/. Embryol. exp. Morph. 76, 235-250 (1983)
235
Printed in Great Britain © The Company of Biologists Limited 1983
Quantitative regulation of acetylcholinesterase
development in the muscle lineage cells of cleavagearrested ascidian embryos
By J. R. WHITTAKER 1
From the Boston University Marine Program and the Marine Biological
Laboratory, Woods Hole
SUMMARY
Some embryos of Ciona intestinalis which were permanently cleavage-arrested with
cytochalasin B at the 1-cell, 4-cell, or 8-cell stages produced, after 12 or 16 h of development
time (18 °C), a level of muscle acetylcholinesterase activity equal to that found in normal early
and later larval stage embryos of the same age. Enzyme activity was measured quantitatively
in single whole embryos by a colorimetric procedure using microdensitometry. Quantitative
regulation of a differentiation end product indicated that the usual transcriptional and translational control mechanisms for that histospecific protein continued to operate normally in the
cleavage-arrested embryos. Acetylcholinesterase expression was apparently regulated independently of the usual cell cytoplasmic volume in the muscle lineage cells and possibly also
independently of the normal nuclear number in the lineage. There is an egg cytoplasmic
determinant that is segregated into the muscle lineage cells during cleavage and which appears
to specify the pathway of larval muscle development. Quantitative control of muscle acetylcholinesterase is possibly one of the consequences of how the agent releases genetic expression
in the presumptive muscle cells. Quantitative regulation was not, however, a general functional activity of cleavage-arrested embryos. Mitochondrial cytochrome oxidase, an enzyme
whose development is believed to be unaffected by cytoplasmic determinants, was not
regulated quantitatively in cleavage-arrested embryos. Cytochrome oxidase activity of
cleavage-arrested embryos, measured in single whole embryos by a colorimetric microdensitometry assay, increased only slightly during 16 h of development time whereas the
activity in normal control embryos doubled during that time.
INTRODUCTION
There appears to be an egg cytoplasmic determinant that regulates larval
muscle differentiation in ascidian embryos (subphylum Urochordata, class
Ascidiacea). This determinant, or perhaps group of determinants, is localized in
the egg and becomes segregated during early cleavages of the zygote into the
muscle lineage cells where it eventually plays some role in initiating differentiation. Such an interpretation is implicit in Conklin's (1905) classic observation
1
Author's address: Boston University Marine Program, Marine Biological Laboratory,
Woods Hole, Massachusetts 02543, U.S.A.
236
J. R. WHITTAKER
that a specific yellow crescent region of cytoplasm in the fertilized egg (of some
species) becomes segregated into the muscle lineage cells. A determinant
hypothesis is supported also by cell lineage fate maps and the exceptional
developmental autonomy expressed by isolated lineage cells in conforming to the
predictions of these fate maps (reviewed by Whittaker, 1979a).
Experimental techniques combined with histochemical and ultrastructural
observations provide results that suggest even more strongly that such an
ascidian larval muscle determinant exists; it is associated with development of a
histospecific larval muscle acetylcholinesterase and other features of muscle
differentiation. Surgically isolated blastomeres from the muscle cell lineage of
4-cell- and 8-cell-stage embryos eventually develop acetylcholinesterase in
division products of the isolated blastomeres (Durante, 1957; Whittaker,
Ortolani & Farinella-Feruzza, 1977). In addition, embryos which are cleavagearrested with cytochalasin B at various stages up to the 64-cell stage develop
enzyme only in the muscle lineage blastomeres (Whittaker, 1973,1919b; Satoh,
1979). Recently, we have found that partial embryos resulting from isolated
muscle lineage blastomeres as well as whole embryos cleavage arrested with
cytochalasin B both develop myofilaments and partially organized myofibrils in
the appropriate cells (Crowther & Whittaker, 1983). Cleavage-arrested ascidian
embryos also acquire electrical excitability in membranes of their muscle lineage
blastomeres (Takahashi & Yoshii, 1981).
Perhaps the most convincing evidence in favour of a determinant hypothesis
is the result of those experiments which move presumptive myoplasm to new
locations in the embryo. When yellow crescent myoplasm is moved mechanically
into extra blastomeres at the 8-cell stage by altering the plane of third cleavage,
it causes these extra cells to produce acetylcholinesterase (Whittaker, 1980).
Presumptive myoplasm that is shifted into ectodermal lineage blastomeres by a
direct microsurgical technique also initiates acetylcholinesterase development in
some of the epidermal cell progeny (Whittaker, 1982).
Because cleavage-arrested embryos develop tyrosinase and melanin pigment
only in the two melanocyte lineage cells of the larval brain, this observation
suggests a segregation at each division in the two lineages of an egg cytoplasmic
determinant that regulates melanocyte differentiation (Whittaker, 1973). Since
cleavage-arrested embryos produce the same actual amounts of differentiation
end products in the larger multinucleate cells as in the terminally differentiated
normal melanocytes (Whittaker, 1979c, 1981), the determinant may possibly be
exercising quantitative as well as qualitative control over melanocyte development. The present paper reports that muscle lineage cells of some cleavagearrested embryos developed the same quantities of acetylcholinesterase activity
as normal embryos. This finding also raises the prospect that the egg cytoplasmic
agent involved in muscle expression might be exerting a quantitative control over
enzyme development.
Ascidian muscle lineage
237
MATERIALS AND METHODS
Embryos
Ciona intestinalis (L.) was collected in the vicinity of Woods Hole,
Massachusetts, June through October, and maintained in tanks with running sea
water under conditions of constant light. Eggs and sperm were obtained surgically from the gonoducts of animals. Embryos were cultured in filtered sea water
at 18 ± 0-1 °C using a refrigerated constant temperature water bath (WilkensAnderson Lo-Temp). Some embryos were reared for periods of time in 2 |Ug/ml
cytochalasin B (Sigma Chemical Company). Cytochalasin B was dissolved in a
stock solution at 1 mg/ml in dimethyl sulfoxide (DMSO). The resulting sea water
solutions contained 0-2 % DMSO, a concentration that had no detectable effect
on normal embryonic development.
Histochemistry
Acetylcholinesterase (E.C. 3.1.1.7) was localized in embryos by the Karnovsky & Roots (1964) procedure after 2-3 min fixation in cold (5 °C) 80 % ethanol.
Incubation was for 60 or 120 min at 18 ± 0-1 °C; older embryos were incubated
only 60 min to avoid over reaction. Various substrate and inhibitor controls for
the identity of the Ciona enzyme are described elsewhere (Whittaker et al. 1977;
Meedel & Whittaker, 1979).
Cytochrome oxidase (E.C. 1.9.3.1) was localized by the 3,3'-diaminobenzidine (DAB) reaction of Seligman, Karnovsky, Wasserkrug & Hanker (1968)
containing cytochrome c substrate (0-5 mg/ml). Peroxidase activity was prevented by including 20/ig/ml Sigma C-40 catalase in the reaction medium. Fixation
was for 10 min at 5 °C in Karnovsky's (1965) fixative, but with formaldehyde and
glutaraldehyde each reduced to 1-5%. Embryos were rinsed afterwards for
10 min (5 °C) in the cacodylate-buffered washing solution recommended by Karnovsky (1965), and incubated for 90 min at 18±0-l °C in the incubation medium.
After the respective histochemical reactions, embryos were dehydrated in
ethanol, cleared in xylene, and mounted in damar resin. The two histochemical
methods produced essentially permanent colour reactions. Embryos were
handled throughout the histochemical and mounting procedures in 11mm
diameter open glass tubes covered on one end with No. 21 Standard silk bolting
cloth. All histochemical reagents were obtained from the Sigma Chemical Company.
Quantitative measurements of acetylcholinesterase activity
Acetylcholinesterase activity can be measured quantitatively in similarly
treated histochemical preparations by colorimetric assay of the relative rates of
reaction product accumulation. Concentrations of the insoluble reaction
product of the Karnovsky & Roots (1964) procedure, cupric ferrocyanide, obey
238
J. R. WHITTAKER
the Beer-Lambert law. The amount of this product can, therefore, be measured
in histochemically stained specimens by techniques of microdensitometry
(Storm-Mathison, 1970; Wenk, Krug & Fletcher, 1973). Small spherical embryos which have not hatched from their chorions provide an almost ideal geometry for microdensitometry measurements provided, as in the case of Ciona,
that measurements can be done adequately with lower-power objectives where
there is a reasonable depth of focus, and where the size of the embryo conforms
to a significant portion of the densitometer scanning area in the optical field
(Whittaker, 1981).
Background staining from the Karnovsky & Roots (1964) direct colouring
reaction for cholinesterase activity is not a serious difficulty for microdensitometry studies. Staining of enzyme-containing Ciona embryos with a
reaction medium lacking the acetylthiocholine substrate or one in which
butyrylthiocholine was substituted for acetylthiocholine revealed no localized
enzyme activity. The background colouration in these embryos was no different
from that found in non-muscle regions of normally stained embryos, nor from
the background in normally stained embryos fixed at times before localized
staining develops. Consequently, early embryos (taken at 2-6h of development before the occurrence of enzyme activity) were stained and used as
'reagent' subtraction blanks in the spectrophotometric measurements, on the
assumption that optical density readings after such subtractions accurately
express the real enzyme activity. Such reagent blank embryos were always
removed from the same group being studied, placed in a refrigerator at 5 °C,
and mixed with normal and experimental embryos at the appropriate later time
of development so that the reagent blanks underwent identical fixation and
reaction conditions.
A Vickers M85 scanning and integrating microdensitometer (Smith, Moore &
Goldstein, 1975) was used to measure the reaction product in damar-mounted
ascidian embryos. Measurements were made at 485 nm by the subtractive
method. A 20x objective and a scanning spot of 2/im diameter were used in
conjunction with a mask of 212 fjm diameter (C-2) and a 400 x 400/im (3 x 3)
scanning frame size. The fixed and stained embryos are 120-170 fim in diameter;
shrinkage is variable according to embryonic stage used and the dehydration
time sequence. Each slide contained embryos fixed at a time before normal
acetylcholinesterase production (embryos 2-6h of age); the mean optical density measurement of 25 such embryos was used as a subtractive blank value for
each measurement of the experimental and control embryos on the same slide.
Final results were based on the mean value of 25 embryos selected as having the
appropriate orientation for accurate measurements to be made. This selection
was essentially random. Machine optical density readings of the enzyme activity
were converted to absolute integrated optical density units by calibrating the
instrument against a No. 96 Kodak 1-00 Neutral Density filter (10-0% transmission).
Ascidian muscle lineage
239
Measurements of cytochrome oxidase activity
Identical settings of the Vickers instrument (as above) were used to measure
optical density of the oxidative polymerization product of DAB in a quantitative
cytochrome oxidase assay, except that a different wavelength (460 nm) was selected (Marinos, 1978). Subtractive blanks for these measurements consisted of
embryos from the stages being studied which were incubated for 90 min in parallel reaction mixtures containing 10mM-KCN, a potent inhibitor of cytochrome
oxidase activity. Such embryos were then mixed with normally stained embryos
and mounted on the same slide. Suitability of the DAB reaction for quantitative
histochemical measurements of cytochrome oxidase activity has been confirmed
by Frasch, Itoiz & Cabrini (1978).
RESULTS
Quantitative measurements of acetylcholinesterase development
Two kinds of test indicated that the microdensitometry method of measuring
acetylcholinesterase activity was capable of detecting significant differences in
enzyme activity. Quantity of reaction product formed (absolute optical density
units) in whole embryos was proportional to length of incubation time for the
enzymatic reaction (Fig. 1). Also, enzyme activity in the whole embryos as
measured by microdensitometry increased linearly with development time (Fig.
2).
At early development stages acetylcholinesterase occurs in either a posterior
30 - 60
90
120
Reaction time (min)
20
40
60
80
Reaction time (min)
Fig. 1A. Acetylcholinesterase activity in Ciona intestinalis embryos as a function of
in vitro incubation time. Microdensitometry measurements of the histochemical
reaction product in 10 h embryos (•) and 12 h embryos (O). Points are means based
on 25 embryos; bars are standard errors of the mean.
Fig. IB. Acetylcholinesterase activity as a function of in vitro incubation time in 16 h
embryos. Points are means based on 25 embryos; bars are standard errors of the
mean.
240
J. R. WHITTAKER
100-
37
1 2
d - 75i
b i7t
5 o
T
c
75
50"
50-
< E
W 25-
25-
X)
X)
12
15
18
h after fertilization
12
15
h after fertilization
18
Fig. 2A. Acetylcholinesterase activity in Ciona embryos measured by microdensitometry of the histochemical reaction product after 60min incubation time.
Two series of embryos (•, O) at various ages postfertilization. Points are means
based on 25 embryos; bars are standard errors of the mean.
Fig. 2B. Acetylcholinesterase activity measured in homogenates of Ciona embryos
by a technique that liberates [14C]acetate from [l-14C]acetylcholine. Embryos from
a single series. Enzyme activity units are jumoles of acetate formed per min under
standard assay conditions. Data are from Meedel & Whittaker (1979).
crescent of cells or in two thick muscle bands in the gradually developing tailbud.
Orientations of the early embryo were selected for measurement such that the
densitometer could be focused directly on either the crescent or the muscle bands
(e.g., Fig. 3). At later 'larval' stages, when the tail had grown out and become
thinner, measurements were made after focusing the microdensitometer on the
central notochordal level of the embryo tail in lateral orientation (Figs 4,5). All
normal embryos of appropriate age produced a localized histochemical reaction
for acetylcholinesterase; only orientation of the embryo on the slide (Figs 3-5)
served as a basis for selecting the embryos to be measured.
The relationship between product formed and enzyme incubation time was
tested at three developmental stages: 10 h, 12 h, and 16 h. In 10 h embryos,
product accumulation was proportional to time for 120 min (Fig. 1A). At later
stages accumulation was proportional to time for about 60min (Figs 1A, IB).
Extensions of the time intervals (not shown) all indicated that enzyme activity
declined in relation to time after 60-120min. Activity measurements were
based, therefore, on a 60-120min incubation period, depending on the age of
embryo used.
Results of enzyme activity measurements in a series of developmental stages
were consistent with the likelihood that activity is proportional to amount of
enzyme present. There was a linearly increasing enzyme activity during 10-18 h
of development, and activity was proportional during this time to age of the
embryos (Fig. 2A). This activity closely paralleled specific activity measurements made on Ciona embryo homogenates by a radiometric assay of enzyme
Ascidian muscle lineage
241
activity (Meedel & Whittaker, 1979), as shown in Fig. 2B. These curves are
directly comparable because there is no change in total protein content per
embryo during embryonic development (Meedel & Whittaker, 1979). A given
weight of protein (1 mg) is equal to a fixed number of embryos at all developmental stages up to hatching. Ciona larvae begin hatching at 18 h, with the first
few larvae breaking free of the chorionic membrane at that time.
Acetylcholinesterase in cleavage-arrested embryos
Many cleavage-arrested early embryos eventually develop acetylcholinesterase activity in blastomeres of the muscle lineage, but the total number of
embryos doing this is usually small and variable (Whittaker, 1973). Some
cytochalasin-treated 1-cell-stage embryos developed enzyme in the whole egg
(Fig. 6); cleavage-arrested 4-cell- (Fig. 7) and 8-cell-stage (Fig. 8) embryos
frequently developed acetylcholinesterase in each of their two muscle lineage
blastomeres. Often the relative intensity of staining appeared to be the same in
both blastomeres. Microdensitometry measurements were made on the experimental embryos by focusing the microdensitometer at a median level of the
stained blastomeres and by selecting only embryos with two equal-staining
blastomeres where the blastomeres occurred in approximately the same plane of
focus. Except for these restrictions of orientation and blastomere pairs having
approximately equal-staining intensity (in the 4-cell and 8-cell stages) embryos
were measured as encountered during a progressive horizontal and vertical
examination of the slides. Selection was assumed to be random but no statistical
test of randomness was applied to the data.
Enzyme activity in cleavage-arrested embryos was examined at two time
periods: 12 h and 16 h embryos. Since the ontogeny curve for acetylcholinesterase activity was linear with time during the later part of embryonic development (Figs 2A, 2B), it was probably not important which later time was selected
for comparison between normal and cleavage-arrested embryos. One correction
factor was incorporated directly into the experimental design. Cleavage-arrested
embryos were about 30min slower than the time at which enzyme was first
observed histochemically in control embryos. Consequently, experimental embryos were arranged so that they were 30min older than the normal control
embryos to which they would be compared.
Embryos from the same collection of eggs (three to four animals) were taken
for experimental, control, and subtraction blank measurements. Experimental
embryos were fertilized first (with a mixed sperm suspension) and placed in
2 jug/ml cytochalasin B immediately after fertilization for 1-cell stages and shortly after 90 and 120 min for 4-cell and 8-cell stages. Control embryos were fertilized 30 min later; subtraction blank embryos were removed from control group
dishes 2-3 h after fertilization and refrigerated at 5 °C. When the control group
reached the appropriate age, all three groups were recombined in the same
fixation tube, fixed and incubated together, and mounted on the same
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J. R. WHITTAKER
6
&-;
•"4
8
Figs 3-8
?:*<
Ascidian muscle lineage
243
Table 1. Microdensitometry measurements of acetylcholinesterase activity in 12 h
embryos and in early cleavage stages arrested with cytochalasin B until the embryos were 12-5 h of age
Embryos
1-cell cleavage-arrested
12 h control
Acetylcholinesterase activity
(absolute O.D. units embryo"1)
Series 1
Series 2
4-cell cleavage-arrested
12 h control
5318 ± 393*
4720 ± 350
5102 ± 413
4920 ± 359
5680 ± 799
5499 ± 269
5433 ± 582
4928 ± 252
8-cell cleavage-arrested
12 h control
4989 ± 299
5089 ± 553
5164 ± 280
5238 ± 349
* Mean value of 25 embryos ± standard error of the mean ; enzyme incubation of 60min.
microscope slide. The possibility of inadvertently creating non-specific differences in enzyme content between experimental and control embryos was
greatly reduced by using the same blanks and by keeping as many other variables
as constant as possible.
The first comparisons were made at 12 h of normal development. Two sets of
data are shown for each of the cleavage-arrested stages (Table 1). There was no
large difference in enzyme activity between means of the cleavage-arrested and
normal embryos; activity in normal and experimental embryos appeared to be
the same.
Since it is possible that an accelerated enzyme synthesis might occur in experimental embryos for only a relatively short time, activities were also compared at
a later development stage (16 h). Such measurements showed the higher activity
levels of 16 h embryos and also revealed no difference in acetylcholinesterase
Fig. 3. 10 h embryo of Ciona intestinalis stained histochemically for acetylcholinesterase activity with the 'direct colouring' thiocholine method. Embryo viewed from
the dorsal aspect with the plane of focus at the enzyme-containing muscle bands.
Fig. 4. 12 h embryo stained for acetylcholinesterase activity and showing the
enzyme-containing tail muscle. Side view with plane of focus at the notochord.
Fig. 5. 16 h embryo stained for acetylcholinesterase activity. Orientation with plane
of focus along the notochord of the tail.
Fig. 6. 1-cell-stage embryo cleavage-arrested with cytochalasin B (2,ug/ml) and
stained histochemically for acetylcholinesterase activity at 16 h after fertilization.
Fig. 7. 4-cell-stage embryo cleavage-arrested with cytochalasin B and stained for
acetylcholinesterase activity at 16 h after fertilization.
Fig. 8. 8-cell-stage embryo cleavage-arrested with cytochalasin B and stained for
acetylcholinesterase activity at 16 h after fertilization.
Embryos in Fig. 3 incubated histochemically for 120 min; others incubated 60min.
All Figures are of the same magnification; bar is 50 ytm.
244
J. R. WHITTAKER
Table 2. Microdensitometry measurements of acetylcholinesterase activity in 16 h
embryos and in early cleavage stages arrested with cytochalasin B until the emr
bryos were 16-5 h of age
Embryos
1-cell cleavage-arrested
16 h control
4-cell cleavage-arrested
16 h control
8-cell cleavage-arrested
16 h control
Acetylcholinesterase activity
(absolute O.D. units embryo"1)
10743 ± 781*
11515 ±497
10407 ± 663
10750 ±356
9220 ± 462
9498 ±442
* Mean value of 25 embryos ± standard error of the mean; enzyme incubation for 60 min.
activity between normal and cleavage-arrested embryos (Table 2). Since enzyme
activity was still proportional to developmental time up to 18 h (Fig. 2A), the 16 h
stage activity does not represent a saturation level for measurements by this
technique.
Cytochrome oxidase in cleavage-arrested embryos
Another enzyme, cytochrome oxidase, did not follow the same pattern of
increase in cleavage-arrested embryos as it does in normal embryos. Cytochrome
oxidase activity was measured in normal and cleavage-arrested embryos by a
Fig. 9. 4-cell-stage Ciona intestinalis embryo stained histochemically for cytochrome
oxidase activity with the diaminobenzidine method.
Fig. 10. 16 h embryo stained for cytochrome oxidase activity. Embryos incubated
histochemically for 90 min. Figs 9 & 10 are of the same magnification; bar is 50 |Um.
Ascidian muscle lineage
245
Table 3. Microdensitometry measurements of cytochrome oxidase activity in
cleavage stage and 16 h embryos and in early cleavage stages arrested with
cytochalasin B until the embryos were 16 h of age
Embryos
4-cell control
4-cell cleavage-arrested
16 h control
8-cell control
8-cell cleavage-arrested
16 h control
Cytochrome oxidase activity
(absolute O.D. units embryo"1)
3282 ± 134*
3920 ± 226
7071 ± 254
4900 ± 280
5010 ± 348
9602 ± 436
% increase
0
19
115
0
2
96
* Mean value of 25 embryos ± standard error of the mean; enzyme incubation for 90 min.
microdensitometry method. The DAB reaction for cytochrome oxidase resembles the acetylcholinesterase assay in being a simple, easily controlled reaction
that yields very little background staining (Figs 9, 10). Enzyme activity in 16 h
embryos was double that of normal 4-cell and 8-cell embryos (Table 3). This
result agrees closely with that found by D'Anna (1966) where he measured
cytochrome oxidase activity in homogenates of Ciona embryos by a manometric
technique and found a doubling of activity during the same period of development. Comparison of the two results shows the microdensitometry method to be
quite adequate for measuring changes of cytochrome oxidase activity during a
2-16 h time.
Cytochrome oxidase activity in cleavage-arrested embryos increased only
slightly during a 16 h development period (Table 3). Other series (not shown)
had activity changes within the same 2-19 % range. Obviously, no quantitative
regulation of cytochrome oxidase development occurred. All embryos,
cleavage-arrested as well as control, produced a localized cytochrome oxidase
reaction. The only basis used in selecting embryos to measure quantitatively was
their orientation on the slide (Figs 9, 10), such that strongly staining regions
would fall within a median plane of focus of the microdensitometer.
Cleavage-arrested and normal 4-cell- and 8-cell-stage embryos could not be
combined on the same slides because they were indistinguishable in their staining
characteristics. Cleavage-arrested and cleavage-stage control embryos were incubated separately with 16 h control embryos from the same group. The
measurement of each cytochalasin-arrested embryo was normalized to the 16 h
value of the cleavage-stage control set by applying the ratio of the two 16 h mean
(N = 25) values. A secondary problem is a variability in staining introduced by
slight solubility of the enzyme reaction product in alcohols during the dehydration of embryos. This accounts for the differences in the 16 h measurements seen
in Table 3, and necessitated the inclusion of 16 h control embryos in each group
246
J. R. WHITTAKER
of embryos processed so that results could be calculated relative to the 16 h
control value (% increase).
DISCUSSION
Microdensitometry measurements show clearly that some cleavage-arrested
early Ciona embryos produced an amount of acetylcholinesterase activity similar
in range to that of normal embryos developing for the same length of time. A
serious bias, however, has been introduced into selection of the cleavagearrested embryos to be measured. Many cleavage-arrested embryos make no
enzyme, and others do not produce (by visual estimation) equivalent amounts
of enzyme reaction product in the two lineage blastomeres at 4-cell and 8-cell
stages; non-reactive embryos and these partially reactive embryos were disregarded in making the measurements reported in Tables 1 & 2. The acetylcholinesterase results presented in this paper are offered with the caveat that one can
ignore 'non-developing' cleavage-arrested embryos and those in which development does not appear to be symmetrical or otherwise 'normal'. Findings of future
studies may conceivably invalidate this assumption.
The reasons why some cleavage-arrested embryos fail to produce enzyme in
some or all of the muscle lineage cells are not known, but the most probable
explanation is that a certain number of nuclear divisions (or DNA replications)
must occur before the cell is competent to undergo differentiation (Satoh, 1982).
Preliminary experimental results of my own are consistent with this explanation.
In Ciona embryos dechorionated at fertilization, one can observe easily which
of them become multinucleate during development in cytochalasin B. Those
which are not obviously multinucleate in appearance do not later give reactions
for acetylcholinesterase, whereas multinucleate embryos do. Continuity of
nuclear divisions seems to fail early in some cytochalasin-treated embryos; such
embryos are 'non-developing'.
Regulation of acetylcholinesterase expression in cleavage-arrested embryos
occurs independently of the normal cytoplasmic volume associated with larval
muscle cells. The terminal number of larval muscle cells is 36 (Berrill, 1935), but
most of the muscle lineage volume is already established at the 64-cell stage
(Reverberi, 1971; Whittaker, 1979a). The volume of the lineage cells at the
64-cell stage represents approximately the relative cytoplasmic volume of larval
muscle: l/8th (8 cells out of 64) of the original egg cytoplasmic volume (Fig.
11A). Cleavage-arrested 1-cell stages have, therefore, about eight times the
volume of normal muscle lineage cell cytoplasm. One can calculate that the two
muscle lineage cells of cleavage-arrested 4-cell stages have approximately four
times the cytoplasmic volume of the normal larval muscle mass. Inspection of the
muscle lineage territory at the 8-cell stage (Fig. 11B), as determined by the
Ortolani (1954) marking experiments, shows that half of the two muscle lineage
247
Ascidian muscle lineage
A
B
Fig. 11. Diagrams of the 64-cell-stage embryo (A) indicating the eight muscle
lineage cells into which the future myoplasm has become segregated and an 8-cellstage ascidian embryo (B) showing fate map of the cytoplasm (diagonal lines) destined to enter tail muscle cells. Figures are according to Ortolani (1955).
cells is future myoplasm: the two cells in cleavage-arrested 8-cell stages have
about twice the volume of normal myoplasm.
Quantitative acetylcholinesterase expression is not so obviously exclusive of
a fixed nuclear number. Cleavage-arrested embryos are very clearly multinucleate (Crowther & Whittaker, 1983), but without counting the nuclei histologically at the time of first enzyme expression one cannot know whether expression in cleavage-arrested embryos occurs in the presence of many more than
the maximum number (36) usually associated with myoplasm. Perhaps there are
never more than an average of 36 nuclei associated with myoplasm at the time
of first expression in cleavage-arrested embryos. This seems an unlikely coincidence. Given a normal progression of nuclear divisions, 1-cell, 4-cell, and 8-cell
cleavage-arrested embryos should have significantly different numbers of nuclei
per myoplasm-containing cells. Certainly during early stages of treatment with
cytochalasin B, nuclei are observed to divide regularly on approximate schedule
(Brachet & Tencer, 1973; Whittaker, 1973).
Another example of end-product regulation is found in the melanocyte
lineages of cleavage-arrested ascidian embryos: regulation of tyrosinase activity
levels and amounts of melanin pigment occurs independently of cytoplasmic
volume and also of nuclear number (Whittaker, 1979c, 1981). A cytoplasmic
determinant seems to be segregated in the melanocyte lineages (Whittaker,
1973). Perhaps quantitative regulation in cleavage-arrested embryos occurs only
in relation to differentiation pathways governed by egg cytoplasmic determinants.
Quantitative regulation of end products is not a general property of expression
in cleavage-arrested ascidian embryos. Mitochondrial cytochrome oxidase, a
universal rather than histospecific protein, changes only slightly in cleavagearrested Ciona embryos. Large numbers of mitochondria become segregated
into the muscle lineage cells of ascidian embryos, but mitochondrial enzymes are
not known to be regulated by a differentially segregated determinant (Whit-
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J. R. WHITTAKER
taker, 19796). Since biosynthesis of cytochrome c oxidase requires participation
of both mitochondrial and cytoplasmic translation systems (Poynton, 1980), its
regulation in cleavage-arrested embryos may fail to occur for reasons unrelated
to lack of involvement with a determinant factor.
The nature of the cytoplasmic determinant for muscle differentiation is unknown, but the hypothesis that determinants are the differential segregation of
preformed maternal messenger RNA (mRNA) for histospecific proteins (Reverberi, 1971; Brachet, 1974) could explain most features of muscle determinant
behaviour, including quantitative regulation of end product. A fixed amount of
maternal mRNA for muscle acetylcholinesterase might, for example, give rise
to a very definite quantity of enzyme irrespective of cytoplasmic volume or
nuclear number. Substantial contrary evidence, however, denies that the acetylcholinesterase determinant is enzyme mRNA.
Results of experiments with actinomycin D, an inhibitor of RNA synthesis,
identify a period of new RNA synthesis beginning at mid-gastrulation in various
ascidian species as essential for later expression of muscle acetylcholinesterase
(Whittaker, 1973, 19796; Meedel & Whittaker, 1979; Satoh, 1979). Meedel &
Whittaker (1983) have now found that RNA extracted from Ciona embryos at
mid-gastrula and later stages elicits synthesis of immunospecific Ciona acetylcholinesterase in Xenopus laevis oocytes; RNA extracted from stages earlier
than mid-gastrula does not. The mRNA for muscle acetylcholinesterase is apparently first synthesized at mid-gastrulation as implied by the results of
actinomycin D experiments. Such findings also preclude occurrence of a maternally preformed but inactive proenzyme.
Similar amounts of acetylcholinesterase activity in normal and cleavagearrested embryos indicate that some processes of genetic transcription and
translation proceed normally in cleavage-arrested embryos. Possibly this quantitative control of differentiation end product is evidence of simple and as yet
unappreciated properties of the transcriptional and translational control systems
operating during embryonic development. More likely, however, quantitative
control is a feature of development in those 'mosaic' embryos which have
maternally preformed elements directing later pathways of development. Such
control may be linked directly to the manner in which these egg cytoplasmic
determinants function.
This work was supported by Grant HD-16547 from the National Institute of Child Health
and Human Development, DHHS, and March of Dimes Birth Defects Foundation Grant
1-780. I thank Dr Vincent J. Cristofalo for permission to use the Vickers M85 microdensitometer at the Wistar Institute of Anatomy and Biology in Philadelphia.
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{Accepted 11 February 1983)