Download The relative rates of synthesis of DNA, sRNA and rRNA in the

/. 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. Graham for having made available to them unpublished material showing the frequency of DNA synthesis in different regions of X. laevis
embryos, to Dr D. D. Brown for comments on the manuscript and to Miss J. Rooney and
Miss V. Speight for technical assistance. They are very grateful to the Medical Research
Council for supporting this work.
REFERENCES
R. & DAVIDSON, E. H. (1966). Nuclear activation at the onset of Amphibian
gastrulation. /. exp. Zool. 163, 285-95.
BACHVAROVA, R., DAVIDSON, E. H., ALLFREY, V. G. & MIRSKY, A. E. (1966). Activation of
RNA synthesis associated with gastrulation. Proc. natn. Acad. Sci. U.S.A. 55, 358-65.
BARTH, L. G. & BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula in
unconditioned medium. /. Embryol. exp. Morph. 7, 210-22.
BACHVAROVA,
BELITSINA, N. V., AYTKHOZHIN, M. A., GAVRILOVA, L. P. & SPIRIN, A. S. (1964). Informa-
tional ribonucleic acids of differentiating animal cells. Biokhimiya 29, 363-74.
D. D. (1965). RNA synthesis during early development. In Developmental and Metabolic Control Mechanisms and Neoplasia, pp. 219-36. Baltimore, U.S.A.: Williams and
Wilkins Co.
BROWN, D. D. & CASTON, J. D. (1962). Biochemistry of amphibian development. II. High
molecular weight RNA. Devi. Biol. 5, 435-44.
BROWN, D. D. & GURDON, J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 51, 139-46.
BROWN, D. D. & GURDON, J. B. (1966). Size distribution and stability of DNA-like RNAsynthesized during development of anucleolate embryos of Xenopus laevis. J. molec. Biol.
19, 399^22.
BROWN, D. D. & LITTNA, E. (1964). Variations in the synthesis of stable RNA's during
oogenesis and development in Xenopus laevis. J. molec. Biol. 8, 688-95.
BROWN, D. D. & LITTNA, E. (1966). Synthesis and accumulation of low molecular weight
RNA during embryogenesis of Xenopus laevis. J. molec. Biol. 20, 95-112.
COMB, D. G., KATZ, S., BRANDA, R. & PINZINO, C. J. (1965). Characterization of RNA species
synthesized during early development of sea urchins. /. molec. Biol. 14, 195-213.
DAWID, I. (1965). Deoxyribonucleic acid in amphibian eggs. J. molec. Biol. 12, 581-99.
DEUCHAR, E. M. (1958). Regional differences in catheptic activity in Xenopus laevis embryos.
J. Embryol. exp. Morph. 6, 223-37.
ELSDALE, T. R., FISCHBERG, M. & SMITH, S. (1958). A mutation that reduces nucleolar number
in Xenopus laevis. Expl Cell. Res. 14, 642-3.
ELSDALE, T. R., GURDON, J. B. & FISCHBERG, M. (1960). A description of the technique for
nuclear transplantation in Xenopus laevis. J. Embryol. exp. Morph. 8, 437-44.
BROWN,
FLICKINGER, R. A., GREENE, R., KOHL, D. M. & MIYAGI, M. (1966). Patterns of synthesis
of DNA-like RNA in parts of developing frog embryos. Proc. natn. Acad. Sci. U.S.A.
56, 1712-18.
FLICKINGER, R. A., MIYAGI, M., MOSER, C. R. & ROLLINS, E. (1967). The relation of DNA
synthesis to RNA synthesis in developing frog embryos. Devi. Biol. 15, 414-31.
GRAHAM, C. F. & MORGAN, R. W. (1966). Changes in the cell cycle during early Amphibian
development. Devi. Biol. 14, 439-60.
GRANT, P. (1958). The synthesis of DNA during early embryonic development of Rana
pipiens. J. cell. comp. Physiol. 52, 227-48.
GURDON, J. B. (1967 a). Control of gene activity during the early development of Xenopus
laevis. In Heritage from Mendel (ed. R. A. Brink), pp. 203-41. University of Wisconsin
Press.
Embryonic nucleic acid synthesis
385
J. B. (19676). Xenopus laevis (Daudin), the South African Clawed Frog. In Techniques in Developmental Biology, pp. 75-84, ed. N. Wessells and F. Wilt. New York:
Thomas Crowell Publishing Co.
GURDON, J. B. & BROWN, D. D. (1965). Cytoplasmic regulation of RNA synthesis and nucleolus formation in developing embryos of Xenopus laevis. J. molec. Biol. 12, 27-35.
HAY, E. D. & GURDON, J. B. (1967). Fine structure of the nucleolus in normal and mutant
Xenopus embryos. J. Cell. Sci. 2, 151-62.
MANDELL, J. D. & HERSHEY, A. D. (1960). A fractionating column for analysis of nucleic
acids. Analyt. Biochem. 1, 66-77.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis {Daudin). Amsterdam:
North Holland Publishing Company.
OTAKA, E., MITSUI, H. & OSAWA, S. (1962). On the ribonucleic acid synthesized in a cell-free
system of E. coli. Proc. natn. Acad. Sci. U.S.A. 48, 425-30.
SraoKAWA, K. & YAMANA, K. (1965). Demonstration of 'polyphosphate' and its possible role
in RNA synthesis during early development of Rana japonica embryos. Expl Cell Res.
38, 180-6.
STOCKDALE, F. E. & TOPPER, Y. J. (1966). The role of DNA synthesis and mitosis in hormonedependent differentiation. Proc. natn. Acad. Sci. U.S.A. 56, 1283-9.
SZE, L. C. (1953). Changes in the amount of DNA in the development of Rana pipiens.
J. exp. Zool. 122, 577-601.
TENCER, R. (1961). Incorporation of Tritium-labelled thymidine in Bufo $ x Rana temporaria
cJ hybrid embryos. Nature, Lond. 190, 100-1.
WADDINGTON, C. H. & PERKOWSKA, E. (1965). Synthesis of RNA by different regions of the
early Amphibian embryo. Nature, Lond. 207, 1244-6.
WARNER, A. H. & FINAMORE, F. J. (1962). Nucleotide and nucleic acid metabolism in
developing Amphibian embryos. III. Metabolism of acid-soluble nucleotides. Comp. Biochem. Physiol. 5, 233-40.
GURDON,
{Manuscript received 14 August 1967, revised 23 October 1967)