/. Embryol. exp. Morph., Vol. 17, 2, pp. 367-374, April 1967
Printed in Great Britain
367
Programming of gastrulation
during the second cleavage cycle in Limnaea
stagnalis: a study with lithium chloride and
actinomycin D
By W. L. M. GEILENKIRCHEN 1
From the Zoological Laboratory, State University of Utrecht
It is well known that treatment with LiCl can cause abnormal embryonic
development, but its mode of action is still uncertain (Gustafson, 1954; Geilenkirchen, 1961; Lallier, 1964). It also remains to be shown whether LiCl enters
the egg cell at early developmental stages and acts internally, or does not penetrate into the cell and is active only at the outer surface (Elbers, 1959).
LiCl treatment changes the rate of oxygen uptake during the early cleavage
cycles in Limnaea. The rate of oxygen consumption varies between the first and
the third cleavage in a cyclic fashion with maxima at mitotic prophases
(Geilenkirchen, 1961). Continuous treatment with LiCl disturbs the pattern of
periodic changes in the rate of uptake (Fig. 2). The influence of LiCl, however,
is not the same at all times. It does not start until shortly before prophase of
the second mitotic cycle, and this may indicate that until prophase of the
second division no processes in which LiCl interferes occur. Treatment of early
developmental stages of Limnaea with LiCl causes abnormalities much later in
development; most prominent are exogastrulae and head malformations. The
observation that LiCl does not interfere with oxygen consumption before the
prophase of the second division prompted us to compare the influence of LiCl on
morphogenesis after the treatment of eggs before and after this time.
It can be surmised that a treatment during early cleavage stages which affects
morphogenesis at a later stage causes interference with processes of specific
importance to later development (Geilenkirchen, 19646,1966). Therefore it is of
interest to study whether successive cleavage cycles and successive stages in one
cycle are equally susceptible to LiCl as judged by its effects upon later morphogenesis.
MATERIALS AND METHODS
Eggs of Limnaea stagnalis were obtained by exposing snails to plant leaves
and to a slight temperature rise (Geilenkirchen, 1961).
1
Author's address: Zoologisch Laboratorium, University of Utrecht, Holland.
368
W. L. M. GEILENKIRCHEN
In each experiment one egg mass only was used. The cleavage divisions of the
eggs in one mass, however, are not synchronous. Groups of about ten almost
synchronously dividing eggs can be obtained by selecting eggs which start the
first or the second division within 1 min. The first and the last group of each
batch selected in this way were used as controls. Treatment groups A-E were
treated with LiCl from 0, 20, 40, 60 and 80 min after the start of first cleavage
and group F at the start of second cleavage. In a second set of experiments the
period between second and third cleavage was tested in the same way. In a third
set of experiments the period between oviposition and first cleavage was tested.
In these last experiments the egg mass was divided into groups of ten eggs
without regard to synchronization because of the lack of a reference point. It
was observed that the first and second maturation divisions and the first cleavage
of the eggs in control groups took place within 15-20 min. Before treatment the
eggs were kept at a room temperature of about 20 °C.
The aim was to treat distinct stages in the early cleavage cycles with a LiCl
solution. Here two problems arise: first to stop or slow down development at
the start of treatment, and secondly to remove the treatment agent before development is resumed. In order to meet these requirements the treatment was given
at low temperature. At 6 °C development is slowed down to a great extent and
ultimately stops. After treatment the eggs were washed in tap-water at 6 °C.
After being washed, the eggs were transferred to tap-water at 25 °C.
The treatment procedure was as follows. Control group I was kept in tapwater of 6 °C for 4 h, beginning at first or second cleavage. The eggs were then
transferred to tap-water of 25 °C to continue development. Control group II was
kept in tap-water of 25 °C. Groups A-F were treated for 2 h in 5 x 10~2 M-LiCl at
6 °C, washed for 2 h in tap-water at 6 °C, and then reared in tap-water at 25 °C.
No differences in development were observed between controls kept at 25 °C
and those given a cold treatment for 4 h. The possible effect of a cold treatment
was also checked in separate experiments. A treatment before the second maturation division affects development; about 50% of the eggs developed normally
and 50% were retarded and ultimately died. After the second maturation
division no effects were observed after cold treatment.
After treatment the eggs were kept in tap-water for 48 h. Then the capsules,
each containing one egg, were laid out on moist agar in Petri dishes and cultured
at 25 °C.
The development of each embryo was followed from day to day until it
reached a stage at which the eyes in normal embryos are well developed. In all,
five grades of developmental disturbances were distinguished, namely firstperiod death (death before gastrulation), second-period death (death after
gastrulation), exogastrulation, non-specific malformations, head malformations
(Geilenkirchen, 19646).
About 1800 eggs in all were used in these experiments.
Gastrulation in Limnaea
369
RESULTS
Treatment with LiCl
First to third cleavage
Groups of synchronously dividing eggs were exposed to LiCl at different
times after first cleavage and then cultured in the normal way. The results are
shown in Fig. 1A and B.
In Fig. 1 B the percentage of exogastrulation and first-period death plus
exogastrulation are plotted against the time after first or second cleavage at
which treatment is initiated. Between first and third cleavage the treatment
causes first-period death only rarely. Between first cleavage and 40 min later
the proportion of exogastrulation is about 15%. From the stage 60 min after
first cleavage (mitotic prophase) the number of exogastrulating embryos rises
sharply and a maximum of almost 100% is reached at second cleavage. The
period in which the number of exogastrulae increases covers the period from
mitotic prophase to telophase, as is indicated in the figure. The succession of
mitotic stages has been studied in separate experiments, yet to be published.
After second cleavage the number of exogastrulae produced decreases continuously and at 60 min after second cleavage the number is down to 15-20%.
At this time prophase of third division is due. Instead of a rise in effect after
prophase as found in the second mitotic cycle, a further decrease is observed
in the third mitotic cycle. Treatment starting at the onset of the third cleavage
does not cause exogastrulation.
From these data it is obvious that between prophase of the second division
and prophase of the third division the sensitivity to LiCl is high, whereas before
and after this period a very low sensitivity is observed. After the third cleavage
the sensitivity for exogastrulation after LiCl treatment remains low (never
exceeding 5 %). When the embryo has reached the 24-cell stage, LiCl no longer
causes exogastrulation. This has been found in comparable experiments by
N. H. Verdonk (unpublished results).
In Fig. 1A cumulative percentages of normal, non-specifically malformed,
head-malformed and second-period death embryos are plotted against the time
after first and second cleavage at which treatment is initiated. It is obvious from
the figures that at stages of low LiCl sensitivity with respect to exogastrulation
most embryos develop normally. A small percentage of embryos dies after
gastrulation or becomes non-specifically malformed. It is seen that head malformations are produced at all stages between the first and third cleavage but
that between the second and third cleavages the number increases.
First maturation division to first cleavage
Groups of eggs from one egg mass were treated at intervals of 20 min between
oviposition and first cleavage. Fig. 1A and B show the results. In Fig. 1B the
percentages of exogastrulation and first-period death plus exogastrulation are
r
180
160
140
I—I
1st mat. div.
»Q
160
140
I—I
1st mat. div.
o—
180
Q
Q—..
100
80
I—I
2nd mat. div.
120
100
80
I—I
2nd mat. div.
120
60
60
40
40
K
20
0
20
M.
1st cleavage
T
•
1st cleavage
0
40
40
Time of treatment (min)
20
20
K
60
60
P
80
80
K
20
0
20
I—I
2nd cleavage
I—I
2nd cleavage
0
PM MA T T
40
40
PM
60
60
I—I
3rd cleavage
100
80
100
I—I
3rd cleavage
P PM MA T
80
PM MA T
Fig. 1. The cumulative percentages of normal and abnormal development in relation to time of treatment before first
cleavage, after first cleavage and after second cleavage. A, Ordinates: cumulative percentages of normal (O), non-specific
malformations ( • ) , head malformations (O) and second-period death ( x ) . Abscissae: time of onset of treatment with
regard to stage of development. B, Ordinates: percentages of exogastrulae (O) and first-period death + exogastrulae
( x ) . Abscissae: time scale as in A. T = telophase; K = karyomere nucleus; PM = polymorphic nucleus; P = prophase;
M = metaphase; A = anaphase.
20
40
60
80
100
20
40
60
80
100 r-
T
o
O
to
3
Gastrulation in Limnaea
371
plotted against the time before first cleavage at which treatment is initiated.
Between the second maturation division and first cleavage hardly any exogastrulation is observed. It is questionable whether the exogastrulae scored between
the first and second maturation divisions are in fact exogastrulated embryos.
This needs further histological proof. The exogastrulae found after treatment
at later stages are at first compact cell masses in which a few cells are swollen.
Later they swell and become dumb-bell shaped. The exogastrulae found after
9-6
9-5
9-4
93
9-2
9-1
90
8-9
88
8-7
86
8-5
8-4
83
8-2
8-1
7-9
x
d
7-8
7-7
76
100
75
7-4
80
73
72
7-1
60
70
6-9
40
68
6-7
20
66
•
, ^ ^ ,
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 100
1st cleavage
2nd cleavage
3rd cleavage
1 K
PM
P PM MA TT K
PM
P PM MA T
65
Time
Fig. 2. Rate of oxygen uptake (left ordinate) of normal ( 0 ) and lithium-treated ( • )
eggs in relation to cleavage stages between first and 3rd cleavage. Percentages of
exogastrulae (O) and exogastrulae plus first-period death ( x ) (right ordinate)
against time of onset of treatment.
372
W. L. M. GEILENKIRCHEN
treatment before first cleavage remain compact and never become dumb-bell
shaped. In this period, too, the percentage of first-period death is larger. As
already stated above, this period of development is also sensitive to cold treatment. Thus it is not possible to distinguish between LiCl effects and temperature effects.
In Fig. 1A the cumulative percentages of normal, non-specifically malformed,
head-malformed and second-period-death embryos are plotted. No stage is
especially sensitive to the treatment. It is, however, striking that before first
cleavage not a single head malformation was produced.
Treatment with actinomycin D
For reasons set forth in the discussion, twenty experiments were made with
actinomycin D at concentrations varying between 5 y/ml and 200 y/ml. No
effect on cleavage or development was observed if treatment was ended within
6 h after first cleavage.
It was not possible to check the penetration of actinomycin D in the eggs,
because with Limnaea eggs this would be possible only with labelled actinomycin
D, which was not available to us.
DISCUSSION
The results clearly show that the eggs are exceptionally sensitive to the LiCl
treatment between prophase of the second and prophase of the third division
as shown by exogastrulation. During comparable stages of the first and third
divisions they are not susceptible. During a period of 3 h after the third cleavage
the sensitivity is low (5 % exogastrulae, development at 25 °C). After that period
exogastrulae are no longer produced by LiCl (N. H. Verdonk, to be published).
This emphasizes the susceptibility in the period around second division.
After treatment, development proceeds in a normal way up to the time of
gastrulation. This indicates that during the mitotic stages immediately preceding
and following the second division a specific process occurs which is related to
gastrulation. The process is not apparently repeated. In this respect the LiCl
effect resembles the effect of ethionine treatment of sea-urchin eggs between
fertilization and 3rd cleavage. Bosco & Monroy (1960) found that such a treatment allowed further development but caused degeneration of the primary
mesenchyme cells.
It is obvious that the changes in sensitivity have a symmetrical course with a
maximum around second cleavage. Fig. 2 clearly shows that the increase in
sensitivity at about prophase of the second division is coincident with the LiClinduced change in oxygen consumption. Apparently LiCl first inhibits an oxygenconsuming process.
The high LiCl sensitivity around second cleavage only, points to damage of
processes specifically preparing for gastrulation. The nature of these processes
Gastrulation in Limnaea
373
remains unknown. Selective syntheses may take place and thus we have tried
the possibility of selective m-RNA production using suppression with actinomycin D. The results were inconclusive. Interference with DNA metabolism
is another possibility. In eggs of Limnaea DNA synthesis starts shortly after
each cleavage. The replication of DNA after second cleavage is, however,
marked by the fact that incorporation of exogenous thymidine is now possible,
whereas after first cleavage it is not (J. A. M. van den Biggelaar, to be published).
One possibility is then that LiGl interferes with thymidine kinase synthesis prior
to DNA synthesis after second cleavage.
Thus far we have only considered the effects of LiCl on gastrulation. With
regard to head malformations it is evident that they never occur on treatment
before first cleavage. After treatment between the first and second cleavage a
few cases are found and between the second and third cleavage the number may
increase to 10%. After the third cleavage three additional periods are found at
which LiCl causes head malformations (namely 1, 7 and 8-12 h after third
cleavage, temp. 25 °C; Verdonk, 1965). Apart from this it was observed that the
cell pattern of the head region specifically varied with the cycle treated. Similar
results have been obtained by a heat treatment. By applying a heat treatment or
centrifugation of short duration at successive stages of the early cleavage cycles,
it appeared that treatment around the time of the actual cleavage disturbed
development at a later stage. Development is not influenced by the same treatment of the stages in between cleavages (Geilenkirchen, 19646, 1966). With
regard to head malformations these are again found only after first cleavage has
passed. After first cleavage similar sensitive stages are found for heat treatment
and LiCl. Furthermore, heat treatment, like LiCl, causes specific head malformations, depending on the period of treatment (Verdonk, 1965; Geilenkirchen,
1966).
The data provide evidence that during the early cleavage cycles in Limnaea
processes occur, the products of which as metabolites, or structural entities, have
specific functions in later development. These processes are restricted to definite
cleavage cycles and definite phases of one cycle.
SUMMARY
1. Eggs of Limnaea stagnalis were treated with LiCl at successive stages
between oviposition and third cleavage.
2. The treatment of stages between prophase of the second division and prophase of the third division causes exogastrulation, whereas before and after
this period of development LiCl rarely causes exogastrulation.
3. The increase in the morphogenetic effect of LiCl after prophase of the
second division can be correlated with an effect of LiCl on oxygen consumption.
4. Head malformations never occur after treatment of eggs before the first
cleavage.
374
W. L. M. GEILENKIRCHEN
5. Actinomycin D treatment of early developmental stages of Limnaea had
no effects on development.
6. It is concluded that during second cleavage a process occurs preparing
specifically for gastrulation.
RESUME
La programmation de la gastrulation pendant la deuxieme division de
Limnaea stagnalis, etudie par Vaction du LiCl et de Vactinomycin D
1. Des oeufs de Limnaea stagnalis sont traites au chlorure de lithium a des
stades successives entre l'oviposition et la troisieme division.
2. Un traitement entre les prophases de la deuxieme et la troisieme division
entraine l'exogastrulation, ce qui est rarement le cas pour un traitement en
dehors de cette periode.
3. On peut trouver une correlation entre cet effet morphogenetique et la
consommation d'oxygene de l'oeuf.
4. Jamais des malformations de la tete de l'embryon sont trouvees a la suite
d'un traitement des oeufs avant la premiere division.
5. Un traitement a l'actinomycine D au debut n'a jamais d'effet sur le developpement ulterieur des oeufs.
6. Pendant la deuxieme division un processus specifique est en cours, qui
prepare l'ceuf a la gastrulation.
Thanks are due to Dr H. B. Woodruff of Merck, Sharp and Dohme, Rahway, New Jersey,
for a gift of actinomycin D.
REFERENCES
Bosco, M. & MONROY, A. (1960). Inhibition of the differentiation of the primary mesenchyme
in the sea-urchin embryo caused by ethionine. Ada Embryol. Morphol. exp. 3, 53-65.
ELBERS, P. F. (1959). Over de beginoorzaak van het Li-effect in de morphogenese. Thesis,
Utrecht.
GEILENKIRCHEN, W. L. M. (1961). Effects of mono- and divalent cations on viability and oxygen
uptake of eggs of Limnaea stagnalis. Thesis, Utrecht.
GEILENKIRCHEN, W. L. M. (1964C). The action and interaction of calcium and alkali chlorides
on eggs of Limnaea stagnalis and their chemical interpretation. Expl Cell Res. 34, 463-87.
GEILENKIRCHEN, W. L. M. (19646). Periodic sensitivity of mechanisms of cytodifferentiation
in cleaving eggs of Limnaea stagnalis. J. Embryol. exp. Morph. 12, 183-95.
GEILENKIRCHEN, W. L. M. (1966). Cell division and morphogenesis of Limnaea eggs after
treatment with heat pulses at successive stages in early division cycles. / . Embryol. exp.
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GUSTAFSON, T. (1954). Enzymatic aspects of embryonic differentiation. Int. Rev. Cytol. 3,277.
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embryo. Adv. Morphogen. 3, 147.
VERDONK, N. H. (1965). Morphogenesis of the head region in Limnaea stagnalis L. Thesis,
Utrecht.
{Manuscript received 10 October 1966)