J. Embryol. exp. Morph. Vol. 44, pp. 281-295,1978
Printed in Great Britain © Company of Biologists Limited 1978
281
The effects of high hydrostatic pressure on
microfilaments and micro tubules in Xenopus laevis
By PAUL-EMIL MESSIER 1 AND C. SEGUIN 1
From the Departement cTAnatomie, Universite de Montreal
SUMMARY
Xenopus laevis embryos of stages 14-20 were subjected, for periods of 5-330 min, to
hydrostatic pressures ranging from 500 to 10000 psi. The specimens were fixed under corresponding pressures and their neuroepithelium was studied under light and electron microscopy.
A pressure of 3000 psi, maintained for as long as 180 min, did not inhibit neurulation
though it induced slight deformities of the neuroepithelium. A pressure of 4000 psi, applied
for 180 min, disrupted the apical ring of microfilaments and blocked neurulation. The cells
lost their dissymmetry. The effect was reversible. Lengthening the duration of treatment to
330 min caused the neuroepithelial cells to loose their microtubules and to become round.
This situation was not reversible. Our results indicated that microfilaments are more sensitive
than microtubules, that both organelles became increasingly sensitive as the exerted pressure
was increased and that microtubules of older embryos exhibited a better resistance. Finally,
we showed a correlation between the presence of microfilaments and the constricted state of
the cellular apices and a relationship between the presence of microtubules and cell elongation.
INTRODUCTION
During neurulation the neuroepithelium invaginates thereby transforming
the flat neural plate into a neural tube. Two major modifications in the cell
shape are observed during the process. Firstly, the cells elongate; it has been
said that they become asymmetric. Secondly, they acquire a slender apical
portion and a broad base; this has been referred to as an apico-basal
dissymmetry (Messier, 1916 a).
More than 10 years ago Waddington & Perry (1966) suggested that microtubules might be involved in neurulation in that they could act as shape
generating structures capable of inducing cell elongation. Since then many
reports, covering an impressive variety of cell systems, have shown the existence
of a correlation between the presence of microtubules and the production
and/or maintenance of cell asymmetry. Such a correlation has been tested
experimentally and established in the cells making up the neuroepithelium of
Xenopus (Karfunkel, 1971) and chick embryos (Auclair & Messier, 1974).
Concerning the second feature, the acquisition of an apico-basal dissymmetry,
1
Authors' address: Departement d'Anatomie, Faculte de Medecine, Universite de
Montreal, C.P. 6128, Succ. A, Montreal, P.Q. H3C 3J7, Canada.
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P.-E. MESSIER AND C. SEGUIN
it was proposed by Baker & Schroeder (1967) and Schroeder (1970) that once
the cells acquire their columnar shape (asymmetry), an apical ring of microfilaments would contract thus reducing the surface area of the cellular apices.
This production of an epithelium, made up of dissymmetric cells each endowed
with a slender apex and a broad base, would create part of the forces that lead
the neural plate into invagination, a prerequisite for neurulation.
To this concept were added a number of findings showing that the setting-in
of an apico-basal dissymmetry also involves the phenomenon of interkinetic
nuclear migration which induces the enlargement of the base of the cell by
placing the nucleus in the basal region of the cell for the longer part of the cell
cycle (Messier & Auclair, 1973, 1975; Messier, \976a).
The role of microfilaments in constricting the cell apices was investigated by
Karfunkel (1972). The author treated neurulating chick embryos with colchicine
and cytochalasin B. Both chemicals, it was shown, blocked neurulation.
Karfunkel reports that colchicine breaks down microtubules thus inducing a
loss of asymmetry and that cytochalasin B selectively disrupts the ring of microfilaments thereby causing the loss of the apical construction and the resulting
inhibition in neurulation. However, cytochalasin B, it is now most apparent, is
a substance whose effects and cell target(s) are quite controversial (Burnside &
Manasek, 1972; Holzer & Sanger, 1972; Auclair & Messier, a review, 1977).
For instance, it is known now that cytochalasin B inhibits interkinetic nuclear
migration in the chick embryo; thereby jeopardizing the apico-basal dissymmetry (Messier & Auclair, 1974). Also, it has been shown in the same
tissue that the chemical greatly alters the morphology of the cell surface
(Messier, 19766). Finally, cytochalasin B allows intercellular leakage of electron
opaque tracers, thus suggesting that it may weaken the epithelial cohesiveness
necessary for neurulation to proceed normally (Messier & Auclair, 1977).
Because of the uncertainties linked to experimentations with cytochalasin B,
it was decided to reinvestigate the role of microfilaments and microtubules in
neurulation firstly, via a different avenue and secondly, using a different species.
Hence, we used high hydrostatic pressure as a means and the neurulating embryo
of Xenopus laevis as a model. High hydrostatic pressures were selected because
they are known to depolymerize microtubules and microfilaments (Zimmerman,
1971, 1970 a).
MATERIALS AND METHODS
Ovulation in Xenopus laevis was induced by injections of chorionic gonadotropin according to Gurdon's method (1967). The gastrulae were chemically
dejellied by treating them 10-15 min with a mercaptoacetic acid solution
(0-7 % in 50 % Steinberg solution) brought to pH 8-5 with NaOH (Karfunkel,
1971). They were then kept at 23 °C in a 10 % Steinberg solution until they had
reached stages 14-20 (according to Nieuwkoop & Faber, 1967), whence they
were used for experimentation. A pressure-fixation chamber identical with that
Pressure effects on micro filaments and microtubules
283
designed by Landau & Thibodeau (1962) was employed for pressurizing and
fixation of specimens under hydrostatic pressure. The embryos were subjected
to pressures of 500, 1500, 2000, 3000, 4000, 4500, 5000, 6000 and 10000 psi*
for intervals of 5, 15, 60, 90, 120, 180, 240 and 330 min. For each of the
pressure/time combinations used a minimum of six embryos were analysed
microscopically. However, for combinations (psi/min) of 4000/180, 4000/330,
4500/180 and 5000/90 at least 15 embryos were analysed.
All specimens were fixed with 4 % glutaraldehyde buffered with 0-1 M phosphate (pH 7-2). Fixation was carried out for 15 min at each of the pressures
used. Upon decompression, specimens and fixative were transferred to Petri
dishes where, to enhance fixation, the upper halves of the eggs were dissected with
iris scissors. Fixation was pursued on the isolated embryos for an additional
3-6 h. Other embryos, used as controls, were grown at atmospheric pressure
(14,7 psi) and fixed at intervals where they had reached stages 14, 16, 18 and
20. In order to control the effect of a possible oxygen depletion (see Tilney &
Gibbins, 1969) 20 embryos were inserted in the pressure bomb and kept there
under atmospheric pressure for 330 min. These controls neurulated exactly as
those grown under atmospheric pressure. All the controls were fixed as above.
Following fixation, the specimens were washed overnight in the phosphate
buffer and post-fixed for 90 min in 0-1 M phosphate buffered osmium tetroxide.
Specimens were dehydrated and embedded in Epon. Using an LKB ultrotome,
sections covering an average length of 50/*m were cut transversely to the
cephalocaudal axis in the mid-trunk region. For light microscopy, the sections
were 1 /.cm thick and stained with a 1 % aqueous solution of toluidine blue;
thin sections for electron microscopy were stained with uranyl acetate and lead
citrate (Reynolds, 1963) and examined with a Siemens 1A microscope.
The response exhibited by microfilaments and microtubules was always
observed in more than 95 % of the neuroepithelial cells of each individual
embryo. Furthermore, in most cases, at each of the pressure/time combinations
used, 100 % of the embryos reacted in the same way; cases making exception
are mentioned in the Results.
RESULTS
Preamble
The histology and cell ultrastructure of the neuroepithelium of early Xenopus
laevis embryos have already been described (Schroeder, 1970; Tarin, 1972).
Moreover, morphometric data related to this tissue is also available (Mathieu
& Messier, 1977). Therefore, we will only recall here that which is pertinent to
the present title.
In Xenopus laevis the neuroepithelium is made up of two layers of epithelial
cells. They are termed the superficial and deep layers. The cells whose ultrastructure we will be concerned with are those touching the floor of the neural
* 703070xl02kg/m2.
284
P.-E. MESSIER AND C. SEGUIN
Pressure effects on microfilaments and microtubules
285
groove. They belong to the superficial layer and occupy the topographical zone
termed median by Schroeder (1970) or those zones labelled proximal superficial
and suprachordal superficial by Mathieu & Messier (1977). The cells making
up these zones always exhibit a narrow apex and a broad base (dissymmetry)
and show asymmetry in that they are either slightly elongated (suprachordal
superficial) or highly elongated (proximal superficial) (Fig. 11). In the literature,
these cells have been referred to as 'bottle cells', 'flask cells' and 'wedgeshaped cells'. They contain an apically situated ring of microfilaments (Fig.
2A) and numerous microtubules oriented parallel to the cell's longer axis
(Fig. 2B).
The fate of the neuroepithelium, as it progresses from the neural plate to the
neural tube, is illustrated in Figs. 1A-D. This series of micrographs shows the
degree of development attained under atmospheric pressure, starting from
stage 14 or stage 16, which were the ones selected as starting points in our
experimentation. The series helps in evaluating how much a given pressure,
applied for a given length of time, disturbed neural organogenesis. For instance,
pressurizing stage-14 embryos (Fig. 1A) for 180 min at 4000 psi impeded the
invagination of the neuroepithelium and gave rise in 75 % of the cases, to
specimens whose neural groove was wide open (Fig. 1G). By comparison, an
embryo developed to approximately stage 14 and grown for 180 min under
atmospheric pressure reached stage 18; at which time its neural groove was
deep and narrow (Fig. 1C). In any of the pressure/time combinations mentioned
later, such comparisons could be made provided that the treatment is not so
deleterious as to render comparison unnecessary. The effects that the various
combinations used had on microfilaments and microtubules are summarized
in Table 1.
FIGURE 1
(A-D) A series of light micrographs of sections cut transversely to the longer axis
of control embryos. It shows four consecutive stages in neurulation. At stage 14 (A)
the neuroepithelium (N) is in the form of a neural plate. It progressively invaginates
(B, C) forming a neural groove and eventually (D) a neural tube. The neural
groove (#), somites (5) and notochord (n) are shown.
(E, F) These light micrographs show the aspect taken by the neuroepithelium in
embryos developed for 3 h under a pressure of 3000 psi. The groove is formed. In
(F) abnormalities are evident as epidermal cellsinvade the groove, x 750.
(G) Section of an embryo exposed for 3 h to a pressure of 4000 psi. The neural groove
is wide open, x 750.
(H) Section of an embryo exposed for 2 h to a pressure of 4500 psi. x 750.
(I) Enlargement of a region depicted in (C). It shows the cell asymmetry (highly
elongated cell marked x) and its dissymmetry (large base and narrow apices),
x 2500.
(J) Illustration of the aspect of a few neuroepithelial cells from an embryo exposed
to 4000 psi for 3 h. Note cell marked x exhibiting an apex (right-hand side) as
large as its base (loss of dissymmetry), x 2500.
19
EMB
44
286
P.-E. MESSIER AND C. SEGUIN
Table 1. Effect of high hydrostatic pressure (psi) exerted for various
durations (min)
The outcome of the various treatments in terms of the presence (+) or absence (—)
of microfilaments (mf) or microtubules (mt) is indicated. The stage at which each
treatment began is also shown.
Pressure (psi)
A
3000
Duration
(min)
5
Sta|
mf
4000
mt
mf
4500
mt
mf
5000
mt
mf
mt
6000
mf
mt
10000
mf
mt
16
15
16
45
16
90
120
16
16
180
14
330
14
Effects on microfilaments and microtubules
It was found that a pressure of 3000 psi did not inhibit the invagination of
the neural plate. Stage-14 embryos subjected to such a pressure for as long as
180 min developed to stage 18 (Fig. 1E) just as controls did (Fig. 1C). However,
although neurulation always occurred, some deformities in the neuroepithelium
were frequently noted (Fig. 1F). Most often these deformities took the aspect
of an abnormal mediad convergence of the epidermis which caused a crowding
in of the neurocoele. Raising the pressure to the 4000 psi range disturbed the
invagination. For instance stage-14 embryos which showed a flat neural plate
(Fig. 1 A) exhibited, in 75 % of the cases, a wide open neural groove following
a 180 min exposure at 4000 psi (Fig. 1G). Raising the pressure still more
(4500 psi) while shortening the pressurizing period (120 min) induced less of
an accentuated widening of the groove (Fig. 1H). At a pressure of 4000 psi,
maintained for 180 min, the so-caHed bottle-shaped cells (Fig. II) lost their
dissymmetry; their apices widened and assumed a width equal to that of the
base (Fig. 1J).
Ultrastructuratly, these cells lost the apical ring of microfilaments (Fig. 3 A)
FIGURE 2
(A) Electron micrograph of parts of a few neuroepithelial cells taken from a stage-18
control embryo. It shows the ring of microfilaments (arrows) situated at the cellular
apex, x 21000.
(B) Electron micrograph of the apical region of a cell belonging to an embryo which
has been exposed to 4000 psi for 3 h. No microfilaments are observed apically.
x 21000.
Pressure effects on microfilaments and microtubules
287
>
19-2
288
P.-E. MESSIER AND C. SEGUIN
Pressure effects on microfilaments and microtubules
289
which is normally encountered in untreated embryos (Fig. 2 A). These effects
were reversible. Indeed, in all stage-14 embryos treated at 4000 psi for 180 min,
the invagination resumed upon decompression. Filaments reappeared in the
cells, dissymmetry was achieved anew and neurulation proceeded though it led
to the appearance of deformities in the neural tubes. One typical example of
such deformities was the production of an abnormally large neurocoele (Fig.
4F). When a pressure of 4000 psi was applied to stage-14 embryos for as long
as 330 min, 75 % of the pressurized specimens showed rounded neuroepithelial
cells in a completely disrupted epithelium (Fig. 3B). Similar results were
obtained in 88 % of the stage-16 embryos kept at 4500 psi for 180 min (Fig.
3C). At pressure/time combinations of 4000 psi/330 min and 4500 psi/180 min,
microtubules were no longer found in the neuroepithelial cells, which, we
emphasize, became round. This situation was not reversible upon decompression. However, the general aspect of the epithelium could resist higher
hydrostatic pressures provided the duration of treatment was made shorter.
For instance, a pressure of 5000 psi, held for 90 min, induced in 10 % of the
embryos a widening of the neural groove (Fig. 3D). In such specimens, the
cells, though devoid of microfilaments, still contained microtubules (Fig. 3F).
In the remaining 90 % of the embryos exposed to 5000 psi/90 min, both
microfilaments and microtubules were lost and the neuroepithelium was
unrecognizable (Fig. 3E).
A pressure of 6000 psi, maintained for 15 min, disturbed only slightly the
general aspect of the neuroepithelium (Fig. 4 A). Here, the neuroepithelia.1 cells
lost their filaments but they still contained some microtubules as small portions
of these could occasionally be found (Fig. 4C). Following a 45-min exposure
to this same pressure, the neuroepithelium became unrecognizable, all cells
assumed a round shape (Fig. 4B) and they lost all of their microtubules.
Finally, a pressure of 10000 psi applied for 5 min, barely modified the epithelial
FIGURE 3
(A) Electron micrograph of cellular portions from a control embryo. Numerous,
long microtubules are easily observed, x 42000.
(B) Effect of a pressure of 4000 psi applied for 330 min. The epithelium is disrupted
and cells are round, x 750.
(C) Effect of a pressure of 4500 psi lasting for 180 min. All neuroepithelial cells
assume a round shape, x 750.
(D) In 10 % of the embryos exposed for 90 min to a pressure of 5000 psi the neural
groove widened and the cellular apices lost their constriction, x 750.
(E) In 90% of the embryos exposed for 90 min to a pressure of 5000 psi all neuroepithelial cells became round and the epithelium was broken up.
(F) Small part of a neuroepithelial cell from an embryo maintained for 90 min
under 5000 psi and belonging to the category described in (D) above. Note some
microtubules (arrows) are still present in the cell, x 38000.
19-3
290
P.-E. MESSIER AND C. SEGUIN
H
Pressure effects on micro filaments and microtubules
291
nature of the neuroepithelium (Fig. 4D). The treatment did not rid the cells of
all of their microtubules. In most cells, very short segments of microtubules,
though admittedly quite scarce, could still be found. Prolonging the treatment
to 15 min induced the cells to become rounded (Fig. 4E) and a total loss of
microtubules.
Effects on cell organelles in general
In cases where the pressure used destroyed the apical bundle of microfilaments, yolk platelets, mitochondria and pigment granules were found
closer to the apical surface of the cell. The apical surface became smooth and
devoid of its characteristic microvilli.
Treatments which induced a rounding of the cells also induced cytolysis in
some cells. The degree of cytolysis observed was related to the pressure/time
combination employed. The cells from the superficial layer were more prone
to cytolysis just as they were to rounding and dissociation. Generally, rounded
cells displayed denser cytoplasm and round nucleus; their usual perinuclear
clumps and islets of heterochromatin were virtually absent. Under high pressure
(e.g. 4000 psi/180 min) a swelling of the perinuclear space and of the endoplasmic
reticulum was regularly observed (Fig. 4G, H).
DISCUSSION
High hydrostatic pressures have been reported to depolymerize cytoplasmic
microtubules and to produce shape changes in several cell types (Zimmerman,
1971; a review). The pressure/time combinations reported to be needed to
FIGURE 4
(A) Section of an embryo treated for 15 min at a pressure of 6000 psi. Although the
apical constrictions are lost the general aspect of the neuroepithelium is preserved.
x750.
(B) A treatment of 45 min at 6000 psi destroys the epithelial nature of the neuroepithelium and induces the cells to bscome rounded, x 750.
(C) Small portions of microtubules (arrows) can still be found in the cells of embryos
exposed for 15 min to 6000 psi. x 35000.
(D) A pressure of 10000 psi, held for 5 min, barely modifies the epithelial nature
of the neuroepithelium. x 750.
(E) A pressure of 10000 psi, held for 15 min, induces the cells to become rounded.
x750.
(F) Section of a stage-14 embryo first held for 180 min at 4000 psi and then left for
330 min at atmospheric pressure. Upon decompression neurulation resumed.
However, the neurocoele (lumen of the tube) is abnormally large (compare with
Fig. 1D). x 750.
(G. H) Electron micrographs of small portions of cytoplasm from a control embryo
(G) and from an embryo exposed for 45 min to 6000 psi (H). Note in (H) the enlarged perinuclear space (s) and the dilated aspect of the endoplasmic reticulum
(er). N, nucleus; M, mitochondria. Both x9000.
292
P.-E. MESSIER AND C. SEGUIN
destroy these organelles vary somewhat depending on the experimental model
used. Depolymerization is achieved at 4000 psi/10 min in Actinosphaerium
(Tilney, Hiramoto & Marsland, 1966) while it occurs at 10000 psi/10 min in
HeLa cells (Salmon, Goode, Maugel & Bonar, 1976). It is also known that
high pressure affects microfilaments. For instance, in the eggs of Arbacia,
microfilaments are disrupted and furrowing is inhibited by pressure/time
combinations ranging from 5000 psi/4 min (Marsland, 1956) to 6500 psi/
0-1 min (Marsland, 1970).
Although most of the work done on the effects of high pressures dealt with
protozoa, isolated cells and marine eggs, some data are available concerning
metazoa. Along that line, Ebbecke (1944) indicated that pressures of 1500020000 psi are required for the glandular cells of the frog's pharynx to become
round. Also, Tilney & Cardell (1970) showed that a pressure of 6500 psi,
applied for 30 min, causes the disruption of the microfilaments contained in
the microvilli of the cells forming the small intestine of the salamander. Finally,
O'Connor, Houston & Samson (1974) did not succeed in depolymerizing
neuronal microtubules in adult frogs even after a 30 min exposure to 10000 psi.
We found that the microfilaments which are usually assembled in the form
of a ring at the apex of the neuroepithelial cells in Xenopus, were more sensitive
to high pressure than were microtubules. Microfilaments were always lost
before microtubules disappeared. For a pressure treatment lasting a given
period of time (e.g. 180 min), a small increment of 12 % over 4000 psi (bringing
the pressure to 4500 psi) induced the disappearance of microtubules (Table 1).
Yet, at this pressure of 4500 psi, 120 min did not suffice to rid the cells of their
microtubules; whereas an increment of 10% over that 4500 psi achieved it,
even if the duration of treatment was reduced by 25 %. Clearly, microtubules
became increasingly sensitive as the pressure exerted was increased. The same
applied to microfilaments.
The pressure-induced disappearance of microfilaments was always accompanied by a release of the apical constriction, that is to say by a loss of cell
dissymmetry. Furthermore, in the reversibility tests, we have always observed
the reappearance of microfilaments together with a return to dissymmetry. In
this respect, our observations concur with Karfunkel's view (1972) derived
from work done on chicken embryos exposed to cytochalasin B, that there
exists a relationship between microfilaments and apical constrictions. Although
our approach avoided the pitfalls of cytochalasin B, it gave no indications as
to the cellular processes that might have been disturbed by the high pressures
used. On that account, the effects of pressure on a variety of cell functions have
been studied extensively (Zimmerman, \970a, 1971, reviews). However, such a
study has not been found for an amphibian. In essence, the study of 'primitive
systems' has shown that pressures less than 6000 psi usually have a negligible
effect on the rate of cell metabolism. Higher pressures are required to delay
significantly DNA, RNA and protein synthesis. Moreover, cellular permeability
Pressure effects on micro filaments and microtubules
293
in Arbacia is not affected at 4000 psi (Zimmerman, 1970b); while it seems that
it is affected in the onion Allium cepa at a pressure of 7500 psi (Murakami, 1963).
Our work confirmed the existence of a relationship between the elongated
state of neuroepithelial cells and the presence of microtubules. Indeed, conditions that rid the cells of their microtubules induced a loss of cell asymmetry.
Additionally, we showed that the pressure-induced disintegration of microtubules was, in most cases, not reversible. This is of interest, for in the neuroepithelial cells of the chick embryo the depolymerization of microtubules under
cold exposure was shown to be reversible (Auclair & Messier, 1974). Here, it
appears as though once dissymmetry is gone, the additional loss of asymmetry
will leave the cells incapable of reverting to their original shape. The difficulty
is not one of geometry only, for upon releasing those pressures that destroy
microtubules neither they, nor microfilaments, will reappear. Our results
suggest that there may exist a factor responsible for the polymerization of
microfilaments and microtubules which is irreversibly affected beyond a given
pressure/time combination. A similar view is held by O'Connor et al. (1974)
who have suggested that pressure could cause depolymerization of microtubules
indirectly either by a pressure stimulus response or by a pressure effect on some
stabilizing or control factor. Forer & Zimmerman's (1976) recent finding agrees
with this suggestion.
As part of our experiments, relatively young embryos (stage 15-16) were
subjected for 15min to a pressure of 6000 psi. In these, the neuroepithelial
cells maintained their elongated shape and microtubules were present. Older
embryos (stage 20) submitted to the same conditions also showed elongated
cells containing microtubules. It follows that, in the domain of the stages
analysed, microtubules were not less sensitive from one stage to the other.
However, when stage-15-16 embryos were pressurized for 90min at 5000 psi
their cells became round and they lost their microtubules; whereas stage-20
embryos treated in the same way had slightly elongated cells showing microtubules. Thus, it appears that microtubules in older specimens were more
resistant than those found in younger embryos. More work is needed to determine whether the increased resistance derived from the fact that (a) the subunits
microtubules in older specimens possess a greater degree of macromolecular
bonding and therefore are better protected or (b) more differentiated cells host
stabilizing factors capable of counteracting the effect of pressure or (c) the
inclusion of a cell in a more differentiated (more cohesive) tissue endows it
with better protection.
Our results underline, once again, the role of microfilaments and microtubules inneurulation. The loss of one of the organelles is sufficient to jeopardize
neurogenesis. In previous studies we have said much on the possibility that the
phenomenon of interkinetic nuclear migration might also have something to do
with neurulation. In that sense, it would have been interesting to learn how the
nuclear movements are affected by high pressures. However, regrettably, our
294
P.-E. MESSIER AND C. SEGUIN
present experimentation offers no arguments concerning interkinetic nuclear
migration. These nuclear movements cannot be followed with precision in the
neuroepithelial cells of Xenopus. A similar study is presently being carried out
on the better suited neural epithelium of Triturus viridescens.
RESUME
Effet des pressions hydrostatiques eleve'es sur les microfilaments et les microtubules
de Xenopus laevis
Des embryons de Xenopus laevis, ayant atteint les stades 14 a 20, ont ete soumis, pour des
periodes variant de 5 a 330 min, a des pressions hydrostatiques allant de 500 a 10000 psi.
Les specimens ont ete fixes a des pressions correspondantes et leur neuroepithelium a ete
etudie en microscopie photonique et electronique. La pression de 3000 psi, maintenue pour
aussi longtemps que 180 min, n'a pas empeche la neurulation de se produire bien qu'elle ait
induit des malformations mineures du neuroepithelium. La pression de 4000 psi, appliquee
pendant 180 min, a detruit l'anneau de microfilaments normalement observe a l'apex des
cellules et a inhibe aussi la neurulation. Les cellules ont perdu leur dissymetrie. L'effet
etait reversible. Le fait d'augmenter la duree du traitement jusqu'a 330 min a entraine la
perte des microtubules et provoque l'arrondissement des cellules. Cet etat n'etait pas reversible.
Nos resultats indiquent que les microfilaments sont plus sensibles aux pressions que ne le
sont les microtubules, que les deux organites deviennent de plus en plus labiles au fur et a
mesure qu'augmente la pression et que, finalement, les microtubules des embryons les plus
ages resistent mieux aux pressions. Enfin, on a montre une correlation entre la presence des
microfilaments et l'etat de constriction des apex cellulaires de meme qu'une correlation entre
la presence des microtubules et la forme allongee des cellules.
This work was supported by the Medical Research Council of Canada. We are grateful
to Mme Genevieve Anglade and to Mile Lucie Heroux for their technical assistance and to
Mrs Barbara Lafreniere for her help with the English presentation of this text.
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{Received 13 September 1977, revised 24 October 1977)