/. Embryol. exp. Morph. Vol. 26, 3, pp. 367-391, 1971
367
Printed in Great Britain
Timing of the phases of the cell cycle
during the period of asynchronous division up
to the 49-cell stage in Lymnaea
By J. A. M. VAN DEN BIGGELAAR 1
From the Zoological Laboratory, University of Utrecht
SUMMARY
The duration of the phases of the cell cycle (M-G!-S-G2) has been determined from the 8up to the 49-cell stage in eggs of Lymnaea, using autoradiography and cytophotometry of
Feulgen-stained nuclei. Division asynchrony of corresponding cells in different quadrants is
primarily caused by unequal lengthening of the G2 phases. In general it appeared that in the
vegetative cells lengthening of the cell cycles is chiefly due to an extension of the G2 phases,
whereas in the cells of the animal half the duration of both the S and the G2 phases are
extended. DNA synthesis is not blocked in cells which stop dividing and start to differentiate.
A conspicuous lengthening of the cell cycles is observed in the 16- and 24-cell embryo; this
is accompanied with the reappearance of distinct nucleoli. Supporting evidence has been
obtained for the assumption that bilateral symmetry at the animal pole of the embryo is
induced by cells from the vegetative hemisphere, presumably by the macromere 3D, during
the 24-cell stage.
INTRODUCTION
Up to the formation of the first quartet of micromeres the blastomeres divide
synchronously. Beyond this stage division synchrony is lost, and the derivation
of the quartets of micromeres and the macromeres have different cleavage
rhythms. As a rule corresponding cells of the four quadrants divide synchronously.
At the 4-cell stage the blastomeres show an almost similar cytoplasmic
composition. Within the individual cells the components of the cytoplasm
exhibit an animal-vegetative gradient. At the animal side the blastomeres contain
dense protoplasm, which is formed by the confluence of the animal pole plasm,
the perinuclear plasm and the subcortical plasm (Raven, 1946). This dense
protoplasm is rich in mitochondria and compact yolk granules (beta-granules).
The vegetative side contains yolk granules surrounded by a vacuole (gammagranules) and fat droplets (Elbers, 1957; Bluemink, 1967; Raven, 1967). As a
consequence, at the 8-cell stage the composition of the macro- and micromeres
differs significantly. This cytological differentiation coincides with the disappearance of division synchrony. The differences in the cytoplasmic composition
1
Author's address: Zoologisch Laboratorium der Rijksuniversiteit, Utrecht, Janskerkhof 3,
The Netherlands.
368
J. A. M. VAN DEN BIGGELAAR
appearing during subsequent cleavages might represent a prerequisite for the
development of division asynchrony.
When divisions become asynchronous, generally the interphase is prolonged
in time, whereas the duration of mitosis remains rather constant (Agrell, 1964;
Dettlaff, 1964; Graham & Morgan, 1966; Kauffman, 1968). Obviously, a
differentiation in the duration of division cycles is controlled by the duration
of interphase. Zeuthen (1951) suggested that the nucleus has the alternative
choice of reorganizing itself on the one hand, or controlling growth processes of
the whole cell on the other hand. In connexion with this idea Agrell (1962,1964)
assumed that the degree of differentiation of the blastomeres may be related to
the duration of interphase. Only during interphase does the nucleus have the
possibility of directing the differentiation by the production of messenger RNA
for the synthesis of specific proteins (Agrell, 1962). For this reason the duration
of the phases of the cell cycles has been studied during the asynchronous
divisions up to the 49-cell stage of the Lymnaea egg.
MATERIALS AND METHODS
The DNA synthetic period was determined with tritiated thymidine ([3H]Tdr)
and autoradiography and by means of microspectrophotometric determinations
of the dye content of Feulgen-stained nuclei (van den Biggelaar, 1971 a). In the
autoradiographic experiments the eggs were cut at 2 /mi thickness. Finally each
egg was reconstructed to denote the different cell types. In the spectrophotometric experiments whole mounts were prepared. The eggs were carefully
oriented with the vegetative pole facing the slide. With the aid of the pattern
of the spiral cleavage and the differences in the height of the nuclei within the
embryo, it was possible to denominate each nucleus. Auto radiographs were
photographed as described by Flight, Van Kooten and Snelleman (1970).
RESULTS
Cell-lineage
The cell-lineage of the eggs of three egg masses was studied up to the 49-cell
stage. The results are illustrated in Fig. 1, and generally confirm those obtained
by Verdonk (1965), with the exception that neither the cells lax-ldx nor the cells
Ia2-ld2 divided synchronously. The positions of the nuclei in representative
whole mounts of eggs at the 8-, 12-, 16-, 24-, 44- and 49-cell stage are shown in
Figs. 2A-F, respectively.
The duration of the different mitotic stages after the 8-cell stage has not been
studied in detail. No differences were observed in the duration of mitosis during
the synchronous and asynchronous divisions.
Timing of the phases of the cell cycle in Lymnaea. / /
369
The cell cycle of the macromeres 1A-1D and the micromeres la-Id
The average cell cycle of the macromeres 1A-1D was 80 ± 5 min. The average
cell cycle of the cells of the first quartet of micromeres la-Id was 96 ± 7 min.
DNA synthesis in these cells, deduced from the determination of the Feulgenfa 11 . 1b",
1c"
fa'-fd 1
fa 12 , 1b'2, 1c'2
fa-fd
fa21, fb21 (Pr.)
1c2\ fd21 (H.V.)
faMd 2
1c2\ fd«(H.V.)
Ja22, Jb 22 (Pr.)
A-D
2a11, 2c", 2d" (H.V.)
2b" (Pr.)
2a'-2d'
2a 12 -2d 12
2a-2d
2a 21 -2d 21
2a2-2d2
2a 22 -2d 22
1A-1D
3a-3d
3a2-3d2
2A-2D
4a, 4b, 4c
M(4d)
3A-3D
4D
4A, 46, 4C
0
1
2
3
4
5
6
7
8
9
Time after 3rd cleavage (h)
Fig. 1. Cell-lineage of the Lymnaea egg. Cells of the head vesicle (H.V.) and the
prototroch (Pr.) stop dividing.
dye content, is shown in Figs. 3A and B. It will be clear that, although the microand macromeres did not divide synchronously, they reduplicated their DNA
simultaneously. DNA synthesis started when the nuclei were in telophase 7 min
370
J. A. M. VAN DEN BIGGELAAR
FIGURE 2
Position of the nuclei in successive cell stages. For convenience, the nuclei of
corresponding cells in different quadrants are interconnected, demonstrating the
alternation of dextral and sinistral cleavages.
(A) Eight-cell stage.
(B) Beginning of the 12-cell stage. The micromeres la-Id are in metaphase. The
macromeres 1A-1D just start to divide into the micromeres 2a-2d and the macromeres 2A-2D (telophase).
Timing of the phases of the cell cycle in Lymnaea. / /
371
FIG. 2 (cont.)
(C) End of the 16-cell stage. The nuclei of the micromeres 2a-2d are in prophase;
the nuclei of the macromeres 2A-2D are in metaphase.
(D) End of the 24-cell stage (resting stage). The nuclei of the central macromere 3D
and the micromeres 2ax-2dx are in metaphase or in anaphase.
24
E M B 26
372
J. A. M. VAN DEN BIGGELAAR
after the formation of the first quartet of micromeres. Hence, the cells 1A-1D
and la-Id did not pass through a Gx phase. Reduplication was completed after
about 30 min.
(E) Forty-four cell stage, at which division asynchrony in the first quartet of
micromeres becomes visible. The cell Id1 has just divided and the primary trochoblasts la2 and 1b* are in metaphase, whereas the corresponding cells 7c2 and ldz
are still in interphase. The primary mesoblast 4d is in interphase, whereas the cells
4a, 4b and 4c are just formed.
The interval during which DNA synthesis takes place was also determined
with [3H]Tdr and autoradiography. The results have been summarized in
Table 1. It can be concluded that the reduplication of DNA within macro- and
micromeres took place between 7 and 30 min after the beginning of the third
division, which is consistent with the spectrophotometric data of Fig. 3. In
comparison with the preceding cycles the duration of mitosis remained constant.
Therefore, the extension of the cell cycle of the celts of the first quartet of
Timing of the phases of the cell cycle in Lymnaea. / /
373
micromeres la-Id was nearly restricted to the G2 part of interphase. The duration
of the G2 phase in the macro- and micromeres was 19 and 32 min, respectively.
The cell cycle of the macromeres 2A-2D and the micromeres Idi-ld,
The average duration of the division cycle of the macromeres 2A-2D and of
the micromeres 2a-2d was 75 ± 5 and 85 + 5 min, respectively. The position of
the nuclei of these cells is shown in Figs. 2B and C. The spectrophotometric
FIG. 2 (cont.)
(F) Position of the nuclei in a 49-cell embryo. Nuclei of corresponding sister cells in
different quadrants are represented in the same way.
measurements for the determination of the S phases have been plotted in Figs.
4A and B. In comparison with the preceding stages, the pattern of DNA synthesis remained practically unaltered. Reduplication of DNA started at late telophase, and was completed in both cell types about 30 min after the beginning
2+-2
374
J. A. M. VAN DEN BIGGELAAR
4x-
2x--
10
20
30
40
60
t I s
90
70
H
Formation
of 1A-1D
100 min
f
Formation
of 2A-2D
4x-
2x-
10
t I
Formation
of la-Id
20
S
30
I
40
5Q
G2
60
I
70
80
M
90
100 min
I
Division
of la-Id
Fig. 3. Reduplication of DNA in the macromeres JA-1D (A) and in the micromeres
la-Id (B). The amount of DNA in this and subsequent figures is expressed in
multiples of the relative amount of DNA measured in a haploid nucleus, x (sperm
head). Means and standard errors of the means of three experiments. Each point
represents about 15 eggs.
of the formation of the second quartet of micromeres. Again changes in the
duration of the cell cycle were mainly reflected in the duration of the G2 phase.
The duration of DNA synthesis in the cells 2A-2D and 2a-2d can also be
derived from the results of an experiment in which eggs were exposed to [3H]Tdr
at different intervals from the 8- up to the 24-cell stage. The results of this
experiment are displayed in Fig. 5. The eggs of groups 1, 2 and 3 were incubated
Timing of the phases of the cell cycle in Lymnaea. / /
375
from 14 min before to 5, 10 and 15 min, respectively, after the beginning of the
formation of the second quartet of micromeres. In group 1 none of the nuclei of
the cells 2A-2D and 2a-2d were labelled. In group 2 a small number of grains
could be observed over the nuclei of both cell types. As in this group the nuclei
were in telophase, the cells 2A-2D and 2a-2d did not pass through a G1 phase. In
Table 1. Incorporation of [3H]Tdr at the 8-cell stage
(Actual incubation times have been converted into the corresponding times of a cleavage
cycle of the average duration. The cleavage cycle of the macromeres was 80 ± 5 min, the
cleavage cycle of the micromeres was 96 ± 7 min.)
Relative
incubation
time (min)
after the onset of:
,
A
stage
57566057565658—
—
—
—
—
^
stage
-0
-5
-6
-7
-10
-11
-12
1-17
6-22
8-25
12-32
22-42
25-46
27-50
30-50
33-50
34-56
35-59
No.
of
eggs
Eggs
label lee
(%)
34
10
38
18
17
—
—
—
—
59
73
57
100
100
100
100
80
38
6
—
—
—
—
n
14
4
3
12
46
10
21
16
11
8
14
16
Eggs in mitosis (%)
A
\
Macromeres
lOOt
lOOt
lOOt
83 t
24 t
9 t
—
—
—
—
—
—
48 p
69 p
82 p
75 p
100 p
100 p
Micromeres
lOOt
lOOt
lOOt
83 t
24 t
9t
—
—
—
—
—
—
—
—
—
—
—
—
t = telophase; p = prophase
group 3, a relative high number of grains could be counted. Two representative
sections through the nuclei of 2a-2d and 2A-2D are shown in Figs. 6 A and B,
respectively. In group 4 the eggs were incubated from 22 to 42 min after the
formation of the second quartet of micromeres. In comparison with group 3 the
number of grains was strongly reduced. In the eggs of group 5, incubated from
35 to 55 min after the beginning of the cycles of 2A-2D and 2a-2d, the nuclei of
these cells were unlabelled. This implies that the S phase of both types of cells
must have been completed before the beginning of incubation in group 5. This
agrees with the spectrophotometric data displayed in Figs. 4A and B. It may be
concluded that the macromeres 2A-2D and the micromeres 2a-2d reduplicate
their DNA synchronously between 5 and 35 min after the onset of the cell cycle.
376
J. A. M. VAN DEN BIGGELAAR
The cell cycle of the macromeres 3A-3D and the micromeres 3a-3d
During the first part of the cell cycle the macromeres 3A-3D are arranged
symmetrically around the vegetative pole. At a later stage the macromere 3D is
shifted towards the centre of the vegetative pole (Verdonk, 1965) and as a result
the macromeres 3A, 3B and 3C move to a more peripheral position (Fig. 2D).
This is the first indication of bilateral symmetry of the embryo.
4x-
2x-
10
I
Formation
of 2A-2D
20
S
30
40
| Ga |
50
60
M
70
80
90
100 min
(
Formation
of 3A-3D
4x-
2x-
100 min
|
Formation
of 2a-2d
Division
of 2a-2d
Fig. 4. Reduplication of DNA in the macromeres 2A-2D (A) and in the micromeres
2a-2d(B). Means and standard errors of the means of three experiments. Each point
represents about 15 eggs.
Timing of the phases of the cell cycle in Lymnaea. / /
377
As in most gastropods, the 24-cell stage is a resting stage in the development
of the embryo. During a period of about 3 h no divisions can be observed
(Verdonk, 1965). As a consequence, the cycles of the cells constituting the 24-cell
embryo are significantly extended. The division of the cell 3D into the macromere 4D and the primary mesoblast 4d or M is the first exception to the rule
that corresponding cells in different quadrants divide synchronously.
Incubation
periods
0-0-0
33
fa'-Jd
32
10
0
Grains
0
1
60
40
20
1a-1d
80
100
Minutes
faMd2
0-0-0
18
22
Grains
30
Grains
48
43
2a1-2d1
0-10-103
74
0
Grains 0
40
20
2a-2d
Minutes
2a2-2d2
46
1A-1D
20
40
60
42
80
34
Grains
Minutes
72
41
Grains
20
40
60 Minutes
71
39
Grains
3a-3d
2A-2D
0-12-102
67
0
Grains 0
3A-3D
21
3
Fig. 5. Incorporation of [ H]Tdr at different intervals from the 8- up to the 24-cell
stage. Incubation periods of the different groups of eggs indicated in the upper part of
the figure. Number of grains over the nuclei of the different cell types are listed
vertically below the number of each group.
The cell cycle of 3D was about 200 min, whereas the macromeres 3A, 3B and
3C divided 90 min later. The average cycle of the cells of the third quartet of
micromeres 3a-3d was about 270 min. During the first part of the 24-cell stage
the macromeres 3A-3D cannot be distinguished from each other. As in the
individual eggs no differences could be observed in the Feul gen-dye content of
378
J. A. M. VAN DEN BIGGELAAR
(A)
(B)
Fig. 6. Two sections through an egg of group 4, incubated in [3H]Tdr during the
period indicated in Fig. 5. (A) Nuclei of the macromeres 2A-2D. (B) Nuclei of the
micromeres 2a-2d. The micromeres lax-ld} extend in the blastocoel.
Timing of the phases of the cell cycle in Lymnaea. / /
379
the four nuclei, it can be concluded that notwithstanding the different durations
of the cell cycles of 3A, 3B and 3C in comparison with 3D, reduplication was
synchronous. From Figs. 7 A and B it can be concluded that DNA synthesis in
10
20
30
20
30
1
i
i
40
50
60
70
40
50
60
70
i
80
—*-
90
100 min
80
90
100 min
Formation
of 3A-3D
2x-
10
(I
S
|
Ga
—
Formation
of 3a-3d
Fig. 7. Reduplication of DNA in the macromeres 3A-3D (A) and in the micromeres
3ci-3d (B). Means and standard errors of two experiments. Each point represents
five eggs.
the macromeres 3A-3D is also simultaneous with reduplication in the micromeres 3a-3d. As the nuclei were in late telophase at the onset of DNA synthesis,
the cells do not pass through a Gx phase.
380
J. A. M. VAN DEN BIGGELAAR
By means of the experiment shown in Fig. 5, the S phases of 3A-3D and
3a-3d can be determined partially. The eggs of group 6, incubated from 14 min
before up to 10 min after the formation of the third quartet of micromeres, were
labelled. This indicates that DNA synthesis started during late telophase and
that the cells 3A-3D and 3a-3d do not pass through a Gx phase. Both types of
cells reached the maximum labelling intensity in group 7, incubated from 10 up
to 30 min after the beginning of the cycle. The number of grains observed over
the nuclei in group 8, incubated from 28 up to 48 min after the onset of the cycle,
was much reduced. Apparently, during the incubation period of this group the
greater part of the DNA had already been replicated.
Summarizing, it may be concluded that the macromeres 3A-3D and the
micromeres 3a-3d do not pass through a Gx phase and reduplicate the DNA
synchronously. As the S phase is only slightly extended, the extension of the cell
cycle is almost restricted to the G2 part of interphase.
The cell cycle of the derivatives of the first quartet of micromeres,
l a M d 1 and l a M d 2
According to Verdonk (1965) the first sign of bilateral symmetry in the
localization of the blastomeres and in time of cleavage is formed by the macromere 3D. The first sign of bilateral symmetry at the animal pole would become
apparent at the division of the basal cell of the dorsal arm of the cross, Id121.
However, a transient indication of bilateral symmetry at the animal pole could
already be observed at the division of the cells lax-ldx and la*-ld2 (Fig. 2E).
The cells Ia1-ld1 divided asynchronously, as could be observed in 35 eggs. In 24
of these eggs (69 %) the blastomeres la1,lb1 and 7c1 were in mitosis, started to
divide or had already divided whereas the cell Id1 was still in interphase, entered
mitosis or was in a less advanced mitotic stage. In seven eggs (20 %) two or one
of the cells la1, 1b1 or 7c1 were in an advanced stage. In six of these la1 was in
advance, in five eggs lb1, and 1c1 in one egg only. In two other eggs (6 %) the
cells la1,1b1 and Id1 had divided whereas 1c1 was still in mitosis. Finally, in one
e
gg (3 %) the cell 1c1 had divided whereas the cells la1, 1b1 and Id1 were in
mitosis.
A second deviation to the rule of synchronous division of corresponding cells
within the first quartet of micromeres could be observed at the division of the
cells Ia2-ld2. In 37 eggs this asynchrony could be analysed. In 25 of these eggs
(68 %) either the blastomeres la2 and lb2 were in mitosis or had already divided
whereas the cells 7c2 and Id2 were still in interphase (Fig. 2E), or la2 and lb2 had
divided whereas the cells 7c2 and Id2 were in mitosis. In four eggs (11 %) the cell
lb2 was in advance. In four other eggs (11 %) the cell la2 was in advance. In one
egg (3 %), only the cell 7c2 was delayed. In three eggs (8 %) the four cells divided
synchronously.
The average cell cycle of the cells la1, lb1 and 7c1 was about 300 min, the cell
1
Id generally divided 30 min later. The average cell cycle of the cells 7a2 and lb2
Timing of the phases of the cell cycle in Lymnaea. / /
2
381
2
was about 340 min, whereas the cells 7c and Id divided about 60 min later.
Reduplication of DNA in the cells lax-ldx and Ia2-ld2 is shown in Figs. 8 A and
B. Although the cells lax-ldx did not divide synchronously, a simultaneous
4x-
2x- -
10
20
I
30
40
50
S
60
70
80
I
90
100
110 120 min
G,
—
Formation
of Ja'-fd'
2x-
-U
10
t I
20
30
40
50
60
S
70
80
90
100 110 120 min
I•
G2
-
Formation
of fa2— 1d2
Fig. 8. Reduplication of DNA in the micromeres lax-ldx (A) and in the micromeres
Ja*-Jd2 (B). Means and standard errors of the means of five experiments. Each
point represents about 20 eggs.
increase in the Feulgen stain could be observed. The same phenomenon was
perceived in the cells Ia2-ld2. DNA synthesis started about 10 min after the
beginning of the cycle, when the nuclei were in late telophase. This indicates the
absence of a Gx phase. In the cells lax-ldx DNA synthesis was completed after
382
J. A. M. VAN DEN BIGGELAAR
about 55 min and in the cells Ia2-ld2 about 110 min after the beginning of the
cycle. Apparently, reduplication of DNA in the primary trochoblasts Ia2-ld2
takes place at a much lower rate than in the cells la^-ld1.
1d
20fim
Fig. 9. Section through the nuclei of the micromeres Ic^-ld1 (in the centre) and
Ja2-Jd2 (at the periphery) of an egg of group 6, incubated in [3H]Tdr during the
period indicated in Fig. 5.
This observation has been confirmed by means of the incorporation of
[ H]Tdr (Fig. 5). The eggs of group 6 were incubated from 40 up to 66 min
after the beginning of the cell cycle of the cells lax-ldx and Ia2-ld2. It may be
assumed that the cells la^-ld1 completed DNA synthesis before the end of the
incubation period, as in group 7 grains could no longer be observed. The cells
Ia2-ld2 acquired the highest labelling intensity in group 6 (Fig. 9), and the low
number of grains observed over the nuclei after the incubation periods of group
7 and 8 indicate a low rate of DNA synthesis from 66 up to about 110 min after
the beginning of the cycle (compare Fig. 8B).
3
Timing of the phases of the cell cycle in Lymnaea. / /
x
x
2
383
2
It may be concluded that the cells la -ld and Ia -ld do not pass through a
Gx phase. The rate of DNA synthesis is much higher in the former than in the
latter. The extension of the cell cycles of lax-ldx and Ia2-ld2 is partly due to an
extension of the S phase, but predominantly of the G2 phase.
The cell cycle of the derivatives of the second quartet of
micromeres, 2a1-2d1 and 2a2-2d2
The duration of the division cycles of the cells 2a1-2d1 and 2a2-2d2 was 190 and
230 min, respectively. Time and duration of the S phases are shown in Figs.
10 A and B. Again DNA synthesis started when the nuclei were in late telophase,
about 10 min after division of 2a-2d. Therefore, a Gx phase must be absent. In
both types of cells DNA synthesis was completed after about 50 min. The
extension of the cell cycles was reflected in a minor extension of the S phases
but mainly by a lengthening of the G2 phase.
DNA synthesis in the cells constituting the 49-cell stage
Of the blastomeres constituting the 49-cell embryo (Fig. 2F) only the onset of
DNA synthesis could be determined. With the spectrophotometric technique
used, it was not possible to investigate the increase of the Feulgen-dye content of
the individual cells owing to the great number of nuclei. However, by means of
the incorporation of [3H]Tdr into DNA it could be demonstrated that all
blastomeres of the 49-cell embryo started reduplication of DNA during late
telophase. Thus, up to and including the 49-cell stage the blastomeres do not pass
through a Gx phase. Of particular importance is, that this implies that DNA
synthesis is not blocked in cells which stop dividing when the 49-cell stage is
reached and start to differentiate into cells of the prototroch (7a21, lb21, la22
and lb22) and of the head vesicle (ic 21 , Id21, lc22, Id22, 2a11, 2c11 and 2dir).
Cytological observations
From the 8- to the early 24-cell stage strongly pyroninophilic subcortical
plasm could be observed as has been described for Lymnaea by Raven (1946,
1967, 1970) and Minganti (1950). In this subcortical plasm, located around the
cross furrow of the macromeres (Fig. 11), pyroninophilic granules are formed
which move towards the central part of the macromeres during the 24-cell stage
(Fig. 12). At the same developmental stage a strongly pyroninophilic subcortical plasm was observed in the central parts of the micromeres la^-ld1
(Fig. 13).
During the 24-cell stage another remarkable observation can be made. The
cell bodies of the cells la^-ld1 extend deeply into the cleavage cavity (Figs. 6B,
14). The cleavage cavity disappears and especially the cells la^-ld1 come into
close contact with the macromeres 3A-3D. The same phenomenon has already
been described for Physa by Wierzejski (1905) and for Lymnaea by Raven (1946)
and Minganti (1950).
384
J. A. M. VAN DEN BIGGELAAR
Prior to the 16-cell stage no distinct nucleoli could be observed. During the
early cleavages the nuclei possessed a number of small, weakly pyroninophilic
granules. From the 16-cell stage the cells lax-ldx and Ia2-ld2 do not divide until
several hours later, at the end of a prolonged resting stage. Only in these cells
distinct nucleoli could be observed. The remaining cells 2a-2d and 2A-2D soon
divide into 2a1-2d1 and 2a2-2d2, and 3a-3d and 3A-3D, respectively. By these
divisions the embryo reaches the 24-cell stage and then nucleoli appeared in all
blastomeres.
4x-
2x_
10
I
20
30
S
40
50
|
60
70
80
90
G2
100 min
—
Formation
of 2a'-2d'
4x-
2x-
90
100 min
Formation
of 2a2-2d2
Fig. 10. Reduplication of DNA in the micromeres 2a1-2d1 (A) and in the micromeres 2<f-2d2 (B). Means and standard errors of two experiments. Each point
represents five eggs.
Timing of the phases of the cell cycle in Lymnaea. / /
385
Fig. 1.1. Concentration of pyroninophilic granules (rich in RNA) along the crossfurrow of the macromeres 2A-2D, 16-cell stage.
Fig. 12. Concentration of pyroninophilic granules along the cell walls of the
macromeres 3 A, 3B and 3C adjacing the central macromere 3D in which these
granules can only be observed in more animal sections.
386
J. A. M. VAN DEN BIGGELAAR
1a2
1b2
20/im
Fig. 13. Autoradiogram of an egg incubated with [3H]Tdr (group 4 of Fig. 5),
showing strongly pyroninophilic subcortical plasm along the inner cell membranes
of the cells ltf-ld1. 16-cell stage.
Fig. 14. The primary trochoblast cells lcP-ld2 are connected with the cells 3a-3d.
The central part of the cell bridge extending in the blastocoel is formed by the cells
Itf-ld1. Early 24-cell stage.
Timing of the phases of the cell cycle in Lymnaea. / /
387
DISCUSSION
During the early embryonic development of Lymnaea the divisions succeed
each other rapidly without concomitant cell growth. It has been assumed that
within an embryo divisions take place synchronously as long as the cells are
essentially alike as regards their nucleus and cytoplasm (Agrell, 1964). It will be
evident that this supposition only holds true with respect to those components
which are important for cell division. Cytological differences are not necessarily
associated with division asynchrony. For instance, in eggs of Crepidula (Conklin,
1897) and Ilyanassa (Clement, 1952) division synchrony persists up to the 8-cell
stage. Nevertheless, at the 2-cell stage the composition of the blastomeres AB
and CD is already different as in both species the polar lobe substances are
segregated into the CD and then into the D blastomere. Apparently, the polar
lobe substances have no connection with the preparations for cell division
during the early cleavages.
In Lymnaea, as in other molluscs, the third cleavage is unequal and gives
rise to cytologically different macro- and micromeres. This unequal division
coincides with the end of division synchrony within the embryo as a whole. Both
the micromeres and the macromeres from which they have been split off, divide
asynchronously. However, division synchrony is maintained within corresponding cells in the different quadrants (e.g. the cells la-Id divide simultaneously;
similarly the cells 2a2-2d2, etc.). The first division of the first, second and third
quartet of micromeres is not synchronous with the division of the macromeres
from which they have been split off. Nevertheless, reduplication of DNA, one of
the preparative steps for cell division, takes place synchronously (Figs. 3 A, B;
4 A, B; 7 A, B). It is, therefore, very unlikely that asynchronous divisions are the
result of differential distribution of precursors for DNA synthesis. A differentiation in the pattern of DNA synthesis can only be demonstrated during the
second division of the cells of the first and second quartet of micromeres (Figs.
8A, B; 9 in comparison with Fig. 7A, B). A differential distribution of the prerequisites for DNA synthesis at that moment cannot be excluded.
According to Mazia (1961), Wilt, Sakai & Mazia (1967) the blastomeres of
sea-urchin eggs contain precursors for the mitotic apparatus. During division
asynchrony this supply was supposed to become exhausted (Mazia, 1961). It
may be possible that in Lymnaea the minor differences in the duration of the
cleavages prior to the 24-cell stage are brought about by an unequal distribution
or an unequal rate of synthesis of precursors for the mitotic apparatus.
As in most gastropods, the 24-cell stage of the Lymnaea embryo is called a
resting stage. During a period of about 3 h no divisions can be observed, and as
a consequence each cell type shows a long interphase. It is highly improbable
that this sudden extension of the cell cycle of all blastomeres at one particular
stage is caused by an exhaustion of reserve materials. The scarcity of available
data precludes a well-founded explanation for this phenomenon.
25
E M B 26
388
J. A. M. VAN DEN BIGGELAAR
An extension of the cell cycles in the 24-cell embryo is probably of great
morphogenetic importance. If a nucleus exerts its controlling influence upon
differentiation by transcription of its genetic information, an increasing control
of the nuclei upon morphogenesis will be accompanied by a lengthening of those
parts of the cell cycle in which DNA is capable to transcribe its genetic information into messenger RNA and the gene products can be transported to the cytoplasm. The reappearance of nucleoli in the 16- and 24-cell embryos corresponds
to conspicuously long G2 phases. It is not impossible that the appearance of the
nucleoli heralds the activation of the genetic information of the nuclei. Neyfakh
(1964) reported that radiation-induced damage of nuclei prior to or at the 12- to
16-cell stage does not disturb the development of snails (Lymnaeae) earlier than
the 22-cell stage. Unfortunately, neither the 22-cell stage has been described nor
the nature of the disturbances observed. Neyfakh concluded that in snails,
nuclear activity commences at the 12- to 16-cell stage.
At the end of the resting stage all blastomeres divide and the embryo reaches
the 49-cell stage (the M cell divides once more) (Fig. 2F). Some of these cells
stop dividing and develop into larval structures: the dorsal blastomeres lc21,
lc22, Id21 and Id12 with an interradial position develop into head vesicle cells; the
ventral blastomeres la21, la22, lb21 and lb22 develop into cells of the prototroch.
The dorsal tipcell 2dn and the lateral tipcells 2a11 and 2c11 also develop into
head vesicle cells, whereas the ventral tipcell 2b11 will divide once more and the
daughter cells 2b111 and 2b112 differentiate into prototroch cells (Verdonk, 1965).
Inhibition of cell division in these larval cells is not associated with an inhibition
of DNA synthesis, for all cells constituting the 49-cell embryo incorporated
[3H]Tdr into the DNA.
In Biomphalaria glabrata the cells of the larval liver have lost the capacity to
divide without losing the capacity to reduplicate the DNA (Schreiber & Carney,
1966). In Biomphalaria and in Bulla striata a high DNA content is correlated
with a large volume of the nuclei (Fallieri, Carney, Schreiber & Schreiber, 1969).
For this reason it is not unlikely that in Lymnaea the cells of the apical plate,
head vesicle, prototroch, larval liver and protonephridia with their relatively
large nuclei are polytenic or polyploid. Further investigations will be required
to determine the amount of DNA and to look for a possible relationship
between the DNA content and the function of these cells.
Division asynchrony is probably caused by a differential segregation of the
ooplasm. Clement (1952) presented evidence that in Ilyanassa division asynchrony between the macromeres 3A-3D is determined by means of a specific
segregation of cytoplasmic substances. The substances of the polar lobe are
sorted out into the macromere 3D. After removal of the polar lobe at the trefoil
stage, the cell 3D divides simultaneously with the macromeres 3A, 3B and 3C.
Although the Lymnaea egg does not show a polar lobe, a specific segregation of
subcortical plasms at the vegetative side has been demonstrated by Raven (1946,
1967, 1970) and Minganti (1950). Exactly the same phenomenon has already
Timing of the phases of the cell cycle in Lymnaea. / /
389
been observed in Physa by Wierzejski (1905). Therefore it may be postulated that
the precocious division of 3D in Lymnaea is also determined by the presence of
specific cytoplasmic substances. This assumption implies that in some way or
other these substances only affect the cleavage rhythm beyond the formation
of the third quartet of micromeres, as it is only the macromere 3D that does not
divide simultaneously with the macromeres 3A, 3B and 3C.
At the animal pole of the egg a second deviation to the rule of synchronous
division of corresponding cells in the different quadrants appears at the moment
when the cross figure becomes visible. In Lymnaea the cells that develop into
the ventral (lb1) and lateral arms (la1 and 1c1) divide 30 min earlier than the cell
Id1 that develops into the dorsal arm of the cross. On either side of the cell Id1
the interradially located primary trochoblasts lc2 and Id2 can be found. These
cells differentiate into head vesicle cells and divide 60 min later than the primary
trochoblasts la2 and lb2, that develop into cells of the prototroch (Fig. 2E). A
comparable situation can be observed during the development of Crepidula
(Conklin, 1897) and Physa (Wierzejski, 1905). Therefore the bilateral symmetry
of the head region is adumbrated by the division asynchrony of the cells of the
first quartet of micromeres, which form the head of the snail. The division
asynchrony between these cells seems to be related to the future dorsal-ventral
axis of the embryo instead of being restricted to a certain quadrant. Also at
later stages in the development of Lymnaea the bilateral symmetry is not only
expressed in the dorsal arm of the cross, but also in the development of the
innermost cells of the lateral arms (Verdonk, 1965, 1968). Clement (1952)
reported that the micromeres of the D quadrant only ' differ from their counterparts in the other quadrants in size, division tempo, visible composition or some
combination of these features'. If the polar lobe is removed, these differences
disappear and four similar quadrants, representing a radial plan of organization,
are produced. However, Clement did not investigate the division pattern of the
primary trochoblasts, Ia2-ld2. So it would be of interest to reinvestigate the
cleavage pattern of these cells in control eggs and in lobeless eggs of Ilyanassa. If
the differences would appear not to be limited to the D quadrant, as is the case
in Lymnaea, Physa and Crepidula, it is more obvious to assume that the division
asynchrony in the cells of the first quartet of micromeres is rather achieved by an
inductive influence, e.g. emanating from the cell 3D, than by a segregation of
special substances into the first quartet cells. In this context it must be emphasized
that after the formation of the third quartet of micromeres, i.e. in the 24-cell
embryo, the cleavage cavity disappears and the micromeres of the first quartet
come into contact with the inner side of the macromeres, especially with 3D
(Wierzejski, 1905; Raven, 1946,1970; Minganti, 1950). In Physa and Lymnaea it
has been observed that during the 24-cell stage irregular complexes of RNA
containing granules, located around the cross furrow of the macromeres, move
towards the innermost parts of the macromeres (Raven, 1970), and probably
pass to the micromeres (Wierzejski, 1905; Minganti, 1950). Apparently, the
25-2
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J. A. M. VAN DEN BIGGELAAR
morphological requirements are present for an inductive influence of the
vegetative macromeres upon the animal micromeres.
Additional information about the development of division asynchrony in the
first quartet of micromeres and the development of bilateral symmetry, and
the question whether this originates in the animal half of the embryo by
means of induction or ooplasmic segregation, will be discussed elsewhere (van
den Biggelaar, 19716).
RESUME
Determination des phases du cycle cellulaire pendant laperiode
de division asynchrone jusqu'au stade de 49 blastomeres chez Lymnaea
La duree des phases du cycle cellulaire (M-Gj-S-Ga) a ete determinee du stade a 8 jusqu'au
stade a 49 blastomeres dans les oeufs de Lymnaea, en utilisant la methode autoradiographique
et la determination photometrique de la teneur relative en ADN. La division asynchrone des
cellules correspondantes dans differents quadrants ne se manifeste comme une extension du
phase G2. En general il paraissait que dans les cellules vegetatives l'extension du cycle est
realisee principalement par une prolongation du phase G2, tandis que dans les cellules du
pole animal la duree des phases S et G2 est prolongee. La synthese de l'ADN n'est pas bloquee
dans les cellules qui ne se divisent plus et dans lesquelles la differentiation definitive est
commences. Une prolongation considerable du cycle cellulaire peut etre observee dans les
embryons du stade a 16 et a 24 blastomeres, ce qui est accompagnee par la reapparition des
nucleoles distinctes. Des indices ont ete obtenus que la symmetrie bilaterale a l'hemisphere
animal est induit par des cellules de l'hemisphere vegetatif, probablement par le macromere
3D au stade a 24 cellules.
This paper was prepared from a part of a doctoral thesis. The author is greatly indebted to
Professor Dr Chr. P. Raven and to Dr W. L. M. Geilenkirchen for sympathetic interest
and for reading the manuscript.
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