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/. Embryo!, exp. Morph. Vol. 21, 2, pp. 295-308, April 1969
295
Printed in Great Britain
RNA synthesis in the early embryogenesis of a fish
(Misgurnus fossilis)
By C. A. KAFIANI, 1 M. J. TIMOFEEVA, 1 A. A. NEYFAKH, 2
N. L. MELNIKOVA 1 & J. A. RACHKUS 1
From the Institute of Molecular Biology and Institute of Developmental
Biology of the Academy of Sciences of the U.S.S.R., Moscow
Synthesis of ribonucleic acids in early embryos has been extensively studied
during recent years in a number of laboratories and has been shown to begin
shortly after fertilization (Kafiani, Tatarskaya & Kanopkayte, 1958; Wilt, 1963;
Brown & Littna, 1964; Decroly, Cape & Brachet, 1964; Glisin & Glisin, 1964;
Kafiani & Timofeeva, 1964, 1965; Nemer & Infant, 1965). Early embryos of
Xenopus (Brown & Gurdon, 1964; Brown & Littna, 1964, 1966) and of sea
urchins (Wilt, 1963; Glisin & Glisin, 1964; Nemer & Infant, 1965) synthesize
up to gastrula stage predominantly or exclusively polydisperse RNA of a nonribosomal nature usually referred to as DNA-like RNA (dRNA).
The occurrence of continuous dRNA synthesis in early embryogenesis is in
apparent conflict with the periodicity of the 'morphogenetic function' of cell
nuclei found by one of us (Neyfakh, 1959, 1961, 1964, 1965) in embryos of a
number of animal species. In fact, for a certain time after ferilization of eggs
with a regulatory type of development, the nuclei remain morphogenetically
inactive. Microsurgical (Harvey, 1940; Briggs & King, 1959), chemical (Gross
& Cousineau, 1963; Lallier, 1963; Gross, Malkin & Moyer, 1964; Neyfakh,
1965) or radiation-induced (Neyfakh, 1959, 1961; Neyfakh & Rott, 1958;
Shapiro & Lander, 1960) enucleation of the egg does not visibly affect early
development up to the late blastula stage. After the initial period of inactivity,
a period of 'morphogenetic activity' of the nuclei begins which assures the
process of gastrulation. The occurrence of dRNA synthesis starting immediately
or shortly after fertilization is hard to reconcile with the proposal of a direct
relationship between the morphogenetic and the biochemical (dRNA-synthesizing) functions of cell nuclei. This work seeks to analyse this apparent
contraction, to further understanding of the molecular basis of early
embryogenesis.
1
Authors' address: Institute of Molecular Biology, Academy of Sciences of USSR, Vavilov
Street 32, Moscow B-312, USSR.
3
Author's address: Institute of Developmental Biology, Academy of Sciences of USSR,
Vavilov Street 26, Moscow B-133, USSR.
296
C. A. KAFIANI & OTHERS
To approach this problem we used the method of quantitative evaluation of
RNA synthesis in embryos described previously (Kafiani & Timofeeva, 1964,
1965). The relative rate of dRNA synthesis ('dRNA-synthesizing activity') was
estimated at different stages of development in normal embryos of loach
(Misgumusfossilis) and in embryos partially or nearly wholly deprived of nuclear
genetic material (haploid and 'anucleate' embryos obtained by inactivation of
one or both of the gametes with X-rays).
It was with loach embryos that morphogenetic function of nuclei was studied
for the first time using the method of radiation-induced inactivation of nuclei;
it was shown that the nuclear activity controlling the onset and the progress of
gastrulation begins at the mid-blastula stage (Neyfakh, 1959). RNA synthesis
was found to be sharply accelerated at about the same time (Kafiani &
Timofeeva, 1964, 1965).
The present work shows that the activation of dRNA synthesis occurs not
only on a per embryo but also on a per cell base. The extent of activation depends
on the quantity of genetic material in the nuclei. These facts confirm the occurrence of the true activation, or the onset of genome transcription in the cell nuclei
at the mid-blastula stage. On the other hand RNA synthesis during the preceding
period of development (synchronous cleavage of the egg) exhibits peculiarities
which suggest that this 'early' synthesis of RNA is at least partly independent
of the nuclei.
MATERIAL AND METHODS
Material. Mature eggs of loach were obtained 40 h after injecting the females
with 200 i.u. of Choriogonin (Gedeon Richter, Hungary). Eggs were fertilized,
left to develop in tap water at 21 °C and staged as described previously (Neyfakh,
1959).
X-ray irradiation. Eggs were irradiated with a dose of 40 kr, and sperm with
80 kr using apparatus RUP-1 (190 kV, 15 mA, 5 kr/min).
Introduction of the label. Since egg membranes are poorly permeable to usual
precursors RNA synthesis was studied using [14C]carbonate. It was introduced
in the eggs as described by Cohen (1954) and Flickinger (1954). Equal quantities
of eggs at desired stages (from the same clutch of developing eggs) were incubated
in stoppered flasks in a slightly acid medium (pH 6-0-6-5) containing Na214CO3.
In different series of experiments isotope concentration varied from 20 to
40/^c/ml of final medium. Temperature (21 °C) and duration of incubation
(60 or 90 min) were kept constant in each series. Penicillin and streptomycin
(100 and 50 i.u./ml respectively) were added to prevent bacterial contamination.
Extraction and purification of RNA. Total RNA of embryos was extracted
with sodium dodecylsulphate (SDS) and phenol in the cold by a procedure
similar to that used by Brown & Littna (1964). Embryos were quickly homogenized in ice-cold tris-HCl buffer, 0-01 M (pH 6-5) containing 0-01 M-MgCl2,
the homogenate made 1 % in respect to SDS and mixed for £-1 min at 10-15 °C.
RNA synthesis in a fish embryo
297
The lysed material was deproteinized three times with water-saturated phenol
at 3-5 °C. Nucleic acids were precipitated from the aqueous phase with ethanol
after addition of a small amount of potassium acetate.
For the determinations of RNA-synthesizing activity, the total nucleic acid
precipitate was dissolved in a small volume of cold water and made 2 M with
sodium chloride. The precipitate of 'salt-insoluble RNA' (siRNA) formed
overnight (—10 °C) contained ribosomal RNA (rRNA) and other kinds of
RNA (including dRNA) except transfer RNAs (tRNA). The latter, as well as
DNA, polysaccharide and other contaminants, remained in the supernatant.
The siRNA precipitate was freed of traces of these contaminants by three more
reprecipitations and washings with 2 M-NaCl. The RNA preparations contained
less than 0-3 % protein (Lowry, Rosebrough, Farr & Randall, 1951) and were
free of DNA. The label in siRNA preparations was 90-95 % sensitive to RNase
treatment. A lower proportion of counts (60-80 %) was, however, sensitive to
RNase in siRNA from early cleavage eggs.
For sucrose gradient centrifugations, total RNA preparations were used. To
obtain these, initial crude nucleic acid preparations were treated for 30 min at
37 °C with DNase (Worthington), 5/*g/ml, in 001 M tris-HCl buffer (pH 7-8)
containing 0-0lM-MgCl2. DNase was then eliminated with three successive
phenol-SDS deproteinizations, and total RNA precipitated with ethanol.
Sucrose-gradient centrifugation (Britten & Roberts, 1960). Two to 3 mg RNA
dissolved in 0-5 ml of 0-1 M acetate buffer (pH 5) with 001 M - E D T A was
layered on a 5-20% sucrose gradient prepared in the same buffer. After 12 h
centrifugation at 24000 rev./min in the SW-25 rotor of the Spinco L ultracentrifuge at 10-12 °C, the bottom of the tube was pierced and fractions collected.
Radioactivity measurements. After determining RNA content in aliquots or
fractions from u.v.-absorption (at 260 m/£) RNA was precipitated with cold
5 % trichloroacetic acid (TCA). The precipitate was collected on nitrocellulose
filters (RUFS, Czechoslovakia, pore diameter 0-9-1-2 fi), and washed with cold
TCA and ethanol. Radioactivity was counted in a liquid-scintillation spectrometer (Lie Belin, France) with a non-polar scintillation mixture: 0-4% 2,5diphenyloxazole and 001 % l,4-bis-2-(5-phenyloxazolyl)benzene in toluene.
RESULTS
Figure 1, A and B, show typical sedimentation patterns of total RNA
preparations from loach eggs at mid-gastrula stage, obtained after 14CO2
incorporation during 1 h (A) and 5 h (B). It is seen that the bulk of RNA (u.v.absorption, solid line) forms three peaks, two of which represent the large and
small components of rRNA (28 S and 18 S), while the third peak (4-5 S) corresponds to low molecular weight RNAs, mainly tRNA.
The distribution of labelled RNA (dotted line) is quite different and reveals
the existence of a poly disperse population of newly formed RNA molecules
298
C. A. KAFIANI & OTHERS
sedimenting along the entire gradient with a few distinct peaks. The first peak,
in the upper portion of the gradient, contains labelled RNAs of low molecular
weight including transfer RNA. Since the latter rapidly turn over their terminal
nucleotides, the incorporation of the label in this region of the gradient probably
does not wholly correspond to the synthesis of RNA. However, a significant
part of the labelled RNA in this peak sediments faster than u.v.-absorbing
material. This suggests that this broad peak includes some other labelled RNA
species besides tRNA. This is confirmed by the sedimentation pattern of a
siRNA preparation obtained at the same developmental stage (Fig. 2). Here,
10
05
10
20
30
40
Fraction no.
x
28S
10
4I
0-5
2 o
10
20
30
40
Fraction no.
Fig. 1. Sucrose-gradient centrifugation patterns of total RNA isolated from loach
eggs at the mid-gastrula stage after incubation with [14C]carbonate (24 /tc/ml) for
l h (A) and 5h (B).
, u.v.-absorption (at 260 m/t);
, TCA-precipitable
radioactivity.
RNA synthesis in a fish embryo
299
the 4-5 S peak of u.v.-absorption is lacking due to elimination of tRNA, while
there is a significant amount of labelled RNA of somewhat higher molecular
weight.
Most of the radioactive RNA sediments move rapidly, the 'heaviest' fractions
being found in greater proportions at shorter durations of 14CO2 incorporation
(cf. Figs. 1A and B). But even after prolonged incorporation, the distribution
of the label does not correspond to that of rRNA. This is true also for earlier
stages of development (Timofeeva & Kafiani, 1965,1966).
30
0-6
6-8S
28S
0-5
24
0-4
18
0-3
12 o
0-2
0-1
10
20
30
40
Fraction no.
Fig. 2. Sucrose-gradient centrifugation pattern of a 'salt-insoluble' RNA preparation from loach eggs at the mid-gastrula stage after 2 h incubation with [14C]carbonate (20 jtic/ml).
From the sedimentation behaviour of labelled RNAs of early loach embryos
we conclude that, up to mid-gastrula stage, rRNA is not synthesized to any
significant degree. Consequently siRNA preparations freed of tRNA contain
the label predominantly, if not exclusively, in polydisperse RNA, or dRNA,
which may include messenger RNA. A typical sedimentation pattern of an
siRNA sample shown in Fig. 2 illustrates the character of the RNA preparations
used throughout the present work.
In order to study the dynamics of transcription during early development in
loach, we followed variations in the rate of dRNA synthesis at cleavage and
blastula stages, up to the onset of gastrulation. Figure 3 summarizes the results
of three separate experiments showing the peculiar course and reproducibility
of the curves obtained. In each experiment equal quantities of eggs from the
same batch were pulsed at different stages with 14CO2 under identical conditions,
then siRNA isolated from each egg sample and specific radioactivity determined
20
J E E M 21
300
C. A. KAFIANI & OTHERS
as described in Methods. In Fig. 3 (as well as in Figs. 4 and 5) the values obtained
are plotted against hours of development, the latter representing mid-points
between the beginning and the end of incubation with [14C]carbonate.
£
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2 3 4 5 6 7 8
Hours of development at 21 °C
Fig. 3. Incorporation of 14CO2 into siRNA of loach eggs at different stages of
development. The curves illustrate the results of three separate experiments in
which equal quantities of eggs were incubated at stages indicated under following
conditions: (1) 45 min., 20/*c/ml; (2) 60min., 40/iC/ml; (3) 60min., 20/tc/ml.
Since incorporation conditions were similar for all developmental stages,
since siRNA content (2 /tg per egg) remains constant over the period studied
(Timofeeva & Kafiani, 1964), and since, finally, the rate of RNA labelling in
conditions defined is not limited by 14CO2 incorporation into free nucleotides
of developing eggs (Kafiani & Timofeeva, 1964, 1965) we consider observed
changes in specific radioactivities of siRNA to be a measure of variations in the
rate of RNA synthesis on a per embryo basis. The capacity of an embryo to
incorporate a given quantity of the label into siRNA per unit time (e.g. 1 h) is
therefore designated as'dRNA-synthesizing activity'.
The curves in Fig. 3 have a clear-cut break at about 6 h of development. Up
to this time dRNA-synthesizing activity remains very low, increasing abruptly
within a narrow time interval between the 6th and 7th h. It was conceivable
that the increase in the over-all rate of dRNA synthesis in the embryos was due
to a corresponding increase in the number of nuclei rather than to their activation.
RNA synthesis in a fish embryo
301
Figure 4 shows, however, that the rates of increase of dRNA synthesis and of
cell number are quite different. In fact, from the 3rd until the 6th h, when
cleavage proceeds in a synchronous manner with a 30 min period (Neyfakh &
Rott, 1958; Rott & Sheveleva, 1967) the cell number increases about 30-fold
while dRNA-synthesizing activity increases only twofold. After the 6th h the
rate of dRNA synthesis increases much faster than the cell number. As a result
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Fig. 4. Changes in dRNA-synthesizing activity and in cell number in early development of loach. (1) dRNA-synthesizing activity of the embryos (specific radioactivity
of siRNA at the end of 60 min pulses with 14CO2, 20 /*c/ml); (2) number of cells per
embryo (from Rott & Sheveleva, 1967); (3) dRNA-synthesizing activity calculated
per cell.
the dRNA-synthesizing activity calculated per cell (see curve 3, Fig. 4) shows,
at about the 7th h, a marked rise indicating a real activation of dRNA synthesis.
The left-hand, descending part of curve 3 of Fig. 4 requires special consideration, as it implies that RNA-synthesizing activity is very high at the time of
morphogenetic inactivity of cell nuclei. This would seem paradoxical if the
nuclei are supposed to be the sole site for RNA synthesis. This paradox might
be resolved, however, if the 'early' RNA synthesis were partly or entirely independent of the cell nuclei.
We attempted to clarify this point by measuring the rate of RNA synthesis
in embryos with a reduced quantity of nuclear genetic material. The dRNAsynthesizing activities were compared in diploid embryos, gynogenetic haploids
(obtained by fertilizing normal eggs with X-ray-irradiated sperm) and 'anucleate' embryos (obtained either by combining X-ray-irradiated gametes or by
irradiating the zygotes). Morphologically, haploid embryos develop normally
302
C. A. KAFIANI & OTHERS
at least till late gastrula, the 'haploid syndrome' appearing considerably later.
In 'anucleate' embryos cleavage still occurs, resulting in a blastula-like mass of
cells virtually devoid of chromosomes (Shapiro & Lander, 1960).
18 -
16
.E 12
E
10
4
5
6
7
8
9
Hours of development at 21 °C
10
Fig. 5. Incorporation of 14CO2 into siRNA of diploid (2/?), gynogenetic haploid (1/;)
and 'anucleate' (On) embryos. The latter were obtained either by irradiating zygotes
or by combining irradiated gametes. The eggs were given 90 min pulses of [14C]carbonate (20 /*c/ml) at stages indicated.
Figure 5 and Table 1 show that until 5-6 h diploid and haploid embryos
incorporate 14CO2 into siRNA at nearly the same rate while at 6-8 h diploid
embryos incorporate label into siRNA about twice as fast as do haploid ones.
This difference is not due to the smaller number of cells in the latter, as they
contain at this time even more cells than do diploid embryos (Rott & Sheveleva,
1967). This higher rate of cell multiplication probably accounts for the compensation in the over-all production of dRNA shown by the reapproachment
of the In and In curves in Fig. 5 by the beginning of gastrulation.
Appearance of a direct dependence of RNA-synthesizing activity of embryos
on their ploidy testifies to the onset of nucleus-directed RNA synthesis. The
lack of such correlation over the first 6 h of development suggests that RNA
synthesis is independent of the nuclei at this time. The sites for the' early' RNA
RNA synthesis in a fish embryo
303
synthesis might reside in the cytoplasm, which would account for the decrease
of the left-hand part of curve 3 in Fig. 4 as a result of division of egg cytoplasm
into progressively smaller units.
This proposal is supported by experiments with 'anucleate' embryos. The
data summarized in Fig. 5 and Table 1 show a quite definite incorporation of
14
CO3 into siRNA in such embryos. Early RNA synthesis rises gradually,
increasing fivefold by 10 h, but fails to display the activation characteristic of
nucleated embryos. Despite the low level of incorporation most part of it
appears to reflect actual RNA synthesis since it is 80 % sensitive to RNase in
control and 60 % sensitive in anucleate embryos.
Table 1. dRNA synthesis in diploid, haploid and'anucleate' embryos
(At stages indicated, 6 ml portions of eggs were incubated with 3 ml of 0-01 M
phosphate buffer (pH 6-0-6*5) containing Na214CO3 (30 /*c/ml), penicillin (100 i.u./
ml) and streptomycin (50 i.u./ml) for 1 -5 h as indicated in column 1. Radioactivity was
counted in aliquots of siRNA preparations (see Methods) before and after treating
them with 'guanylic' RNase from actinomycetes. Specific radioactivities were calculated by dividing RNase-sensitive cold TCA-insoluble counts by the quantity of
siRNA present initially in aliquots.)
Specific radioactivity
(counts/min per mg siRNA)
Time of introduction
of"CO 2
A
Hours of
development
at 21 °C
M-2J
4-5*
6-74
7i-9
9H1
\
'Anucleate' embryos
obtained by irradiating:
f '
Stage
1 st-3rd division
Morula-early
blastula
Mid-blastula
Mid-late blastula
Early gastrula
Diploid
embryos
Haploid
embryos
Both gametes
Zygote
117
159
87
127
—
79
90
81
4060
14980
29350
1690
10600
24350
143
—
233
538
—
444
In order to get some information on the nature of the early incorporation
into siRNA we tested its sensitivity to actinomycin D. The antibiotic could be
introduced in the eggs only if applied at high concentrations (up to 100/ig/ml)
immediately after fertilization (at the time of swelling). Cleavage occurred in
such eggs but development was arrested at late blastula stage. 14CO2 incorporation into the RNase-sensitive portion of siRNA preparations isolated from the
eggs at 6 h of development after 3 h incubation with the isotope was found to
be inhibited with actinomycin by about 50% both in normal (diploid) and
'anucleate' embryos, when compared to respective untreated controls. This
suggests that at least a part of the 14CO2 incorporation into the siRNA of normal
early cleavage eggs represents DNA-dependent RNA synthesis. Furthermore
the result supports the proposal about the cytoplasmic location of early RNA
304
C. A. KAFIANI & OTHERS
synthesis since a comparable RNase and actinomycin-sensitive incorporation
can be demonstrated in embryos virtually devoid of nuclear genetic material.
The results obtained with actinomycin D should, however, be interpreted with
caution because of uncertainty of the actual concentration of the antibiotic in
the cells and its possible indirect effects.
DISCUSSION
The present paper gives some characteristics of RNA synthesis in early
embryogenesis of a teleost fish (Misgurnus fossilis).
Sucrose-gradient analysis showed that synthesis of ribosomal RNAs does
not occur to any significant extent at least until mid-gastrula stage, and the
embryos synthesize predominantly or exclusively polydisperse RNAs of a nonribosomal nature designated as dRNA. Selective inhibition of expression of
ribosomal cistrons seems to be a general phenomenon in early embryonic development since it has been demonstrated also in pre-gastrula stages of Xenopus
laevis (Brown & Gurdon, 1964; Brown & Littna, 1964, 1966) and sea urchins
(Glisin & Glisin, 1964; Nemer & Infant, 1965; Guidice & Mutolo, 1967).
A quantitative approach (Kafiani & Timofeeva, 1964) was applied here for
evaluating changes in the activity of the embryonic genome in transcription of
genetic information during pregastrular development. It consists of comparing
specific radioactivities of RNA of the eggs after pulsing them, at different stages,
under standard conditions, with a precursor (14CO2) which is rapidly incorporated into the free nucleotide pool. In view of the non-specific nature of the
precursor and possible terminal incorporation into tRNA, thoroughly purified
RNA preparations were used freed of tRNA and other contaminants by
repeated precipitations with sodium chloride. Such RNA preparations contained
rRNA and dRNA of which only the latter was labelled. Specific radioactivities
of the RNA preparations corresponded to the approximate rate of dRNA
synthesis and were considered a measure of 'dRNA-synthesizing activity' of
the embryos.
dRNA-synthesizing activity changes strikingly during the early development
of loach. It remains very low for the first 6 h of cleavage and increases abruptly
at 6-7 h of development (mid-blastula), suggesting operation of a trigger
mechanism. The large extent of the activation suggests, furthermore, that the
triggering involves a large part of the cell population.
The sharp activation of nuclear RNA synthesis in loach at the mid-blastula
stage has been shown previously (Kafiani & Timofeeva, 1964, 1965). In the
present paper additional evidence is given that this activation really involves
nuclear genetical material: (a) the increase in dRNA-synthesizing activity calculated per cell, and (b) the appearance of a correlation between the rate of dRNA
synthesis and ploidy of embryos.
Similar activation of RNA synthesis has been found in Rana pipiens
RNA synthesis in a fish embryo
305
(Bachvarova, Davidson, Allfrey & Mirsky, 1966) and in X. laevis (Brown &
Littna, 1966) at the late blastula stage, which correlates with the onset of morphogenetic activity (Neyfakh, 1964). In starfish, transcription of templates supporting gastrulation is reported to begin at early mid-blastula stage (Barros,
Hand & Monroy, 1966). An abrupt activation of RNA synthesis was found in
mouse embryos at the morula-early blastocyst stage (Monesi & Salfi, 1967).
Stimulation of genome transcription appears therefore to be a characteristic
event in early embryogenesis.
In loach, the activation of RNA synthesis coincides with important cytological
changes: the beginning of fusion of caryomeres (Pankova, 1963), and a drop
in the mitotic index and lengthening of mitotic cycle due to lengthening of
interphase (Rott & Sheveleva, 1967). Since RNA synthesis occurs mainly during
interphase our data are in accord with the cytological observations.
By the 7th h of development an important change in the developmental
potency of loach embryos occurs, the blastoderms becoming able to differentiate
in artificial media (Kostomarova & Neyfakh, 1964).
Finally, at the 6th h of development morphogenetic function of the cell
nuclei starts (Neyfakh, 1959). Concomitant activation of dRNA synthesis in
the nuclei is unlikely to be a coincidence and suggests that the beginning of
active gene transcription forms a biochemical basis for nuclear function in
directing gastrulation.
The correlation between the dRNA-synthesizing and morphogenetic activity
of the nuclei seems, however, to be confined to the time of their commencement.
Morphogenetic activity exhibits periodicity, the first period lasting for 2-^h
(from 6 to 8^ h) followed by an 'interruption of morphogenetic function' which
lasts until the 14th h. This is not so, however, with RNA synthesis, which goes
on with increasing speed after being switched in at the 6th h. The reasons for
the discrepancy between biological and biochemical function remain obscure.
As for the first period of morphogenetic inactivity of the nuclei (up to 6 h
postfertilization), the observed limited RNA synthesis seems to be at least partly
nuclear-independent. This view is supported by the fact that the rate of RNA
synthesis in early cleavage eggs correlates neither with cell number nor with
the ploidy of embryos. The 'early' RNA synthesis is presumed to occur in some
cytoplasmic DNA-containing structures. Such a possibility has been suggested
for anucleate halves of sea-urchin eggs (Baltus, Quertier, Ficq & Brachet, 1965).
Loach-egg cytoplasm contains sufficient DNA to support measurable RNA
synthesis, since as shown previously the DNA content of an egg is 6-10 x 10~3 fig
while that of a sperm cell is 2-4 x 10~6/*g, the 'extra' DNA corresponding to
the nuclear DNA of about 1000 diploid cells (Timofeeva & Kafiani, 1964). We
have, however, no direct data concerning the exact localization of nuclearindependent 'early' RNA synthesis nor are we able to answer the question
whether cell nuclei are totally inactive during early cleavage.
In conclusion, the data presented show that there are two clearly distin-
306
C. A. KAFIANI & OTHERS
guishable functional states of cell nuclei during early embryonic development
of loach. The nuclei are virtually inactive in transcription of genetic information
during early cleavage, and at this time they are also inactive morphogenetically.
At the mid-blastula stage the nuclei progress into another state characterized
by a high rate of gene transcription and morphogenetic activity.
SUMMARY
1. RNA synthesis during early embryogenesis of loach (Misgurnus fossilis)
was studied using 14CO2 incorporation into the 2 M-NaCl-precipitated fraction
of total embryonic RNA.
2. From fertilization until mid-gastrula stage the RNA preparations contain
label in non-ribosomal RNAs (dRNA) which have a high degree of size
heterogeneity.
3. A quantitative method was used for evaluating relative dRNA-synthesizing
activities of embryos at different stages of development by measuring specific
radioactivities of NaCl-precipitated RNA preparations isolated from eggs
pulsed with 14CO2 under standard conditions.
4. The dRNA-synthesizing activity is very low for early (synchronous) cleavage
stages and increases slowly compared to cell division. At this time the rate of
RNA synthesis is nearly equal in diploid and haploid embryos. A detectable
RNA synthesis occurs also in 'anucleate' embryos. It is concluded tentatively
that at least a part of the 'early' RNA synthesis occurs independently of the
cell nuclei, in some DNA-containing cytoplasmic structures.
5. At the mid-blastula stage, dRNA synthesis is sharply accelerated per
embryo as well as per cell, the rate of synthesis becoming directly dependent on
cell ploidy. It is concluded that after an initial period of cleavage an activation,
or onset of gene transcription, occurs in the cell nuclei which may be the basis
of their morphogenetic function.
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Mepe qacTb ,,paHHero" CHHTe3a P H K npoHCxoflHT He3aBHCHMO OT
H^ep, B KaKiix-TO ^HK-co^epmamHx D,PiTonjia3MaTHHecKHx CTpyKTypax.
5. Ha CTa^HM cpeflHeii SjiacTyjiti ciiHTes p;PHK pe3KO ycKopneTCH Kan B
pacqexe na sapojrbnn, Tan II Ha KJieTKy, npn^eivi TeMn CHHTe3a npnoSpeTaeT
npHMyio saBiiciiMOCTb OT njioiiaHOCTH KJieTOK. ^ejiaeTCH BbiBop;, HTO nocjie
iia i iajibnoro nepnofla p,po6jieHiiH B a^pax KJieTOK HMeeT MecTO aKTHBan;HH HJIH
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(Manuscript received 14 September 1967, revised 23 August 1968)