In order to determine whether or not a crucial number of DNA replications are prerequisite for cellular differentiation, we have studied development of a tissue-specific enzyme, muscle acetylcholinesterase (AChE) in the presumptive muscle cells of cleavage-arrested ascidian embryos. Embryos were cleavage-arrested with cytochalasin B (an inhibitor of cytokinesis) and aphidicolin (an inhibitor of DNA synthesis). The 64-ceIl-stage embryos which had been permanently cleavage-arrested with cytochalasin B developed AChE in all the eight presumptive muscle cells, but the same stage embryos which had been prevented from undergoing further divisions by simultaneous treatment with aphidicolin and cytochalasin did not produce AChE at all. Cytochalasin-arrested 76-cell-stage embryos were able to differentiate AChE in the ten presumptive muscle cells, while aphidicolin-cytochalasin-arrested 76-cell stages in as many as four cells. The early gastrulae which had been arrested with cytochalasin B produced AChE in all the sixteen presumptive muscle cells, while the same stage embryos arrested with aphidicolin and cytochalasin in as many as twelve cells. Cytochalasin-arrested late gastrulae developed AChE in twenty blastomeres, while aphidicolin-cytochalasin-arrested late gastrulae in eighteen cells.

The presumptive muscle cells at these four stages consist of those of three different (seventh, eighth, and ninth) generations, and the relative positions of the blastomeres in the cleavage-arrested embryos remained fixed. Judging from the relative positions of the blastomeres, the AChE-producing cells in aphidicolin-cytochalasin-arrested embryos were always at eighth or ninth generation, while those with no AChE activity were certainly at seventh generation. Based on these findings it was supposed that aphidicolin-sensitive cell-cyclic events, presumably DNA replication, are closely associated with AChE development and that the eighth cleavage cycle may be ‘quantal’ for the histospecific enzyme development.

At the time a cell line is segregated it is made up of a small number of cells, far smaller than the number of cells eventually to constitute the final tissue or organ. Hence the lineage cells are bound to divide a number of times before the phenotypic character of the differentiated state is overtly expressed. This raises the important question as to whether or not the lineage cells have to undergo a definite number of DNA replication cycles or a definite number of cell divisions in order to express their differentiated condition. Since the answer to this question may have important implications for the interpretation of the regulatory mechanisms of gene expression in differentiation, the roles and significance of DNA replication in cellular differentiation are worthy of intense scrutiny (Holtzer, Weintraub, Mayne & Mochan, 1972; Rossi, Augusti-Tocco & Monroy, 1975).

Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of the tail of the developing ascidian embryos (Durante, 1956; Whittaker, 1973; Ohmori & Sasaki, 1977; Meedel & Whittaker, 1979). AChE activity is first detected histochemically and biochemically in the presumptive muscle cells of the peurula, and the enzyme activity increases dramatically with development time (Whittaker, 1973; Meedel & Whittaker, 1979). An inhibitor of protein synthesis (puromycin) prevents the occurrence of AChE activity in embryos treated continuously with it from the neural-plate stage onwards, while those treated continuously with it from the neurula stage onwards develop slight traces of enzyme activity (Whittaker, 1973, 1977; Meedel & Whittaker, 1979; Satoh, 1979a). Since this puromycin-sensitivity period coincides with the time that the enzyme is first detected, embryos probably begin to synthesize the enzyme at this time (cf. Fig. 1). Actinomycin D inhibits the development of AChE in embryos reared in the drug from the early gastrula stage onwards, while those from the late gastrula stage onwards eventually develop a low level of the enzyme activity (Whittaker, 1973, 1977; Meedel & Whittaker, 1979; Satoh, 1979a). This result suggests that new RNA synthesis, presumably including mRNA synthesis, which begins the early and late gastrula stages is needed for AChE development (Fig. 1). Since embryos which are permanently cleavage-arrested with cytochalasin B or with colchicine can develop the enzyme, neither cytokinesis of the blastomeres nor the so-called nuclear division is required for AChE development (Whittaker, 1973; Satoh, 1979a; Satoh & Ikegami, 1980).

Fig. 1.

Diagram summarizing acetylcholinesterase development in the presumptive muscle cells of the ascidian embryo. (Upper) Relationship between the time of first synthesis of acetylcholinesterase, its actinomycin D-sensitivity period, aphidicolin-sensitivity period and the embryonic stages of Halocynthia roretzi. (Middle) The number of presumptive muscle cells examined in cleavage-arrested embryos. (Lower) The timing and frequency of cell divisions in the muscle lineage blastomeres (left half embryo). The division of B8-15 to B9-29 and B9-30 is arbitrary. Vertical dotted lines indicate the presumptive muscle cells at the four stages subjected to the present inquiry. Constructed according to data of Conklin (1905), Ortolani (1955), Whittaker (1973), Satoh (1979a, b), Satoh & Ikegami (1980) and the present study. The lineage of muscle cells is discussed in the text.

Fig. 1.

Diagram summarizing acetylcholinesterase development in the presumptive muscle cells of the ascidian embryo. (Upper) Relationship between the time of first synthesis of acetylcholinesterase, its actinomycin D-sensitivity period, aphidicolin-sensitivity period and the embryonic stages of Halocynthia roretzi. (Middle) The number of presumptive muscle cells examined in cleavage-arrested embryos. (Lower) The timing and frequency of cell divisions in the muscle lineage blastomeres (left half embryo). The division of B8-15 to B9-29 and B9-30 is arbitrary. Vertical dotted lines indicate the presumptive muscle cells at the four stages subjected to the present inquiry. Constructed according to data of Conklin (1905), Ortolani (1955), Whittaker (1973), Satoh (1979a, b), Satoh & Ikegami (1980) and the present study. The lineage of muscle cells is discussed in the text.

As to the significance of DNA synthesis, aphidicolin (an inhibitor of DNA synthesis) prevents the development of AChE in embryos treated continuously with it from the 64-cell stage onwards, but those from the 76-cell stage onwards eventually differentiate the enzyme, suggesting that DNA replications might be prerequisite for the histospecific enzyme development (Satoh & Ikegami, 1980; Fig. 1). The aim of the present study is to determine whether or not a crucial number of DNA replication cycles are needed for the enzyme development. Fortunately, muscle AChE development of ascidian embryos offers a very advantageous experimental system for studying this question. The presumptive muscle cells of the gastrula consist of those of three different generations. If, in the gastrulae which are prevented from undergoing further DNA replication by continuous treatment with aphidicolin, the presemptive muscle cells of a certain generation do not develop AChE activity, but the cells of the next generation produce the enzyme, then the critical number of DNA replications which are inevitable for the enzyme synthesis must be determined. In other words, if AChE development is closely associated with a definite number of DNA replication cycles, both presumptive muscle cells with AChE activity and those without AChE activity will appear in one gastrula which is treated with aphi-dicolin.

Embryos. Halocynthia roretzi (Drasche) was obtained during two breeding seasons (November through April) in the vicinity of the Marine Biological Station of Asamushi, Aomori, Japan. Naturally spawned eggs, about 280 μm in diameter, were fertilized artificially and reared in filtered sea water at 15 ±0·2 °C using a constant temperature water bath. Only batches in which cleavage occurred in more than 95 % of the eggs were used. Development of eggs from different animals fertilized at the same time is essentially synchronous. The timing of the developmental stages at 15 °C is shown in Fig. 1.

Enzyme histochemistry. Acetylcholinesterase activity was detected histochemically in whole embryos by the direct-colouring thiocholine method of Karnovsky & Roots (1964). During Halocynthia embryogenesis histochemical staining of AChE activity was first detected at 12 h of development, and staining intensity of the cells increased dramatically with development time (Satoh, 1979a). Since enzyme activity in cleavage-arrested embryos was examined usually at 26–28 h of development, the distinction between occurrence and non-occurrence of AChE reaction was clear enough to exclude the possibility of misjudgement (Fig. 2). In addition, because the presumptive muscle cells of the gastrulae are large, about 50 μm in diameter, the number of AChE-producing blastomeres could be counted exactly using a dissecting microscope.

Fig. 2.

Acetylcholinesterase (AChE) development in the presumptive muscle cells of cleavage-arrested embryos, (a, c, e, and g) AChE activity in cytochalasin-arrested embryos. Eight blastomeres of the arrested 64-cell stage (a), ten cells of the arrested 76-cell stage (c), sixteen blastomeres of the arrested early gastrula (e), and twenty cells of the arrested late gastrula (g), respectively, develop AChE activity, (b, d,f, and h) AChE development in embryos cleavage-arrested by simultaneous treatment with aphidicolin and cytochalasin B. The arrested 64-cell stage does not develop AChE activity (b). However, four cells of the arrested 76-cell stage (d), twelve blastomeres of the arrested early gastrula (f), and eighteen cells of the arrested late gastrula (A), respectively, produce the enzyme. The four AChE-containing cells of the arrested 76-cell stage consist of two groups of cells apart from each other (d), and the twelve reacting blastomeres of the arrested early gastrula two clusters of cells between which some cells of no-AChE activity are always present (f). Embryos which are treated with aphidicolin and cytochalasin do not become so flattened as those arrested with cytochalasin.

Fig. 2.

Acetylcholinesterase (AChE) development in the presumptive muscle cells of cleavage-arrested embryos, (a, c, e, and g) AChE activity in cytochalasin-arrested embryos. Eight blastomeres of the arrested 64-cell stage (a), ten cells of the arrested 76-cell stage (c), sixteen blastomeres of the arrested early gastrula (e), and twenty cells of the arrested late gastrula (g), respectively, develop AChE activity, (b, d,f, and h) AChE development in embryos cleavage-arrested by simultaneous treatment with aphidicolin and cytochalasin B. The arrested 64-cell stage does not develop AChE activity (b). However, four cells of the arrested 76-cell stage (d), twelve blastomeres of the arrested early gastrula (f), and eighteen cells of the arrested late gastrula (A), respectively, produce the enzyme. The four AChE-containing cells of the arrested 76-cell stage consist of two groups of cells apart from each other (d), and the twelve reacting blastomeres of the arrested early gastrula two clusters of cells between which some cells of no-AChE activity are always present (f). Embryos which are treated with aphidicolin and cytochalasin do not become so flattened as those arrested with cytochalasin.

Cleavage inhibition. Cytochalasin B (Aldrich Chem. Co.) and aphidicolin were used as cleavage inhibitors. Cytochalasin B (2 μg/ml) completely prevents cytokinesis of the blastomeres, but the nucleus in the cell of cytochalasin-treated embryos divides in good synchrony with that of normal embryos. Aphidicolin at 2 μg/ml blocks ascidian development, presumably due to inhi-bition of DNA synthesis by interfering with the activity of DNA polymerase-a (Ikegami et al. 1978). But aphidicolin allows one more cleavage in eggs kept in aphidicolin and stops divisions thereafter, no matter how soon a cleavage it is applied (Satoh & Ikegami, 1980). In cytochalasin-arrested embryos the relative positions of the blastomeres remain fixed. The presumptive muscle cells of aphidicolin-arrested gastrulae, however, do not remain stationary as they do in cytochalasin-arrested gastrulae, but tend to migrate the inside of an embryo. Therefore, it was difficult to count precisely the number of AChE-producing blastomeres in aphidicolin-arrested embryos (Satoh & Ikegami, 1980). In this study, to get rid of this difficulty, aphidicolin was always used together with cytochalasin B.

Twelve separate experimental series of total 829 embryos were examined.

A ChEdevelopment in embryos cleavage-arrested with cytochalasin B andfrequency of cell divisions in the muscle lineage blastomeres

Whittaker (1973) has successfully shown that ascidian embryos which are permanently cleavage-arrested with cytochalasin B differentiate AChE activity only in their muscle lineage blastomeres. The maximum numbers of AChE-producing blastomeres at each cleavage-arrested stage are one at 1-cell stage, two at 2-cell stage, two at 4-cell stage, two at 8-cell stage, four at 16-cell stage, six at 32-cell stage, and eight at 64-cell stage, respectively. Applying this method to the later stages, the numbers of presumptive muscle cells at the 64-cell stage, 76-cell stage, early gastrula stage, and late gastrula stage were determined respectively. As shown in Table 1, all of cytochalasin-arrested 64-cell and later stages developed the enzyme in some blastomeres. Cytochalasin-arrested 64-cell-stage embryos produced AChE usually in eight blastomeres (Figs 2a, 3d), and in arrested 76-cell stages as many as ten blastomeres could be found differentiating the enzyme (Figs 2 c, 3 b). Cytochalasin-arrested early gastrulae usually developed AChE in sixteen blastomeres (Figs 2e, 3 c), and arrested late gastrulae formed the enzyme in up to twenty blastomeres (Figs 2 g, 3d).

Table 1.

Acetylcholinesterase development in embryos cleavage-arrested with cytochalasin B or by simultaneous treatment with aphidicolin and cytochalasin B

Acetylcholinesterase development in embryos cleavage-arrested with cytochalasin B or by simultaneous treatment with aphidicolin and cytochalasin B
Acetylcholinesterase development in embryos cleavage-arrested with cytochalasin B or by simultaneous treatment with aphidicolin and cytochalasin B

The cell lineage for larval muscle-cell development in ascidian embryos has been studied (Conklin, 1905; Ortolani, 1955; Mancuso, 1969; Whittaker, 1973). Based on Conklin’s description (1905), Ortolani’s correction (1955) and the present result, the timing and frequency of cell divisions in the muscle lineage blastomeres are summarized in Fig. 1. As is noticeable in Fig. 1, eight presumptive muscle cells of the 64-cell-stage embryo are all at the seventh generation, counting the unsegmented egg as the first generation according to Conklin (1905). The presumptive muscle cells of the 76-cell-stage embryo, early gastrula and late gastrula, however, consist of those of different generations; i.e. the 76-cell-stage embryo has ten presumptive muscle cells of two different (seventh and eighth) generations, while the early and late gastrulae those of three different (seventh, eighth, and ninth) generations.

An early gastrula which contains 16 presumptive muscle cells of three different generations is shown in a scanning electron micrograph (Fig. 4; Satoh, 1979b).

Fig. 3.

Frequency of embryos relating to the number of blastomeres containing acetylcholinesterase.

Fig. 3.

Frequency of embryos relating to the number of blastomeres containing acetylcholinesterase.

Fig. 4.

A scanning electron micrograph of an early gastrula of Halocynthia roretzi showing that sixteen presumptive muscle cells consist of those of seventh (B7·5 and B7·6), eighth (B8·15 and B8·16), and ninth (B9·13, B9·14, B9·15, and B9·16) generations. (From Satoh, 1979 b.)

Fig. 4.

A scanning electron micrograph of an early gastrula of Halocynthia roretzi showing that sixteen presumptive muscle cells consist of those of seventh (B7·5 and B7·6), eighth (B8·15 and B8·16), and ninth (B9·13, B9·14, B9·15, and B9·16) generations. (From Satoh, 1979 b.)

AChE development in embryos cleavage-arrested by simultaneous treatment with aphidicolin and cytochalasin B

The 64-cell-stage embryos which had been permanently cleavage-arrested with aphidicolin and cytochalasin did not develop AChE activity at all (Table 1, Figs 2b, 3a).

The 76-cell-stage embryos treated with these drugs, however, developed a distinct enzyme reaction in about 40 % of the embryos, although the number of reacting embryos varied from one batch of eggs to another (Table 1, Fig. 2d). There was a range in the number of enzyme-producing blastomeres, but about a half of the reacting embryos produced four enzyme-containing blastomeres (Figs 2d, 3 b). The four AChE-producing cells usually consisted of two clusters of blastomeres, each with two cells, apart from each other (Fig. 2d).

All of the cleavage-arrested embryos in the early and late gastrula stages developed AChE activity (Table 1), but the number of AChE-producing blastomeres in aphidicolin-cytochalasin-arrested gastrulae was quite different from that of cytochalasin-arrested gastrulae (Fig. 3 c, d). In most of aphidicolin-cytochalasin-arrested early gastrulae twelve blastomeres could be found differentiating AChE (Figs 2 f, 3 c). The twelve blastomeres, almost without exception, consisted of two rows of blastomeres, each containing six cells, between which several cells with no AChE activity were present (Fig. 2f). Aphidicolin-cytochalasin-arrested late gastrulae usually produced AChE in as many as eighteen blastomeres (Figs 2h, 3d).

Interpretation of the results

Cytochalasin B completely prevents cytokinesis of the blastomeres, but not nuclear division or DNA replication. Aphidicolin blocks ascidian development, but allows one more cleavage in eggs kept in aphidicolin and stops divisions thereafter; i.e. if embryos are treated with aphidicolin soon after ‘n-l’th division, ‘n’th division occurs but not ‘w + l’th division. This may presumably be due to an overlap in time of ‘n -1’th mitosis with ‘n’th S-phase. However, aphidicolin must block ‘n + l’th DNA synthesis, because ‘n + l’th division is blocked with it.

The 64-cell stage

As shown in Figs 1 and 5 a, eight presumptive muscle cells of the 64-cell-stage embryos are all at the seventh generation (i.e. the cells have divided six times up to this stage). The 64-cell-stage embryos which had been permanently cleavage-arrested with cytochalasin B developed AChE activity in the eight blastomeres, while the same stage embryos which had been continuously arrested with aphidicolin and cytochalasin B did not develop AChE at all. It is probable that seventh DNA replication might be completed in the aphidicolin-cytochalasin-arrested blastomeres (Fig. 5 a). Therefore, the result seems to indicate that seven times DNA replications are not enough for AChE development.

Fig. 5.

Illustration demonstrating the relationship between the number of DNA replication cycles and acetylcholinesterase (AChE) development in the presumptive muscle cells at three embryonic stages of the ascidian embryos. The numerals within blastomeres indicate the supposed number of DNA replications. Blastomeres containing AChE activity are dotted. See the text for details.

Fig. 5.

Illustration demonstrating the relationship between the number of DNA replication cycles and acetylcholinesterase (AChE) development in the presumptive muscle cells at three embryonic stages of the ascidian embryos. The numerals within blastomeres indicate the supposed number of DNA replications. Blastomeres containing AChE activity are dotted. See the text for details.

The 76-cell stage

Among ten presumptive muscle cells of this stage, B8·7, B8·7, B8·8, and B8·8 are at the eighth generation, while the other cells are at the seventh generation (Figs 1, 5 b). The 76-cell-stage embryos which had been arrested with cytochalasin B developed AChE activity usually in ten blastomeres. On the other hand, the same stage embryos which had been arrested by simultaneous treatment with aphidicolin and cytochalasin produced enzyme in as many as four blastomeres. Judging from the relative positions of the AChE-producing blastomeres in aphidicolin-cytochalasin-arrested embryos, the reacting cells must be B8·7, B8·7, B8·8, and B8·8 (Fig. 5b)=. These four blastomeres might have finished eighth DNA replication cycle, while the six other cells might have completed but seventh DNA replication (Fig. 5b). Therefore, it is likely that eight times DNA replications may lead to develop AChE in the presumptive muscle cells.

The early gastrula

The early gastrula has sixteen presumptive muscle cells of three different generations; B7·5, B7·5, B7·6, and B7·6 are on the seventh generation, B8·15, B8·15, B8·16, and B8·16 on the eighth generation, and B9·13, B9·13, B9·14, B9·14, B9·15, B9·15, B9·16, and B9·16 on the ninth generation, respectively (Figs 1, 4, 5 c). The early gastrulae which had been arrested with cytochalasin B were able to differentiate AChE activity in all these sixteen blastomeres. If the early gastrulae are prevented from further development by concomitant treatment with aphidicolin and cytochalasin B, the arrested embryos form 12 AChE-producing blastomeres. Judging from their relative positions of the AChE-containing blastomeres in aphidicolin-cytochalasin-arrested embryos, it is sure that the presumptive muscle cells of eighth and ninth generations developed AChE activity, while the cells of seventh generation did not differentiate the enzyme (Figs 2f, 5 c). The cells of eighth and ninth generations might have completed eighth DNA replication in aphidicolin-cytochalasin-arrested embryos, while those of seventh generation seventh DNA replication. The result strongly suggests that eight times DNA replications are required for the enzyme development.

The late gastrula

The late gastrulae which had been arrested with cytochalasin B developed AChE activity usually in as many as twenty blastomeres, while the same stage embryos which had been arrested with aphidicolin and cytochalasin showed as many as eighteen AChE-containing blastomeres. Conklin (1905) described that B7·6 and B7·6 do not divide until at least the late gastrula stage. If B7·6 and B7·6 in aphidicolin-cytochalasin-arrested embryos do not develop AChE, thus one can explain the difference in the number of AChE-producing blastomeres between cytochalasin-arrested embryos and aphidicolin-cytochalasin-arrested embryos.

These results are consistent in implying that eight times DNA replications may be needed for AChE development in the presumptive muscle cells of the ascidian embryos.

Details of the lineage for larval muscle-cell development in ascidian embryos have not fully been established yet. Originally Conklin (1905) described that the blastomeres derived from B6-3 were mesenchyme cells. However, Ortolani (1955) followed the muscle cell lineage by marking the blastomeres with granules of coloured chalk and she concluded that the descendants of B6·3 (i.e. both B7·5 and B7·6) were muscle cells. On the other hand, based on the ultrastructural characteristics of the presumptive muscle cells such as existence of numerous mitochondria, Mancuso (1969) asserted that B7·5 was a muscle cell, while B7·6 was a mesenchyme cell. The results of the present study, support Ortolani’s proposal (1955), and the interpretation of the results has been done according to it.

A significant concept in cellular differentiation is that a ‘quantal cell cycle’, proposed by Holtzer and his co-workers (Holtzer et al. 1972, 1975). The basic tenet of this model is that a unique cell-cyclic event, called a quantal cell cycle, occurs when a cell becomes programmed from an undifferentiated to a differentiated state. During this quantal cell cycle, extensive changes in the genetic programme can occur which are needed for differentiation. The decision to differentiate is presumably made during a critical S phase of the cell cycle. The finding reported here may be a demonstration of a quantal cell cycle for cellular differentiation during early embryonic development. In addition, the present results imply that the quantal DNA replication may occur after a definite number of DNA replications. If so, the time at which the changes in the genetic programme responsible for this differentiation begin, may be counted by the cycle of DNA replication and determined by a crucial number of DNA replication cycles. Relating to this clock mechanism, Holliday & Pugh (1975) presented a model that DNA itself may count the number of its replications by the enzymatic modification of specific bases in repeated DNA sequences. However, details of properties of the clock mechanism are subjects of further studies.

It has been proposed that the eggs of some animal groups have cytoplasmic determinants localized in particular regions of the egg and that these agents are segregated by a determinate cleavage pattern into certain cell lineages where they appear to play a role in programming the differentiation pathways of the cells (Wilson, 1925; Davidson, 1976). Since the cell lineage studies of ascidian embryonic development by Conklin (1905) and his direct observations on segregation during cleavage of visible yellow crescent region of myoplasm into muscle lineage blastomeres, the muscle cell development of ascidian embryos may be one of the best-known examples of cytoplasmic determinants for cellular differentiation. Whittaker’s discovery (1973) that cells which showed AChE activity in cleavage-arrested embryos are always, at each cleavage stage, the presumptive muscle cells, strongly suggests the presence of cytoplasmic information for the tissue-specific enzyme development. Therefore, the changes in the state of the genome during the supposed quantal cell cycle might involve the interaction of the cytoplasmic determinants to the genome (Davidson & Britten, 1971 ; Whittaker, 1973, 1979). Very recently, Whittaker (1980) has successfully induced AChE development in extra cells by changing the distribution of myoplasm during the third cleavage of the ascidian egg, suggesting that nuclear lineages are not responsible for muscle AChE development. However, this result (Whittaker, 1980) does not essentially mean that DNA replication is not needed for AChE development. At present, we may speculate that muscle AChE development is as follows: (a) The cytoplasmic determinants for larval muscle-cell development are localized in particular region of the egg and segregated by a determinate cleavage pattern into the muscle lineage blastomeres, (b) The egg counts the time of initiation of differentiation by the cycle of DNA replication, (c) During the eighth DNA replication cycle, the interaction between the cytoplasmic determinants and the genome occurs which brings about activation of the genome responsible for muscle cell development, (d) Following the gene activation, mRNA synthesis related to the protein synthesis occurs from the early gastrula stage onwards, and AChE synthesis begins from the neurula stage onwards.

We wish to thank Dr T. Numakunai and all other members of Asamushi Marine Biological Station of Tohoku University for affording us opportunities to utilize their facilities. Thanks are also due to Profs V. Mancuso and G. Ortolani of Palermo University for discussing the lineage for larval muscle-cell development.

This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.

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