/ . Embryol. exp. Morph., Vol. 16, 2, pp. 321-337, October 1966
Printed in Great Britain
321
Cell division and morphogenesis of Limnaea eggs
after treatment with heat pulses at successive
stages in early division cycles
By W. L. M. GEILENKIRCHEN 1
From the Zoological Laboratory, State University of Utrecht, Holland
Cellular reproduction is related to a number of apparently independent processes of which the integrated results are bound to produce cell division. In eggs
with determinate cleavage the results of division are daughter cells of a different
prospective significance.
It has been observed furthermore in Limnaea eggs that morphogenesis is
related to periodically recurring cell activities in the first, second and third
cleavage cycle (Geilenkirchen, 1964a, b). These activities of unknown nature are
dissociable from the factors involved in cell division. Obviously in the course of
one division cycle the egg discriminates between processes for the preparation
of the next division and processes involved in morphogenesis and differentiation
later on.
The data published in this paper carry the notion that successive divisions
represent well-defined steps of different significance for later development and
differentiation. Three questions are asked: (a) are successive cleavages of equal
importance to later development; (b) are factors of morphogenetic importance
present as a result of oogenesis, or do they arise during early cleavages; (c) are all
steps in one cell cycle equally important to cell division and to later development?
These problems are studied by the simple technique of giving eggs at successive
stages of a cleavage cycle a heat pulse. The criteria for disturbance used are
(a) retardation of the cleavages which follow treatment, and (b) the ultimate
stage of development reached by the embryo after a pulse has been given.
MATERIAL AND METHODS
Eggs of Limnaea stagnalis were obtained by exposing the snails to stimulating
plant leaves and a mild temperature rise (Geilenkirchen, 1961).
In each experiment one egg mass only was used. The cleavage divisions of the
eggs of one egg mass are not synchronous. In order to obtain treatment groups
of synchronously dividing eggs, groups of eggs (8-10), which started first
1
Author's address: Zoologisch Laboratorium der Rijksuniversiteit, Utrecht, Holland.
322
W. L. M. GEILENKIRCHEN
cleavage almost simultaneously (within 1 min) were selected. The first group
selected was used as a control. Treatment groups A, B, C, D, etc., were given a
heat-pulse (10 min at 38 °C), at 0, 10, 20, 30, etc., or 0, 15, 30, 45, etc., minutes
after the eggs started first cleavage. The pulse was started by transferring the
eggs to tap-water of 38° C and ended 10 min later by transferring the eggs to
tap-water of 25 °C. The last group selected was used as a second control. Before
and after the treatment the eggs were kept in tap-water at 25 °C. In the same way
experiments were done in which the developmental stages before first cleavage
were also tested. In these experiments the eggs were sampled for synchronicity
at the first maturation division.
After treatment the time for the next cleavage or the next two cleavages to
occur was determined in each sample and the cleavage delay with respect to the
control groups was calculated. The development of each embryo was then
followed until it reached a stage at which the eyes in normal embryos were well
developed. Separately, a number of experiments were done in which only
development was studied and not delay of cleavage.
Abscissae
Representation of results
The graphical representation of the results of a number of experiments poses
several difficulties, because eggs of different egg masses differ considerably with
respect to duration of the cleavage cycles. The duration of cleavage cycle 2,
i.e. first to second cleavage, is on an average 94 min, with as extremes 85 and
105 min for different egg masses. Within one egg mass the variability between
individual eggs amounts to 5 min for the second cleavage cycle. In the third
cleavage cycle, i.e. second to third cleavage, these figures are 84 min (extremes
78 and 98 min) and 5 min.
In the experiments the times of treatment are preset at 0, 10, 20, 30, etc.,
minutes after a cleavage. Due to the variability in the duration of the cleavage
cycles different stages of the cell cycle will be affected in different experiments.
The variation may amount to 20 min, which is more than the spacing of the
treatment groups. In order to combine in the best way the different groups
treated at the same stage in a given cleavage cycle the following procedure was
used. The time from first to second cleavage is set at 100 % in each experiment.
This ' 100 %' thus represents a figure between 85 and 105 min. The preset times
of treatment are then expressed in percentages of the duration of the cleavage
cycle in any experiment.
The scatter of points thus obtained is regrouped in 5 % periods and given in the
graphs of Text-fig. 2 A, B and C. For the calculation of averages in Text-fig. 2 A,
B and C the single points in successive 10 % periods are taken together.
The same procedure is used in the experiments for stages between second and
third cleavage and for stages between first maturation division and first cleavage.
With respect to the latter period in the graphs of Figs. 2A and B, Fig. 3 and
Fig. 5 a percentage scale is used x 1-5 the scale for the two cleavage cycles to
Morphogenesis of Limnaea eggs
323
follow. This has been adopted because the time between first maturation
division and first cleavage (on an average 141 min) is about x 1-5 the time of
the second cleavage cycle. In this way the actual time relations are nearly preserved in the graphs.
In this procedure the tacit assumption is made that if a cleavage cycle takes
a shorter or longer time all stages of the cycle are proportionately shorter or
longer.
Ordinates
On the ordinates in Figs. 1 and 2 delays of cleavages are given. In Figs. 3,4 and
5 the percentages of normal and abnormal development are given.
In one set of experiments, represented in Fig. 1, a delay of the forthcoming
cleavage after heat treatment was studied.
Delay
120
100-
80-
60-
\
\
40PM
20-
0
PM M ATT
PM
PM.M AT
10 20 30 40 50 60 70 80 90 1 0 0 % 20 30 40 50 60 70 80 90 1 0 0 %
A
1st Cleavage
A
2nd Cleavage
A
3rd Cleavage
Fig. 1. Retardation of second or third cleavage (ordinate) after a heat pulse (10 min.,
38 °C), in relation to time after first or second cleavage at which the pulse started
(abscissa). Abscissae, time scale: first-second cleavage, time 100% = 94 min on
average; second-third cleavage, time 100% = 84 min on average. (For explanation
see text.) T = telophase; K = karyomere nucleus; PM = polymorphic nucleus;
P = prophase;M = metaphase;/4 = anaphase;S = period of DNAreduplication.
In a second set of experiments, represented in Figs. 2A, B and C, not only
the delay of the forthcoming cleavage after treatment was determined but also
the delay of the second cleavage following the heat pulse in so far as this delay
comes on top of the delay of the former cleavage.
In subsequent sets of experiments the development of the embryos rather than
the delay caused by the treatment was studied (Figs. 3, 4, 5).
Each point in Figs. 1, 3 and 4 represents about 70 eggs and in Fig. 2 and Fig. 5
about 50 eggs.
324
W. L. M. G E I L E N K I R C H E N
Delay of first cleavage
140120100io
-
a so3
|
6040200 10 20 30 40 50 60 70 80 90100%
1st Mat. div.
2nd Mat. div. 1st Cleavage
Time of treatment
Extension of the second cleavage cycle
172
140120100-
2 80-
|
604020-
J
0 10 20 30 40 50 60 70 80 90100 20 40 60 80 100%
1st Mat. div. 2nd Mat. div. 1st Cleavage
2nd Cleavage
Time of treatment
Extension of the third cleavage cycle
100
8010
2 60-
\
20-
0
0 20 40 60 80 100 20 40 60 80 100%
1st Cleavage
2nd Cleavage
3rd Cleavage
Time of treatment
Fig. 2. For legend see opposite.
Morphogenesis of Limnaea eggs
325
Developmental disturbances
After the third cleavage has passed, the eggs remained in tap-water at 25 °C
for 48 h. Then the capsules, each containing a single 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 and recorded until a stage at which the eyes in normal
embryos are well developed. In all, seven grades of developmental disturbance
were distinguished.
First-period death. Under this heading were included all those embryos which
died before or during the gastrula stage. In these experiments this category plays
quite an important role. It is noteworthy that more than 95 % of all embryos
scored under this heading develop into blastulae or gastrulae. They are alive
for 3 or 4 days and die without showing any further development.
Second-period death. All embryos which developed farther than the gastrula
stage, but died without showing specific morphological abnormalities.
Exogastrulation. Exogastrulae are vesicular embryos in which the invagination of the archenteron was suppressed. These embryos die within a few days.
Aspecific malformations. In this group were included all embryos which showed
an abnormal development and which could not be described as exogastrulae,
hydropic embryos, shell malformations or head malformations.
Hydropic embryos. In general outline normally developed embryos which are
partly or totally swollen to a considerable degree.
Shell malformations. Embryos with an abnormal shell as the only abnormality.
Head malformations. All microphthalmic, monophthalmic, synophthalmic,
riophthalmic, cyclopic and anophthalmic embryos were classed in this group.
RESULTS
(1) Delay of cleavage
In the first set of experiments eggs of Limnaea differing in age after first
cleavage by periods of 15min were heat-pulsed for lOmin at 38 °C between
first and third cleavage. After treatment the time for the next cleavage to occur
was determined in each sample and the cleavage delay with respect to a cbntrol
Fig. 2. A: Retardation of first cleavage (ordinate) after a heat pulse (10 min, 38 °C),
in relation to time after first maturation division at which the pulse is given (abscissa).
B: Extension of the second cleavage cycle (ordinate) after a heat pulse (10 min,
38 °C), in relation to time after first maturation division orfirstcleavage at which the
pulse is given (abscissa). T = telophase; K = karyomere nucleus; PM = polymorphic nucleus; P = prophase; M = metaphase; A = anaphase; S = period of DNA
reduplication. C: Extension of the third cleavage cycle (ordinate) after a heat pulse
(10 min, 38 °C), in relation to time after first or second cleavage at which the pulse
is given (abscissa). Ordinate, time in minutes. Abscissa, time scale. First maturation
division to first cleavage, time 100 % = 141 min on average. First cleavage to second
cleavage, time 100% = 94 min on average. Second cleavage to third cleavage, time
100% = 84 min on average. (For explanation see text.)
326
W. L. M. GEILENKIRCHEN
group was noted. Fig. 1 shows the results. After a pulse at first cleavage the
delay of second cleavage is maximal. The sensitivity to a pulse decreases rapidly
in the following 30 % of the cleavage cycle, then a plateau in the curve is
observed over the next 30 % of the cycle. From 60 % onwards delay of second
cleavage decreases rapidly to zero. A similar cycle of sensitivity is observed
between second and third cleavage. The onset of the definite decrease at 60 %
coincides with the end of interphase and the beginning of prophase for both
cleavage cycles.
In a second set of experiments the complete period of development from
oviposition to third cleavage was tested for sensitivity in terms of extension of
cleavage cycles (Fig. 2 A, B, C). A heat pulse applied shortly before the first
maturation division causes only a slight retardation of the first cleavage (Fig. 2 A)
and the additional delay of the second cleavage cycle is small (Fig. 2B). A retardation of the maturation divisions has not been found. Right after the first maturation division sensitivity to a pulse increases. The first cleavage is delayed and
likewise (Fig. 2 A, B) an extension of the second cleavage cycle is found. An
obvious maximum in sensitivity is found after a pulse a short time before the
second maturation division is due. The sensitivity then decreases but a plateau
in the curve is observed between 50 and 70 %. As can be seen, the curves for
delay of the first cleavage and lengthening of the second cleavage cycle parallel
each other up to a stage at which 70 % of the time between first maturation
division and first cleavage has passed. From this stage onwards the delay of
first cleavage decreases to zero, but the extension of the second cleavage cycle
increases and reaches a maximum again for a pulse at first cleavage. From first
cleavage onwards a decrease in sensitivity in terms of extension of the second
cleavage cycle is first observed followed by a slight increase which represents a
plateau in the curve between 30 and 60 %. The results of Fig. 1 and Fig. 2B
parallel each other. When the course of sensitivity is followed in each single
experiment it is seen that in each case the same pattern in sensitivity differences
is found.
The sensitive period for extension of the third cleavage cycle starts half-way
between first and second cleavage (Fig. 2C). A maximum is again observed
around second cleavage and a plateau in the curve between 30 and 60 % of the
third cleavage cycle.
It is obvious from Figs. 1 and 2 that a given heat pulse causes a much longer
delay if applied around first cleavage than if applied around second cleavage.
The shape of the curve over one division cycle suggests that it is the resultant
of two curves, one with a maximum around cleavage and one with a maximum
between 30 and 60 %.
(2) Morphogenetic effects
In a first series of experiments, groups of synchronously dividing eggs from
a single egg mass were treated at regular intervals after first and second cleavage
and then cultured in the normal way.
r
10
1
x
20
1
\
f
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30
CT
40
1
O
/
\
\
i
50
/
1st Mat. div.
2nd Mat. div.
Time of treatment
0
Q
1
*
60
1
^> — -""
A
a
v
3
70
I
80
1
^
/ / x
I
I
€fen
x
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1 1
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P i PMM
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•
100% 20 30 40 50 60 70 80 90 100% 20 30 40 50 60 70 80 90 100%
2nd Cleavage
1st Cleavage
3rd Cleavage
90
I
If
V
o
AA \
\ -'V
1f
Head malf.
Shell malf.
Hydropic embr.
Death second period
T K
Fig. 3. The percentages of normal and abnormal development after a heat pulse (10 min, 38 °C), in relation to time
after first maturation division, first, second and third cleavage at which a pulse is given (abscissa). Ordinate: percentages
of normal or abnormal development. Abscissa: time-scale. First maturation division—first cleavage, time 100% =
141 min on average; first-second cleavage, time 100% =103 min on average; second-third cleavage, time 100% = 86 min
on average. (For explanation see text.) T = telophase; K = karyomere nucleus; PM = polymorphic nucleus; P = prophase; M — metaphase; A — anaphase.
o-
20-
40-
60-
80-
-
100-
O Normal
X Death first period
Exogastrulae
O Aspecific malf.
to
CD
328
W. L. M. GEILENKIRCHEN
In Fig. 3 percentages of normal development, first-period death, exogastrulation, aspecific malformations, head malformations, second-period death, etc.,
are plotted against the time of treatment. The eggs are rather insensitive to the
pulse up to the time that 70 % of the cell cycle after first and second cleavage
has passed; normal development lies between 80 and 100 %. After this period
0/
/o
uu O
x
•
O
A
a
80 •_
60-
-
Normal
Death first period
Exogastrulae
Aspecific malf.
Head malf.
Death second period
40-
20-
X
B
Q?
0-
1
Qfi 03
1
0
2nd Cleavage
1
10
A*<
1
20
30
Time in minutes
Fig. 4. The percentages of normal and abnormal development (ordinate) after a heat
pulse (10 min, 38 °C), in relation to time of treatment after second cleavage
(abscissa).
100
o Normal
x Death first period
0
Aspecific malf.
Q Death second period
v Hydropic embr.
80604020-
g
20
^0
60
1st Mat. div. 2nd Mat. div.
Time of treatment
-40
-20
80
1st Cleavage
Fig. 5. The percentages of normal and abnormal development (ordinate) after a heat
pulse (10 min, 38 °C), in relation to time after first maturation division at which a
pulse is given (abscissa). Abscissa: time-scale. First maturation division to first
cleavage, time 100% = 141 min on average. (For explanation see text.)
a decrease of normal development is found and a corresponding increase in
first-period death and exogastrulation sets in. A minimum in normal development and a maximum in abnormal development is observed when about 85 %
of the duration of a cleavage cycle has passed. Whether the curve for normal
Morphogenesis o/Limnaea eggs
329
development shows two maxima, as is suggested in the third cleavage cycle,
needs further investigation.
In a second set of experiments the morphogenetic effect of a heat pulse at
stages between oviposition and first cleavage has been studied. The results are
also shown in Fig. 3. It is evident that the heat pulses applied right after oviposition are quite noxious. A maximum in death-rate is found at a stage between
the extrusion of the two polar bodies.
In a third series of experiments the sensitivity of the period immediately after
first and second cleavage has been studied in detail, with small time-intervals.
The results, Fig. 4, confirm the foregoing experiments. It stresses the observation
that the sensitivity has declined substantially at the time of cleavage. Peak sensitivity is always found when treatment starts at a stage before cleavage, during
mitotic metaphase-anaphase.
In the last sets of experiments done 1 year later, the sensitivity of development
to heat pulses given at stages between oviposition and first cleavage was studied
in more detail (Fig. 5). Two sets of experiments are involved, one covering the
stages between oviposition and first maturation division and the second set
the stages between first maturation division and first cleavage. In the figure
the percentages of normal development and first-period death are given on the
ordinates. Other malformations occur only in very small numbers. The abscissa
is again a time-scale in percentages. The time before first maturation division
is given in negative values of the same scale. As seen from the first-period death
curve sensitivity is high following oviposition, decreases rather sharply a short
time before the first maturation division, rises when the first polar body is
formed and reaches a maximum half-way between the maturation divisions.
After that point the sensitivity decreases fast but rises again during mitosis of the
first cleavage.
(3) Cytological observations
For cytological observations eggs were fixed in Bouin's fixative, sectioned and
stained with iron-haematoxylin-eosin. Eggs treated during the 10 minutes following first, second and third division were studied. The nuclei and cytoplasm of the
heat pulsed eggs differed in no way from the controls.
The succession of mitotic stages indicated in the figures has been studied in
separate experiments (to be published).
DISCUSSION
(1) Delay of cleavage
Heat pulses applied between the first and the second maturation division cause
a delay of first cleavage and in addition an extension of the second cleavage
cycle. These cleavage delays exceed by far the duration of treatment. One very
sensitive period is found which lies shortly before the moment that the second
maturation division is due (Figs. 2A, B). The process to which damage is done
330
W. L. M. GEILENKIRCHEN
by a heat pulse during maturation is apparently linked in a direct way to first
and second cleavage.
Cytologically the two maturation divisions show differences with regard to
(a) chromosomal events—separation of chromosome pairs versus separation of
single chromosomes—and (b) the formation of the mitotic figures—asters and
spindle. In the first maturation division the spindle and both asters with centrioles
are provided by the oocyte. In the second maturation division, however, one
centriole + aster (against the surface) are provided by the oocyte and the second
deep aster is a sperm aster (Raven, Escher, Herrebout & Leussink, 1958). One
may deduce from these data that for maturation to proceed to this point no
splitting and reduplication of centrioles need take place. At the end of the second
maturation division, however, a splitting and reduplication of the centriolar
structure of the sperm aster can be expected, intended for the following divisions
of the egg. A direct relation between heat pulse sensitivity and centriole splitting
and reduplication can be assumed. This assumption is strengthened by the
consistent observation that more than 50 % of the embryos heat-pulsed around
peak sensitivity before the second maturation division divide at first cleavage
in three blastomeres, and consequently to six at the second cleavage. This effect
may also explain the fact that both first and second cleavage are delayed in this
way.
Between the second maturation division and third cleavage similar sensitivity
curves are found in each division cycle (Figs. 1, 2). Sensitivity rises at prophase,
reaches a maximum around cleavage, decreases and rises again in interphase
and decreases eventually to zero between prophase and telophase of the coming
division. The system responding to heat shock clearly goes through a cycle from
prophase to prophase, with two maxima, one at cleavage and one during mitotic
interphase. The maximum delay after a pulse around first cleavage is almost
double the delay found after a pulse at second cleavage.
The heat-pulse sensitivity around cleavage may be correlated with the splitting
and the duplication of the centrioles, as is held above for the second maturation
division. According to the findings of Mazia, Harris & Bibring (1960) with sea
urchin eggs, centrioles are paired structures, which split apart and each generate
a daughter centriole at about mitotic anaphase-telophase. Split parts of the
paired structures are each capable of forming poles. Thus, without conception
of daughter centrioles the cleavage at hand can come to an end and also the
cleavage to follow, because each cell has still two potential centres available for
mitosis.
Heat pulses given at the time of anaphase-telophase may influence both processes : splitting and duplication. If there exists no causal relations between the
two processes and their significance for later divisions we can only assume that
heat pulses influence the splitting apart of the paired structure, for the cleavage
following the one at hand is delayed.
Another possibility is a direct effect on the synthesis of proteins related to
Morphogenesis 0/Limnaea eggs
331
division. If this holds true protein synthesis for a given division has already
started during prophase of the preceding division. There are several arguments
which favour such a hypothesis. The first argument is provided by the extensive
studies on division delay by heat treatment and blocking of protein synthesis in
Tetrahymena (Zeuthen, 1964). It is concluded that heat shocks work at the level
of the synthesis of proteins concerned with the assembling of structures needed
for division. Specific blocking of protein synthesis has the same delaying effects
as heat.
A second argument is that the heat pulse delay curves for Limnaea eggs are
identical to curves found for pulse treatments of Limnaea eggs with chloramphenicol, a well-known suppressor of protein synthesis (Geilenkirchen, to be
published).
A third argument is provided by the data on heat-pulse sensitivity of the
cleavage cycles of Arbacia eggs. First of all it is seen that the first, second and
third cleavage cycles show a similar sensitivity pattern to that of Limnaea eggs.
It appeared that heat pulses given before fertilization delayed first cleavage. If
fertilization was postponed for 30 min after shock no delay effects were found.
This showed that the eggs recovered in 30 min from a shock. Furthermore it was
found that such a pulse evokes intensive 3H-leucine incorporation in the unfertilized egg (Geilenkirchen, to be published), whereas normally no incorporation is found in unfertilized eggs. In the case of unfertilized Arbacia eggs heat
pulses apparently degrade a unit which has to be repaired via protein synthesis.
This may also be the case in Limnaea eggs if in each cleavage cycle an unidentified 'cleavage unit' is again assembled via protein synthesis.
With respect to the plateau in the curves between 30 and 60 %, a correlation
with DNA doubling is obvious. DNA doubling occurs in egg cells after the
second maturation division and then after each division again. In Limnaea DNA
doubling after the second and third cleavage occurs between 25 and 55 % in the
time-scale on the abscissae in Fig. 2B and C, (J. A. M. van den Biggelaar,
personal communication). This coincides exactly with the second period of
heat-pulse sensitivity in the cycle. For reasons of symmetry it also seems acceptable to correlate the plateau found between second maturation division
and first cleavage and between first cleavage and second cleavage with DNA
synthesis. Another argument for assuming a correlation with DNA synthesis
is provided by the observation that this period in the cell cycle is sensitive to
X-rays as regards cleavage delay. This was found in a number of pilot experiments. There are to our knowledge no data available which can explain the
nature of such heat sensitivity during DNA doubling.
(2) Morphogenetic effects
The cleavage delaying effects of heat pulses are reversible, in so far as in all
cases mitosis and cleavage are resumed at the normal rate. Development seems
to be rather normal up till 24-48 h after first division. The egg cells, however,
332
W. L. M. GEILENKIRCHEN
which have been treated around metaphase and anaphase of first, second and
third division stop development, stay alive for several days at a blastula or
gastrula stage, and then die. A high percentage of egg cells shocked at other
stages of the cell cycle develop into perfectly normal young snails.
The heat-pulse effects in Limnaea as related to general development resemble
closely the heat-pulse effects in Arbacia eggs (Geilenkirchen, 19646). These data
lead to the conclusion that the egg cell is able to distinguish between processes
for the preparation of the next divisions and processes involved in development
and differentiation later on.
After mild centrifugation (500g for 10 min) a pattern of sensitivity is found
similar to that for heat pulses (Geilenkirchen, 1964«). The peak in these curves,
however, is found during cleavage, whereas after a heat pulse the peak is found
a short time before cleavage. Whether and how the effects of centrifugation and
heat pulses are related is being studied.
The cycle of enhanced heat-pulse sensitivity carries on after third cleavage
(De Groot, data in Verdonk, 1965). Three sensitive periods were found in addition: 1 h after third cleavage (formation of the second micromere quartette,
stage of 12 cells), 7 h after third cleavage (division of the cells \ax-\dx is due),
and 8-11 h after third cleavage (division of the cells 1 a12-! d12 is due). In addition
to a general suppression of development around gastrulation, as is usually
found, heat treatment may cause malformations of the head region only, in
otherwise completely normal snails (Raven, De Roon & Stadhouders, 1955).
The study of De Groot revealed that heat treatment at the periods listed above
caused head malformations of differing severity specific for the period treated.
After treatment before first cleavage not a single head malformation has been
found in the experiments presented. After treatment of stages between first and
third cleavage embryos with a teratomorphic head region are found (Raven et al.
1955; Verdonk, 1965). After a heat treatment at 1 h after third cleavage 50 % of
the embryos are acephalic, the apical plate and the cephalic plates are missing,
and the head region is strongly reduced. After treatment at 7 h after third
cleavage, the apical plate and one or both cephalic plates are reduced, but
acephalic embryos are never found (Verdonk, 1965)
After treatment between 8-11 h after third cleavage, cyclocephalic head malformations can be produced, characterized by a bridge of small cells connecting
the left and right cephalic plates. It is noteworthy that after a heat treatment at
this period of development all cell types which are normally found are present
in a normal number and arrangement, except for the bridge between the cephalic
plates. Thus a specific and reproducible decrease in severity of head malformations is observed when the heat pulses are applied at successively later stages of
development. This specificity of head malformations after treatment at specific
cleavage cycles supports the hypothesis that successive divisions represent welldefined steps of different significance to later development and differentiations.
Factors of morphogenetic importance are thus added in each cleavage cycle,
Morphogenesis o/Limnaea eggs
333
which are not yet present before first cleavage. Furthermore the results support
the view that specific points in each cell cycle, e.g. metaphase and anaphase, are
of particular importance to later development at least in so far as high heat-pulse
sensitivity is a correct measure.
0
2
4
6
8
10
12
14
16
18
20
22
24
26
1c12112 (cp.)
1c1122 (cp.)
—^1c1121 (cp.)
1b 1222 (pr.)
1b'
2122
6ib 1221 (pr.)
(cp.)
VK
1212 < - > - • -
'
1o 12122 (cp.)
1o12121 (cp.)
12i;
cp.)<->!t 122(Cp)
Ala 1121 (a.p.)
1
6
8
10
12
14
16
18
Time in hours after first cleavage
20
22
1
24
1
1
26
Fig. 6. The history of the first quartette of micromeres. h.v. = head vesicle cell;
a.p. = apical plate cell; pr. = prototroch cell; cp. = cephalic plate cell. The
arrows indicate the cells which go on dividing; the other cells derived from the first
quartette of micromeres undergo no further divisions. (After Verdonk, 1965.)
Part of the delay effects were considered to be related to centriole splitting and
reduplication and to protein synthesis. Both may be related phenomena in so
far as centrioles and spindle-aster fibre synthesis are regarded as being dependent.
The increase in delay, not of the forthcoming cleavage, but of the next one after
treatment (heat pulses and chloramphenicol) during mitosis of the forthcoming
division may be indicative in this respect. The 'factors of morphogenetic importance added in each cleavage cycle' are apparently added between mitotic
prophase and telophase, during that phase of the cycle in which an increase of
sensitivity in terms of delay of cleavages is observed.
334
W. L. M. GEILENKIRCHEN
The question arises whether a disturbance of 'the factors of morphogenetic
importance' can also be correlated with or identified as disturbance of centriole
splitting and duplication and related processes. Such a correlation is possible if
we accept the hypothesis that in early development only a definite number of
generations of centrioles become available. A permanent loss of one generation of
centrioles by heat treatment may then cause a permanent loss of a generation
of cells. The decreasing impact of heat treatment with respect to head malformations, while the number of cells of the embryo increases, may support
such a hypothesis.
The dose of heat given is such that it will or will not cause lasting deleterious
effects in the blastomeres, depending on the stage of the cell cycle and the given
susceptibility of different blastomeres and different eggs.
ma.
Fig. 7. Normal trochophore and hippo of Limnaea stagnalis. a.p. = apical plate;
c.p. = cephalic plate; e = e y e ; / = foot; h.v. = head vesicle; m = mouth; m.a. =
mantle; pr. = prototroch; sh = shell; t = tentacle; v = velum. (After Verdonk,
1965.)
With increasing partitioning of the egg cell into blastomeres, the synchrony
of divisions is soon lost. Due to an increasing asynchrony of the blastomeres
and a lengthening of the cell cycles, the chance that many blastomeres are hit
for a given treatment at a sensitive stage in their cycle decreases. This accounts
for the fact that the developing embryo becomes less and less susceptible to a
heat treatment of short duration.
Cell lineage studies of Molluscs, e.g. Physa (Wierzejski, 1905), Planorbis
(Holmes, 1900) and Limnaea (Verdonk, 1965), show that asynchrony starts after
Morphogenesis o/Limnaea eggs
335
third cleavage (Fig. 6). It is furthermore observed that certain cell lines derived
from the cells 1 a-\ d soon stop dividing and give rise to the larval organs prototroch, head vesicle and apical plate (Fig. 7). It is in these short cell lines that we
may (and must) observe loss of a cell generation in terms of the hypothesis
given above. After treatment shortly before a given division the forthcoming
cleavage and the next one will not be influenced by loss of a generation of centrioles. For, according to Mazia, Harris & Bibring (1960), doubleness of the
centrioles is not required for their mitotic operation. Thus the cleavage at hand
and the next one can always come to an end if a heat shock interferes with reduplication only. Thus:
(1) A treatment shortly before third cleavage (division of A-D, Fig. 6) may
lead to a loss in the cell lines deriving from the cells 1 a1-! d1 and 1 a2-! d2.
(2) A treatment shortly before the cells 1 a-\ d divide may lead to a loss in the
cell lines derived from the cells 1 a11-! d11 and 1 a12-l d12. The cells derived from
1 a2-! d2 are no longer influenced, they are fully differentiated.
(3) When treatment is given shortly before the cells 1 a1-! d1 divide, we may
expect loss in the cell lines deriving from 1 aXX2-l d112,1 a 121 -l d121 and 1 b122. There
are no further derivatives of 1 a111-! d111 nor of 1 a122, 1 c122 and 1 d122.
(4) Treatment given shortly before the division of the cells 1 a12-\ d12 may
cause a loss in the cell lines derived from 1 a1211, 1 c1211, 1 a1212, 1 b1212 and 1 c1212.
Loss of a cell generation in these four groups may lead to:
ad (1): loss of prototroch, apical plate, head vesicle and cephalic plate cells in
varying numbers. When these predicted losses are compared with the actual
abnormalities found in head malformations in the experiments of Verdonk
it shows that at this stage of treatment a strongly reduced prototroch, head
vesicle and apical plate are found, but the different cell types are mostly found.
The cephalic plate reductions are obscured because pretrochal and posttrochal
regions of small-celled ectoderm fuse when they are no longer separated by the
prototroch cells (N. H. Verdonk, personal communication). They strongly resemble head malformations found after LiCl treatment.
ad (2): loss of one prototroch cell, loss of a varying number of apical plate
cells, loss of maximally four head vesicle cells, loss of cephalic plate cells. In the
experiments all head malformations have a well-developed prototroch, but a
reduced head vesicle, apical plate and cephalic plates.
ad (3): loss of one prototroch cell and one head vesicle cell; loss of 1, 2 or 3
apical plate cells and loss of cells of the cephalic plates. In the experiments head
malformations are always found in which the prototroch and head vesicle are
well developed, in which a number of apical plate cells are missing, and in which
the cephalic plates show varying degrees of reduction.
ad (4): loss of cells in the cephalic plates only; prototroch, apical plate and
head vesicle are fully formed. When these predicted losses are compared with the
actual abnormalities found in head malformations, it shows that at this stage of
treatment only abnormalities of the cephalic plates are obtained. It also appeared,
336
W. L. M. GEILENKIRCHEN
however, that in this case a number of cephalic plate cells are obtained by a
division of 1 d1212 and 1 d1211 from origin head vesicle cells. This can be explained
if it is accepted that these cells are potential cephalic plate cells which are
delayed in normal development. They may become actively dividing cells
again later in development when the larval structures, namely head vesicle,
apical plate and prototroch become eliminated. Then a heat shock at this
period would, by eliminating one generation of centrioles, bring these cells
from an arrested state directly into the state of cells of the adult structures
because a later generation of centres becomes activated.
The analysis of the cell pattern in head malformations clearly supports the
hypothesis that heat treatment causes a loss of a generation of centrioles which
then leads to a loss of a generation of cells.
SUMMARY
1. Eggs of Limnaea stagnate, of ages differing by 10 min intervals between
oviposition and third cleavage division, have been heat shocked at 38 °C for
10 min.
2. The heat treatment causes two effects: (a) extension of cleavage cycles,
and (b) morphogenetic effects.
3. With respect to extension of cleavage cycles, sensitivity to a heat shock
goes through a cycle from prophase to prophase. Two maxima can be observed:
one during mitotic metaphase-anaphase and one during interphase. The cleavage
delaying effects are reversible; mitosis and cleavage are resumed at the normal
rate.
4. Strong morphogenetic effects are observed after treatment between first
and second maturation division and around metaphase-anaphase of first, second
and third division.
5. Arguments are given for the hypothesis that the peak in sensitivity for
cleavage delay and for morphogenetic effects around mitotic metaphase-anaphase can be considered as indicative of an effect of the heat treatment on
splitting and reduplication of centrioles.
RESUME
Effets sur la division et la morphogenese d'oeufs de Limnaea d'un traitement par
ondes calorifiques a des stades successifs des premiers cycles mitotiques
1. Des oeufs de Limnaea stagnalis ont ete, a des stades echelonnes de 10 en
10 minutes entre la ponte et la troisieme division, soumis a un traitement par
choc thermique de 38 °C pendant 10 minutes.
2. Ce traitement produit deux effets: (a) extension du cycle mitotique, et
(b) consequences morphogenetiques.
3. En ce qui concerne le premier de ces effets, la sensibilite au choc passe par
Morphogenesis of Limnaea eggs
337
un cycle de prophase en prophase. On peut observer deux maxima, l'un durant
la meta-anaphase, l'autre pendant l'interphase. L'effet d'extension du clivage
est reversible; mitose et clivage reprennent leur rythme normal.
4. De puissants effets morphogenetiques sont observes apres traitement entre
la premiere et la deuxieme division de maturation, et durant la meta-anaphase
des premiere, deuxieme et troisieme divisions de segmentation.
5. Des arguments sont avances en faveur de l'hypothese selon laquelle
l'existence d'un maximum de sensibilite pendant la meta-anaphase (pour la
production de retards de clivage et d'effets morphogenetiques) indiquerait que
le choc thermique agit sur la reduplication des centrioles.
Grateful acknowledgement is made to Professor Dr E. Zeuthen, Biological Institute of the
Carlsberg Foundation, Copenhagen, for his suggestions and discussions, and to Mrs C.
Jansen-Dommerholt for skilful technical help.
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