/ . Embryo!, exp. Morph. Vol. 54, pp. 131-139, 1979
Printed in Great Britain © Company of Biologists Limited 1979
On the aclock' mechanism determining the time of
tissue-specific enzyme development during
ascidian embryogenesis
I. Acetyleholinesterase development in cleavage-arrested embryos
By NORIYUKI SATOH1
From the Department of Zoology, Kyoto University
THIS PAPER IS DEDICATED TO THE MEMORY OF
D R JEAN C. D A N
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.
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 32cell-stage embryos was tested at various development times. Embryos that were
Clock mechanism for cellular differentiation
133
Table 1. Development of Halocynthia roretzi embryos at 15 °C
Embryonic stage
Time (h) after
fertilization
2-cell
4-cell
8-cell
16-cell
32-cell
64-cell
Early gastrula
Late gastrula
Neural plate
Neurula
Tail bud
1-75
2-5
3-25
4
5
6
7-5
9
10-5
12
13-5
Swimming larva (hatching)
30
Table 2. Effect of substrates and a specific enzyme inhibitor
on the cholinesterase activity in Halocynthia embryos
Substrates
Acetylthiocholine iodide (2 x 10~2 M)
Butyrylthiocholine iodide (2 x 10~a M)
Acetylthiocholine iodide+ eserine sulphate
(10- 3 M)
Reaction
+
—*
—f
* 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 24-26 h after fertilizationSince 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 1-0-1-5 ^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.
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 24-26 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-
Clock mechanism for cellular differentiation
(a)
135
(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 64cell 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.
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
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
9
10-5
15
8
(middle (late (neural
12
13-5 (young
gastrula) gastrula) plate) (neurula)(tail bud) tadpole)
Control
Normal embryo
Cytochalasinarrested 32-cell
stage
—
-
—
-
—
-
±
±
+
+
++
++
Puromycin*
(200/ig/ml)
Normal embryo
—
—
—
±
+
++
Cytochalasinarrested 32-cell
stage
-
-
-
±
+
++
Actinomycin D* Normal embryo
(20/<g/ml)
Cytochalasinarrested 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 20-22 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 [3H]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
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. 374232).
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{Received 28 January 1979, revised 13 July 1979)
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