On the a clock' mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis

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1 /. Embryo!, exp. Morph. Vol. 54, pp , 1979 Printed in Great Britain Company of Biologists Limited 1979 On the a clock' mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis I. Acetyleholinesterase development in cleavage-arrested embryos By NORIYUKI SATOH 1 From the Department of Zoology, Kyoto University THIS PAPER IS DEDICATED TO THE MEMORY OF DR JEAN C. DAN SUMMARY During ascidian embryogenesis a tissue-specific enzyme, muscle acetylcholinesterase (AChE) may first be detected histochemically in the presumptive muscle cells of the neurula. [n order to investigate the 'clock' or counting mechanism that is determining the time when AChE first appears, Whittaker's experiment (1973) has been repeated using eggs of the ascidian, Halocynthia roretzi. Embryos that had been permanently cleavage-arrested with cytochalasin B were able to differentiate AChE in their muscle lineage blastomeres. The time of first AChE occurrence in embryos that had been cleavage-arrested in the 32-cell stage with cytochalasin B was about the same as in normal embryos. This result indicates that the clock is not apparently regulated by the events of cytokinesis. The early gastrulae which had been arrested with colchicine or with colcemid could develop AChE activity, although no histochemically detectable AChE activity was observed in the cleavage-stage embryos that had been arrested with either drug. Therefore the clock does not seem to be controlled by the mitotic cycle of the nucleus. It is suggested that the cycle of DNA replication may be related to the regulation of the clock that is determining the time of development of histospecific protein. INTRODUCTION Because acetylcholinesterase (AChE) is found histochemically only in the muscle cells of the tail of the developing ascidian larvae, AChE is thought to be a tissue-specific enzyme of the muscle cells (Durante, 1956; Whittaker, 1973; Whittaker, Ortolani & Farinella-Ferruzza, 1977). In Ciona intestinalis AChE activity is first detected histochemically in the presumptive muscle cells of the neurula; localized staining is seen at 8 h after fertilization but not at 7 h (Whittaker, 1973). Recently, Whittaker (1973) has shown that cleavagearrested Ciona embryos were also able to differentiate the histospecific protein. If cleavages at the early cleavage stages up to the 64-cell stage were permanently 1 Author's address: Department of Zoology, Faculty of Science, Kyoto University, Kyoto 606, Japan.

2 132 N. SATOH arrested by placing the embryos in cytochalasin B, only muscle lineage blastomeres in the cleavage-arrested embryos at each cleavage stage developed AChE activity. This result implies the presence of specific positional information in the egg cytoplasm that is segregated during cleavage. The time of first AChE occurrence in the 8-cell stage embryos cleavage-arrested with cytochalasin B was almost the same as in normal embryos (Whittaker, 1973). He has reported also that embryos cleavage-arrested with mitotic inhibitors such as colchicine, colcemid and podophyllotoxin also could develop AChE activity. Puromycin prevented the occurrence of AChE activity in embryos treated continuously with it from 7 h onwards, while those treated continuously from 8 h onwards developed slight traces of enzyme activity. Actinomycin D inhibited development of AChE activity in embryos reared in the drug from 5 h onwards, while those from 6 h onwards eventually developed a low level of the enzyme activity. From these results he has pointed out that the segregated information is apparently neither the enzyme protein nor an RNA template for the enzyme synthesis, but is probably concerned with activation of appropriate genes (Whittaker, 1973). With respect to the clock system, he has noted that 'since enzyme-related RNA synthesis and subsequent enzyme synthesis occur at about the same times in cytochalasin-arrested as in normal embryos, a "clock" or counting mechanism of some kind must be determining the time at which cytoplasmic information can interact with the genome...the clock is not apparently regulated by the events of cytokinesis nor, judging from similar results with colchicinearrested cells, does it seem to be controlled by the mitotic cycle of the nucleus' (Whittaker, 1973, p. 2099). If the result of his experiment and its interpretation are entirely true, what does control the clock? As a first attempt to elucidate the clock mechanism for tissue-specific enzyme development I have repeated Whittaker's experiment (1973). MATERIALS AND METHODS Naturally spawned eggs of the ascidian, Halocynthia roretzi, were used in this study. The eggs, about 280 /mi in diameter, are yellowish and semitranslucent. The fertilized eggs were raised in filtered sea water at a room temperature maintained at 15 C. The timing of the developmental stage listed in Table 1 was quite consistent among groups of fertilized eggs. Enzyme histochemistry. Embryos for AChE reactions were fixed for several mins in 5-10 % formalin sea water and treated for 2-4 h at 37 C by the directcolouring thiocholine method (Karnovsky & Roots, 1964). Preliminary experiments with the use of a specific cholinesterase inhibitor and substrates have demonstrated that the cholinesterase activity in Halocynthia embryos is an AChE (Table 2). Enzyme activity in normal embryos and in cytochalasin-arrested 32- cell-stage embryos was tested at various development times. Embryos that were

3 Clock mechanism for cellular differentiation 133 Table 1. Development of Halocynthia roretzi embryos at 15 C Embryonic stage 2-cell 4-cell 8-cell 16-cell 32-cell 64-cell Early gastrula Late gastrula Neural plate Neurula Tail bud Swimming larva (hatching) Time (h) after fertilization Table 2. Effect of substrates and a specific enzyme inhibitor on the cholinesterase activity inhalocynthia embryos Substrates Reaction Acetylthiocholine iodide (2 x 10~ 2 M) + Butyrylthiocholine iodide (2 x 10~ a M) * Acetylthiocholine iodide+ eserine sulphate f (10-3 M) * Indicates that the enzyme activity is attributable to the presence of true acetylcholinesterase and not to pseudocholinesterase. f Rules out the possibility that a non-specific esterase is contributing to the cholinesterase reaction. reared in cleavage inhibitors were examined usually at h after fertilization- Since Halocynthia embryos are large (about 280 /tm in diameter), the reaction products could be detected using a dissecting microscope. The distinction between occurrence and non-occurrence of AChE reaction was clear enough to exclude the possibility of misjudgement (Fig. 1). Some stained specimens were dehydrated in ethanol, cleared in xylene, and embedded in balsam for permanent whole mounts. Cleavage inhibition. Cytochalasin B (Aldrich Chem. Co.) at ^g/ml, colchicine (Merck AG) at 200 ^g/ml, and colcemid (demecolcin, Nakarai Chem. Co.) at 20/^g/ml were used as cleavage inhibitors. Cytochalasin B interferes with microfilaments (Schroeder, 1970; Wessells et ah 1971) and possibly other structural components of the cell. The interference prevents cytokinesis but does not inhibit nuclear divisions. Colchicine and colcemid are inhibitors of microtubule formation (Borisy & Taylor, 1967). The drugs are generally considered to prevent both nuclear mitotic activity and cytokinesis.

4 134 N. SATOH Inhibition of protein and RNA synthesis. Puromycin di-hcl (Makor Chem. Ltd) was completely effective at 200/tg/ml in blocking the appearance of histochemically detectable enzyme. Puromycin at this concentration has been shown to inhibit 99 % of labelled valine incorporation into the acid soluble fraction in Ciona embryos (Whittaker, 1966). Actinomycin D (Sigma) at a concentration of 20 /*g/ml inhibits the occurrence of histochemically detectable enzyme. Actinomycin D at 20 /*g/ml causes maximal (70 %) inhibition of labelled uridine incorporation in ascidian embryos; the uninhibited fraction was found to be low-molecular-weight RNA (Smith, 1967). RESULTS In Halocynthia roretzi embryos AChE activity was first detected histochemically in the presumptive muscle cells of the neurula; localized staining was seen at 12 h of development but not at 10-5 h (Tables 1 and 3; Fig. la). The staining was slight at 12 h (Fig. la), but distinct staining was seen at 13-5 h (Fig. 16). Staining intensity of the cells increased progressively with development time. Figs. 1 (a-c) illustrate the location and relative staining intensity of developing muscle cells of the tail at three different embryonic stages. As noted previously in other ascidian embryos (Durante, 1956; Whittaker, 1973), there was no histochemically detectable localization of AChE activity in the Halocynthia nervous system. AChE development in cytochalasin B. Embryos that were placed in cytochalasin B at various cleavage stages and examined histochemically at h of development time had shown AChE activity in some of the blastomeres. Whittaker (1973) reported that in Ciona intestinalis about 10% of cleavagearrested embryos at the 1-, 2- and 4-cell stage, respectively, developed AChE activity, while from the 8-cell stage onward, almost all of the cleavage-arrested embryos developed AChE activity in some blastomeres. In the case of Halocynthia embryos, AChE activity was not detected in most of cytochalasinarrested embryos at the 1-, 2- or 4-cell stage. From the 8-cell stage onward almost all of the cleavage-arrested embryos developed the enzyme activity. Cleavage-arrested embryos at the 8-cell stage showed the occurrence of enzyme activity in two blastomeres in most cases (Fig. 1 d), and arrested embryos at the 16-cell stage formed the enzyme in four blastomeres (Fig. 1 e). In cytochalasinarrested embryos at the 32-cell stage usually six blastomeres could be found differentiating AChE (Fig. 1/), and in cleavage-arrested embryos at the 64-cell stage eight blastomeres produced the enzyme (Fig. lg). As discovered by Whittaker (1973), the numbers and location of AChE-containing blastomeres at each cleavage-arrested stage between the 8- and 64-cell stages followed precisely the known pattern of cell lineage for larval muscle cell development in ascidian embryos (Conklin, 1905; Ortolani, 1955). The times of first AChE occurrence in normal embryos and in cytochalasin-

5 Clock mechanism for cellular differentiation 135 (a) (b) if) (9)1 (h) FIGURE 1 Acetylcholinesterase (AChE) activity in normal and cleavage-arrested embryos. (a-c) Development of AChE activity in normal embryos at 12 h (a), 13-5 h (b), and 15 h (c). AChE activity is seen only in the presumptive muscle cells, and staining intensity increases progressively with development time, (d-g) AChE activity in cytochalasin B-arrested embryos. Two cells of the 8-cell stage (d), four blastomeres of the 16-cell stage (e), six cells of the 32-cell stage (/), and eight blastomeres of the 64- cell stage (#), respectively, show AChE activity. The numbers and location of AChE containing cells at each cleavage stage follow precisely the known pattern of cell lineage for larval muscle cell development in ascidian embryos, (h) AChE activity in colchicine-arrested early gastrula.

6 136 N. SATOH arrested embryos at the 32-cell stage are shown in Table 3. The first histochemical detection of AChE activity in cytochalasin-arrested embryos was at about the same time as in normal embryos. These results of AChE development in cytochalasin-arrested embryos almost confirm Whittaker's report (1973). Puromycin and actinomycin D inhibition of AChE development. The result summarized in Table 3 also confirms Whittaker's report (1973). Puromycin prevented the occurrence of AChE activity in embryos treated continuously with it from 8, 9 and 10-5 h onwards, respectively. Embryos treated continuously from 12 h onwards developed slight traces of the enzyme activity. The later puromycin treatment was begun, the more histochemical staining developed for the enzyme (Table 3). The time of first AChE occurrence, as well as of puromycin sensitivity, was also between 10-5 and 12 h in cytochalasin-arrested 32-cell stages. Actinomycin D prevented development of AChE activity in both normal embryos and cleavage-arrested embryos at the 32-cell stage reared in the drug from 8 h onwards. Normal embryos and cytochalasin-arrested 32-cell stages treated at 9 h eventually developed low amounts of the enzyme activity in some embryos. Those treated from 12 h developed a large amount of activity. Embryos stopped further development soon after puromycin treatment, whereas those reared in actinomycin D continued to develop for a while after the treatment. AChE development in colchicine and colcemid. Whittaker (1973) reported that Ciona embryos which had been arrested with colchicine at 200 /*g/ml or with colcemid at 20 /*g/ml in the 2-cell stage and later cleavage stages could develop AChE activity and showed exactly the same segregation pattern for the development of enzyme as was seen with cytochalasin B. In Halocynthia embryos most of the embryos cleavage-arrested with colchicine at 200 /tg/ml or with colcemid at 20 /tg/ml between the 2- and 64-cell stages did not develop AChE activity. However, the early gastrulae and later stage embryos cleavage-arrested with these drugs could develop AChE activity (Fig. 1 h). The cells of embryos treated with these drugs did not remain rounded up and stationary as they do with cytochalasin B, but became distorted in shape and followed their normal morphogenetic pattern, as noticed by Whittaker (1973). The time of initiation of cell arrangement like gastrulation in colchicine- or colcemid-arrested 32-cell stages was at about 7-5 h of development and this time was the same as in normal embryos (Table 1). DISCUSSION As reported originally by Whittaker (1973) and confirmed by the present study, embryos which had been permanently cleavage-arrested with cytochalasin B were able to differentiate AChE in their muscle lineage blastomeres. The time of first AChE occurrence in cytochalasin-arrested embryos was about

7 Clock mechanism for cellular differentiation 137 Table 3. Effect of puromycin and actinomycin D on development of AChE in normal and cytochalasin-arrested embryos Development time (h) Drugs Embryos (middle (late (neural (young gastrula) gastrula) plate) (neurula)(tail bud) tadpole) Control Normal embryo ± Cytochalasin ± arrested 32-cell stage Puromycin* (200/ig/ml) Normal embryo ± Cytochalasin ± arrested 32-cell stage Actinomycin D* Normal embryo - ± (20/<g/ml) Cytochalasin- ± arrested 32-cell stage + +, Occurrence of intensive AChE activity; +, distinct AChE activity; ±, slight AChE activity; -, no AChE activity. * Embryos were treated continuously with drug from each time onwards and examined histochemically at h of development time. the same as in normal embryos. This result indicates that the clock which must be determining the time when AChE first appears is not apparently regulated by the events of cytokinesis. In Halocynthia embryos no histochemically detectable AChE activity was observed in the cleavage-stage embryos that had been arrested with colchicine or with colcemid. This favours a possibility that the nuclear division activity is a prerequisite for tissue-specific enzyme development and that the number of nuclear divisions may be related to the regulation of the clock. However, the early gastrulae which had been arrested with these drugs could develop AChE activity. Tn addition, cleavage-stage Ciona embryos which had been permanently arrested with colchicine or with colcemid were able to differentiate AChE in their muscle lineage blastomeres (Whittaker, 1973). Therefore the nuclear division activity is not always essential to histospecific protein development. Since [ 3 H]thymidine is incorporated into colchicinearrested Ciona embryos, DNA synthesis seems to continue in nuclei of the cells of colchicine-arrested embryos (Whittaker, personal communication). These results imply that the cycles of DNA replication may be the clock mechanism for tissue-specific enzyme development inascidian embryos. At present, however, it cannot exclude a possibility that cytoplasmic element(s) the function of which

8 138 N. SATOH would not be disturbed with cytochalasin B or with colchicine may be related to control the clock. In the present investigation AChE development in the muscle cells during ascidian embryogenesis has been studied to explore the clock mechanism that is controlling the time of initiation of cellular differentiation. It is generally accepted that cellular differentiation depends on restriction of a specific protein to only one type of cell and that the differentiated state of a given type of cell is associated with the activity of a particular set of genes (Rutter, Pictet & Morris, 1973; Davidson, 1976). Although the function of AChE in the muscle cells of the tail of the ascidian embryos is not thoroughly studied, there has been experimental evidence of tissue specificity of AChE in the muscle cells (Durante, 1956; Fromson & Whittaker, 1970). Particularly, Whittaker's experiment (1973) that cells which showed AChE activity in cleavage-arrested embryos were always, at each cleavage stage, the presumptive muscle cells, strongly suggests that AChE is a tissue-specific enzyme of the muscle cells. This result also implies the presence of specific positional information in the egg cytoplasm that is segregated during cleavage. As confirmed by the present study, there were distinct and separate puromycin- and actinomycin D-sensitivity periods for the occurrence of AChE during development of normal and cleavage-arrested embryos. The segregated information is apparently neither the enzyme protein nor an RNA template for enzyme synthesis, but is probably concerned with activation of appropriate genes (Whittaker, 1973). Therefore the clock must be determining the time at which cytoplasmic information can interact with the genome. It is possible that the 'clock gene' first learns the time for initiation of the cellular differentiation by the number of times the DNA replicates and then it informs the cytoplasmic regulatory element, which then becomes able to interact with the genome. I wish to express my gratitude to Dr T. Numakunai of the Asamushi Marine Biological Station, Tohoku University, for supplying the materials and for general advice during the research. Thanks are also due to Prof. M. Yoneda of Kyoto University for his comments on the manuscript and to Dr A. M. Anderson for a reading of the manuscript. I am heartily grateful to Dr J. R. Whittaker for his suggestion and encouragement. This study was supported in part by a Grant-in-Aid from the Ministry of Education of Japan (no ). REFERENCES BORISY, G. G. & TAYLOR, E. W. (1967). The mechanism of action of colchicine. Binding of colchicine- 3 H to cellular protein. /. Cell Biol. 34, CONKLIN, E. G. (1905). The organization and cell-lineage of the ascidian eggs. /. Acad. nat. Sci. 13, DAVIDSON, E. H. (1976). Gene Activity in Early Development, 2nd ed. New York: Academic Press. DURANTE, M. (1956). Cholinesterase in the development of Ciona intestinalis (Ascidia). Experientia 12,

9 Clock mechanism for cellular differentiation 139 FROMSON, D. & WHITTAKER, J. R. (1970). Acetylcholinesterase activity in eserine-treated ascidian embryos. Biol. Bull. mar. biol. Lab., Woods Hole 139, KARNOVSKY, M. J. & ROOTS, L. (1964). A 'direct-coloring' thiocholine method for cholinesterases. J. Histochem. Cytochem. 12, ORTOLANL, G. (1955). The presumptive territory of the mesoderm in the ascidian germ. Experientia 11, R UTTER, W. J., PICTET, R. L. & MORRIS, P. W. (1973). Toward molecular mechanisms of developmental processes. Ann. Rev. Biochem. 42, SCHROEDER, T. E. (1970). The contractile ring. 1. Fine structure of dividing mammalian (HeLa) cells and the effects of cytochalasin B. Z. Zellforsch. mikrosk. Anat. 109, SMITH, K. D. (1967). Genetic control of macromolecular synthesis during development of an ascidian: Ascidia nigra. J. exp. Zool. 164, WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR, E. L., WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmental processes. Science, N.Y. Ill, WHITTAKER, J. R. (1966). An analysis of melanogenesis in differentiating pigment cells of ascidian embryos. Devi Biol. 14, WHITTAKER, J. R. (1973). Segregation during ascidian embryogenesis of egg cytoplasmic information for tissue-specific enzyme development. Proc. natn. Acad. Sci. U.S.A. 70, WHITTAKER, J. R., ORTOLANT, G. & FARINELLA-FERRUZZA, N. (1977). Autonomy of acetylcholinesterase differentiation in muscle cells of ascidian embryos. Devi Biol. 55, {Received 28 January 1979, revised 13 July 1979) KMB 54

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