/. Embryol. exp. Morph. Vol. 16, 1, pp. 203-10, August 1966
Printed in Great Britain
203
An autoradiographic study of RNA synthesis during
primary embryonic induction
By S. K. BRAHMA 1
From the Biology Division, Oak Ridge National Laboratory
Although much work has been done on the inducing factors involved in
primary embryonic induction, we are far from understanding the mechanism of
the phenomenon. Information concerning the changes in the synthetic patterns
in the reacting tissue, which may be related to the induction, must be accumulated before we will be able to formulate a hypothesis on the mechanism of
embryonic induction at a subcellular level. The work reported in this paper
represents an effort to obtain preliminary information concerning the possible
changes in RNA synthesis correlated with primary embryonic induction using
the autoradiographic technique.
According to earlier cytochemical data of Brachet (1942), when the prospective neural ectoderm is underlaid by the archenteric roof during gastrulation in
Triturus embryos, cytoplasmic basophilia of ectodermal cells increases, while in
the prospective epidermal ectoderm cytoplasmic basophilia remains relatively
weak. The basophilia was interpreted by Brachet to be due to RNA on the basis
of its ribonuclease sensitivity. Takata (1953) made an estimation of the amount
of RNA per mg nitrogen of different areas of Triturus embryos at the beginning
of gastrula and neurula stages, isolating the areas surgically. He found a significant increase in the amount in the neural ectoderm between the two stages, and
a small, statistically insignificant, increase in the epidermal ectoderm. Pfautsch
(1960) cultured the ectoderm of different regions of gastrula and neurula in vitro
and estimated the RNA content per mg dry weight in Triturus and Amblystoma.
An increase was demonstrated in the isolated neural plate, differentiating into
neural tissue, as well as in the isolated gastrula ectoderm, developing into
epidermal cell aggregate. From the data of the two latter workers it appears
rather likely that the RNA content of the ectoderm increases during gastrulation,
and the increase is higher when the ectoderm is induced to form neural tissue by
the archenteric roof than when it is unaffected by the archenteric roof and
differentiating epidermis. Data of Rounds & Flickinger (1958) showing no
increase in the ratio of RNA to DNA during gastrulation in Rana ectoderm
certainly do not contradict the above data, since in the developmental stages in
1
Author's address: Dept. of Medical Anatomy and Embryology, University of Utrecht,
Janskerkhof 3A, Utrecht, Netherlands.
204
S. K. BRAHMA
question DNA per embryo increases without increase in N or dry weight per
embryo. An increase in the amount of RNA per amphibian embryo during
gastrulation has been demonstrated by a number of workers (Steinert, 1951;
Krugelis, Nicholas & Vosgian, 1952; Chen, 1960; Bristow & Deuchar, 1964;
Decroly, Cape & Brachet, 1964). Further, some studies with isotopes demonstrate synthesis of RNA in the ectodermal cells during gastrulation of amphibian embryos (Denis, 19646; Karasaki, 1965; Tiedemann, Born & Becker,
1965).
The present paper reports autoradiographic studies in which RNA synthesis
in the Triturus ectoderm developing in vitro under the influence of the dorsal
mesoderm was compared with that in the control ectoderm without an inductive
influence. Since our present interest is in the mechanism of embryonic induction
or the determination of differentiation pathways, but not in the differentiation
processes themselves, the study was confined to the gastrulation phase, during
which inductive determination of the differentiation pathway occurs.
MATERIAL AND METHOD
Embryological techniques
Fertilized eggs of Trituruspyrrhogaster obtained after injection of Antuitrin-S
into females were reared in spring water at 18°C until they reached the early
gastrula stage. The embryos in capsules were immersed in 70 % ethanol for
about 40 s, washed several times in the culture medium, and decapsulated with
needles. After removal of vitelline membrane the presumptive ectoderm was
isolated with tungsten needles. The piece of ectoderm was cut into halves. One
was used to wrap a piece of the dorsal mesoderm from early gastrula, and the
other half was folded upon itself. A pair of such explants with ectoderm derived
from the same gastrula was used for comparison of grain counts. The culture
medium was composed of sterile Holtfreter saline buffered to pH 7-3 with
Tris-HCl, and contained penicillin G and streptomycin sulfate.
Autoradiographic techniques
The explants kept at 18°C for different time intervals from the beginning of
culture were exposed to 50 /<c/ml [3H]uridine (New England Nuclear Corporation, specific activity 3-84 c/mM) in culture medium for 3 h and fixed in the
mixture of acetic acid and alcohol. Sections were cut at 6 ju, thickness after
embedding in Paraplast under vacuum. Before being covered with NTB 3 Kodak
liquid emulsion, all sections were treated with 5 % trichloroacetic acid for
10 min at 4° C. Some slides were treated with ribonuclease solution (Sigma
ribonuclease 5X crystallized) 1 mg/ml at 37 °C for 90 min in 0-01 M phosphate
buffer (pH 7-1) and then with cold trichloroacetic acid before being covered
with emulsion. Some other slides were treated in the same buffer without the
enzyme (Series II) for 90 min at 37 °C and then with cold trichloroacetic acid.
RNA synthesis during primary induction
205
Since the data presented in this paper depend on comparison of grain counts
of two halves of the prospective ectoderm of one young gastrula, one cultured
with and the other without the dorsal mesoderm, a series of control autoradiographs was conducted in which two halves of the same ectoderm both cultured
without the mesoderm were compared for grain counts. No significant difference
in counts was observed between two ectodermal pieces belonging to the same
pair after culturing for 3 h.
RESULTS
Throughout all autoradiographs to be reported in this work, silver grains are
localized over the nucleus and no significant grain counts are obtained over the
cytoplasm. Unless otherwise indicated, grain counts are based on the unit area
of the nucleus.
Table 1. Grain counts per unit nuclear area after cold trichloroacetic acid treatment in the different pairs of ectodermal pieces at different time intervals from
contact with the early dorsal mesoderm
3h
22 h
6h
A
r
Experimental
Control
3-44 ±0-20 *4-62iO-25
*5-8410-29 3-46 ±0-25
2-88 ±0-17 2-97 ±0-29
*4-1810-25 2-90±0-25
M-4610-25 3-54 + 0-27
41210-25 4-30 + 0-27
3-64 ± 0 1 7
40410-25
3-541017
3-5610-23
*4-26i0-23 3-3210-17
Experimental
*616 + 0-32
*3-4410-17
*5-3010-23
*3-6610-27
•3-77 + 0-25
*7-7610-32
*6-1810-30
*6-4210-23
*5-5810-25
Control
2-9810-14
2-0610-14
1-7210-10
3-1410-17
2-3210-14
5-2010-28
4-9410-20
3-6210-27
3-9610-32
Experimental
Control
*2-98±0-10 2-301016
*2-6510-10 1-971014
*3-15iO-17 1-7010-14
*3-2410-20 2-3610-14
3-1510-25 *4-3510-23
*3-7810-14 2-7410-14
*3-32±0-14 2-0410-14
*3-30i014
1-8310-10
i
d-32)t
0-61)t
(l-ll)t
* Difference was significant with P < 005.
t The ratio of averages of experimentals and controls of each group.
Series I. When grain counts of the autoradiographic sections treated with cold
trichloroacetic acid but not with ribonuclease or buffer were studied, the data
summarized in Table 1 were obtained. The data were evaluated first by comparing grain counts of experimental and control ectoderm derived from one
donor embryo, which are indicated on the same line in the table, and secondly
by the ratio of average experimental and control counts of each group. In the
3 h group, a slight significant difference of counts in favor of experimental
ectoderm was found in four out of nine pairs, but in other pairs the difference
was either insignificant or significant in favor of the control. The ratio of the
averages of experimental and control counts of this group was close to unity.
Thus the 3 h group as a whole did not show a consistent difference between
206
S. K. BRAHMA
experimental and control. In the 6 h group, all pairs showed significantly higher
counts in the experimental ectoderm, and the ratio of averages deviated more
from unity than in the 3 h group in favor of the experimental counts. The 22 h
group revealed significantly higher grain counts in the experimentals except in
one pair in which an opposite result was obtained. That the difference between
two types of ectoderm of the 22 h group was smaller than that in the 6 h group
is revealed in the ratio of average experimental and control counts, which was
intermediate between those of the 3 and 6 h groups.
Series II. Part of the ectodermal sections of the three groups were treated with
ribonuclease solution and then with cold trichloroacetic acid. No significant
grain counts were obtained over the nucleus in any of the cases studied.
Table 2. Grain counts per unit nuclear area after buffer
treatment followed by cold trichloroacetic acid wash
3h
22 h
6h
A
j
Experimental
Control
Experimental
1-53 ±005 1-73 ±003 1-13 ±0-01
1-27 ±0-02 1-43 ±004 l-13±0-01
1-80 ±004 1-53 ±003 •1-50 ±0-04
1-50 ±009 1-30 ±003 1-20 ±001
1-47 ±009 1-30 ±002 110 + 001
1-27 ±002 •1-60 ±0-03 113 ±002
1-20 ±003 1-57 ±004 1-27 ±002
1 07 ±001 •1-33 ±0-03 1-37 + 0-01
1-13 + 0-01 •1-60 ±0-04 1-16 ±0-02
(0-918)t
Control
Experimental
1-40 ±004 •1-73 ±0-04
1-33 ±004 1-20 ±002
110±001
1-23 ±002
l-12±002 1-27 ±002
1-27 ±002 1-20 ±003
1-33 ±003 •1-40 ±0-03
1-17 ±0-02 1-27 ±003
1-33 + 0-02 1-20 ±002
l-16±002
Control
1-27 ±003
1-33 ±003
107 + 001
110±001
103 ±001
117±002
1 33 ±003
1-27 ±003
(O-983)t
* Difference was significant with P < 005.
t The ratio of averages of experimentals and controls of each group.
Series III. The sections comparable to the ones used in series I were treated
with the buffer solution at 37 °C for 90 min. The grain counts obtained are
summarized in Table 2. Compared with the corresponding counts of series I,
a definite reduction was obvious in all ectoderms. No consistent difference was
observed in counts of experimental and control sections from one donor embryo
in any of the groups. The tendency is reflected in the ratio of average experimental and control counts of each group, which fluctuated around unity.
DISCUSSION
The first series of autoradiographs shows that under the inductive influence of
the dorsal mesoderm the ectoderm cells indicate higher radioactivity in their
nuclei at 6 and 22 h after beginning of contact with the dorsal mesoderm.
However, in the 3 h group of the same series such a difference is not evident. In
RNA synthesis during primary induction
207
the first series, the fraction of radioactivity was measured which was retained in
the section after fixation and treatment with cold trichloroacetic acid. On the
other hand, if the radioactivity was measured with sections from corresponding
explants, which were treated with buffer solution at 37 °C as well as cold trichloroacetic acid (series III), a substantial part of the radioactivity observed in
the first series was lost in all classes of explants, and this loss was higher in the
experimental than in the control explants. After ribonuclease treatment no
significant radioactivity could be detected in any classes of explants (series II).
Comparison of series II and III shows that it is dangerous to define all substances removable with a ribonuclease treatment as RNA. It is obvious that
ribonuclease treatment involves not only enzyme-specific removal but also
simple extraction with the medium, which can be considerable according to the
situation.
The answer to the question raised at the outset, does embryonic induction
involve a change in RNA synthesis detectable with the present autoradiographic
technique, depends upon the nature of the radioactive substance removed by
incubation with buffer at 37 °C. If this substance is RNA or ribonucleoprotein,
the present data would suggest a positive answer. If, on the other hand, one
assumes that only the fraction of radioactivity which is left in sections after
fixation, cold trichloroacetic acid treatment, and buffer treatment is due to
RNA, then one has to conclude that embryonic induction does not cause a
change in RNA synthesis detectable with the present technique. In any case,
the fact that this fraction of radioactivity, whatever its chemical nature, is
larger in amount in the experimental than in the control explants seems to
suggest its close connexion with the mechanism of embryonic induction.
Some preliminary attempts were made in the Biology Division by F. J.
Finamore to define the nature of the substance removed by buffer treatment at
37 °C. When the autoradiographic procedures adopted in the present work were
simulated on the homogenate of Rana pipiens ovarian eggs, buffer treatment at
37 °C removed some ultraviolet-absorbing material, roughly 50 % of which was
precipitated with ethanol. No decision can be made at the present moment
whether oligonucleotides, transfer RNA, or something else is involved here. It
should be pointed out that the developmental stages studied in the present work
fall into the time period when a rapid synthesis of oligonucleotides (Finamore,
1964) and transfer RNA (Brown & Littna, 1964; Tiedemann et al 1965) has
been indicated.
The fact that treatment of ectoderm with actinomycin D under certain conditions suppresses its reacting capacity to the dorsal mesoderm (Denis, 1964 a)
has been used as evidence for involvement of synthesis of specific RNA in the
mechanism of embryonic induction. The data so far reported, however, do not
rule out the possibility that the actinomycin causes, instead of specific inhibition
of the inductive mechanism, a general suppression of developmental progress
which does not allow the reacting system to form the induced structure. It should
208
S. K. BRAHMA
be kept in mind that development of non-induced ectoderm also requires
synthesis of different kinds of RNA (Tiedemann et al. 1965) at the time the
inductive mechanism is supposed to be operating.
SUMMARY
1. The prospective ectoderm of young gastrula of Triturus pyrrhogaster was
isolated and cut in two halves. One piece was cultured in Holtfreter saline, and
the other piece was fused to the dorsal mesoderm of young gastrula and cultured
as the experimental ectoderm. Three, 6, and 22 h after beginning of culture, each
pair of experimental and control explants was transferred to a solution of
[3H]uridine fixed 3 h later, and processed for autoradiography.
2. In the first series of experiments, silver grains per unit area of nucleus were
counted in the section treated with cold trichloroacetic acid. In the 3 h group,
no consistent difference between experimental and control ectoderm belonging
to each pair was found. However, significant differences were observed in the
6 and 22 h groups in favor of the experimental ectoderm.
3. When the sections were treated with ribonuclease, no significant grain
counts were obtained over the nucleus in any class of ectoderm.
4. When the sections were treated with the buffer used for ribonuclease
treatment in the same way, a considerable fraction of the grain counts registered
in series I was removed, The remaining colmts did not show a consistent
significant difference between experimental and control ectoderms of one pair
in all groups.
5. The fraction of radioactivity not removable with cold trichloroacetic acid
but removable with warm buffer is present in a higher amount in the experimental
than in the control ectoderm in the 6 h group. A possible significance of this
fraction in the mechanism of embryonic induction was suggested.
RESUME
Etude autoradiographique de la synthese de VARN au cows
de V induction embryonnaire primaire
1. On a isole et coupe en deux moities l'ectoderme presomptif de jeunes
gastrulas de Triturus pyrrhogaster. Un fragment a ete cultive dans le milieu de
Holtfreter, l'autre a ete fusionne avec le mesoderme dorsal d'une jeune gastrula
et cultive comme l'ectoderme experimental. Trois, 6 et 22 h apres le debut de la
culture, chaque paire d'explants experimental et temoin aete transportee dans une
solution d'uridine — 3 H, fixee 3 h plus tard et traitee pour l'autoradiographie.
2. Dans la premiere serie d'experiences, on a compte les grains d'argent par
unite de surface nucleaire sur les coupes traitees a l'acide trichloracetitiue froid.
Dans le groupe de 3 h, on n'a pas trouve de difference appreciable entre l'ectoderme experimental et l'ectoderme temoin de chaque paire. Neanmoins, des
RNA synthesis during primary induction
209
differences significatives ont ete observees dans les groupes de 6 et 22 h, en
faveur de l'ectoderme experimental.
3. Quand les coupes ont ete traitees a la ribonuclease, on n'a pas obtenu de
numerations significatives de grains sur les noyaux de n'importe quelle categorie
d'ectoderme.
4. Quand les coupes ont ete traitees avec le tampon utilise pour le traitement
a la ribonuclease, de la meme maniere, une fraction considerable des grains
comptes dans la serie I a ete eliminee. Les autres numerations n'ont pas montre
de differences significatives entre ectodermes experimental et temoin d'une
meme paire, dans tous les groupes.
5. La fraction radioactive non extraite par l'acide trichloracetique froid mais
extraite par le tampon chaud, se trouve en quantite plus elevee dans l'ectoderme
experimental que dans l'ectoderme temoin, dans le groupe de 6 h. On suggere la
possibility que cette fraction possede une signification dans le mecanisme de
l'induction embryonnaire.
I express my gratitude to Dr Tuneo Yamada for his valuable suggestions and criticisms.
I should also like to thank Dr Marvin A. Kastenbaum, Oak Ridge National Laboratory
Mathematics Panel, for the statistical help, and Mrs Lola M. Kyte for technical assistance.
This research was sponsored jointly by the Damon Runyon Memorial Fund and by the
U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.
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{Manuscript received 24 January 1966)