesis. 6. These results show that the synthesis of sRNA, rRNA and DNA is regulated independently at different stages of development and in different regions of an embryo. RESUME Taux de synthese relatifs de I'ADN, de VARNs et de VARNr dans la zone endodermique et dans d'autres regions d'embryons de Xenopus laevis. 1. L'injection de nucleosides trities, suivie de la chromatographie sur colonne MAK, a ete utilisee pour determiner les taux relatifs de synthese de l'ARNs, de 1'ARNr et de I'ADN dans l'endoderme et les autres zones d'embryons de Xenope en cours de developpement. On a utilise des embryons anucleoles (O-nu) pour determiner la quantite d'ARN non-ribosomique dans ce qu'on considere habituellement comme le pic d'elution de l'ARNr. 2. L'incorporation de nucleosides dans I'ADN et les numerations de cellules ont montre que le taux de synthese de I'ADN est beaucoup plus faible dans l'endoderme que dans le reste de l'embryon et que cette difference devient le plus accentuee au cours des stades gastrula et neurula. 3. La synthese d'ARN soluble a ete decelee en premier lieu entre les stades 8£ et 9£ dans les cellules endodermiques comme dans le reste de l'embryon. La synthese d'ARN soluble, exprimee par cellule ou en fonction de I'ADN synthetise, est toujours apparue plus elevee dans l'endoderme que dans le reste de l'embryon. 4. La synthese d'ARNr a ete decelee dans le reste de l'embryon plusieurs heures plus tot que dans l'endoderme, resultat qui etait en correlation avec l'apparence plus tardive des nucleoles dans cette derniere region (appreciee en microscopie photonique). Apres le stade neurula, l'ARNr parait etre synthetise a un taux plus eleve, par cellule et par rapport a la synthese d'ADN, dans l'endoderme que dans le reste de l'embryon. 5. De l'ARN 'heterogene', defini comme l'ARN qui n'est ni soluble, ni ribosomique, est rapidement synthetise a partir du stade 7. II parait s'accumuler a un taux plus eleve dans l'endoderme que dans le reste de l'embryon, a la fois par cellule et par rapport a la synthese d'ADN. 384 H. R. WOODLAND & J. B. GURDON 6. Ces resultats montrent que la synthese d'ARNs, d'ARNr et d'ADN est regulee de maniere independante aux divers stades du developpement et dans les differentes parties de l'embryon. The authors are indebted to Dr C. F. 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