Histone synthesis during development of Triturus embryos

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 (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.

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.

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 μCi 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 expiants. 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.

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-HCl (pH 7 μ 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 (Imoh & 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.

Following the extraction of soluble protein and ribonucleo-protein with Tris-HCl (pH 7-6) and 0-14 M-NaCl in the cold, the basic proteins were extracted with 0-25 N-HCI 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 (f1), arginine- and alanine-rich (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.

The residue of nuclei after extraction of basic protein was washed and treated with pancreatic deoxyribonuclease (Worthington Biochem. Co., 1 × cryst.). 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.

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 24–25) 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 (f1) 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-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, blastula (sts 8b–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 (f1), arginine-rich (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 blastula stages (sts 8b–9), synthesis of histones occurred and the four peaks were observed, f1 or f2b + f2a2 as considerably high peaks and f3 or f2al as slightly positive ones. At the gastrula stage (st. 12), radioactivities in histones 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 f1.

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 f1 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 f1 histone in the relative composition shows wide fluctuation among four regions studied. The ratio of f1 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.

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 the very lysine-rich f1 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 f1) synthesis also doubled. It was also shown that the composition of the histone fractions except f1 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 f1 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 f1 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 f1 than mesodermal or endodermal tissues before the neurula stage. In the later stages of development the rate of f1 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 f1 histone consists of several subfractions (Nelson & Yunis, 1969; Panyim, Bilek & Chalkley, 1971), f1 histone synthesized in different tissues of embryos may belong to different subfractions. At any rate, the f1 histone was different from other histone fractions in its pattern of synthesis.