J. Embryo!, exp. Morph. Vol. 34, 2, pp. 435-449, 1975
Printed in Great Britain
435
'Immobile' (ira), a recessive lethal mutation
of Xenopus laevis tadpoles
By A. DROIN 1 AND M. L. BEAUCHEMIN 2
From the Station de Zoologie experimentale,
Universite de Geneve
SUMMARY
'Immobile' (im) is a recessive lethal mutation discovered in the F 3 of a Xenopus {Xenopus
laevis laevis) originating from a mesodermal nucleus of a neurula transplanted into an enucleated egg. The im embryos do not contract after mechanical stimulation nor do they
present any spontaneous contraction from the neurula stage onwards. Development proceeds
normally during the first days after which deformation of the lower jaw and tail are observed.
The im tadpoles die when normal controls are at the feeding stage. Nervous and muscular
tissues are histologically normal in the mutant tadpoles; at advanced stages, however, an
irregularity in the path of the myofibrils is observed which is especially conspicuous in the
electron microscope. Cholinesterases and ATPase are present in the mutant muscles.
Parabiosis and chimerae experiments have shown that parabionts and grafts behave
according to their own genotype. Cultures of presumptive axial systems with or without
ectoderm lead to the conclusion that, first of all, the abnormality is situated in the mesodermal
cells and secondly that the first muscular contractions in normal Xenopus laevis are of myogenic origin. The banding pattern of the myofibrils is normal as was shown by obtaining
contractions of glycerol extracted im myoblasts with ATP. It seems therefore that in this
mutation, the abnormality is situated in the membranous system of the muscular cell, sarcoplasmic reticulum and/or tubular system as is probably the case in the mdg mutation of the
mouse.
INTRODUCTION
Muscular contraction and nerve-muscle relationships have been extensively
studied in mammals and amphibians, but only a few studies are concerned with
the ontogenesis of these events. Here we present a mutation in which contraction
and movement of the embryo and young tadpole of Xenopus laevis do not
occur and which may help to elucidate the genesis of muscular contraction in
normal development. The morphology of the mutant is described in this paper
as well as some experiments which show that the abnormality resides in the
muscles.
1
Author's address: Station de Zoologie experimentale, Universite de Geneve, 154, Rte de
Malagnou, 1224 Chene-Bougeries, Geneva, Switzerland.
2
Author's address: Departement d'Ophtalmologie, Hopital Cantonal, 1211 Geneva,
Switzerland.
436
A. DROIN AND M. L. BEAUCHEMIN
MATERIAL
AND
METHODS
Nuclear-transplant frogs {Xenopus laevis laevis) as well as laboratory stock
animals were used. Spawning and fertilization were provoked according to
Gurdon (1967). Two hundred blastulae per cross were preserved, separated in
groups of ten in Petri dishes containing aqualab (dechlorinated water) at room
temperature and observed daily. After 7 days, the normal tadpoles were fed
with nettle powder and transferred into large containers (101). The developmental
stages were determined according to Nieuwkoop & Faber (1956).
For light microscopy, the tadpoles were fixed with Zenker's or Gomori
1-2-3 fluid and stained with hemalum-eosin or fuchsin-orange G-fast green
(Humason, 1972); for nerve histology they were fixed in alcoholic saturated
picric acid-formaldehyde and impregnated with silver nitrate according to
Bodian modified by Fitzgerald (Gabe, 1968). Transverse, sagittal and longitudinal sections were cut at 7, 10 and 15 /*m. For electron-microscopic examinations
the embryos were fixed in a solution of glutaraldehyde-formaldehyde-acrolein
in cacodylate buffer, post-fixed in osmium tetroxide, dehydrated with acetone
and embedded in Epon (Kalt & Tandler, 1971); they were cut with a PorterBlum ultramicrotome and observed in a Philips 300 electron microscope. The
histochemical reaction for cholinesterases was carried out on whole embryos
using the azo-dye technique of Lewis (1958). The ATPase was revealed by the
method of Wachstein & Meisel (1957) on frozen sections of tails of stage-46
tadpoles, obtained by using a Cryostat freezing microtome after incubation
in the ATP medium at 23 °C at different times varying from 5 to 60 min. Parabiosis was performed and chimerae were made by operation on neurulae
(stage 20-22) in standard Holtfreter solution.
For cultures of the presumptive axial systems, normal and presumptive im
embryos of stage 14 were liberated from their jelly coats and after removal of
the endoderm the dorsal part of the embryo was put in Ca-Mg-free solution
for about 30 min. The presumptive neural and epidermal ectoderm was then
peeled off and the remaining mesoderm with some underlying endodermal cells
was cultured for 48 h in standard Holtfreter solution. The tissues formed balllike structures which, after a few hours, could be tested with a hair-loop for
contraction. Controls were treated similarly, apart from the ectoderm which
was left undisturbed. In these cases the tissues developed into normal tadpole
backs with complete axial systems and were also tested for motility.
The in vitro effect of ATP was tested with the method of Holtzer & Abbott
(1958) which applied the Szent-Gyorgi model (1949) to chick embryo myoblasts.
Stage-46 tadpoles were prepared by removing head, gut and preferably skin.
The remaining part composed of trunk and tail was extracted with 50 %
glycerol at 0° for 24 h and -20° for 48 h or more. Clusters and isolated myoblasts were then obtained by teasing bits of the extracted trunk or tail somites on
a slide in a drop of 25 % iced glycerol; the slides were covered with a coverslip,
Recessive lethal mutation o/Xenopus laevis tadpoles
D
?
\
437
?
/
Donor • Neurula
+O
70
140-
.c/ 23
72
A 23-4% • 18-6%
• 22-7%
Fig. 1. Genealogy of the 'family' in which the mutation im was discovered. D =
unknown parents of the donor embryo; • -> O = nuclear transplantation;
<$ 70 = o resulting from the nuclear transplantation; ©, B = ? and <3 heterozygous for im; • = im/im tadpoles.
maintained in the freezer for a short time and observed under a phase-contrast
microscope. Two or three myoblasts per preparation were usually measured
with a millimetric ocular; a few drops of an ATP solution (disodium salt,
5raM,pH 6-8) were then added under the coverslip. After about 1 min the
myoblasts were seen contracting. They were measured again at the end of
contraction and the percentage of contraction was calculated on the basis of
these two values.
OBSERVATIONS
Heredity of the mutation
This mutation has been found in the third generation (F3) of a male ( <$ 70)
resulting from a mesodermal nuclear transplantation of a young neurula into
an enucleated egg (Fig. 1). The parents of the donor embryo are unknown. The
mother of the F 2 ($ 72) crossed with her heterozygous sons ( + //m) possesses
the wild-type genotype + / + , the father ($ 23) originating from the laboratory
stock could not be tested. Eleven out of the 28 individuals (39-3 %) tested in the
F 2 are heterozygous; several individuals of one F 3 have already been crossed
and four out of nine of them are heterozygous.
438
A. D R O I N AND M. L. BEAUCHEMIN
Fig. 2. 'Immobile' tadpole (above) and normal tadpole (below) at stage 45.
In the F 2 , 12 crosses have been carried out between heterozygous individuals:
550 homozygous (im/im) tadpoles have been obtained out of 2242 embryos
giving a percentage 24-5 %, thus showing that the mutation is inherited as a
mendelian recessive and that the penetrance is complete.
Some other mutations have been obtained in this same family - 'oedema'
(oe) (Uehlinger & Beauchemin, 1968), 'dwarf II' (dw-II) and 'precocious
oedema' (p.oe) (Droin, 1974) - but no linkage tests have been effected.
Description of the phenotype
External morphology
No difference can be observed between normal and presumptive 'immobile'
embryos from fertilization up to the neurula stage. At the pre-motile stages
(22-24), normal embryos respond to mechanical stimulation with a neck
contraction. From stage 25 (flexure and coil stage) they contract spontaneously
whereas 'immobile' embryos show neither flexure after stimulation nor any
spontaneous contraction. When stimulated for a long time, however, the im
tadpoles sometimes exhibit a very slow bending movement which leaves them
bent at a right angle for a few minutes.
Except for the lack of motility which leaves the mutant tadpoles lying on the
bottom of the dish, general development, heart beating and hatching proceed
normally up to stage 42. From this stage onwards the head becomes narrower
than that of the normal tadpoles due to a deformation of the lower jaws which
either protrude or are folded inside such as are seen in the mutation 'folded
jaw' (Droin, Reynaud & Uehlinger, 1968); no jaw movement is observed.
At stage 43, the chromatophores contract and at stage 44, an elongation of
the back and the tail takes place, giving the mutants a white appearance and a
ventrally curved aspect (Fig. 2).
Recessive lethal mutation o/Xenopus laevis tadpoles
439
The number of somites in the im tadpoles is the same as in the normal ones
but in these advanced stages they appear wider and more pointed, the angle of
the chevron being more acute (cf. Fig. 8). The eyes are fully developed but do
not move; the pharynx, branchial chamber and gut develop normally but the
tadpoles cannot absorb food. They die between the 8th and the 10th day when
the normal tadpoles are at the feeding stage.
Internal morphology
(a) Histology. Light microscopy does not reveal any obvious difference,
during early developmental stages, between the muscle cells of normal and those
of im individuals. However, from stage 38 a slight disorganization is observed
in the myotomal arrangement of the im somite. The extremities of the myoblasts
are more loosely packed at the level of the myocommata as compared with the
tightly packed normal ones. Moreover, inside the myoblasts, several of the ribbon-like strands formed by the myofibrils take a slightly winding course whereas
the myofibrils of the normal muscles are always arranged in straight rows
(Figs. 3, 4).
With the Bodian silver impregnation technique the brain appears normal
and the pattern of innervation of the motor and sensory fibres of the mutant is
quite similar to the normal one. The motor fibres situated laterally to the
neural tube and forming the motor roots innervating the myotomes can be
seen, as well as the oblique fibres traversing the lower part of the neural tube
(Figs. 5-7). The Mauthner neurons which are thought to be involved in swimming coordination develop normally in the im rhombencephalon.
Normal Rohon-Beard cells, constituting the early sensory system of the tadpoles, are observed in the nervous system of the mutant. These cells are easily
recognized by their large size and the reddish colour of the cytoplasm when
stained with eosin; they are situated in the dorsal part of the spinal cord;
their sensory fibres leave the spinal cord and pass at the level of the myocommata
to innervate the epidermis.
(b) Histochemistry. The histochemical reaction for cholinesterases (Lewis,
1958) was performed on skinned embryos. The reaction becomes visible from
stage 31 and is much more conspicuous in the anterior part of the embryo than
in the posterior part. The presence of cholinesterases, which manifests itself by
a brown colouring, is localized in the myocommata where nerve plexuses are
formed (Lewis & Hughes, 1960). The im tadpoles exhibit this reaction as well
as the normal ones indicating that cholinesterases are present in the mutants
(Fig. 8).
Brown deposits revealing the presence of ATPase are also visible in the
mutants' tails. They are scattered along the myofibrils and exhibit the same
pattern as in the normal tails.
(c) Electron microscopy. At an ultrastructural level no difference is observed
before stage 33. The banding of the fibrils as well as the sarcoplasmic and
28
E M B 34
440
A. DROIN AND M. L. BEAUCHEMIN
50 ^m
50/mi
Recessive lethal mutation oj Xeriopus laevis tadpoles
441
tubular systems appear normal in the im muscles. From this stage on, the
arrangement of the fibrils is not as regular in the im fibres as in the normal
ones; slightly irregular branching and splittings of grouped filaments can be
observed (Figs. 9, 10).
At stage 42 these splittings and the deviation of the fibrils are more conspicuous. Often the fibrils are displaced around a nucleus or around yolk platelets
which appear to be more numerous than they are in the controls. The myofibrils
are sometimes cut, in the same section, both obliquely and transversally, indicating what appears to be a rather haphazard arrangement. The banding of the
acto-myosin filaments still shows a normal structure. The sarcoplasmjc reticulum
and T-system do not present any major discrepancy in their structure (Fig. 11).
At stage 45 the fibrils in the trunk of the mutant still keep their normal
appearance along with the sarcoplasmic reticulum and T-structure. The muscles
of the tail region however, are more affected than those of the trunk region.
Isolated myofibrils may be separated by vacuolated spaces of disintegrating
sarcoplasm. When the sarcoplasmic reticulum and T-structure finally break
down, a degeneration of the acto-myosin filaments follows (Fig. 12).
EXPERIMENTAL RESULTS
The above observations were completed by a series of experiments devised
to determine the level of gene action.
FIGURES 3-8, 13, 14
Fig. 3. Longitudinal section of the somites of a stage-38 normal tadpole.
Fig. 4. Longitudinal section of the somites of a stage-38 im tadpole.
Fig. 5. Longitudinal section of the muscles and neural tube of a stage-42 normal
tadpole (silver impregnation).
Fig. 6. Longitudinal section of the muscles and neural tube of a stage-42 im tadpole
showing the motor and transverse nerve fibres. The irregularity of the muscle fibres
is very conspicuous as compared with the regularity of normal ones (cf. Fig. 5)
(silver impregnation).
Fig. 7. Sagittal section of the muscles of a stage-38/39 im tadpole showing the
innervating nerve fibres (silver impregnation).
Fig. 8. Cholinesterase reaction in whole skinned and eviscerated stage-46 normal
(above) and im tadpole (below). The larger width and the more acute angle of the
im somites are especially conspicuous.
Fig. 13. Section through an im presumptive axial system explant (without ectoderm)
aged 48 h showing the beginning of notochord and metameric myoblast differentiation.
Fig. 14. (A) An im glycerol-extracted myoblast prepared for contraction test;
B-D, three stages of contraction of the same myoblast after addition of ATP
(phase-contrast microscope).
28-2
442
A. DROIN AND M. L. BEAUCHEMIN
Recessive lethal mutation o/Xenopus laevis tadpoles
443
Parabiosis
Ten successful parabioses were performed on neurula stages between wildtype and mutant embryos. Both partners react according to their own genotype,
the wild-type parabiont being mobile and the mutant one remaining immobile
and exhibiting the typical abnormalities of the advanced stages. The only
change observed in the 'immobile' partner is the lack of melanophore contraction and a prolongation of survival.
Chimerae
Chimerae were formed at neurula stages before the 'immobile' phenotype
could be recognized, the combination being haphazard. The embryos were
transversally cut through the middle and the halves exchanged. Of a total of
122 grafts, 29 mixed combinations were obtained. They were easily recognizable
because each half behaves according to its genotype. When stimulated, the
head and the anterior part of the trunk moved, the posterior part and tail
remained immobile and vice versa. When the head was immobile it showed the
typical deformation of the mutant and when the tail was immobile it was usually
curled up. With the light microscope one could observe that the neural tube
connexion had been re-established, thus indicating the probable continuity of
the nervous tissue. In these chimerae there was no clear limit of melanophore
contraction.
Cultures of presumptive axial systems
The eggs used for this experiment came from two kinds of crosses. In the
first three series there were only wild-type embryos; in the other series the
embryos were either wild-type (homozygous or heterozygous) or homozygous
mutants (Table 1). As the preparations were made at stage 14, the embryos
were taken at random. The results show that in the first series all the normal
explants with or without ectoderm exhibit contractions after mechanical
FIGURES
9-12
Fig. 9. Electron micrograph of myofibrils in the tail of a stage-38 normal tadpole
showing an orderly array of myofilaments.
Fig. 10. Electron micrograph of myofibrils in the tail of a stage-38 im tadpole
showing a normal ultrastructure with the exception of a slight disarrangement of certain myofilaments.
Fig. 11. Electron micrograph of myofibrils in the trunk of a stage-42 im tadpole.
Re-routing of myofibrils around a nucleus and a yolk platelet can be seen. Note
occasional haphazardly located myofilaments (arrows).
Fig. 12. Electron micrograph of myofibrils in the tail of a stage-45 im tadpole. The
sarcoplasm and mitochondria are undergoing degeneration, followed by the sarcoplasmic reticulum and T-structure (thin arrows) and finally the myofilaments
(thick arrow).
I
11
lira
I lib
Total
IV
Va
Vb
Via
VI b
Total
+ /+ x +/+ or
+ /+ x +lim
+ \im x + \im
Crossed genotypes
Number of
series
9
20
10
20
18
77
11
—
12
21
44
Total
9
21
13
12
55
11
14
15
20
22
82
—
—
—
—
—
22-2
25
30
25
38-8
28-6
—
—
—
—
—
2
5
3
5
7
22
7
15
7
15
11
55
Total
11
—
12
21
44
Contraction
No
contrac- % ofnon
contraction
tion
With ectoderm
8
11
10
16
17
62
9
21
13
12
55
3
3
5
4
5
20
—
—
—
—
—
No
contraction
Without ectoderm
Contraction
Number of cultures
Table 1. Cultures of presumptive axial systems
27-3
21-4
33-3
20
22-2
24-4
% of noncontraction
X
d
m
•
[-1
o
• d
3
O
Recessive lethal mutation o/Xenopus laevis tadpoles
445
Table 2. Effect of ATP
Glycerol-treated myoblasts
+ / + or +jim tadpoles
H2O-treated myoblasts
imjim tadpoles
No. of
prepar.
%of
contract.
No. of
prepar.
%of
contract.
5
10
4
1-20
21-40
41-60
4
II
6
1-20
21-40
41-60
+ / + or +//m tadpoles
No. of
prepar.
3
1
%of
contract.
1-20
21-40
im/itn tadpoles
No. of
prepar.
3
1
%of
contract.
1-20
21-40
stimulation whereas in the second series composed of + / + , +//m and imjim
embryos, the proportions of the cultures that did not react to stimulation are
respectively 28-6 % with ectoderm and 24-4 % without ectoderm. These percentages correspond to the mendelian percentages of the homozygous mutants.
After sectioning the wild-type- and the mutant-cultured explants without
ectoderm, we observe that differentiation has taken place. The notochord is
developed and vacuolated and the myoblasts are arranged metamerically,
forming somite-like units (Fig. 13). In the myoblasts, the myofibrils are also
differentiated and the striation is visible in the more advanced explants.
Effect of ATP
The normal isolated myoblasts obtained for contraction assays are very
regular elongated cells. The banding of the myofibrils is recognizable as an
alternating pattern of light and dark zones. This banding pattern in the im
myoblasts is visible as well as the internal disorganization of these cells (Fig.
14 A). After addition of ATP, however, the im myoblasts contract in the same
manner as the normal ones (Figs. 14B-D). The average length of the myoblasts
is about 150 jtim and the width 30 jam. On contraction they shorten and become
wider. Only the shortening was measured. The percentages of contraction as
well as their variability referred to in Table 2 are quite similar in normal and in
im tadpoles. Each percentage corresponds to the mean value of two or three
isolated myoblasts measured for each preparation; two to three preparations
are made per tadpole. The measurements were made in a comparative way
preparing successively, whenever possible, one normal and one mutant tadpole.
The variability of the results can be explained by several factors: firstly myoblasts very often adhere to the slide or the cover-slip, thus inhibiting a full
contraction; secondly, with the change of the refractive index of the medium
which occurs after the addition of the ATP solution, the measurements are not
always very precise; thirdly, the maintenance of the temperatures of the preparation before contraction may vary slightly; the capacity of contraction
diminishes with higher temperatures.
446
A. DROIN AND M. L. BEAUCHEMIN
A small series of controls has been made using distilled water as the extraction
medium instead of glycerol. Contractions also take place after addition of
ATP but they are less strong than after glycerol extraction. In this case again,
the reaction of normal and im myoblasts is quite similar.
DISCUSSION
The cultures of the presumptive axial systems allow us to draw two main
conclusions. The first one is that the prospective inability for contraction expressed by the mutant gene is laid down intrinsically in the mesodermal cells
at the end of gastrulation. The results show on the one hand that the lack of
contraction of the mesodermal explants depends indeed on the genotype of the
cells. On the other hand, the presence of nerve cells cannot induce contraction
in the ectomesodermal explants of imjim embryos.
The second conclusion is that the + / + or + \im mesodermal explants can
contract in the absence of nerve cells. This last result raises the problem of the
nervous or muscular origin of the first contraction responses in lower vertebrates.
In fishes it is known that the first contractions have a myogenic origin (Leghissa,
1941; Sawyer, 1944; Harris & Whiting, 1954) but in Amphibians this is still a
subject of controversy. On the one hand, Corner (1964) making explants of
neural plate in a jacket of ectoderm and mesoderm of Xenopus ascertained that
no contraction could be evoked after mechanical stimulation when there was
no nervous tissue associated with mesoderm; he concluded that the movements
were of non-myogenic origin. On the other hand, Hughes (1959) and Muntz
(1964) in a detailed description of the differentiation of the motor system in
Xenopus stated that no motor fibres leave the neural tube at the premotile stage
(stage when the first body contractions occur after stimulation) and Muntz
(1964) postulated that it is the direct action of the skin on the muscle cell which
elicits the contraction response. This statement was confirmed by Macklin &
Wojtkowski (1973), who showed, in measuring the electrical activity of Xenopus
embryos, that the first rhythmical spikes have a myogenic origin and are derived
from the direct relation between skin and muscles. This relation was also
confirmed by Roberts & Smith (1974), who demonstrated the excitability of the
skin before innervation, thus allowing the first responses of the embryo after
stimulation. The results obtained from the cultures of the axial systems confirm
the myogenic origin of the first contractions in Xenopus embryos which may
happen even in the absence of skin.
The last experiment (effect of ATP) leads to the third conclusion, namely
the normality of the contractile apparatus of the im myoblasts. Besides the
normal banding pattern of the fibrils observed in the micrographs, the ATPasepositive reaction and the contraction response of the im glycerinated myoblasts
to ATP, entirely comparable to the response of the normal myoblasts, confirm
the normal potentiality for activity and behaviour of the contractile proteins.
Recessive lethal mutation o/Xenopus laevis tadpelos
447
The site of the gene expression must then be looked for in the other cell
components of the myoblasts. Among the numerous reactions ending in the
contraction of the muscle cell, two important steps are concerned with the
membranous cell components. The first is the membrane depolarization which
spreads through the tubular system on to the sarcoplasmic reticulum; the second
is the release by the sarcoplasmic reticulum of Ca 2+ , which is then fixed to the
contractile apparatus, thus allowing the mechanical reaction of contraction to
take place (Ebashi & Endo, 1968; Sandow, 1970; Huxley, 1971). Caffeine is
known to act upon the sarcoplasmic reticulum by increasing the release of Ca2 +
and therefore the contraction reaction (Weber & Herz, 1968; Ebashi & Endo,
1968). An attempt was made to rear normal and immobile embryos in caffeine
solutions of 2-3 and 5-8 mM. In normal individuals, contractions were increased
with the tadpoles becoming restless and twitching continuously. In the im
tadpoles the slow bending movements occurring irregularly after stimulation
were more pronounced in low concentrations while in higher concentrations,
they occurred spontaneously leaving the tadpoles in a bent state. These preliminary results indicate that the im muscles may react to caffeine. It is then
tempting to put forward the hypothesis that the sarcoplasmic reticulum and/or
the tubular system are the sites of the primary gene action. To test this hypothesis, further studies are needed, especially an electrophysiological one in order
to show whether or not the resting and the action potentials of the cell membrane
are normal, a detailed analysis of the Ca 2+ regulation in the myoblasts during
differentiation and a biochemical analysis of the membranous cell components.
The same hypothesis of the abnormality of the membranous apparatus of the
muscle cells was also postulated in the case of the mutation muscular dysgenesis
(mdg) of the mouse. It is interesting to compare the mdg with the im mutation
as they have several features in common, particularly the inability of the mutant
newborn mouse to move, due to defective muscle cells (Pai, 1965*3, 6). The
phenotypic abnormalities, however, are more conspicuous in the mdg mutation,
especially the skeletal ones, which may be compared to a certain extent to the
deformation of the lower jaw of the im mutant. The cytological degeneration
of the mdg myoblasts (abnormal and dilated sarcoplasmic reticulum, atypical
pattern and atrophy of the myonbrils) constitutes another noteworthy aspect
of this mutation (Platzer & Gluecksohn-Waelsch, 1972). When the mdg
myoblasts are analysed in monolayer cell cultures the differentiation proceeds
normally with respect to the development and rate of maturation but they fail
to contract or exhibit partially abnormal, slow contractions. When exposed to
caffeine, a few mutant cells contract normally but localized contractions are the
predominant responses. The normality of the contractile apparatus was confirmed as well by the contraction response of glycerinated myoblasts to ATP
after 8—14 days of culture (Bowden-Essien, 1972). In both mutations (mdg and
im) the functional differentiation of the muscle cells seems to be impeded
primarily.
448
A. DROIN AND M. L. BEAUCHEMIN
RESUME
Immobile (im) est une mutation recessive letale trouvee dans la descendance d'un Xenopus
(Xenopus laevis laevis) issu de la greffe d'un noyau mesodermique de neurula dans un oeuf
enuclee. L'embryon im ne presente aucune contraction musculaire apres stimulation
mecanique ni contraction spontanee des le stade de neurula. Le developpement est normal
pendant les premiers jours puis des deformations apparaissent dans la machoire et la queue.
Les tetards meurent au moment ou les tetards normaux commencent a se nourrir. Le systeme
nerveux et le systeme musculaire sont histologiquement normaux chez les mutants; on
observe cependant, dans les stades avances, une certaine irregularite dans le trajet des
myofibrilles, particulierement visible au microscope electronique. Les cholinesterases et
I'ATPase sont presentes dans les muscles des tetards immobiles.
Les experiences de parabiose et de chimeres ont montre que les parabiontes et les greffes
se developpent selon leur genotype respectif. Des cultures de systemes axiaux presomptifs,
avec ou sans ectoderme, ont permis de conclure, d'une part, que 1'anomalie se situe dans les
cellules mesodermiques et, d'autre part, que les premieres contractions musculaires chez les
Xenopus normaux sont d'origine myogenique. En outre, les myoblastes des tetards immobiles
extraits a la glycerine peuvent se contracter sous l'effet de l'ATP demontrant ainsi la normalite
de la striation fibrillaire. II semblerait done que le gene exerce son effet au niveau du reseau
membranaire de la cellule musculaire, reticulum sarcoplasmique ou systeme tubulaire comme
ce serait le cas dans la mutation muscular dysgenesis (mdg) de la souris.
We thank Professor M. Fischberg, who put the material at our disposal, for his interest
and criticism and the 'Departement d'Ophtalmologie de l'Hopital cantonal' for the use of
the electron miscroscope. We are indebted to Professor Huggel and Dr Benzonana for their
critical reading of the manuscript.
This work was supported by the 'Fonds national suisse de la Recherche scientifique'
(no. 3.60.68).
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(Received 25 February 1975, revised 13 May 1975)