/. Embryol. exp. Morph. Vol. 30, 1, pp. 73-82, 1973
73
Printed in Great Britain
Historic synthesis during development
of Triturus embryos
By HIROSHI IMOH 1 AND IZUMI KAWAKAMP
From the Department of Biology, Miyazaki University,
and the Department of Biology, Kyushu University
SUMMARY
Synthesis of histone fractions and one basic protein fraction, which moved fast on gel
electrophoresis and had been reported to increase in nuclei accompanying a decrease in
cytoplasm during development, were studied with radioactive carbon dioxide as a tracer.
Acid-extractable proteins of nuclei or cytoplasm, isolated from labelled embryos, were
fractionated by polyacrylamide disc electrophoresis and the histone fractions and the fastmoving basic protein were identified. Radioactivities in these fractions and DNA were
determined.
Synthesis of the fast-moving basic protein was not detected throughout the period of
development studied and this fraction was thought to move in from the cytoplasm to the
nucleus during development. Syntheses of histone fractions were observed as early as the
blastula stage. Rates of syntheses of four histone fractions (f3, f2b, f2a2 and f2al) per
embryo increased thereafter, keeping pace with the increase in the rate of DNA synthesis
with advancing development. The rate of the very lysine-rich fl histone synthesis per embryo
did not increase after the gastrula stage and the rate remained almost constant until the late
tail-bud stage.
Compositions of newly synthesized histones, calculated from the radioactivities incorporated into histone fractions, were almost the same during development and among
different regions of neurula or tail-bud-stage embryos, with the exception of the f 1 fraction,
which varied depending on the stage and region of the embryos.
The results are discussed in relation to the possible roles of the histone fractions in
developing embryos.
INTRODUCTION
Studies on histones since their discovery have accumulated a great deal of
information on their natures. The functions of histones in metazoan cell nuclei,
however, still remain ambiguous and knowledge of histone synthesis is incomplete. Although it seems to be established that, in animal cells, histones are
synthesized in the cytoplasm on small polysomes and migrate into the nuclei
(Borun, Scharff & Robbins, 1967; Robbins & Borun, 1967; Nemer & Lindsay,
1969; Kedes, Gross, Cognetti & Hunter, 1969), further work is required to
determine whether histones are synthesized throughout development.
There are two major problems closely related to histone synthesis during
embryonic development. One is the possibility that histones might be supplied
from a cytoplasmic maternal store during development, especially in amphibians
1
Author's address: Department of Biology, Miyazaki University, Miyazaki 880, Japan.
2
Author's address: Department of Biology, Kyushu University, Fukuoka 812, Japan.
74
H. IMOH AND I. KAWAKAMI
(Horn, 1962; Asao, 1969). Examination of the synthesis of histone fractions
would give some information on this problem; and, as it has been reported that
during embryonic development in the Japanese newt a certain fraction of basic
protein (that which moved fastest on electrophoresis) showed a decrease in the
cytoplasm simultaneously with an increase in the nuclei (Imoh & Negami, 1972),
synthesis of this fraction must also be examined. The other major problem is the
possible existence of differences in the rate of synthesis among histone fractions.
Different histone composition among embryonic tissues has been reported by
Asao (1970), even though constancy of histone pattern among adult tissues has
been generally accepted.
In the present experiments, synthesis of basic proteins, histones and the fastmoving basic protein were investigated in whole newt embryos or in separated
embryonic tissues.
MATERIALS AND METHODS
Embryos of Triturus pyrrhogaster were used throughout the experiments and
they were staged according to the tables of Okada & Ichikawa (1947). Except
during labelling, embryos were kept at room temperature, 18-22°C.
Labelling of embryos and explants
In the experiments with whole embryos, 50 at each of the desired stages were
used. After removal of the vitellin membranes from the sterilized embryos, the
latter were labelled for 5 or 3 h with 40/<Ci of 14CO2 by the method originally
reported by Cohen (1954) and modified for newt embryos (Imoh, Sasaki,
Kawakami & Hayashi, 1972) at 22°C. Aseptic conditions were carefully maintained.
For experiments concerning the regionally of embryos, those in neurula
stages (sts 17-18) and late tail-bud stages (sts 31-32) were used. From 100
neurulae, belly ectoderm (Epidermis), neural plate and the chordamesodermal
mantle underlying the neural plate (Archenteron roof) were cut out as explants.
They were separately labelled under the same conditions as in the experiments
on whole embryos. Tail-bud embryos were labelled, also under the above
conditions, before dissection. Seventy labelled embryos were dissected into head
anterior to gill pouches (Head), epidermis of trunk and tail (Epidermis), endodermal mass (Endoderm), and tail and trunk without epidermis and endoderm,
i.e. tissues consisting of spinal cord, notochord, somites, and mesenchyme
(Trunk and tail).
The conditions of labelling, dose of radioisotope, duration of exposure and
temperature of labelling were carefully kept constant for every labelling. Apart
from this the relative amounts of the histone fractions synthesized from the
same precursor pool were considered in the present report. Thus possible
differences in the permeability of embryos to 14CO2, depending on stages and
regions, were not relevant.
Histone synthesis in Triturus
75
Isolation of nuclei
At the termination of labelling, embryos or tissue isolates were washed with
Holtfreter's solution and homogenized with 1 ml of 0-88 M sucrose solution in
Tris-HCI (pH 7-2) containing 3 mM-CaCl2. To the tissue isolates, unlabelled
embryos at the corresponding stages were added as a carrier before homogenization. From the homogenate, nuclei and cytoplasmic material, in the case of
whole embryo experiments, were isolated by centrifugation on a discontinuous
density gradient of sucrose (lmoh & Negami, 1972). In the preliminary experiments the DNA contents of the homogenate, the cytoplasmic fraction and the
nuclear fraction were determined by the diphenylamine method. Recovery of
DNA in the nuclear fraction was more than 85 %, and while the nuclear fraction
was almost free from contamination by yolk or cytoplasmic granules, the cytoplasmic fraction was contaminated with a small amount of aggregated nuclei or
unbroken intact cells.
Extraction and fractionation of nuclear basic proteins
Following the extraction of soluble protein and ribonucleo-protein with
Tris-HCI (pH 7-6) and 0-14 M-NaCl in the cold, the basic proteins were extracted
with 0-25 N - H C I for 1 h in the cold from isolated nuclei or from recovered
cytoplasmic material. The 0-01 ml aliquot of the sample was fractionated on
15 % polyacrylamide disc electrophoresis according to the method of Shepherd
& Gurley (1966) with glycine buffer (pH 4-0) instead of valine buffer. At the
termination of electrophoresis, gels were stained with amidoblack 10 B and
destained electrically.
Basic proteins from the nuclei consisted of unidentified slow-moving fractions,
histones, a few unidentified fractions which ran faster than histones, and a
fraction that ran fastest of all basic proteins which was referred to as the fastmoving basic protein. Histone was separated into five fractions and these were
identified by running marker histones which had been prepared from calf
thymus with the second method of Johns & Butler (1962). The five components
were, in order of increasing mobility, very lysine-rich (f 1), arginine- and alaninerich (f3), two slightly lysine-rich (lysine- and serine-rich f2b and alanine- and
leucine-rich f2a2), and arginine- and glycine-rich (f2al) histones.
For the determination of radioactivities in the protein bands, the gel was cut
into discs 2 or 1 mm wide and liquefied with 30 % H2O2 at 60°C. To the lysate
were added ethanol and toluene-scintillator to count in a liquid scintillation
counter. Preliminary experiments had shown that these procedures gave reproducible and quantitative results.
Measurement of DNA synthesis
The residue of nuclei after extraction of basic protein was washed and treated
with pancreatic deoxyribonuclease (Worthington Biochem. Co., 1 x cryst).
76
H. IMOH AND I. KAWAKAMI
1000 -
20
30
40
50
Slice number
Fig. 1. Cytological localization of newly synthesized basic protein. Basic protein
extracted with 0-25 N-HCI from isolated nuclei or cytoplasm of labelled embryos
(40/tCi 14CO2 for 5 h, 50 embryos) was electrophoretically fractionated and stained
with amidoblack 10B. Sample applied to each gel corresponded to ten embryos.
After the destained gel was sliced into 2 mm-width pieces and liquefied with 30 %
H2O2, radioactivities were counted in a liquid scintillation counter. For peak
number I and II, see the text, (a) Gastrula, (b) tail-bud stage. Abscissae, slice
number; ordinates, radioactivity in cpm. O
O, Nuclear fraction; •
#,
cytoplasmic fraction.
Radioactivity in DNA was calculated as the difference of radioactivities in the
cold trichloroacetic-acid-insoluble fraction before and after the enzyme treatment. It had been established, in the preliminary experiment using the method
of Schmidt & Thannhauser (1945), that the DNase treatment of de-histoned
nuclei rendered more than 90 % DNA acid-soluble.
RESULTS
Syntheses of basic protein in the whole embryo during development
Fig. 1 shows patterns of radioactivities revealed by electrophoresis of nuclear
and cytoplasmic basic proteins labelled at gastrula (st. 12) or at tail-bud (sts 2425) stages. There were two main peaks, I and II, both high in patterns of nuclear
basic protein and low in cytoplasmic patterns. The peak I represents the very
lysine-rich (fl) histone and the peak II includes arginine-rich (f3) and slightly
lysine-rich (f2a and f2b) histones. Under the electrophoretic conditions used,
the fast-moving basic protein localized at slice numbers 48-52, where radio-
77
His tone synthesis in Triturus
50
10
20
30
40
10
20
30
40
Slice number
Fig. 2. Electrophoretic pattern of basic protein newly synthesized in developing
embryos. Nuclei were isolated from embryos labelled with 40/tCi of 14CO2 for 3 h.
Basic protein extracted from the nuclei was fractionated on polyacrylamide disc
electrophoresis. Sample applied to each gel corresponded to ten embryos. The
gel was stained, electrically destained, and sliced into 1 mm pieces. Radioactivity,
in the slice liquefied with H2O2 was counted in a scintillation counter, (a) Blastula,
(b) gastrula, (c) neurula, and (<-/) tail-bud stage embryos. Abscissae, slice number;
ordinates, radioactivity in cpm.
78
H. IMOH AND I. KAWAKAMI
8000
800
2 600
6000 t
• i 400
4000 •£
o
•5
200
2000
2
I
4
1
1
1
1
9b
12
IS
25
"•&•
• " " " " '
i
8
i
6
Davs
Stauc
Fig. 3. Histone and D N A syntheses during development. Embryos were labelled
with 40 /*Ci of 14 CO 2 at biastula (sts 8 6-9), gastrula (st. 12), neurula (sts 17-18), or
tail-bud stage (sts 24-25) for 3 h. Radioactivity incorporated in each histone
fraction or DNA was determined as described in the text. Abscissa, days after the
eggs were laid or stage; ordinate, radioactivity in each histone or in DNA in cpm per
ten embryos. O
O, Very lysine-rich (f 1); A
A, arginine-rich (f3); A - - A,
slightly lysine-rich (f2b + f2a2); x - • - x , and arginine-rich (f2al) histone
fractions; •
# , DNA.
activities were not observed. Actually, it should be stressed that synthesis
of the fast-moving basic protein was not detected at any developmental stages
studied - that is, biastula (sts 86-9), gastrula (st. 12), neurula (sts 17-18) and
tail-bud (sts 24-25) stages.
For study of the synthesis of histone fractions, 50 embryos at a particular
stage of development were chosen and labelled. From their isolated nuclei, the
basic proteins were extracted and radioactivities in the fractions of basic
proteins were determined after electrophoresis. The results are shown in Fig. 2.
The amount of the sample used for electrophoresis corresponded to ten embryos
at every stage. In Fig. 2 the four peaks observable in slice numbers 17-32 show
radioactivities incorporated in histones. They are very lysine-rich (f 1), argininerich (f3), slightly lysine-rich (f2b and f2a2 as one peak), and arginine-rich (f2al)
histones from left to right. Several peaks which ran faster or slower than
histones were observed though their natures have not been identified.
At biastula stages (sts 86-9), synthesis of histones occurred and the four peaks
were observed, fl or f2b + f2a2 as considerably high peaks and f3 or f2al as
slightly positive ones. At the gastrula stage (st. 12), radioactivities in histones
250
175
245
90
760
cpm
305
100
0-70
100
0-35
Ratiot
320
210
360
80
970
cpm
2-73
0-88
0-60
100
0-25
Ratiot
Archenteron roof
780
275
185
250
70
cpm
110
0-74
100
0-30
314
Ratiot
Neural plate
251
845
570
855
240
cpm
0-99
0-67
100
0-30
2-96
Ratiot
Total
1-25
0-63
1 00
0-30
318
50
80
25
255
Ratiot
100
cpm
125
280
310
100
815
0-56
100
0-30
2-82
75
135
40
380
cpm
0-96
Ratiot
130
cpm
Endoderm
0-32
2-62
100
0-90
0-40
Ratiot
]Head
105
1360
540
265
450
cpm
0-20
2-53
100
0-50
0-83
Ratiot
Trunk and tail
270
2810
1065
670
805
cpm
0-25
2-63
100
0-63
0-75
Ratiot
Total
* Figures are the mean of two determinations. The deviation of each value fronl the mean was less than ± 1 0 % of the mean value.
t Ratio when radioactivities in lysine-rich (f2b + f2a2) histones are taken arbitrarily as unity.
Very lysinerich (fl)
Argininerich (f3)
Lysine-rich
(f2b + f2a2)
Argininerich (f2al)
Total
Histone fraction
Epidermis
A .
Regions
Table 2. Radioactivities incorporated in each histone fraction and their relative composition
in four regions of tail-bud embryos*
* Figures are the means of two determinations. The deviation of each value from the mean was less than ± 10 % of the mean value.
t Ratio when radioactivities in lysine-rich (f2b + f2a2) histones are arbitrarily taken as unity.
Very lysine-rich (fl)
Aiginine-rich (f3)
Lysine-rich (f2b + f2a2)
Arginine-rich (f2al)
Total
Histone fraction
Epidermis
Regions
Table 1. Radioactivities incorporated in each histone fraction and their relative composition
in three regions ofneurula embryos*
51
d
B
H
5»"
80
H. IMOH AND I. KAWAKAMI
were much higher than those at the blastula stages and those in basic proteins
other than histones became positive at the gastrula stage. Radioactivities in
histones except fl increased further through neurula (sts 17-18) to tail-bud (sts
24-25) stages. Incorporation of radioactivity in fl did not increase after the
gastrula stage. From the figure, the radioactivities in each histone fraction were
calculated and mean values for each fraction were obtained from three independent but identical analyses. The deviation of each value from the mean was less
than ± 7 % of the mean value. The results are shown in Fig. 3 together with
those for DNA. It is quite apparent that patterns of increase in the rate of
synthesis are identical for the DNA and histone fractions other than fl.
Regional difference in composition of newly synthesized histones
Radioactivities incorporated into each histone fraction at regions of neurula
(sts 17-18) or late tail-bud stage (sts 31-32) embryos were calculated as described
above and the relative composition of newly synthesized histones was calculated
by taking radioactivities in slightly lysine-rich histones (f2b + f2a2) as unity.
Mean values of two experiments are shown in Table 1 and Table 2. Table 1
shows the results for three regions of neurula embryos. Though the ratio of f 1 in
archenteron roof is slightly lower than that in the other regions, there are no
conspicuous differences in the composition among three regions studied.
The results of similar experiments on four regions of tail-bud embryos are
shown in Table 2. It is noticed that the ratio of fl histone in the relative composition shows wide fluctuation among four regions studied. The ratio of fl
became lower, in the order: epidermis, endoderm, trunk and tail, and head. On
the other hand, the relative composition was rather constant among regions for
f3, f2b + f2a2, and f2al, though the ratio of f3 in the head region is exceptionally
high.
DISCUSSION
From the appearance of histones in the nucleus during development, the
transfer of intact histones from a cytoplasmic 'maternal pool' has been suggested (Horn, 1962; Asao, 1969). The hypothesis, however, was not well
grounded in that some observations were not supplemented by the identification of the transferring materials as histones; and others lacked proof of
transfer from cytoplasm to the nucleus. Imoh & Negami (1972) reported that
cytoplasmic basic protein having identical electrophoretic mobilities with
histones was too small in amount to cover the increase in the nuclei during
early development, while a fraction of basic protein, which moved fastest of all
basic proteins on electrophoresis and was referred to as the fast-moving basic
protein, increased in the nuclei when the content of identical basic protein
decreased in the cytoplasm during development. The present data show that
histones were synthesized in the newt embryo as early as the blastula stage and
histone synthesis was closely related to DNA synthesis, with the exception of
Histone synthesis in Triturus
81
the very lysine-rich fl histone. Synthesis of the fast-moving basic protein was
detected neither in cytoplasm nor in nuclei at any of the developmental stages
studied.
In view of the fact cited above, the absence of cytoplasmic basic protein
having identical electrophoretic mobilities with histones, and the present
observation that histones were synthesized in embryos as early as the blastula
stage, it is suggested that histones are not supplied from a 'maternal pool' but
are synthesized probably in a manner correlated with DNA synthesis. It is also
suggested that a fraction of basic protein, which runs fast on gel electrophoresis,
may move in from cytoplasm to nucleus during development.
It was observed that when the rate of DNA synthesis per embryo increased
with development, the rate of slightly lysine-rich (f2b + f2a2) or arginine-rich
(f3, f2al) histone synthesis also increased and the relationship between DNA
and the histones was almost linear; that is, when the rate of DNA synthesis
doubled, the rates of histone (except fl) synthesis also doubled. It was also
shown that the composition of the histone fractions except f 1 was relatively
constant among regions of neurula or tail-bud-stage embryos. From these
observations it seemed probable that newly synthesized DNA was always
supplied with constant amounts of the histones f2a2, f3 and f2al, irrespective of
the developmental stages or regions of the embryos. This suggestion may be
best understood on the assumption that these histones are the invariable
structural elements of nuclear components containing DNA, i.e. of chromatin.
The rate of fl histone synthesis per embryo was highest of all histones before
neurulation but did not increase after the gastrula stage as repeatedly confirmed,
although the rate of DNA synthesis per embryo increased with advancing
development. As a result, cells which proliferate before neurulation would be
expected to show a higher f 1 content in their nuclei than cells which proliferate
after neurulation. This expectation was in good agreement with the results of
Asao (1970) that ectodermal neural plate or epidermis showed a higher content
of f 1 than mesodermal or endodermal tissues before the neurula stage. In the
later stages of development the rate of fl synthesis differed among tissues;
neural plate was slightly higher than other tissues at the neurala stage and
epidermis showed the highest rate while head tissue was lowest in tail-bud
embryos. As it has been reported that f 1 histone consists of several subfractions
(Nelson & Yunis, 1969; Panyim, Bilek & Chalkley, 1971), fl histone synthesized
in different tissues of embryos may belong to different subfractions. At any
rate, the f 1 histone was different from other histone fractions in its pattern of
synthesis.
E M B 30
82
H. IMOH AND I. KAWAKAMI
REFERENCES
ASAO, T. (1969). Behavior of histones and cytoplasmic basic proteins during embryogenesis
of the Japanese newt, Triturus pyrrhogaster. Expl Cell Res. 58, 243-252.
ASAO, T. (1970). Regional histone changes in embryos of the newt, Triturus pyrrhogaster,
during development. Expl Cell Res. 61, 255-265.
BORUN, T. W., SCHARFF, M. D. & ROBBINS, E. (1967). Rapidly labelled polysome-associated
RNA having the properties of histone messenger. Proc. tmtn. Acad. Sci. U.S.A. 58, 1977—
1983.
14
COHEN, S. (1954). The metabolism of CO2 during amphibian development. J. biol. Chem.
211, 337-354.
HORN, E. (1962). Extranuclear histone in the amphibian oocyte. Proc. natn. Acad. Sci. U.S.A.
48, 257-265.
IMOH, H. & NEGAMI, M. (1972). Studies on basic proteins in developing embryos. 1. Existence
of small basic protein which moves from cytoplasm to nucleus. Sci. Rep. Miyazaki Univ.
31, 1-7.
IMOH, H., SASAKI, N., KAWAKAMI, I. & HAYASHI, H. (1972). Changes in macromolecular
synthesis in Triturus presumptive epidermis stimulated by inducing agent. J. exp. Zool.
182, 31-40.
JOHNS, E. W. & BUTLER, J. A. V. (1962). Further fractionation of histones from calf thymus.
Biochem. J. 82, 15-18.
KEDES, L. H., GROSS, P. R., COGNETTI, G. P. & HUNTER, A. L. (1969). Synthesis of nuclear
and chromosomal proteins on light polyribosomes during cleavage in the sea urchin
embryo. /. mol. Biol. 45, 337-351.
NELSON, R. D. & YUNIS, J. J. (1969). Species and tissue specificity of very lysine-rich and
serine-rich histones. Expl Cell Res. 57, 311-318.
NEMER, M. & LINDSAY, D. T. (1969). Evidence that the s-polysomes of early sea urchin
embryos may be responsible for the synthesis of chromosomal histones. Biochem. biophys.
Res. Commun. 35, 156-160.
OKADA, Y. & ICHJKAWA, M. (1947). Normal table of normal developmental stages of
Triturus pyrrhogaster. Jap. J. exp. Morph. 3, 1-6.
PANYIM, S., BILEK, D. & CHALKLEY, R. (1971). An electrophoretic comparison of vertebrate
histones. /. biol. Chem. 246, 4206-4215.
ROBBINS, E. & BORUN, T. W. (1967). The cytoplasmic synthesis of histones in HeLa cells and
its temporal relationship to DNA replication. Proc. natn. Acad. Sci. U.S.A. 57, 409-416.
SCHMIDT, G. & THANNHAUSER, S. J. (1945). A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animals tissues. /. biol. Chem. 161,
83-91.
SHEPHERD, G. R. & GURLEY, L. R. (1966). High-resolution disc electrophoresis of histones.
1. An improved method. Analyt. Biochem. 14, 356-363.
{Received 5 December 1972)