/. Embryol exp. Morph. Vol. 53, pp. 305-314, 1979
Printed in Great Britain © Company of Biologists Limited 1979
305
Correction of the maternal effect
linked to the ac mutation, by injury of the egg, in
the salamander Pleurodeles waltlii
By MARIA FERNANDEZ 1
From the Laboratoire de Biologie Generate, Universite Paul-Sabatier,
Toulouse, France
SUMMARY
At the gastrula stage, deep irregular furrows appear on the animal hemisphere in embryos
arising from ac/ac females and characterize a maternal effect (Beetschen, 1970). Disturbed
morphogenetic movements (epiboly and invagination) frequently elicit exogastrulation or
anomalies of later embryonic development. Pricking the animal hemisphere of the uncleaved
egg with a micropipette prevents the occurrence of anomalies during gastrulation and
neurulation in many embryos. Injection of normal oocyte nuclear sap or of normal egg
cytoplasm into mutant female eggs does not improve that result. The partial correction of the
maternal effect therefore appears to be consecutive to the injury of the cortical cytoplasm
and/or the plasma membrane. Assuming that activation could be deficient, an electric shock
was applied to freshly laid fertilized mutant eggs. It did not correct ectodermal anomalies but
nevertheless seemed to increase the corrective effect of pricking when both treatments were
applied.
INTRODUCTION
Several maternal effects have been discovered in the offspring of various
mutant female axolotls (for reviews see Briggs, 1973, and Malacinski &
Brothers, 1974). In the salamander Pleurodeles waltlii, the principal characteristics of another maternal effect have been described by Beetschen (1970,
1976) in the progeny of ac/ac females. After normal cleavage, except for a few
rare cases, the + /ac or ac/ac embryos arising from this type of female, all
normally show the same syndrome from the very beginning of gastrulation: the
ectoderm of the animal hemisphere becomes furrowed with irregular grooves,
the depth and frequency of which depend on the individual. In many embryos,
as gastrulation proceeds, the furrows become continuous giving the animal
hemisphere a 'brain-like' appearance (Fig. 1). It is clear that, during this phase,
epiboly movements of the ectoderm become more disturbed as the syndrome
becomes more intense. This is also very often true for invagination movements
because of an abnormally large sized yolk plug and this can be seen in stages
1
Author's address: Laboratoire de Biologie generate, Universite Paul-Sabatier, 118, route
de Narbonne, 31077 Toulouse Cedex, France.
306
M. F E R N A N D E Z
Fig. 1. Animal hemisphere of a stage-11 gastrula from ac/ac mother, with a strong
maternal effect. Scanning electron micrograph, x 35.
Fig. 2. Another stage-11 gastrula with prominent yolk plug (exogastrulation) added
to ectodermal syndrome. Side view, animal pole upward. Scanning electron micrograph, x 35.
11 and 12 of the normal table of development (Gallien&Durocher, 1957). Under
these conditions, many embryos exogastrulate (Fig. 2), sometimes even when
the ectoderm shows no apparent anomalies, and die either at the end of gastrulation or during neurulation without having withdrawn their yolk plug. In the
least abnormal neurulae, the lateral and ventral epidermis is often wrinkled and
irregular. These anomalies become progressively restricted to the most ventral
region in the rare embryos that survive until the tail-bud stage.
In a preliminary series of experiments aimed at the correction of the maternal
effect, the eggs of ac/ac females received injections of cytoplasm from normal
eggs (Romanovsky & Beetschen, 1970). We have revised and completed this
investigation to make it analogous to the studies on the o mutation in the
axolotl (Briggs & Cassens, 1966; Briggs, 1972).
MATERIALS AND METHODS
The eggs for this study came from $ ac/ac x <£ ac/ac crosses. The Petri dishes
used for the operation were agar-bottomed (Difco agar, 15%0) and filled with
Barth's medium pH 7-6 (Barth & Barth, 1959) containing penicillin (10 mg/1),
streptomycin (10 mg/1) and Elkosin Ciba (0-5 g/1). The injection and pricks
were carried out using an 'Oxford' micromanipulator. The micropipettes used,
bevelled and ground, had an internal diameter from 10 to 17 /im depending
Correction of maternal effect in the salamander
307
Figs. 3-6. Living gastrulae stage 11. Animal hemisphere. Fig. 3. Strong maternal
effect. Fig. 4. Average maternal effect. Fig. 5. Weak maternal effect. Fig. 6. TV/7
maternal effect (similar to a normal gastrula). x 35.
on the type of injection. The quantity of normal oocyte nuclear sap introduced
in the animal hemisphere was 6-8 nl, i.e. about 0-5% of the receiving egg
volume; the quantity of cytoplasm injected, its source being the animal hemisphere of normal, fertilized eggs, was larger (25-30 nl) and approximated 2 %
of the receiving egg volume. An electric shock, from a condenser (8 /.iF, 70V),
308
M. F E R N A N D E Z
Fig. 7. Dorsal view of a neurula with open neural plate and wrinkled lateral
ectoderm below the neural folds. Scanning electron micrograph, x 35.
Figs. 8-10. Side views of living neurulae at stage 19. Fig. 8. Right side; average
maternal effect, conspicuous on the most ventral ectoderm. Fig. 9. Right side;
weak maternal effect. Fig. 10. Left side; nil maternal effect (similar to a normal
neurula). x 30.
Correction of maternal effect in the salamander
309
was inflicted on certain freshly laid fertilized eggs that had been dejellied and
immersed in 10% Barth's medium.
The injections of normal nuclear sap and cytoplasm were carried out on
eggs from the same spawning ($ ac/ac CS-71-25) which made comparison
easier by eliminating certain variables. It should be pointed out that the intensity
of the manifestation of the maternal effect varies between females and, sometimes, between spawnings from the same female.
After the operations, the eggs were transferred into 10 % Barth's solution for
24 h, then into dechlorinated filtered tap water, and were kept at 17-18 °C.
The results are presented taking into consideration both the normal or
abnormal gastrulation and neurulation of the embryos and the severity of the
ectodermal syndrome, which is evaluated as follows: strong, when the whole
of the animal hemisphere has a 'brain-like' appearance, i.e. is covered with a
series of deep continuous furrows (Figs. 1 and 3); weak, when small depressions
are observed (Fig. 5); average, when it is between these two conditions, i.e.
with clear furrows that are occasionally interconnected (Fig. 4); nil, when the
surface of the animal hemisphere is smooth (Fig. 6).
Development is considered abnormal if there is clear exogastrulation or if,
at the time of neurulation, the yolk plug is not completely withdrawn.
RESULTS
First series of experiments
The results are given in the upper half of Table 1. It can be seen that all the
'control' gastrulae (from dejellied eggs) showed ectodermal anomalies to
varying degrees whereas a fair proportion of the embryos that were simply
pricked (26-5 %), or pricked and injected with nuclear sap (16-5 %) or cytoplasm
(25%) were perfectly normal; four other embryos had a smooth animal
hemisphere but showed signs of exogastrulation. Similarly, embryos with a
normal sized yolk plug and 'weak' ectodermal syndrome were 3-6 times more
numerous in the experimental groups (22-40%) than in the control group
(6-5%): the comparison between percentages is significant (e ^ 2-3).
However, in the majority (an overall 72-5%) of the dejellied controls,
irrespective of the normal or abnormal progression of gastrula imagination,,
deep furrows deformed the animal hemisphere, whereas this category comprised
less than 20 % of embryos when the eggs were either pricked or pricked and
injected. In all groups of eggs, the number of embryos that developed normally
during neurulation was lower than the number of gastrulae that were apparently
normal at stage 12 (small yolk-plug stage); this is due to the fact that many
embryos started neurulation without having totally withdrawn their yolk plugs.
Neural tube closure was nevertheless completed in some of them. Most of the
embryos that arose from very abnormal gastrulae died, generally at the
beginning of stage 14, when the outline of the neural plate started to becom&
visible.
40
36
20
28
Electric
shock
+ prick
Prick into
animal
hemisphere
Electric
shock
90
89
98
99
225
249
2nd series of experiments
139
Dejellied
156
controls
Nuclear sap
injection
68
79
62
1st series of experiments
Dejellied
72
controls
Prick into
animal
hemisphere
Cytoplasm
injection
tulae
Ul
of*
eggs
liU.
M^/"\
No.
1
5%
± 9-6
11
1
5%
± 9-6
7
18
20%
± 8-3
34
38%
±101
± 7-9
16
18%
11-5%
± 4-2
7-5%
± 3-5
10
11%
± 6-5
26
18
13%
± 5-9
17
± 8-9
71
51%
±15
30-5%
± 8-1
± 8-8
19-5%
±12-3
9
13%
11
16%
13
21 %
± 101
56-5%
±12-3
++
35
+++
± 9-8
34-5%
31
8
9%
± 5-9
77
34%
± 9-4
26
29%
± 2-2
1
1%
± 6-2
± 1-4
59
26%
± 5-7
1
1%
16-5%
±12-2
6
5
25%
±19
26-5%
±10-5
—
—
—
18
0
± 4-7
8-5%
12
±13-6
8
22%
±21-5
8
40%
± 9-9
15
22%
6-5%
± 61
4
+
Ectodermal syndrome!
NORMAL-SIZED YOLK PLUG (St. 12)
10
16%
—
—
—
17
19%
± 8-2
2-5%
± 21
6
± 7-2
35
25%
± 4
—
—
—
—
—
—
2
3%
± 9-2
r
GASTRULATION
± 3
T 0/
-£ /o
2
3-5%
± 3-8
3
4
2%
± 1-7
—
—.
—
±103
4
11%
1
5%
± 9-6
1-5%
± 2-9
1
—
—
—
++
0/
2
2 /o
± 3
7
8%
± 5-6
± 3-3
O -> / o
£.C
15
—
—
—
—
—
±13-1
2
10%
± 2-9
1-5%
1
—
—
—
+
Ectodermal syndromej
ABNORMAL
0/
/o
3-5%
+ 3-8
3
± 2-2
1
1%
± 2-8
11
± 2
1-5%
2
—
—
±131
2
10%
± 4
1
J
—
—
—
2
0
Table 1. Results of experiments aimed at the correction of the maternal effect on eggs of ac/ac female
F
Dead
—
—
—
—
—
4-5%
± 2-7
10
—
—
—
—
—
—
—
—
± 8-1
—
—
—
9
13%
St. 12
Dei ore
1
U)
N
o
>->
C
28
40
Cytoplasm
injection
Nuclear sap
injection
36
20
59
62
90
89
215
5
z
25
28%
± 9-3
41
45-5%
±10-3
10
11%
± 6-5
1
1%
± 2-2
34
16%
± 4-9
26
29%
± 9-5
E
—
2
3-5%
± 4-6
2
10%
±13-1
17
19%
± 8-2
8
22%
±13-6
11
18-5%
± 9-9
2
10%
±13-1
—
0
17
12%
± 5-5
84
39%
± 6-5
12
13-5%
± 7-1
3
15%
±15-6
18
30-5%
±11-8
6
9-5%
± 7-4
+
15
11%
± 5-2
33
15-5%
± 4-8
24
17-5%
± 6-3
6
3%
± 2-2
—
± 7-1
1
5%
± 9-6
o.co/
6
9-5%
± 7-4
++
+++
2
2%
± 3
24
27%
± 9-2
1
1%
± 2-2
6
16-5%
±12-2
z
2
3-5%
± 4-6
11
18%
± 9-5
++
13
9-5%
± 4-8
11
5%
± 2-9
5
5-5%
± 4-8
35
25%
± 7-2
6
3%
± 2-2
19
30-5 %
±11-5
2
3-5%
± 4-6
3
15%
±15-6
10
28%
±14-6
c
PERSISTANT YOLK PLUG
4
4-5%
± 4-3
8
6%
± 3-9
15
7%
± 3-4
2
2%
± 31
4
6-5%
± 61
7
12%
± 8-3
1
5%
± 9-6
6
16-5%
±12-2
+
Ectodermal syndrome f
2
2%
± 3
—
i
3%
± 2-4
E
I
—
3
5%
± 5-6
—
0
+ + , strong; + •f, average; +, weak; 0, nil(cf. text); * Percentages calculated on living gastrulae, ± standard deviation.
99
Electric
shock
+ prick
•f
98
Electric
shock
t
249
+++
4
6-5%
± 61
i
NORMAL
Ectodermal syndromef
4
4-5%
± 4-3
3
3-5%
± 3-8
27
19-5%
± 6-6
19
9%
± 3-8
12
19-5%
± 9-8
9
15%
± 9-2
8
40%
±21-5
6
16-5%
±12-2
Dead
at
St. 14
H—»
U)
3
s
1
ofmaternat effect i'n the salt
Prick into
animal
hemisphere
2nd series of experiments
Dejellied
156
139
controls
79
Prick into
animal
hemisphere
1st series of experiments
Dejellied
72
controls
No.
No. of of living
eggs gastrulae
NEURULATION*
Table 1 {cont.)
Correcthm
312
M. FERNANDEZ
It should be pointed out next that the percentage of neurulae with no apparent
yolk plug in the pricked batch (an overall 61 %) was significantly larger
(e = 3-91) than that of the control batch (25-5 %). It should also be noted that
the overall proportion of embryos with a 'weak' or '«//' ectodermal syndrome
and with normal morphology, was of the same order in the three experimental
batches (20-22 %), the control batch showing approximately half this proportion
(9-5%).
These observations therefore lead us to suppose that the partial correction of
the maternal effect has no relationship to the injection of normal nuclear sap
or cytoplasm, but rather relates to the modifications brought about by injury.
This is not the case in the o mutant axolotl (Briggs & Cassens, 1966).
Second series of experiments
Injections of nuclear sap or cytoplasm were consequently forsaken and the
eggs from three spawnings of different females were all pricked. It was thus
possible to check the reproducibility of the preceding results. We assumed that
the activation of eggs from ac/ac females following sperm penetration might
be too slow or incomplete. In amphibians and especially in Pleurodeles,
activation of virgin eggs can be induced by an electric shock (Signoret &
Fagnier, 1962). A similar treatment was applied in the present experiments,
not to virgin eggs, but to freshly laid fertilized mutant eggs, half of them then
being subjected to a prick in the cortex of the animal hemisphere.
The observations concerning this second series of experiments are shown in the
lower half of Table 1; they deal with four categories of egg. The first batch of
eggs was simply dejellied and was used as a control; the second batch was
pricked; the third batch received an electric shock; the fourth batch received
an electric shock and was then pricked.
Here again, as in the preceding experiments, correction of the ectodermal
syndrome only occurred in pricked or in shocked and pricked batches, in
which 34 % and 29 % respectively of the gastrulae were perfectly normal. With
dejellied eggs, however, or eggs that had only received a shock, only 1 %
developed normally. In the fourth batch, which was shocked and pricked, the
proportion of embryos in which gastrular invagination occurred normally
(92-5 %) was much higher (e = 3-57) than that observed for the eggs that were
just pricked (79%), just shocked (68-5%, e = 3-99), or only dejellied (73-5%,
e = 3-53).
During neurulation (Figs. 7-10), 77 completely normal gastrulae, from pricked
eggs, gave rise to 43 embryos in which slight depressions of the epidermis
progressively appeared; the other 34 developed normally. Among the shocked
and pricked embryos however, out of 26 normal looking gastrulae (ectodermal
syndrome 'nil'), all but one developed into neurulae showing no signs of
ectodermal alteration. On the other hand, withdrawal of the yolk plug was
normal in more than 60 % of the neurulae obtained after an electric shock, a
Correction of maternal effect in the salamander
313
percentage which is significantly higher (e = 3-16) than that obtained in
neurulae developing from control dejellied embryos (40%), though the same
electric shock did not modify the ectodermal syndrome.
DISCUSSION
The injury inflicted by the prick of a micropipette on the uncleaved egg of
an ac/ac female has a clear curative effect on the expression of the maternal
effect in certain embryos. Furthermore, it would seem possible to improve the
transitory nature of this recovery in other embryos by preceding the prick with
an electric shock.
This situation seems to be quite different from that which is known for
various maternal effects in the axolotl. The o maternal effect can be cured by a
macromolecular factor which is present in the nuclear sap of the normal oocyte
(Briggs & Justus, 1968). A similar situation occurs for the nc maternal effect
(Raff, Brothers & Raff, 1976). On the contrary, the cl maternal effect, which
affects plasmalemma formation during cleavage, is not corrected by such
injection experiments (Carroll & Van Deusen, 1973). Investigations into the
causes of the maternal effect correction in Pleurodeles must be now oriented
towards the consequence of a wound on cortical cytoplasmic ultrastructures.
Previous transmission electron microscopy studies on wounded fertilized eggs
of Ambystoma mexicanum (Luckenbill, 1971) and Xenopus laevis (Gingell,
1970; Bluemink, 1972), seem to indicate that microfilaments are involved in
the healing process. Contractile microfilaments seem to be necessary for morphogenetic movements (Spooner et al. 1973) as well as for shape changes in cells
or cell sheets (Perry, 1975). Such microfilaments are already conspicuous in
ectoderm cells of Triturus alpestris early gastrulae (Perry, 1975). We may
therefore expect to find such contractile microfilaments in the bottom of the
ectodermal furrows in abnormal Pleurodeles gastrulae.
We wish to thank Dr J. Cl. Beetschen for valuable comment and criticism during the course
of this work.
The scanning electron micrographs were performed at the 'Centre de Morphologie
experimental du C.N.R.S.' in Bordeaux-Talence.
This investigation was part of the programme of the ERA no. 327, Centre National de la
Recherche Scientifique.
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M. FERNANDEZ
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