A definite number of aphidicolin-sensitive cell-cyclic events are required for acetylcholinesterase development in the presumptive muscle cells of the ascidian embryos

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 cylochalasin in as many as twelve cells. Cytochalasin-arrested late gastrulae developed AChE in twenty blastomeres, while aphidicolin-cytochalasinarrested 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 cleavagearrested embryos remained fixed. Judging from the relative positions of the blaslomeres, 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.

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On the ‘clock’ mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis

Development ◽

10.1242/dev.54.1.131 ◽

1979 ◽

Vol 54 (1) ◽

pp. 131-139

Author(s):

Noriyuki Satoh

Keyword(s):

Ache Activity ◽

Mitotic Cycle ◽

Cytochalasin B ◽

Specific Enzyme ◽

Cleavage Stage ◽

Halocynthia Roretzi ◽

Cell Stage ◽

Tissue Specific ◽

Enzyme Development ◽

Clock Mechanism

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 theascidian, 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.

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Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications

Development ◽

10.1242/dev.87.1.1 ◽

1985 ◽

Vol 87 (1) ◽

pp. 1-12

Author(s):

Izumi Mita-Miyazawa ◽

Susumu Ikegami ◽

Noriyuki Satoh

Keyword(s):

Dna Replication ◽

Specific Inhibitor ◽

Muscle Cells ◽

Acetyl Cholinesterase ◽

Specific Enzyme ◽

Isolated Cells ◽

Cell Stage ◽

Simultaneous Treatment ◽

Tissue Specific ◽

Ascidian Embryos

The presumptive muscle cells (B5.1 blastomeres) were isolated from 16-cell-stage embryos of the ascidian, Ciona intestinalis. The isolated cells were allowed to divide either twice or three times thereafter. Then further divisions of the cells were continuously inhibited by a simultaneous treatment with aphidicolin (a specific inhibitor of DNA synthesis) and cytochalasin B (an inhibitor of cytokinesis). When development of muscle-specific acetyl-cholinesterase in these division-arrested progeny cells of B5.1 blastomeres was examined histochemically, the B5.1 blastomeres which had been allowed two further divisions did not produce any detectable acetyl-cholinesterase activity. Whereas those which had been allowed three further divisions showed the tissue-specific enzyme activity. These results provide further evidence for the presence of a quantal DNA replication cycle for the tissue-specific enzyme development, which is qualitatively different from the other DNA replication cycles.

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On the ‘clock’ mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis

Development ◽

10.1242/dev.64.1.61 ◽

1981 ◽

Vol 64 (1) ◽

pp. 61-71

Author(s):

Noriyuki Satoh ◽

Susumu Ikegami

Keyword(s):

Dna Replication ◽

Nuclear Envelope ◽

Ache Activity ◽

Specific Inhibitor ◽

Specific Enzyme ◽

Cell Stage ◽

Tissue Specific ◽

Nuclear Envelope Breakdown ◽

Enzyme Development ◽

Clock Mechanism

Acetylcholinesterase (AChE) is a tissue-specific enzyme of the muscle cells of ascidian embryos and its synthesis begins at the neurula stage. Embryos which had been permanently cleavage-arrested with cytochalasin B could develop AChE activity. The time of first AChE occurrence in embryos which had been arrested in the 32-cell stage with cytochalasin was about the same as in normal embryos. The nucleus in the cell of cytochalasin-arrested embryos divided in good synchrony with that of normal embryos. Embryos which had been continuously arrested with colchicine could also produce AChE activity at nearly the same time as did normal embryos. In the cell of colchicine-arrested embryos normal nuclear divisions did not occur, but the cell showed repeated cycles of nuclear envelope breakdown and nuclear envelope reformation in almost parallel with cell cycles of normal embryos. The cell of colchicine-arrested embryos incorporated [3H]thymidine. Aphidicolin, a specific inhibitor of DNA synthesis, prevented cleavages of ascidian eggs. Embryos which had been permanently arrested with aphidicolin in the cleavage stages up to the 64-cell stage did not develop AChE activity, while embryos which had been treated with it from the 76-cell stage onwards were found to be able to differentiate AChE activity. Based on these findings it was proposed that DNA replication is prerequisite for development of the histospecific protein and that the cycle of DNA replication is closely associated with the clock mechanism which is determining the time of initiation of the enzyme development.

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Acetylcholinesterase development in extra cells caused by changing the distribution of myoplasm in ascidian embryos

Development ◽

10.1242/dev.55.1.343 ◽

1980 ◽

Vol 55 (1) ◽

pp. 343-354

Author(s):

J. R. Whittaker

Keyword(s):

Cytochalasin B ◽

Continuous Treatment ◽

Functional Specificity ◽

Cell Stage ◽

Division Plane ◽

Styela Plicata ◽

Developmental Fate ◽

Ascidian Embryos ◽

Functional Autonomy ◽

Ascidian Embryo

This research shows that myoplasmic crescent material of the ascidian egg has both functional autonomy and functional specificity in establishing the differentiation pathway of muscle lineage cells. The cytoplasmic segregation pattern in eggs of Styela plicata was altered by compression of the embryos during third cleavage. This caused a meridional division instead of the normal equatorial third cleavage; first and second cleavages are meridional. Since eggs of S. plicata have a pronounced yellow myoplasmic crescent, one observes directly that third cleavage under compression resulted in a flat 8-cell stage with four cells containing yellow myoplasm instead of the two myoplasm-containing cells that would be formed by normal equatorial division at third cleavage. If such altered 8-cell-stage embryos were released from compression and kept from undergoing further divisions by continuous treatment with cytochalasin B, some embryos eventually developed histospecific acetylcholinesterase in three and four cells instead of in just the two muscle lineage cells found in cleavage-arrested normal 8-cell stages. The wider myoplasmic distribution effected by altering the division plane at third cleavage apparently caused a change in developmental fate of the extra cells receiving myoplasm. This meridional third cleavage also resulted in a changed nuclear lineage pattern. Two nuclei that would ordinarily be in ectodermal lineage cells after third cleavage were now associated with yellow myoplasm. Acetylcholinesterase development in these cells demonstrates that nuclear lineages are not responsible for muscle acetylcholinesterase development in the ascidian embryo.

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DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Differentiation ◽

10.1111/j.1432-0436.1982.tb01192.x ◽

1982 ◽

Vol 21 (1-3) ◽

pp. 37-40 ◽

Cited By ~ 19

Author(s):

NORIYUKI SATOH

Keyword(s):

Dna Replication ◽

Specific Enzyme ◽

Tissue Specific ◽

Enzyme Development ◽

Ascidian Embryos

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Early embryonic expression ofFGF4/6/9gene and its role in the induction of mesenchyme and notochord inCiona savignyiembryos

Development ◽

10.1242/dev.129.7.1729 ◽

2002 ◽

Vol 129 (7) ◽

pp. 1729-1738 ◽

Cited By ~ 16

Author(s):

Kaoru S. Imai ◽

Nori Satoh ◽

Yutaka Satou

Keyword(s):

Cell Differentiation ◽

Nuclear Localization ◽

Target Genes ◽

Muscle Cells ◽

Cell Stage ◽

Ciona Savignyi ◽

Mesenchyme Cell ◽

Notochord Cells ◽

Mesenchyme Cells ◽

Endodermal Cells

In early Ciona savignyi embryos, nuclear localization of β-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of β-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 was transient and the transcript was seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene was then expressed again in cells of the nervous system after neurulation. When the gene function was suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells was completely blocked, and the fate of presumptive mesenchyme cells appeared to change into that of muscle cells. The inhibition of mesenchyme differentiation was abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of β-catenin nuclear localization resulted in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in β-catenin-downregulated embryos evoked mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. On the other hand, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction was inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes were expressed later to form a partial notochord.

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Postimplantation development of tetraploid mouse embryos produced by electrofusion

Development ◽

10.1242/dev.110.4.1121 ◽

1990 ◽

Vol 110 (4) ◽

pp. 1121-1132

Author(s):

M.H. Kaufman ◽

S. Webb

Keyword(s):

Cytochalasin B ◽

Histological Analysis ◽

Mouse Embryos ◽

Normal Embryo ◽

Craniofacial Abnormalities ◽

Success Rates ◽

Cell Stage ◽

Extraembryonic Membranes ◽

Mammalian Embryonic Development ◽

Viable Embryo

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell ‘fused’ embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.

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An analysis of the response to gut induction in the C. elegans embryo

Development ◽

10.1242/dev.121.4.1227 ◽

1995 ◽

Vol 121 (4) ◽

pp. 1227-1236 ◽

Cited By ~ 7

Author(s):

B. Goldstein

Keyword(s):

Body Wall ◽

Cell Lineage ◽

Muscle Cells ◽

The Other ◽

Cell Stage ◽

Pharyngeal Muscle ◽

Cell Cycles ◽

Sister Cell ◽

C Elegans ◽

Cell Diversity

Establishment of the gut founder cell (E) in C. elegans involves an interaction between the P2 and the EMS cell at the four cell stage. Here I show that the fate of only one daughter of EMS, the E cell, is affected by this induction. In the absence of the P2-EMS interaction, both E and its sister cell, MS, produce pharyngeal muscle cells and body wall muscle cells, much as MS normally does. By cell manipulations and inhibitor studies, I show first that EMS loses the competence to respond before it divides even once, but P2 presents an inducing signal for at least three cell cycles. Second, induction on one side of the EMS cell usually blocks the other side from responding to a second P2-derived signal. Third, microfilaments and microtubules may be required near the time of the interaction for subsequent gut differentiation. Lastly, cell manipulations in pie-1 mutant embryos, in which the P2 cell is transformed to an EMS-like fate and produces a gut cell lineage, revealed that gut fate is segregated to one of P2's daughters cell-autonomously. The results contrast with previous results from similar experiments on the response to other inductions, and suggest that this induction may generate cell diversity by a different mechanism.

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