Cytoplasmic regulation of RNA synthesis and nucleolus formation in developing embryos of Xenopus laevis

J . M ol. B iol. (1965) 12, 27-35

Cytoplasmic Regulation of RNA Synthesis and Nucleolus Formation in Developing Embryos of Xenopus laevis J. B.

GURDON

Department of Zoology, Parks Road, Oxford, England AND

DONALD D. BROWN

Department of Embryology, Carnegie Institution of Washington Baltimore, Maryland, U.S.A. (Received 21 November 1964) Pronounced cytological changes, as well as changes in t he kinds of RNA synthesized, follow the tranaplantation of nuclei from embryon ic or larval cells of X enopus laevis t o enuc lea t ed unfertilized eggs. N uclei wer e transplanted into en ucleate d eggs which cont a ine d [32P]phosphate, and the resulting nuclear transplant -embryos showed a pattern of RNA synt hesis which was indistinguishable fr om t hat of normal em bryo s dev eloping from fertilized eggs . Although ribosomal RNA is a ctively sy nthesized by neurula endo derm cells and by swimming t adpo le gut cells, no synthesis of ribosomal RNA wa s detected during the cleavage promoted by nuclei transplanted from these kinds of cells. Howeve r, synthesis of ribosomal RNA recommen ces during gastrulation in nuclear-t rans p lan t embryos in t he same way as in em bryos from fertilized eggs . DNA·like RNA and soluble R NA are synthesized during late cleavage by nuclear-transplant em b ryos as well as b y embryos derived from fertilized eggs . Forty minutes after t ra nsplan t ation , nuclei have undergon e a 30·fold in cr eas e in volume, reaching the same size as zygote nuclei during t his period, and their definitive nucleoli d isappear. At gastrulation, definitive nucleoli reappear in the nuclear-transplant embryo s. These results show that the synthesis of RNA is regulated by the type of cy to plas m in which a nucleus lies.

1. Introduction When a cell nucleus is transplanted from an embryo or tadpole into an enucleated unfertilized egg, the transplanted nucleus substitutes functionally for the zygote nucleus and promotes normal embryonic development in 30 to 40 % of cases using blastula or gastrula endoderm nuclei (Gurdon, 1960c). In this study, we have asked whether the functional changes which such a transplanted nucleus undergoes ar e accompanied by a modification of the pattern of RNA synt hesis which the transplanted nucleus and its desc endants support. An analysis of this kind is possible in X enopus laevis, since the quantity and the types of RNA that are sy nthesized vary greatly according to the developmental st age (Brown & Littna, 1964a). The synthesis of D.RNAt and S·RNA is first detected in late cleavage and proceeds at a rapid rate during gastrulation and neurulation (Brown, 1964). In contrast, R·RNA is not synthe-

t Abbreviations used: D·RNA, DNA-like RNA; R.RNA, ribosomal RNA; S-RNA, soluble RNA. 27

28

J. B. GURDON AND D. D. BROWN

sized during cleavage; it begins to be synthesized at the beginning of gastrulation and is synthesized faster in later development (Brown & Littna, 1964b). We have therefore been able to transplant a nucleus from a tissue which is actively synthesizing ribosomal RNA, and to determine whether the resulting nuclear-transplant embryo retains the pattern of RNA synthesis characteristic of the more differentiated donor tissue or whether the synthetic pattern reverts to that of a normal embryo derived from a fertilized egg. We also describe some pronounced cytological changes which take place in nuclei transplanted to unfertilized eggs. These changes are reversible and are correlated with the changes in the pattern of RNA synthesis.

2. Materials and Methods Xenopus laevis embryos and tadpoles were used for these experiments. Ovulation and mating were induced by the injection of 500 international units of chorionic or serum gonadotrophic hormone (Organon Laboratories) into the dorsal lymph sac of females, and of 150 units into males. Embryos were reared at 18 to 20°C in 1/10 Niu & Twitty saline solution (Flickinger, 1949) to which had been added 0·01 mg/ml, of both streptomycin sulphate and of sodium benzylpenicillin. The addition of these antibiotics suppresses bacterial growth on the jelly surrounding the eggs (Brown & Littna, 1964a). When the embryos had reached the required stage they were removed from their jelly and vitelline membrane with forceps, washed in distilled water, and kept at or below - 20°C until chemically analysed. Stages in development are described by numbers according to the normal table of Nieuwkoop & Faber (1956). Nuclear transplantation was carried out as described by Gurdon (1960b) and by Elsdale, Gurdon & Fischberg (1960). Donor cells are dissociated by leaving a piece of tissue for 10 to 20 min in full-strength saline solution from which Ca 2+ and Mg2+ have been omitted and 0·1 mM-EDTA added. A dissociated cell is sucked into a pipette in order to break the cell wall without removing the cytoplasm from around the nucleus. It is then injected into a newly laid, unfertilized, egg which has been irradiated in order to inactivate the egg nucleus. Incorporation of [32 P]orthophosphate and 1100 2 As described by Brown & Littna (1964a), 1 to 2 me of [32P]orthophosphate (Radiochemical Centre, Amersham, Bucks) was injected into a female frog intraperitoneally about 8 hr before eggs were laid, a method developed by Kutsky (1950). 14C0 2 was incorporated by incubating embryos in a small closed bottle with CO2-free air and a small volume of buffered salt solution to which had been added 30 ,..e of Na214COa (Cohen, 1954; Brown & Caston, 1962). Extraction and characterization of RNA The methods for RNA extraction, and for sucrose density-gradient centrifugation and analysis, have been described in detail (Brown & Littna, 1964a). The fractions from sucrose gradients were collected on Millipore filters and counted. To determine the base composition of 32P-labelled RNA, the filters were eluted with 0-1 M-NH 40H and the eluate hydrolysed in 0·3 N-KOH for 18 hr at 37°C. The solutions were acidified with N-HCI at O°C, centrifuged, and the supernatant solutions adsorbed on 25 mg of acid-washed Norit. Norit was washed twice with distilled water and then eluted twice with 50% ethanol containing 3% NH 40H. Hydrolysed yeast RNA was added as carrier and the solutions evaporated to dryness. The [32P]nucleotides were dissolved in a small volume of 0·1 M-NH 40H and separated by descending chromatography according to the method of Lane (1963). The nucleotides were detected with an ultraviolet lamp, cut out and counted directly in a liquidscintillation spectrometer.

3. Results (a) RNA synthesis in nuclear-transplant embryos

We have determined the amount of RNA synthesized by embryos which were derived from transplanted nuclei of embryonic as well as differentiated cells. In the

CYTOPLASMIC REGULATION OF RNA SYNTHESIS

29

first experiment, donor nuclei were taken from the endoderm cells of an embryo at the neural folds stage (stage 18); at this stage, R-RNA is being synthesized (Figs l(d) and 2(b); Brown & Littna, 1964a). The development promoted by nuclei from stage 18 embryos is shown in Table 1 (experiment A). As expected from previous experience (Gurdon, 1960c), 43% of these nuclei promoted regular cleavage of the recipient eggs and more than 40% of the complete blastulae developed into swimming tadpoles. The failure of more than half the transplanted nuclei to promote normal cleavage and development is believed to be due to a number of factors which include the poor quality of some of the eggs laid in the laboratory, damage sustained during transplantation, and the incapacity of nuclei taken at certain stages of the mitotic cycle to divide normally after transplantation (Gurdon, 1962) There is no reason to doubt that the nuclei which promote normal cleavage after transfer are a random sample of those present in the donor tissue. The number of swimming tadpoles obtained from transplanted nuclei is therefore expressed in Table 1 as a percentage of complete blastulae. TABLE

1

Development of embryos obtained from the transplantation of nuclei to enucleated unfertilized eggs As percen.

Experiment Stage of donor nuclei

Developing embryos as percentage of total transfers

tage of complete blastulae Total Complete Early Tailbud Swimming Swimming no. of blastulae neurulae embryos tadpoles tadpoles transfers (st. S) (st. 14) (st. 26) (st. 40) (st. 40)

A

Neural fold endoderm (st. IS)

S76

43

B

Swimming tadpole mid-gut (st. 41)

351

32

C

Serial transfers from a blastula obtained from experiment B

321

57

36

26

17

41 [15]t

53

[6o-S0]t

All percentages have been calculated after allowing for the removal of embryos for chemical analysis. Experiments A, B, C, were done on eggs laid by different frogs. Fertilized eggs from each female were kept for comparison with transplant-embryos from eggs of the same female. t The numbers in square brackets show what proportion of nuclear-transplant embryos from the donor stage used would have been expected (from Gurdon, 1960c) to have reached the swimming tadpole stage, if they had not been used for chemical analysis.

Nuclei from an unlabelled host embryo (stage 18) were transplanted into enucleated eggs containing [32PJorthophosphate. When the resulting nuclear-transplant embryos had become late blastulae (stage 8), 50 were taken for chemical analysis (Fig. l(a)). Some ofthe eggs laid just after those used as the recipients oftransplanted nuclei were fertilized and allowed to develop to stage 8 (about seven hours). At that stage 50 of them were collected and analysed as a control (Fig. l(b)). Figure l(a) and (b) shows that little or no R-RNA was synthesized during the cleavage (up to stage 8) of embryos resulting from fertilized eggs or from eggs injected with nuclei derived from endoderm cells of embryos at the neural folds stage. Thus the nuclei which were

s,

30

B. GURDON AND D. D. BROWN

actively synthesizing R-RNA in the neurula gave rise after transplantation to daughter nuclei which did not synthesize R-RNA during cleavage, but which did synthesize heterogeneous RNA like normal embryos in late cleavage. In this experiment there was not enough [32P]RNA for base analysis of this heterogeneous high molecular weight RNA. However, many other experiments show clearly that this high molecular Transplant

Control Stage 8

(b)

tal ~

s0'"

e

~

Control

Transplant

0

u

Stage 18

1·0

45 53 r---. r - - - l 200

100

Tube no.

(d)

(c)

FIG. 1. Sedimentation patterns of total RNA purified from 32P-Iabelled embryos reared from fertilized eggs, and from nuclear-transplant embryos from stage 18 (neural fold) endoderm nuclei. All embryos were derived from 32P-Iabelled eggs laid by the same female. (a) 50 blastula (stage 8) transplant-embryos. (b) 50 stage 8 embryos reared from fertilized eggs. (c) 26 neural fold (stage 18) transplant.embryos. (d) 26 stage 18 embryos reared from fertilized eggs. In eaah experiment 250 /Lg of non-radioactive RNA isolated from X. laeuis oocytes was added as carrier at the beginning of the purification procedure. The numbers at the top of (c) and (d) show the percentage of guanylic + cytidylic acid of the radioactive RNA which sedimented in the regions indicated by the brackets. Optical density, - 0 - -0-; radioactivity,

-e--e-.

+

weight RNA formed during late cleavage has the low guanylic cytidylic acid content characteristic of D-RNA (see Figs 4o(a) and 5(a». When total RNA is isolated from whole [32P]embryos and subjected to sucrose density-gradient centrifugation, there is a large amount of radioactive material which remains at the top of the gradient (see Figs I, 4 and 5 in Brown & Littna, 1964a). Depending upon the stage, this material consists largely of partially degraded DNA and unknown acid-insoluble substances. However, 32P-Iabelled 4 s RNA is also present in this fraction and it has been shown (Brown & Littna, 1964b) that there is no degradation of R-RNA by this isolation

CYTOPLASMIC REGULATION OF RNA SYNTHESIS

31

+

technique. This RNA has a guanylic cytidylic acid content of about 60% and is probably exclusively S-RNA. The cessation of R-RNA synthesis by transplanted nuclei might be attributed to nuclear damage during transfer. That this suggestion is not tenable is shown by the fact that nuclear-transplant embryos, derived from stage 18 neurula nuclei and allowed to develop to the neurula stage, synthesized R-RNA again (Fig. l(c» just like control embryos derived from fertilized eggs and reared to the same stage (Fig. l(d». The 28 sand 18 s RNA shown in Fig. l(c) and (d) contain D-RNA as well as R-RNA. Thus at the neural folds stage, newly synthesized high molecular weight RNA has an Transplant

Control Stage 18

58

46

63

r----l

1·0

c

~ .....u'"

0,5-

Tube no.

(a)

(b)

FIG. 2. Sedimentation patterns of RNA isolated from ribosome pellets. (a) 27 stage 18 transplantembryos from stage 18 endoderm nuclei. (b) 27 stage 18 embryos reared from sibling fertilized eggs. 500 ",g of non-radioactive whole RNA from X. laeoie ooeytes was added to each ribosome pellet and purification continued from this point. The numbers in the top of each graph show the percentage of guanylic cytidylic acid in the radioactive RNA covered by the brackets. Optical density, - 0 - - 0 - ; radioactivity,

+

-e--e-.

intermediate base composition (Fig. l(d); Brown & Littna, 1964a), suggesting that it is a mixture of R-RNA and D-RNA. The two classes of molecules can be separated by collecting ribosomes by high-speed centrifugation, and then extracting the RNA from them. If this RNA is subjected to sucrose density-gradient centrifugation, the D-RNA is partially degraded (Brown & Littna, 1964a) and can be found sedimenting at rates ranging from 4 s to 18 s, depending upon the extent of its degradation. Radioactive ribosomal RNA, having a characteristically high guanylic + cytidylic acid content, can be obtained in this way from nuclear-transplant embryos at stage 18 (Fig. 2). The cessation of R-RNA synthesis after nuclear transplantation is observed when nuclei are taken from differentiating cells of tadpoles as well as from early embryonic cells. We have shown this by using donor nuclei from the mid-gut of swimming tadpoles (stage 41). In this experiment the number of embryos that could be obtained for chemical analysis was increased by serial nuclear transplantation. Thus, one of the

32

J. B. GURDON AND D. D. BROWN Gut

Dorsal fragment Stage 41

2000

10,00004

20

c

~

~

o

...,

V>

'0

o'"

U

1000

o

Tube no. (b)

(a)

FIG. 3. Comparison of RNA synthesized during a 6-hr pulse at 23°C with 14C0 2 in the gut (b), and in the rest of the tadpole (a), from 160 tadpoles. Total RNA was extracted within 1 hr after the pulse. The results are plotted on the same relative scale (O.D. versus ctsjrnin) to show that the specific activity of the RNA in the gut is similar to that of the rest of the tadpole. Optical density, - 0 - - 0 - ; radioactivity,

-e--e-.

Control

Transplant

55 ,-------,

40

57

39

0·6 100 04

c

'E

::l..

E 0

<, V>

...,

'0

u

o'"

50

0

02

. ~........-.-.........--'..~.-..........

,.

/

10

10

20

20

Tube no. (a)

(b)

FIG. 4. Sedimentation pattern of total RNA synthesized, (a) by 66 blastula nuclear transplantembryos from stage 41 tadpole gut nuclei, and (b) by 66 blastulae reared from fertilized eggs. The dotted line in (a) shows the 32P-labelled RNA content of 66 sibling unfertilized eggs. The numbers at the top of each graph show the percentage of guanylic cytidylic acid in the bracketed radioactive RNA. The greater number of heterogeneous counts in (a) compared to (b) is probably due to the fact that the transplant embryos in (a) were frozen at stage 9, whereas the controls in (b) were frozen at stage 8. Optical density, - 0 - - 0 - ; radioactivity,

+

-e--e-.

CYTOPLASMIC REGULATION OF RNA SYNTHESIS

33

blastulae obtained from the transplantation of a gut-cell nucleus was itself used to provide nuclei for a serial transfer generation of embryos. The neurulae of experiment C in Table 1 were therefore all derived from one original gut-cell nucleus. Gut-cell nuclei are very active in the synthesis of R-RNA (Fig. 3), yet after transplantation the daughter nuclei derived from them do not synthesize a detectable amount of R·RNA during cleavage (Figs 4 and 5(0.)). This is not a result of nuclear damage, since the serial nuclear-transplant embryos (experiment C of Table 1) begin to syntheBlastulae

40

40

Neurulae 58

r-----1

46

54

r------t r - - 1

48

.----,

Tube no. (a)

FIG. 5. Sedimentation patterns of total RNA synthesized, (a) by 100 blastula serial nuclear transplant embryos from an original stage 41 tadpole gut nucleus, and (b) by 100 stage 14 neurula serial nuclear-transplant embryos also derived from the same stage 41 gut cell nucleus. The dotted line in (a) is radioactive RNA from the same number of sibling unfertilized eggs. The numbers at the top of each graph show the percentage of guanylic + cytidylic acid in the radioactive RNA included within the brackets. Optical density, - 0 - - 0 - ; radioactivity,

-e-e-·

size R-RNA if allowed to develop beyond the blastula stage (Fig. 5(b)). The baseratios of the radioactive RNA substantiate these conclusions. In Figs 4(0.) and (b) and 5(0.), the newly synthesized high molecular weight RNA has a low guanylic + cytidylic acid content and is heterogeneous in its sedimentation properties; it therefore contains no detectable R-RNA. In contrast, at neurula stages as shown in Fig. 5(b), the RNA sedimenting at 28 sand 18 s has a higher guanylic + cytidylic acid content, as expected if it contains some R-RNA as well as D-RNA. As would be predicted from the results reported above, we have found that blastula nuclei which would normally be just about to start synthesizing R-RNA do not do so if they are transplanted to enucleated eggs; R-RNA synthesis then commences only when these transplant-embryos begin gastrulation. These experiments clearly demonstrate that the amounts and types of RNA synthesized by a nucleus are precisely and continually controlled by the kind of cytoplasm in which it lies. (b) Oytological changes induced by nuclear transplantation

Certain striking cytological changes take place in transplanted nuclei very soon after they come into contact with the egg cytoplasm. These changes include a great 3

34

J. B. GURDON AND D. D. BROWN

increase in nuclear volume (Gurdon, 1960a; Subtelny & Bradt, 1963; Gurdon, 1964), and the disappearance of nucleoli. An intestinal epithelium cell nucleus has a volume of about 1601-'-3, but 40 minutes after transplantation it has increased to about 4500 JL3 (Gurdon, 1964), as shown in Plate I, c and d. Blastula nuclei of about 1900 JL3 increase to a similar size 40 minutes after transplantation (Plate I, g and h), and sperm nuclei also show a pronounced increase in volume after penetration. Thus nuclei from quite different kinds of cells swell very considerably after exposure to egg cytoplasm; their daughter nuclei become progressively smaller again as cell size decreases during cleavage and in further development. Another change which occurs after transplantation is the disappearance of nucleoli. In normal development, definitive nucleoli are absent from nuclei during cleavage, but appear at the beginning of gastrulation and are particularly pronounced in growing tissues (Plate I, c, k and 1). During cleavage, nuclei of X. laevis contain multiple small blobs, whereas there is never more than one definitive nucleolus per chromosome set from gastrulation onwards. Intestine nuclei have large nucleoli (Plate I, a and c) which can no longer be seen 40 minutes after transplantation (Plate I, d). This disappearance is not due to the cell disaggregation treatment (see Materials and Methods), since dissociated intestine cells still have definitive nucleoli (Plate I, b). During cleavage the nuclei derived from transplanted intestine nuclei have multiple small blobs typical of this stage of development (Plate I, e), and at gastrulation they again develop definitive nucleoli (Plate I, f). Thus the nucleolar changes in transplanted intestine nuclei correspond exactly to those which take place in normal development and which are exemplified in Plate I, g to j, by transplanted blastula nuclei. They are also correlated in time with changes in R-RNA synthesis.

4. Discussion If RNA is a direct gene product and is synthesized primarily in the cell nucleus, then the changes in the amount of different classes of RNA synthesized after nuclear transplantation may represent changes in gene activity. Previous work has shown that the transplantation of a nucleus from one kind of specialized cell gives rise after transplantation to daughter nuclei which promote the formation of several quite different cell types (Gurdon, 1962). This has been thought to be due to changes in gene activity. The results reported here show that nuclear transplantation also leads to a pronounced change in the pattern of RNA synthesis. Nuclei were taken from tissues which were synthesizing predominantly R-RNA. After the transplantation of such nuclei, the resulting nuclear-transplant embryos showed PLATE I. All pictures are of Xenoptu laeei», a and c b d

e f

g h to j k to rn

Section of intestinal epithelium of stage 46 tadpoles. Chemically isolated intestinal epithelium cell (phase-contraat], Intestinal epithelium cell nucleus 40 min after transplantation to an enucleated unfertilized egg. Phase-contrast view of a blastula nucleus derived from a transplanted intestine nucleus. Gastrula nucleus derived from a transplanted intestine nucleus. Nucleus of a stage 8 blastula cell. Nuclei derived from transplanted blastula nuclei, shown at the same stages as in d to f respectively. Brain cell nuclei of swimming tadpoles: k, wild type; I, heterozygous for the anucleolate mutation; m, homozygous for the anucleolate mutation.

a

b

' IOJL

I

e

IOfL

m_ ......

-"'-_......:::.~

[facing p. 34

CYTOPLASMIC HEGULATION OF RNA SYNTHESIS

3ii

a pattern of RNA synthesis which was indistinguishable from that of normal embryos derived from fertilized eggs. They synthesized D·RNA and S·RNA, but showed no detectable R-RNA synthesis. Our results show that R-RNA synthesis is not detectable during the cleavage of embryos resulting from the transplantation of nuclei from tissues which are actively synthesizing R·RNA. This does not tell us how soon after transplantation R-RNA synthesis ceases. R-RNA synthesis by the small number of nuclei present during the first few cleavages after nuclear transfer would not be detected if none were synthesized by the many thousand nuclei present during late cleavage. The cytological changes which follow nuclear transplantation suggest that R-RNA synthesis may cease before the first nuclear division. Thus the transplantation of a nucleus is followed by a pronounced increase in nuclear volume and by the disappearance of definitive nucleoli. The absence of definitive nucleoli is correlated with the lack of detectable R·RNA synthesis. This is shown by the absence ,of nucleoli and of R·RNA synthesis during normal cleavage and in cells of embryos homozygous for the anucleolate mutation of X.laevis (Plate I, m; Brown & Gurdon, 1964), as well as by the appearance of definitive nucleoli and the start of R-RNA synthesis at the beginning of gastrulation (Brown & Littna, 1964a). The disappearance of nucleoli may be connected with nuclear swelling, since a disappearance and reappearance of nucleoli can be induced by the swelling and contraction of isolated HeLa cell nuclei (Fisher & Harris, 1962). We wish to thank Professor H. Harris, Dr I. Dawid, and Dr T. J. King for discussion of the content of this paper, and also Miss Janet Rooney and Miss Elizabeth Littna for expert technical assistance, One of us (J. B. G.) gratefully acknowledges a research grant from the Medical Research Council. REFERENCES Brown, D. D. (1964). J. Exp. Zool. 157, 101. Brown, D. D. & Caston, J. D. (1962). Develop. Biol. 5, 412. Brown, D. D. & Gurdon, J. B. (1964). Proc, Nat. Acad. Sci., Wash. 51, 139. Brown, D. D. & Littna, E. (1964a). J. Mol. Biol. 8, 669. Brown, D. D. & Littna, E. (1964b). J. Mol. Biol. 8, 688. Cohen, S. (1954). J. Biol. Ohem, 211, 337. Elsdale, T. R., Gurdon, J. B. & Fischberg, M. (1960). J. Embryol. Exp. Morph. 8, 437. Fisher, H. W. & Harris, H. (1962). Proc. Roy. Soc. B, 156, 521. Flickinger, R. A. (1949). J. Exp. Zool. 112, 465. Gurdon, J. B. (1960a). Quart. J. Micr. Sci. 101, 299. Gurdon, J. B. (1960b). J. Embryol. Exp. Morph. 8, 327. Gurdon, J. B. (1960c). J. Embryol. Exp. Morph, 8, 505. Gurdon, J. B. (1962). J. Embryol. Exp. Morph. 10, 622. Gurdon, J. B. (1964). Advanc. Morphogen. 4, 1. Kutsky, P. (1950). J. Exp. Zool. 115, 429. Lane, B. G. (1963). Biochim. biophys. Acta, 72, 110. Nieukoop, P. D. & Faber, J. (1956). Normal Table of Xenopus laevis (Daudin). Amsterdam: North-Holland Publ. Co. . Subtelny, S. & Bradt, C. (1963). J. Morph, 112, 45.