/. Embryo/, exp. Morph. Vol. 23, 3, pp. 571-582, 1970
Printed in Great Britain
571
An inverse relation
between the rate of cell division and RNA synthesis
per cell in developing frog embryos
By R. A. F L I C K I N G E R , 1 M. R. L A U T H AND P. J. S T A M B R O O K
From the Department of Biology, State University of New York
Previous work has shown that endoderm cells synthesize more RNA per cell,
including nuclear d-RNA, than do dorsal ectoderm-mesoderm cells (Flickinger,
Miyagi, Moser & Rollins, 1967; Woodland & Gurdon, 1968). Both groups found
lower levels of DNA synthesis in endoderm cells compared to dorsal ectodermmesoderm cells. It has also been found that in developing frog embryos both
LiCl and cytosine arabinoside inhibit DNA synthesis and also inhibit RNA
synthesis. Since RNA synthesis is inhibited less than DNA synthesis, the levels
of RNA synthesis relative to total DNA increase (Flickinger, Miyagi, Moser &
Rollins, 1967). A number of investigators have demonstrated that LiCl can
cause isolated amphibian gastrula ectoderm to form endoderm and mesoderm
(Barth & Barth, 1962; Gebhardt & Nieuwkoop, 1964; Masui, 1966; Ogi, 1961).
Such compounds as NaHC0 3 (Barth & Barth, 1963) and NaCl (Barth, 1966)
can induce isolated frog gastrula ectoderm to form neural tissue.
The present investigation was undertaken to examine quantitative variations
of nucleic acid synthesis in expiants of Rana pipiens embryos undergoing
different rates of cell division. Variations in rates of cell division were obtained
by treating embryos with LiCl and NaHC0 3 or by comparing the rapidly
dividing dorsal ectoderm-mesoderm cells with the more slowly dividing endoderm cells. The lengths of the Gl5 S and G2 periods of cells from frog embryos
cultured in LiCl and NaHC0 3 , and of dorsal ectoderm-mesoderm and endoderm cells was determined by cell counts and autoradiography with [3H]thymidine. Correlations were sought between the lengths of Gl5 S and G2, as well as
the rates of DNA synthesis per cell, and the amounts of RNA synthesized per
cell. In particular, the concept that cells with longer G2 periods can synthesize
more RNA per cell was examined.
1
Author's address: Department of Biology, State University of New York, Health
Sciences Building, Buffalo, N.Y.14214, U.S.A.
37-2
572
R. A. F L I C K I N G E R , M. R. LAUTH AND P. J. STAMBROOK
MATERIALS AND METHODS
The manner of cutting the frog embryos (Rana pipiens) for incubation with
the isotopic compounds in Niu-Twitty saline (1953), isolating nuclei, washing,
homogenizing and fractionating the cells, hydrolyzing the RNA and DNA and
counting these fractions has been described (Flickinger, Miyagi, Moser &
Rollins, 1967). The animal halves of gastrulae, neural plate-dorsal mesoderm
regions of neurulae and the dorsal axial regions of tailbuds are referred to as
dorsal ectoderm-mesoderm regions in the text. The vegetal halves of gastrulae
and belly regions of neurulae and tailbuds are called the endoderm fractions in
the text. The belly endoderm cells are covered by lateral mesoderm and epidermis at the neurula and tailbud stages, but the endoderm cells constitute the bulk
of the isolated belly regions. The culture medium contained penicillin G-streptomycin sulfate to prevent bacterial growth. In the experiments utilizing
[2-14C]uridine, the isotopic activity of total RNA was determined by precipitation of the homogenate with 5 % trichloroacetic acid (TCA) and hydrolysis of
the RNA of the precipitate with 0-3 N - K O H at 37 °C for 18 h. The supernatant
fraction was counted after neutralization of the KOH with perchloric acid and
sedimentation of the DNA. Using samples of labeled RNA of known specific
activity, it was found that 78 % of the counts were recovered after hydrolysis
of the samples in 0-3 N - K O H . The variation in the results of duplicate experiments was 5 % or less. After the hydrolysis of RNA by KOH, the DNA in the
sodium Perchlorate precipitate was hydrolyzed by heating at 90 °C for 20 min
with 5 % trichloroacetic acid. The TCA was removed with ether and aliquots
of the supernatant fractions were evaporated with an infra-red lamp on planchets and counted with a gas flow counter. In order to ensure that results
obtained with [2-14C]uridine were valid, DNA synthesis was examined using
[3H]thymidine and RNA synthesis was checked with [5-3H]uridine. The tritium
label in the 5 position of uridine is released when uridine is converted to
thymidine, and is not incorporated into DNA in frog embryos (Freedman,
Stambrook & Flickinger, 1967). In the tritiated thymidine and [5-3H]uridine
experiments total nucleic acid was hydrolyzed with 5 % TCA at 90 °C for
20 min. The TCA hydrolysate, as well as the acid-soluble pool fractions, was
extracted with ether to remove the TCA, then each hydrolysate was brought to
a 1 ml volume with distilled water. One aliquot was used for the diphenylamine
DNA determinations, while 0-2 ml was added to 10 ml of Bray's solution for
counting in a liquid scintillation spectrometer. For the extraction of RNA from
isolated nuclei by phenol, the nuclei were homogenized in 005M-NaCl, 0 0 1 M sodium acetate, 10/6g/ml polyvinylsulfate and 0-5% sodium laurylsulfate at
pH 50. An equal volume of redistilled water-saturated phenol was added and
extraction was accomplished by shaking for 20 min at 45 °C. The interphase
and phenol layers were re-extracted with the buffer solution until the extract
contained no more RNA as determined by absorption at 260 mjn. The nuclear
Cell division and RNA synthesis
573
DNA-like RNA (d-RNA) remains in the interphase at this temperature
(Flickinger, Miyagi, Moser & Rollins, 1967). Quantitative determinations of
RNA and DNA were made using the orcinol and diphenylamine methods
(Dische, 1955) respectively.
The isotopic activities of the trichloroacetic acid-soluble fractions were
determined by counting the centrifugal supernatant fractions after the addition
of 5 % TCA to homogenates of embryos that had been washed four times in
Niu-Twitty saline. The 5 % TCA centrifugal supernatant fractions were
extracted with ether to remove the TCA, then 0-2 ml aliquots were evaporated
on planchets with an infra-red lamp and counted with the gas-flow counter.
After the cut embryos had been washed four times with Niu-Twitty saline,
further washes did not produce any further significant change in the activity of
the acid-soluble pool.
For the autoradiographic experiments the procedures for squashing cells,
staining the squashes, preparing the autoradiographs, making the nuclear
counts and calculating generation times have been published (Flickinger,
Freedman & Stambrook, 1967). Three squash preparations were made for each
time interval examined and each squash was thoroughly examined for metaphase figures. The number of metaphase figures varied between 15 and 75, but
was usually 40-50. The duration of the G2 period was obtained by noting the
time necessary for ca. 50 % of the metaphase figures to show the first appearance
of silver grains. The time required for maximal labeling of approximately half
of the metaphase figures represented the duration of the G2 plus S periods. The
duration of the S period was calculated by subtracting G2 from G2 plus S and
the generation times were calculated as previously described (Flickinger,
Freedman & Stambrook, 1967) by comparing cell counts of expiants at the start
and at the end of a culture period. Thirty expiants were used for each determination. The length of the Gx period was determined by subtracting G2 plus S
from the generation time. Since the time for mitosis in Xenopus embryos at
comparable stages is one-half hour (Graham & Morgan, 1966), it is likely that
the time required for mitosis in Rana pipiens embryos is similar.
„,,,
.
.
RESULTS
RNA synthesis
Cut parts of 100 gastrulae, neurulae and tailbuds were cultured in Niu-Twitty
saline containing [2-14C]uridine (5 /tCi/ml) for either one or 24 h at 20 °C. Based
on equal amounts of DNA, the levels of synthesis of labeled RNA
total cpm RNA
, . .
_ . T.
. -r- total ßg DNA
- - —\A—.-,-.
total cpm acid-soluble pool
are always higher for the endoderm fraction (Table 1). This ratio represents a
description of the levels of RNA synthesis on a per cell basis by relating the
RNA synthesis to equivalent amounts of total DNA at the end of the
574
R. A. F L I C K I N G E R , M. R. LAUTH AND P. J. STAMBROOK
incubation. Dividing by the total cpm acid-soluble pool takes into account the
differences in penetration of the labeled precursor into the expiants.
Effect ofLiCl
and NaHC03
upon cell division
The effect of LiCl and N a H C 0 3 upon cell division was examined by determining generation times for whole early gastrulae (stage 10, Shumway, 1940)
and animal halves of early gastrulae. Direct counts of nuclei in homogenates were
made after staining intact cells with lactopropionic orcein. In each experiment,
counts were made on an equal number (15) of whole early gastrulae or animal
Table 1. RNA synthesis per cell in regions of developing frog embryos
Parts of 100 cut embryos were cultured in Niu-Twitty Saline containing [2-14C]uridine for 1 h
(5 /tCi/ml) and 24 h (2 /tCi/ml) at 20 °C. Values are averages of three experiments ± S.D.
Total cpm RNA
^ ,
^ X1A
-—:
~——,
,-=- Total /*g DNA
Total cpm acid-soluble pool
Stage
Gastrulae, stage 10
Animal halves
Vegetal halves
Late gastrulae, stage 12
Neural plate-dorsal mesoderm
Bellies
Neurulae, stage 14
Neural plate-dorsal mesoderm
Bellies
Neurulae, stage 15
Neural plate-dorsal mesoderm
Bellies
Tailbuds, stage 18
Dorsal axial regions
Bellies
1 h cultures
24 h cultures
8-46 xl0- 4 ± 0-35 xlO- 4
9-68 xl0- 4 ± 0-24 xlO- 4
0-78 xl0" 2 ± 016 xlO- 2
1 16 xl0~ 2 ± 005xl0- 2
10-5 xl0" 4 ± 0-97 xlO- 4
13-4xl0- 4 ±l-2xl0" 4
14-5 xl0- 4 ± 0-35 xlO- 4
15-7 xlO"4 ±042 xlO"4
1-21 xl0" 2 ± 004 xlO" 2
l-33xl0- 2 ±007xl0- 2
24-7 xl0- 4 ± 0-27 xlO"4
34-7 xl0" 4 ± 0-50 xlO- 4
3-68 xl0- 4 ± 0-54 xlO- 4
7-3xl0" 4 ±l-35xl0- 4
0-73 xl0- 2 ± 008 x 10-2
l-29xl0- 2 ±031xl0- 2
Table 2. Stimulation by NaHCOs and inhibition by LiCI
of cell division in frog embryos as shown by cell counts
Generation times* in h
Niu-Twitty
saline
LiCl
0-25 y,
NaHC0 3
0-25 %
Intact gastrulae developing to early neurulae
20 ± 0-98
25 ± 1 -30
16 ± 0-8
(stage 13) after 24 h of culture
Animal halves of gastrulae (ectoderm-mesoderm)
19 ± 21
26 ± 1-7
14± 0-95
cultured for 24 h
* The generation times were calculated by the method of Flickinger, Freedman &
Stambrook (1967) based on 5 determinations: mean values ±S.D.
Cell division and RNA synthesis
575
hemispheres of early gastrulae obtained from the same female frog just before
and just after a 24 h incubation in Niu-Twitty saline, or in Niu-Twitty saline
plus LiCl or NaHC0 3 . Examination of the data (Table 2) shows that NaHC0 3
decreases the generation time, while LiCl increases the generation time.
Stimulation of cell division during embryonic induction
The positive correlation between changes in differentiation induced by these
salts and their effect upon cell division suggested that changes in rate of cell
division might occur during the process of embryonic induction in the embryo.
The following experiment was performed to determine if cell division was
stimulated in a tissue reacting to an embryonic induction stimulus. The neural
plate-dorsal mesoderm and epidermis-lateral plate mesoderm areas of very
early neurulae (stage 13 + ) were isolated in Niu-Twitty saline. [3H]thymidine
(25 /tCi/ml) was added to these cultures for 2 h and then the expiants were
fixed, squashed and coated with liquid emulsion for autoradiography (Flickinger, Freedman & Stambrook, 1967). The developed autoradiography from
five different experiments were examined for labeled and unlabeled nuclei. The
neural plate-dorsal mesoderm squashes showed 73 % ( ± 4 %) of the nuclei were
labeled, while only 58 % (± 5 %) of the nuclei from the adjacent epidermislateral mesoderm region were labeled.
Effect of NaHC03 upon DNA and RNA synthesis
To study the effect of NaHC0 3 on RNA and DNA synthesis, 1600 stage 18
tailbuds were cut into axial and belly regions and 800 were incubated in NiuTwitty saline plus [2-14C]uridine (1 /tCi/ml) for 24 h at 20 °C. The cut embryos
appeared healthy at the end of the incubation. The other 800 were treated
similarly, but NaHC0 3 was present during the incubation at a concentration of
0-25 %. Aside from estimating the isotopic activity of the total RNA of aliquots
of the homogenates, nuclear fractions from the control and NaHC0 3 -treated
embryos were extracted repeatedly with phenol at 45 °C at pH 5-0 to remove
RNA not bound to DNA. At pH 50 little DNA is extracted in the aqueous
phase (Brown & Littna, 1964). The total RNA, 45 °C-extracted RNA and the
residue containing DNA-bound RNA not extracted by phenol at 45 °C, were
hydrolyzed with 0-3N-KOH. Concentrations of total RNA and the isotopic
activity of [14C]RNA were then determined.
The data (Table 3) show that NaHC0 3 causes an increase in DNA synthesis
as indicated by the ratio of
total cpm DNA
total cpm acid-soluble pool
and the total DNA determinations. That RNA synthesis per cell is inhibited by
NaHC0 3 treatment is shown also by a reduction in the ratio of
cpm RNA
cpm DNA
of NaHCOz
0-268
0008
—
—
0016
—
0-266
0-750
—
2340
0-787
2220
Total cpm RNA
TotäTcpm D N A
incorporation
2-76
2-40
Total cpm D N A
Total cpm
acid-soluble pool
upon [2- C]uridine
and RNA
embryos*
3-2 x 10-*
3-2 xlO- 4
009 x l O - 4
0-742
0022
016x10-*
8-2 x 10- 4
8-6 x l O - 4
Tot_al_cpmRNA^TotalDNA
Total cpm
acid-soluble pool
of tailbud
0-738
0035
1-8
1-9
Total cpm R N A
Total cpm
acid-soluble pool
into DNA
* Incubation: 1600 stage 18 tailbuds were cut and half were incubated in Niu-Twitty saline alone and half with 0-25 % N a H C 0 3
for 24 h at 20 °C. with [2- 14 C]uridine (1 /tCi/ml). The values are averages of duplicate experiments.
NaHCOg 0-25 %
Total R N A
45° phenolextracted RNA
45° phenol
residue RNA
Controls
Total RNA
45° phenolextracted RNA
45° phenol
residue
Total fig D N A
T a b l e 3. Effect
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Cell division and RNA synthesis
577
and the ratios of
total cpm RNA
total acid-soluble pool
and
total cpm RNA
-total DNA
total acid-soluble pool
for total RNA, as well as 45 °C-extractable RNA and 45 °C residue fraction
(d-RNA) of isolated nuclei.
Lack of effect of increase in ribonucleoside pool
One way in which LiCl might increase RNA synthesis per cell would be by
increasing the size of the ribonucleoside pool by inhibiting DNA synthesis. The
lesser conversion of ribonucleotides to deoxyribonucleotides for DNA synthesis
might then allow the greater ribonucleoside pool to augment RNA synthesis
per cell. The following experiments, however, indicate that an increase in size
of the pool does not alter the level of incorporation of isotope. Groups of 50 cut
neurulae (stage 14) were incubated in Niu-Twitty saline or Niu-Twitty saline
plus all four ribonucleosides (adenosine, guanosine, cytidine and uridine) at
concentrations of 10 -4 M and 10~5 M for 24 h at 20 °C, with [14C]uridine present
at levels of 1 /tCi/ml in each of the cultures. In triplicate experiments the levels
of total [14C] RNA/total cpm acid-soluble pool H- total DNA were 2-16 x 10~3
(±013) for the controls, 2-20x 10~3 (±009) for the embryos cultured with
10 -5 M ribonucleosides, and 2-19 x 10 -3 (±0-17) for embryos cultured with a
10~ 4 M concentration of added ribonucleosides. Uridine was added to the
controls to a concentration of 10 -4 M or 10~5 M in order to match the uridine
levels in the cultures with all four ribonucleosides added. In another series of
experiments a higher level of isotopic uridine was used (lO^Ci/ml), but the
levels of total cpm RNA/total cpm acid-soluble pool were similar (2-10 x 10~3)
(±011) to those of cultures with 1 /iCi/ml [14C]uridine.
Autoradiographic experiments with embryo expiants exposed to LiCl andNaHCO^
Cut pieces of ectoderm-mesoderm of stage 10 gastrulae were cultured in
Niu-Twitty (1953) saline alone, or in Niu-Twitty saline plus LiCl (0-25 or
0-2 %) or NaHCOg (0-25 %) for 4 h, followed by 20 h further culture in the
same media with [3H]thymidine (25 ^Ci/ml) continually present at 20 °C.
Expiants were withdrawn at intervals of 1 or 2 h after addition of the [3H]thymidine, squashed, stained and coated with the liquid emulsion in the dark.
After 1 week the slides were developed and examined. The effect of 4 h of
preincubation in LiCl (0-25 %) in Niu-Twitty medium, followed by 20 h of
further culture in the same medium with [3H]thymidine present, is given in
Table 4. There is an increase in generation time for the LiCl cultures, the Gx
decreases and the S and G2 periods are increased. In the NaHC0 3 cultures the
generation time, S and G2 periods are decreased while the length of Gx is
increased.
578
R. A. F L I C K I N G E R , M. R. LAUTH AND P. J. STAMBROOK
Cell cycles of ectoderm-mesoderm
and endoderm of gastrulae and neurulae
Cell counts were made upon 15 isolated ectoderm-mesoderm and endoderm
regions of gastrulae (stage 10) and neurulae (stage 14) at the beginning and after
24 h of culture at 20°Cin Niu-Twitty saline in order to determine the generation
periods (Table 5). The lengths of the G 2 and S periods were determined by
culturing ectoderm-mesoderm and endoderm regions of gastrulae and neural
plate-dorsal mesoderm and belly regions of neurulae in [ 3 H]thymidine (25
/iCi/ml) at 20 °C. The ectoderm cells at the animal pole region and endoderm
cells at the vegetal pole region of the gastrula explants, as well as the neurula
expiants, were excised from the expiants and were squashed at intervals of \ h
for autoradiography. At the gastrula stage the G 2 of the endoderm cells at the
vegetal pole was H h, while the G 2 of the ectoderm cells at the animal pole was
1 h. The length of the G 2 of neurula endoderm cells was 4 h, while the G 2 of
the neural plate-dorsal mesoderm cells was 2^ h (Table 5).
Table 4. Influence ofLiCl and NaHCOz on the cell cycle
of ectoderm-mesoderm of mid-gastrulae
[3H]Thymidine was present the last 20 h of the 24 h of culture. Values are averages of
6 different experiments ± S.D.
Duration in h
Controls
LiCl(0-25%)
NaHC0 3 (0-25%)
A
Percent labeled nuclei
after 1 h
in [3H]thymidine
,
Generation
time
»
Gi
S
G2
88
87
90
18-5 ± 1-8
24 ±2-4
15 ±2-1
10-5
9
12
6±1
9±1
2±1
2±0
6±1
l±0
Table 5. Cell cycle of ectoderm-mesoderm and endoderm
regions of gastrulae and neurulae
Percent labeled nuclei
after 1 h
in [3H]thymidine
Gastrula (stage 10)
Ectoderm-mesoderm
Endoderm
Neurula (stage 14)
Neural platedorsal mesoderm
Endoderm
Duration in h ± S.D.
/
*
Generation
time
Gj
S
N
G2
95
96
19 ±1
24±1±
17
21±
l±i
1 ±0
1±±0
73
70
26 ± 1-y
31 ±2
m
6±i
6+ i
4 ±0
21
Cell division and RNA synthesis
579
DISCUSSION
At concentrations which other investigators (Barth & Barth, 1963; Masui,
1966) have shown can induce neuralization of amphibian gastrula ectoderm,
NaHC0 3 stimulates cell division of Rana pipiens embryos. That cell division is
stimulated is shown by cell counts (Table 2) and increased levels of [2-I4C]uridine incorporation into DNA (Table 3). This suggests that a stimulation of
mitosis may be an important initial event in the normal embryonic induction
process. This idea receives support from our autoradiographic finding that after
2 h culture in [3H]thymidine the induced neural plate-dorsal mesoderm regions
have a higher percentage of labeled nuclei than the adjacent epidermis-lateral
plate mesoderm in which induction has not occurred.
With the increase in DNA synthesis in NaHC0 3 , there is a reduction of the
ratio of labeled RNA/labeled DNA which is expressed by both the RNA extracted at 45 °C and the DNA-bound RNA (45 °C residue) fractions (Table 3).
A decrease in DNA synthesis in LiCl is correlated with an increase in RNA
synthesis per celt (Flickinger, Miyagi, Moser & Rollins, 1967). The endoderm
cells of amphibian embryos divide and synthesize DNA more slowly than the
ectoderm-mesoderm cells and the levels of RNA synthesis per cell are greater
for the endoderm cells than for the ectoderm-mesoderm cells (Flickinger,
Miyagi, Moser & Rollins, 1967; Woodland & Gurdon, 1968). That endoderm
cells synthesize more nuclear d-RNA was shown by extraction of d-RNA
(characterized by base composition) from nuclei with hot phenol (Flickinger,
Miyagi, Moser & Rollins, 1967) or MAK column chromatography (Woodland
& Gurdon, 1968).
The total cpm RNA/total cpm DNA ratio (Table 3) is significant because no
matter how LiCl and NaHC0 3 might affect permeability, the levels of DNA
and RNA synthesis depend on the same supply of [14C]uridine and hence they
would reflect the actual relative levels of synthesis. While LiCl increases and
NaHC0 3 decreases RNA synthesis on a per cell basis, the total levels of RNA
synthesis depend upon the total number of cells. Embryos cultured in LiCl
undergo fewer cell divisions and the lower cell number accounts for the synthesis
of less total RNA than by control embryos. Embryos cultured in NaHC0 3
undergo more divisions, thereby allowing more RNA synthesis by the greater
number of cells.
Experiments were performed to eliminate the possibility that a competition
for precursors determines the extent of DNA and RNA synthesis in individual
cells. The stimulation of RNA synthesis per cell by LiCl could be due to the
larger pool of ribonucleosides present because of a decreased conversion of
ribonucleotides to deoxyribonucleotides due to inhibition of DNA synthesis by
LiCl. The action of NaHC0 3 might be to reduce RNA synthesis per cell by
shunting more of the common precursors into DNA synthesis. However, this
does not seem likely. When experiments were performed with the addition of
580
R. A. F L I C K I N G E R , M. R. LAUTH AND P. J. STAMBROOK
all four ribonucleosides to cut embryos at 10~4 M and 10 -5 M concentrations,
there was no difference in the levels of synthesis of labeled RNA per cell.
LiCl treatment of gastrula ectoderm-mesoderm cells increases the generation
time by lengthening the S and G2 periods, while NaHC0 3 treatment decreases
the generation time by shortening the S and G2 periods (Table 4). Endoderm
cells have longer generation periods than do ectoderm-mesoderm cells in
Xenopus embryos (Woodland & Gurdon, 1968) and our work now demonstrates longer generation periods for Rana pipiens endoderm cells of gastrulae
and neurulae (Table 5). Previous work from our laboratory reporting shorter
generation times for endoderm cells at these stages has proved to be incorrect
(Flickinger, Freedman & Stambrook, 1967). By selecting ectoderm cells from
the animal pole region and endoderm cells from the vegetal pole of gastrulae,
and squashing expiants of gastrulae and neurulae at \ h intervals, longer G2
periods have been detected for the endoderm cells, which divide more slowly
(Table 5). It is possible that a slower rate of division and longer G2 periods
account for the fact that endoderm cells start synthesizing nuclear d-RNA
earlier than ectoderm cells in Xenopus blastulae (Bachvarova & Davidson,
1966). The previous failure to detect differences in length of G2 between
ectoderm-mesoderm and endoderm cells is ascribed to increased resolution
obtained by squashing at \ h intervals instead of at hourly intervals as previously
reported (Flickinger, Freedman & Stambrook, 1967). If the length of time spent
in Gl5 S or G2 relates to the amount of RNA synthesis per cell, then the duration
of the S and G2 periods could play a role in determining the amount of RNA
synthesis per cell in these embryonic tissues. It is possible that longer S and G2
periods could allow more RNA to be synthesized in an individual cell since the
doubling of the DNA during the S period increases the amount of template
DNA for RNA synthesis.
An objection to the suggestion that longer S and G2 periods allow more
RNA synthesis is that levels of heterogenous RNA synthesis per cell decline
from the neurula to the tailbud stage in Xenopus (Woodland & Gurdon, 1968),
while the S and G2 periods increase in length (Graham & Morgan, 1966).
However, the population of dividing cells in Rana pipiens is decreasing during
this developmental period as shown autoradiographically by the decrease in
percentage of nuclei labeled with [3H]thymidine in a given time (Flickinger,
Freedman & Stambrook, 1967). Since d-RNA synthesis in Xenopus is proportional to DNA synthesis (Brown & Littna, 1964), the lower levels of d-RNA
synthesis probably are due to fewer dividing cells. Similar stage embryos were
used for the LiCl-NaHC0 3 experiments and the percentage of dividing cells was
similar (Table 4).
The data showing that more rapidly dividing cells synthesize less RNA per
cell (Tables 1, 3) suggest that this could be a factor accounting for dedifferentiation of cloned differentiated cells under conditions of rapid cell division (Coon,
1966). Furthermore, transdetermination of imaginai discs cultured in adult
Cell division and RNA synthesis
581
Drosophila occurs more frequently with rapid cell division (Hadorn, 1966).
Further work is necessary in order to ascertain if the rate of cell division, or
number of cell divisions, plays a role in determining the kind of differentiation
a cell will undergo.
SUMMARY
1. Endoderm regions of gastrulae, neurulae and tailbuds synthesize more
RNA per cell than do the dorsal ectoderm-mesoderm cells at these stages. The
endoderm cells divide more slowly than do the dorsal ectoderm-mesoderm
cells at the gastrula and neurula stages and have longer G2 periods.
2. Concentrations of LiCl which can direct amphibian embryo gastrula
ectoderm into endoderm and mesoderm differentiation decrease the rate of cell
division, increase the length of S and G2 and increase the amount of RNA
synthesized per cell in Rana pipiens embryos.
3. A concentration of NaHC0 3 which can neuralize amphibian gastrula
ectoderm stimulates the rate of cell division, shortens the S and G2 periods and
reduces the amount of RNA (including nuclear d-RNA) synthesized per cell.
4. There is less total RNA per embryo synthesized after treatment with LiCl
and more total RNA per embryo synthesized after treatment with NaHC0 3
due to the reduction and increase in cell number in LiCl and NaHC0 3 respectively.
5. Autoradiographic experiments suggest that stimulation of mitosis is one
of the first reactions in the induction of neural tissue.
ZUSAMMENFASSUNG
Ein umgekehrtes Verhältnis zwischen der Geschwindigkeit von Zellteilung und
RNA Synthese pro Zelle in der Entwicklung von Frosch Embryos
1. Die Endoderm-Bereiche im Gastrula, Neurula, und Tailbud Stadium
synthetisieren mehr RNA pro Zelle als die dorsal Ectoderm-Mesoderm-Zellen
auf diesen Entwicklungsstufen. Die Zellen des Endoderms teilen sich langsamer
als die dorsal Ectoderm-Mesoderm Zellen in der Gastrula und Neurula-Phase
und haben eine längere G2-Periode.
2. Verschiedene Konzentrationen von LiCl, welche in Amphibien Embryos
zu einer Differenzierung des Gastrula Ectoderms in Endoderm und Mesoderm
führen können, vermindern die Geschwindigkeit der Zellteilung, verlängern die
S-und G2-Periode und erhöhen die Menge an RNA, die in Rana pipiens
Embryos pro Zelle synthetisiert wird.
3. NaHC0 3 in einer Konzentration, welche Gastrula Ectoderm von Amphibien in Neurula Gewebe umwandeln kann, stimuliert die Geschwindigkeit der
Zellteilung, verkürzt die S- und G2-Perioden und verringert die Menge an
RNA (einschliesslich der d-RNA), die pro Zelle synthetisiert wird.
4. Nach der Behandlung mit LiCl wird weniger Gesamt-RNA und nach der
Behandlung mit NaHC0 3 mehr Gesamt-RNA pro Embryo synthetisiert, was
582
R. A. F L I C K I N G E R , M. R. LAUTH AND P. J. STAMBROOK
zurückzuführen ist auf die Verminderung und Erhöhung der Zellanzahl infolge
der Behandlung mit LiCl, beziehungsweise NaHC0 3 .
5. Die autoradiographischen Experimente deuten darauf hin, dass die
Stimulierung der Mitosis eine der ersten Reaktionen bei der Induktion von
Nerven-Gewebe ist.
This research was supported by grants from the National Science Foundation (GB5500)
and the National Institute of Health (GM16236-010).
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{Manuscript received 1 July 1969)