J. Embryol exp. Morph. 88, 333-348 (1985)
333
Printed in Great Britain © The Company of Biologists Limited 1985
The role of microfilaments in cranial neurulation in
rat embryos: effects of short-term exposure to
cytochalasin D
GILLIAN MORRISS-KAY AND FIONA TUCKETT
Department of Human Anatomy, South Parks Road, Oxford OX1 3QX, U.K.
SUMMARY
During the late stages of cranial neurulation in mammalian embryos, the neural epithelium
becomes concave. A thick subapical band of microfilament bundles, attached to junctions which
are both vertical and horizontal in orientation, can be seen by TEM. Prior to this the neural
epithelium is first biconvex and then V-shaped in transverse section, microfilament bundles are
absent, and the subapical junctions are only vertical in orientation.
In order to determine the role of microfilaments in cranial neurulation, rat embryos were
exposed to cytochalasin D (0-15 jUgml"1) for l h at three stages of development: convex neural
fold stage, early concave (prior to midline apposition at the forebrain/midbrain junction:
'preapposition') and later concave ('postapposition'). They were subsequently washed and
cultured in addition-free medium for 5,12, 24 or 36h, then examined alive and by LM, TEM, or
SEM.
The degree of neural fold collapse varied with the stage of development: at the convex stage
there was only slight opening out of the neural groove; early concave (preapposition) neural
folds collapsed laterally to a horizontal position; later concave (postapposition) neural folds
showed widening of the midbrain/hindbrain neuropore and slight neuroepithelial eversion at
the anterior neuropore. Neural epithelium which had been concave prior to cytochalasin D
treatment changed in structure so that the cells were broader and shorter; most of the subapical
junctions were vertical in orientation, and microfilament bundles were represented either as a
mass of amorphous material adjacent to the junctions, or as separated and broken filaments.
Re-elevation of neural folds in 'recovery' cultures was accompanied by regeneration of apical
microfilament bundles and horizontal junctions. Embryos which had been exposed to
cytochalasin D at the convex or later concave stage of cranial neural fold development were able
to complete cranial neural tube closure; none of the early-concave-stage embryos achieved
apposition at the forebrain/midbrain junction, and all had major cranial neural tube defects.
The results suggest that contraction .of apical microfilament bundles plays an essential role in
elevation of the neural folds and in the generation of concave curvature during the later stages of
cranial neurulation. During the convex neural fold stage, microfilaments are important in
maintaining neuroepithelial apposition in the neural groove, but are not crucial to maintenance
of the convex shape.
Successful formation and maintenance of the forebrain/midbrain apposition point at the
appropriate time is considered to be essential for subsequent brain tube closure.
INTRODUCTION
The presence of cytoplasmic microfilaments has been correlated with a wide
variety of morphogenetic movements, suggesting that these are the contractile
Key words: microfilaments, neurulation, cytochalasin D, rat embryo, cranial development.
334
G. MORRISS-KAY AND F. TUCKETT
elements which generate cell movement and epithelial curvature (Wessels et al.
1971; Spooner, 1974). The importance of microfilament contraction in neurulation
in amphibian, avian, and mammalian embryos is now widely accepted (Baker &
Schroeder, 1967; Burnside, 1971; Freeman, 1972; M. Jacobson, 1978), but there is
some disagreement as to whether they are actually responsible for generating
curvature of the neural epithelium, or whether they simply bring about the
observed shrinkage of the neural plate's surface area which, in amphibian embryos
at least, precedes the development of the neural folds (A. G. Jacobson, 1978).
The role and timing of microfilament-mediated contraction during cranial
neurulation in mammalian embryos is not obvious. The cranial neural plate first
forms convex neural folds which subsequently flatten and then become concave
prior to neural tube closure (illustrated in accounts of human, pig, and rat
development: Hamilton & Mossman, 1972; Patten, 1948; Morriss & Solursh,
1978). In the rat, and probably in other mammalian species also, these changes in
neural fold shape are accompanied by changes in cellular shape and organization
within the neural epithelium (Morriss-Kay, 1981). Dense apical microfilament
bundles have been reported only at the concave stage (Morriss & New, 1979). At
the 10-somite (early concave) stage in rodent and human (Hamilton & Mossman,
1972) embryos, a small point of neural fold apposition occurs at the
forebrain-midbrain junction, dividing the anterior neuropore into two parts.
Cytochalasin B has been used in a variety of developing systems, where it has
been found reversibly to inhibit microfilament structure and function (Wessels et
al. 1971; Spooner, 1978). In a preliminary study on cultured rat embryos, the effect
of cytochalasin B on the late stages of cranial neurulation suggested that these
stages are microfilament-dependent (Morriss-Kay, 1981). In the present study we
have used cytochalasin D which is more potent than cytochalasin B (Carter, 1967),
having a higher affinity for the contractile-related binding site (Tannenbaum,
1978), and has an interpretative advantage in that it has little or no effect on
hexose transport (Miranda, Godman, Deitch & Tanenbaum, 1974a; Tannenbaum,
Tanenbaum & Godman, 1977). The aims were as follows: (a) to compare
cytochalasin-D-induced effects with the normal shape and ultrastructure of the
neural epithelium at three stages of neurulation (convex, early concave and late
concave), in order to determine the relative importance of microfilament
contraction at these stages; (b) to monitor recovery of neural fold shape and to
discover whether neural tube closure could occur following short-term exposure to
cytochalasin D at the same three stages; (c) to correlate changes in shape with
ultrastructural changes in the microfilament-rich apical border of the concavestage neural epithelium.
MATERIALS AND METHODS
Wistar-strain rat embryos were explanted in Tyrode's saline at 9-11 p.m. on day 9 of gestation
(2- to 5-somite stage) or 9-11 a.m. on day 10 (9- to 11-somite stage) and Reichert's membrane
was opened (day of positive vaginal smear = day 0). Day-10 embryos were examined under the
dissecting microscope and divided into two groups: 'preapposition-stage' embryos (9-somite to
Cranial neurulation and cytochalasin D
335
early 10-somite stages, in which neural fold apposition at the forebrain-midbrain junction had
not yet occurred, and 'postapposition-stage' embryos (11 somites) in which the open region of
the cranial neural tube was divided into a midbrain/upper hindbrain spindle-shaped opening
and the forebrain anterior neuropore (Morriss & New, 1979, fig. 3). 10-somite-stage embryos
which had just initiated apposition were used as controls.
The embryos were cultured at 38°C in 60ml cylindrical bottles rotating at 30r.p.m. The
culture medium consisted of 2-5 ml immediately centrifuged, heat-inactivated rat serum (Steele
& New, 1974) and 2-5 ml Tyrode's saline containing SOjUgml"1 streptomycin and penicillin and
either cytochalasin D or DMSO. The gas phase was 5 % O2/5 % CO2/90 % N2 (New, Coppola
& Cockroft, 1976a,b). After l h , some embryos were removed, washed in Tyrode's saline and
fixed for Hh in 2-5% cacodylate-buffered glutaraldehyde (0-1 M, pH7-2). They were then
transferred to buffer and viewed with the dissecting microscope. Yolk sac and amnion were
removed from all day-10 embryos and from day-9 embryos which were to be prepared for
scanning electron microscopy (SEM). Neural fold shape and somite number were assessed
before further processing for SEM or for light microscopy (LM) and transmission electron
microscopy (TEM).
The remaining embryos were transferred to fresh medium prepared as described above but
without the addition of cytochalasin D or DMSO, regassed with the same gas mixture, and
continued in culture. Each culture bottle contained a maximum of eleven embryos for the first
hour, and a maximum of six embryos thereafter. Eighteen day-9 embryos and thirteen day-10
embryos (eight preapposition and five postapposition) were removed and fixed after a further
12 h, and eleven day-10 embryos (six preapposition and five postapposition) were removed and
fixed after 5 h. All remaining embryos were cultured until the morning of day 11 (36 or 24 h in the
addition-free medium), being regassed on late day 10 with 5 % CO2 in air (New et al. 1976a,6).
They were then washed, fixed, and transferred to buffer, and the membranes removed. Somite
number, general morphology, and neural tube morphology was recorded for all embryos; some
were then prepared for SEM or LM and TEM.
Cytochalasin D
Cytochalasin D (Aldrich Chemical Co.) was prepared as a 10% aqueous stock solution of
lmg cytochalasin D in lml dimethylsulphoxide (DMSO) (Sigma) and stored frozen. 10%
aqueous DMSO was used for controls. In a series of preliminary cultures, 2-5 to 25 jul of the
cytochalasin D solution was added to 5 ml serum to produce a range of concentrations of 0-05 to
0-5 jug ml" 1 , in each of which four orfiveday-10 embryos were cultured for 1-3 h. Cranial neural
fold shape, which was partly or wholly concave in profile at the start, was examined in the living
state and by SEM, LM, and TEM, and compared with that of controls. Some embryos were
transferred to addition-free medium after exposure to cytochalasin D or DMSO, cultured for a
further 24h, and examined as at 1-3 h. On the basis of these experiments, a cytochalasin D
concentration of O-lSjUgmP1 (3X10~ 6 M; 7-5(A stock solution) and an exposure period of l h
were chosen for all subsequent experiments, being the minimum concentration and time which
produced complete collapse of the neural folds. 7-5 [A of aqueous DMSO was added to 5 ml
medium for control embryos.
Exposure of day-10 embryos to 0-15 /Ugml"1 cytochalasin D had similar morphological effects
to that of O-SjUgmP1 cytochalasin B as used previously (Morriss-Kay, 1981), confirming the
greater potency of cytochalasin D.
Scanning electron microscopy
All embryos not used for LM and TEM were prepared for SEM. They were dehydrated in
graded acetones, critical-point dried, mounted on aluminium stubs with double-sided Sellotape,
coated with gold in a sputter coater, and viewed in a JEOL JSM-T20 scanning electron
microscope.
Light microscopy (LM) and transmission electron microscopy (TEM)
Embryos were photographed whole for reference during subsequent sectioning. They were
then postfixed in cacodylate-buffered osmium tetroxide, washed, dehydrated, and embedded
336
G. MORRISS-KAY AND F. TUCKETT
individually in Spurr resin at an orientation appropriate for cutting transverse sections, l/zm
sections were mounted on glass slides and stained with 0-5 % methylene blue/0-5 % azure II in
1 % borax for light microscopy. Adjacent sections were cut ultrathin for TEM, and stained with
uranyl acetate and lead citrate. Two to four embryos of each of the initial neural fold stages and
from each culture period were prepared for LM and TEM.
RESULTS
The development and appearance with LM, TEM and SEM of control-cultured
embryos used in this study was not detectably different from that of embryos
cultured in addition-free medium in other studies in this laboratory, or from
embryos freshly explanted at equivalent stages. We therefore conclude that the
DMSO added to the culture medium of both control and cytochalasin-treated
embryos had no detectable effect on development.
In the following description, not all of the features described for control
embryos are illustrated. Illustrations of many of the features referred to here may
be found in Morriss & Solursh (1978), Morriss & New (1979) and Morriss-Kay
(1981).
Day-9 embryos
At the start of culture, embryos were at the 2- to 5-somite stage, with convex
cranial neural folds. LM of preculture embryos, and control embryos after 1 hour's
culture, showed a 50/mi-deep neural groove consisting of approximately five
supranotochordal cells and close apical surface apposition of the adjacent ten cells
or so on each side (Fig. 1A).
After 1 hour's culture in medium containing cytochalasin D the shape appeared
unchanged by dissecting microscope (live embryos) and SEM (Fig. 2A,B)
observations. LM revealed that the neural groove had opened out to form a Vshape in the region which was closely apposed in the midline in control embryos
Fig. 1. Transverse sections of day-9 embryos cultured for l h in medium containing
(A) DMSO and (B) cytochalasin D. The control embryo shows apposed
neuroepithelial cells in the midline; this apposition is not present in the treated
embryo. Bar = 0-1 mm.
Cranial neurulation and cytochalasin D
337
(Fig. IB). Otherwise, the LM appearance of neuroepithelial cell shape and
organization was similar to that of control embryos, having a columnar or slightly
pseudostratified form. TEM (not illustrated) showed that the cells were
approximately the same breadth from base to apex, lacking the narrow necks and
apical surface bulges seen at later stages. At their apical border they were joined
by short desmosome-like junctions. Filamentous material was attached to these
junctions, extending for only a few nm in the plane of section, in both control and
cytochalasin D-treated embryos.
Embryos examined after further culture
After a further 12 h culture in addition-free medium (Fig. 2C,D), both control
and cytochalasin D-treated embryos had gained six to seven somite pairs, so that
they ranged in somite number from 8 to 11. All of the treated embryos showed
neural tube/fold development (form of cranial neural folds, spinal neural tube,
and posterior neuropore) appropriate to the previous somite stage, e.g. embryos
with nine pairs of somites had neural tube/fold development resembling that of 8somite-stage controls. Only one treated embryo (with eleven pairs of somites)
showed any concavity of the cranial neural epithelium; this epithelium had the
ultrastructure as well as the shape of that of a 10-somite control embryo, with
apical microfilament bundles and junctions oriented parallel to the apical surface
but not forming the near-continuous line normally seen at the 11-somite stage
onwards. Heart rate, yolk-sac blood island development, and yolk-sac and amnion
expansion were similar in control and treated embryos; 'turning' was slightly
retarded relative to somite number in the treated embryos.
Of the eighteen cytochalasin D-treated embryos cultured from day 9 to day 11
(Table 1), twelve formed neural tubes which were completely closed except for the
small posterior neuropore. These differed from control embryos in being slightly
smaller, and in having a less well developed cranial flexure (Fig. 2E,F). LM and
TEM (not illustrated) showed a normally organized neural epithelium but with
Table 1. Embryos examined on day 11 of development
Stage of exposure
to cytochalasin D
Late day 9
(3-5 somites)
Day 10 preapposition
(9-10 somites)
Day 10 postapposition
(11 somites)
Controls:
Day 9 (3-5 somites)
Controls:
Day 10 (10 somites)
n
closed
neural
tube
cranial
NTD
only
cranial
+ spinal
NTD
spinal
NTD
only
open
otic
pits
18
12
3
3
0
0
14
0
14
0
0
14
15
15
0
0
0
15
16
15
0
0
1
0
0
0
0
0
•
10
10
338
G. MORRISS-KAY AND F. TUCKETT
Fig. 2. Scanning electron micrographs of late day-9 embryos cultured in medium
containing DMSO (A,C,E) or cytochalasin D (B,D,F,G) for l h (A,B), followed by
12 h (C,D) or 36 h (E,F,G) in addition-free medium. xlOO.
many pyknotic cells and less well-expanded brain vesicles and neural canal than
those of controls.
The six embryos with open neural tube defects (Fig. 2G) were all unturned; the
embryonic axis formed a tight U-shape, with the amnion closely applied to the
surface of the neural folds. There was very little amnioticfluidpresent, no yolk-sac
circulation, and the heartbeat was weak. However, it was striking that even in
these embryos the otic pit had sunk beneath the surface to form a closed otocyst.
(Four of these embryos were from one culture bottle which contained no other
embryos, and two were from a bottle which also contained four embryos whose
brain tubes closed.) Since twelve of these embryos succeeded in forming closed
brain tubes, we conclude that failure to do so was not due to a primary effect on
neurulation, and this result will not be discussed further. One control embryo was
also unturned, and compressed into a tight U-shape within a fluid-deficient
amniotic cavity. There were neural tube defects in the midbrain and spinal regions.
Cranial neurulation and cytochalasin D
339
This embryo was normal by all visible criteria when the medium was changed 17 h
after the start of culture in DMSO-free medium, and is therefore assumed to have
been damaged by handling at this stage.
Monitoring during culture showed that all cytochalasin-D-treated embryos
developed a yolk-sac circulation later than controls; six of the twelve embryos with
closed neural tubes had not achieved this at termination.
Day-10 embryos
During the 9- to 11-somite stages, the midbrain/hindbrain cranial neural
epithelium of normal rat embryos begins to develop a concave surface, apposes
and fuses at the forebrain-midbrain junction to form the spindle-shaped
midbrain-upper hindbrain neuropore, which then narrows as the lateral edges
move towards each other (illustrated in Morriss & New, 1979). At the same time,
the two sides of the forebrain (anterior neuropore) move together and become
apposed.
TEM of control embryos of these stages showed that where the neural
epithelium has a flat or only slightly concave apical surface, the cells have narrow
necks with apical bulges, with the subapical junctions predominantly parallel with
the long axis of the cells (i.e. vertical). Microfilaments could be seen attached to
the subapical junctions but only rarely formed a continuous bundle stretching
between two junctions within the plane of section (Figs 3A, 4A). Where the
epithelial surface was more concave, many of the subapical junctions were
orientated parallel to the surface (i.e. horizontal); together with the microfilament
bundles they formed an almost unbroken subapical line, with rounded areas of cell
above it. This pattern was also characteristic of the concave neural epithelium of
later stage embryos (Figs 3E, 4F).
After a l h exposure to cytochalasin D, embryos which had not formed the
forebrain-midbrain apposition point at the start of culture (late 9- or early 10somite stage, Fig. 5A) showed a convex midbrain-hindbrain neural epithelium,
i.e. neural folds which had begun to form concave surfaces had flopped laterally
(Fig. 5B). The lateral forebrain neural epithelium was also slightly everted, but the
deep optic sulci were maintained. LM and TEM showed the neuroepithelial cells
to have much broader surfaces and neck regions than those of equivalent regions
in control embryos (Fig. 4B). Microfilament-like material could be seen attached
to the subapical junctions or free within the cytoplasm; this was either fuzzy and
indefinite in structure (Fig. 4B) or in the form of broken filaments (Fig. 4C).
Embryos which had formed the forebrain-midbrain apposition point at the start
of culture (11-somite-stage embryos) showed a widely gaping midbrain-hindbrain
neuropore, but retained a concavely curved neuroepithelial surface in this region.
The TEM appearance of the apical region was similar to that of unapposed
embryos, with few intact microfilament bundles, horizontal junctions or apical
surface bulges. Maintenance of the concave shape therefore appeared to be due to
the limitation on lateral movement of the neural folds imposed by the spindle-
340
G. MORRISS-KAY AND F. TUCKETT
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Cranial neurulation and cytochalasin D
341
shaped neuropore rather than to the structural organization of the epithelium
itself.
Embryos examined after further culture
After culture in addition-free medium, recovery of neural fold shape had
progressed well in both preapposition- and postapposition-stage embryos, but had
not yet achieved the degree of elevation seen at the start of culture (Fig. 5C,F).
TEM showed some narrowing of the neck region and apical surface of the
neuroepithelial cells, some reappearance of apical microfilament bundles, and
slight bulging of the apical cell surfaces. However, no horizontal junctions were
seen, and the few microfilament bundles observed were less clearly organized and
closer to the apical surface than those of control embryos (Fig. 4D).
After 12 h culture in addition-free medium, control embryos (Fig. 5G) were all
normal and had gained five or six pairs of somites and a yolk-sac circulation.
Cytochalasin-D-treated embryos had gained five to seven somite pairs except for
one 11-somite-stage embryo which increased to nineteen somite pairs. They had a
slightly weaker heartbeat than the controls, and blood islands but no circulation in
the yolk sac. Of the five embryos which were at the postapposition stage at the
start of culture, two had completed cranial neurulation, and the other three had
small inverted-teardrop-shaped midbrain-upper hindbrain openings (Fig. 51,J),
one with a small anterior neuropore also.
None of the eight embryos which were at the preapposition stage at the start of
culture had achieved forebrain-midbrain apposition, even though the widely
collapsed cytochalasin-D-affected neural folds had regained their concave
curvature and in three embryos (with sixteen to seventeen somite pairs) the lateral
edges were very close to each other in the midline (Fig. 5H).
The ultrastructure of the apical region of the cranial neural epithelium which
had regained its concave curvature was similar in all embryos examined, whether
pre- or postapposition at the time of cytochalasin exposure. Microfilament bundles
and horizontal junctions were present, but did not form such a clear subapical line
as that seen in late-preclosure-stage control embryos, though apical surface bulges
were well developed (Figs 3D,E and 4E,F).
After 24 h in addition-free medium (Fig. 6A-C and Table 1) the cranial neural
tube of all embryos of the postapposition group was closed. However, like
cytochalasin-treated day-9 embryos cultured to day 11, the cranial flexure was less
acute than in controls, the brain vesicles less well expanded, and there was much
pyknosis in the neural epithelium and elsewhere. All had turned.
Fig. 3. Low-power transmission electron micrographs showing the apical neuroepithelial surface of day-10 embryos. (A) control embryo, late 10-somite-stage; (B)
preapposition-stage embryo after 1 h exposure to cytochalasin D; (C) embryo exposed
to cytochalasin D at the postapposition stage, after 5h culture in addition-free
medium; (D) 16-somite embryo exposed to cytochalasin D at the preapposition stage,
after 12 h culture in addition-free medium; (E) 13-somite-stage control embryo cocultured with (D). Arrows: microfilament bundles. Bar = 5jum.
342
G. MORRISS-KAY AND F. TUCKETT
Cranial neurulation and cytochalasin D
343
None of the 'preapposition' embryos had succeeded in forming the apposition
point, and the cranial neural tube was wide open from the forebrain to the
metencephalon, i.e. the whole area which had been open at the start of culture.
Five had not completed turning.
The otic pit was wide open in all embryos exposed to cytochalasin D on day 10,
whereas in all of the controls a closed otocyst had formed. Treated embryos were
smaller than controls, the heart rate was slower, and the yolk-sac circulation was
poor or absent. Spinal neural tube closure progressed normally except for the
slight retardation described above.
DISCUSSION
During the 24 h period from late day 9 of gestation, rat embryos undergo a
complex integrated sequence of morphogenetic changes. All of these events
involve cell movement, exocytosis, or endocytosis, and may therefore be assumed
to be dependent to some degree on microfilaments. The possibility of an adverse
effect on some or all of them, and secondary effects through them, must be born in
mind when interpreting the results presented here. Cytochalasin D must have
affected all microfilament-dependent functions, even though its effect on the thick
subapical line of microfilament bundles in the curving neural epithelium is the only
one which was immediately obvious after a l h exposure period. For instance,
during the longer recovery periods, cytochalasin-D-treated embryos were
observed to develop a yolk-sac circulation more slowly than controls, and to have a
slower heart beat. Consequently a less efficient supply of oxygen and nutrients
may have contributed to the pyknosis seen in the neural epithelium with LM and
TEM in these embryos, and to their smaller size.
Yolk-sac-mediated nutrition involves phagocytosis, and embryos of the stages
used here are dependent on this process for their survival, growth, and normal
development (Beck & Lloyd, 1966). Mimura & Asano (1976) observed a 30%
inhibition of phagocytosis in peritoneal macrophages in medium containing
0 - 5 ^ ml" 1 cytochalasin D but no inhibition at 0-1 ^g ml" 1 . Although the
concentration of 0-15/ig ml" 1 used in the present study is low in relation to the
macrophage study, the slightly smaller size of the treated embryos when compared
with controls at day 11 suggests that there may have been some effect on nutrient
Fig. 4. Transmission electron micrographs: subapical microfilament junction region of
the neural epithelium. (A) High-power view of microfilament bundles and junctions
from Fig. 3 (A). (B,C) From embryos exposed to cytochalasin D for lh: (B) fuzzy
cytoplasmic material adjacent to subapical junctions; (C) microfilament bundle-like
structure composed of broken filaments. (D) Microfilament bundle-like structure close
to the apical surface in a cytochalasin D-treated embryo after 5h in addition-free
medium. (E) and (F) High-power views from Fig. 3 (D and E), showing the
microfilament bundle/subapical junction organization characteristic of the concave
late preclosure stage neural epithelium in: (E) an unclosed 16-somite embryo exposed
to cytochalasin D at the preapposition stage, and (F) a 13-somite stage co-cultured
control embryo just completing cranial neural tube closure. Bar =
344
G. MORRISS-KAY AND F. TUCKETT
B
•«**••
Fig. 5. Scanning electron micrographs of day-10 embryos. (A) to (C) Preappositionstage embryos: (A) as at start of culture (early 10-somite-stage); (B) after 1 h exposure
to cytochalasin D; (C) cytochalasin D-treated embryo after 5 h culture in addition-free
medium. (D) to (F) Postapposition-stage embryos at equivalent stages. (G) to (J)
Control (G), 'preapposition' (H), and 'postapposition' (I) and (J) embryos after In
exposure to DMSO or cytochalasin D followed by 12 h in addition-free medium, x 100.
uptake. But while an effect on phagocytosis may have had some effect on growth,
it could not have been responsible for the initial morphogenetic alterations
Cranial neurulation and cytochalasin D
345
brought about by cytochalasin D treatment. Transfer of the breakdown products
of nutrients phagocytosed by yolk-sac endoderm cells at this stage is a relatively
long-term process (Beck et al. 1967), whereas we observed neural fold collapse
within one hour.
The effects of cytochalasin D on day-10 embryos showed a clear correlation
between loss of neuroepithelial shape and loss of microfilament bundles. During
subsequent culture in addition-free medium, the pretreatment cranial neural fold
shape was regained in more than 5h but less than 12 h; ultrastructurally this was
correlated with regeneration of subapical microfilament bundles, suggesting a
causal relationship between the two events.
Studies on the mechanism of action of cytochalasins suggest that they have up to
four effects on microfilament structure, function and organization, all of which are
reversible. Different effects have been observed using different cell types and
different experimental protocols. MacLean-Fletcher & Pollard (1980) observed an
effect on filament-filament interactions, whereas Schliwa (1982) found that sideto-end junctions of actin filaments were intact after cytochalasin treatment.
Cytochalasins can also prevent filament elongation, binding to the fast assembly
end of the microfilament and thereby preventing further polymerization except
from the slow assembly end (Flanagan & Lin, 1980; Lin, Tobin, Grumet & Lin,
1980; Brown & Spudich, 1981). They can also induce rapid depolymerization of
filamentous actin (Casella, Flanagan & Lin, 1981, using platelets in medium
containing O-SjUgml"1 cytochalasin D), although this effect was not observed in
various established cell lines such as HEp-2 and HeLa (Morris & Tannenbaum,
1980; Miranda et al. 1974a; Miranda, Godman & Tanenbaum, 1974b). Disruption
of filaments with the release of filament fragments was the chief effect observed by
Schliwa (1982).
.'*
J
r
Fig. 6. Scanning electron micrographs of embryos cultured for 24 h after exposure to
DMSO or cytochalasin D for l h on day 10. (A) Control, dorsal view: closed neural
tube, closed otocysts; (B) 'postapposition' embryo, dorsal view: closed neural tube,
open otic pits; (C) 'preapposition' embryo, ventral view of cranial region and heart,
dorsal view of tail: open cranial neural tube defect. x65.
346
G. MORRISS-KAY AND F. TUCKETT
Our ultrastructural observations suggest that the effect of cytochalasin D on
neurulation in rat embryos is mediated through microfilament disruption,
involving both depolymerization and fragmentation, and that loss of cross linking
between parallel actin filaments within microfilament bundles may also occur.
Cytochalasin B was first used to study neurulation by Linville & Shepard (1972).
They exposed chick embryos in culture to 2-5, 5, and lO/igml" 1 throughout the
period of neurulation. At the two higher concentrations many of the embryos had
open neural tube defects, but at 2-5/igml" 1 only 15 of 22 viable embryos were
affected (though all were retarded in the timing of closure). These results are
surprising in view of our earlier observation of neural fold collapse in rat embryos
exposed to only 0-5/^g ml" 1 cytochalasin B (Morriss-Kay, 1981). The difference
may be due to methodology or to a species difference; ultrastructural observations
were not made.
Lofberg (1974) observed only retardation of neurulation and an elongated
neural plate in axolotl embryos treated with 1 /igml" 1 cytochalasin B, whereas 2-5
and SjUgml"1 resulted in disaggregation of the neuroepithelial cells and consequent neurulation failure. In this case the differences from our results are more
likely to involve a species difference, since even the disaggregating neural tissue
formed elevating neural folds, whereas in our rat embryos neural fold collapse
occurred at cytochalasin B or D concentrations much lower than those which
induced even minor signs of disaggregation. Our results also suggest that in rat
embryos cranial neurulation is more vulnerable than spinal neurulation to very low
concentrations of cytochalasins.
We saw no indication of any effect on premitotic nuclear migration or
cytokinesis, perhaps due to the short exposure time as much as to the low
concentration used. Webster & Langman (1978) observed these effects, together
with some disaggregation, in the neural epithelium of day-11 mouse embryos
exposed to 10/igml" 1 cytochalasin B for 2h in culture. This concentration is
twenty times that used in our cytochalasin B study.
Studies on the effects of cytochalasins on neurulation in mammalian embryos in
vivo show that neural tube defects can be brought about by ingestion of these drugs
(Shepard & Greenaway, 1977; Wiley, 1980; Austin, Wind & Brown, 1982).
However, the cellular effects associated with exencephaly and encephalocoele did
not involve microfilaments at the lower dose levels, suggesting a maternalmediated effect, to which a direct effect on embryonic microfilaments was added
at the higher dose levels (Wiley, 1980).
Our results suggest that although there may have been minor effects on
phagocytosis and (less likely) exocytosis, direct interference with the role of
microfilaments in epithelial morphogenesis was the major effect of cytochalasin D
on day-9 and day-10 embryos. The relative importance of the effects on the
mechanisms of neurulation, otocyst formation, cranial flexure, and turning
depended on the stage of development at the time of exposure.
Some conclusions may be drawn for neurulation. In late day-9 embryos,
microfilaments are mainly involved in maintaining close apposition of the apical
Cranial neurulation and cytochalasin D
neuroepithelial cell surfaces adjacent to the midline, and do not play a major role
in maintaining shape of the convex neural folds. In day-10 embryos, the subapical
line of microfllament bundles is essential for generation and maintenance of the Vshaped or concave form of the neural folds. Following cytochalasin-induced
collapse of the elevated neural folds, re-elevation can take place as the
microfllament bundles regenerate. But if the forebrain-midbrain midline fusion
(apposition) point has not been achieved prior to cytochalasin exposure, it will not
form after re-elevation even if the neural folds are closely apposed. This
developmental event, involving cell-cell adhesion, appears to be finely timed and
of crucial importance in brain tube formation.
We wish to thank Martin Barker, Beth Crutch and Janet Kilcoyne for technical and
photographic assistance, and the MRC forfinancialsupport.
REFERENCES
W. L., WIND, M. & BROWN, K. S. (1982). Differences in the toxicity and teratogenicity
of cytochalasin D and E in various mouse strains. Teratology 25, 11-18.
BAKER, P. C. & SCHROEDER, T. E. (1967). Cytoplasmicfilamentsand morphogenetic movement in
the amphibian neural tube. Devi Biol. 15, 432-450.
BECK, F. & LLOYD, J. B. (1966). A histochemical study of embryotrophic nutrition in the rat.
J. Anat. 100, 432-433.
BECK, F., LLOYD, J. B. & GRIFFITHS, A. (1967). A histochemical and biochemical study of some
aspects of placental function in the rat using maternal injection of horseradish peroxidase.
J. Anat. 101, 461-478.
BROWN, S. S. & SPUDICH, J. A. (1981). Mechanism of action of cytochalasin: evidence that it
binds to actin filament ends. J. Cell Biol. 88, 487-491.
BURNSIDE, B. (1971). Microtubules and microfilaments in amphibian neurulation. Amer. Zool.
13, 989-1006.
CARTER, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature 210, 261-264.
CASELLA, J. F., FLANAGAN, M. D. & LIN, S. (1981). Cytochalasin D inhibits actin polymerization
and induces depolymerization of actin filaments formed during platelet shape change. Nature
293, 302-305.
FLANAGAN, M. D. & LIN, S. (1980). Cytochalasins block actinfilamentelongation by binding to
high affinity sites associated with F-actin. /. biol. Chem. 255, 835-938.
FREEMAN, B. G. (1972). Surface modifications of neural epithelial cells during formation of the
neural tube in the rat embryo. /. Embryol. exp. Morph. 28, 438-448.
HAMILTON, W. J. & MOSSMAN, H. W. (1972). Hamilton, Boyd & Mossman's Human
Embryology, 4th ed. Williams & Wilkins/MacMillan, London.
JACOBSON, A. G. (1978). Some forces that shape the nervous system. Zoon 6, 13-21.
JACOBSON, M. (1978). DevelopmentalNeurobiology, 2nd ed. New York & London: Plenum Press.
LIN, D. C , TOBIN, K. D., GRUMET, M. & LIN, S. (1980). Cytochalasins inhibit nuclei-induced
actin polymerization by blocking filament elongation. /. Cell Biol. 84, 455-460.
LINVILLE, G. P. & SHEPARD, T. H. (1972). Neural tube closure defects caused by cytochalasin B.
Nature New Biology. 236, 246-247.
LOFBERG, J. (1974). Effects of cytochalasin B on cell adhesion, surface topography, and
microfilaments in neuroectodermal cells of the amphibian neurula. Abstr. Upps. Diss. Fac. Sci.
319.
AUSTIN,
MACLEAN-FLETCHER, S. &POLLARD,T. D. (1980). Mechanism of action of cytochalasin Bon actin.
Cell 20, 329-341.
N. & ASANO, A. (1976). Synergistic effect of colchicine and cytochalasin D on
phagocytosis by peritoneal macrophages. Nature 261, 319-321.
MIRANDA, A. F., GODMAN, G. C , DEITCH, A. D. & TANENBAUM, S. W. (1974a). Action of
cytochalasin D on cells of established lines. I. Early events. /. Cell Biol. 61, 481-500.
MIMURA,
348
G. MORRISS-KAY AND F . TUCKETT
A. F., GODMAN,.G. C. & TANENBAUM, S. W. (19746). Action of cytochalasin D on
cells of established lines. II. Cortex and microfilaments. /. Cell Biol. 62, 406-423.
MORRIS, A. & TANNENBAUM, J. (1980). Cytochalasin D does not produce net depolymerization of
actin filaments in HEp-2 cells. Nature 287, 637.
MORRISS, G. M. & NEW, D. A. T. (1979). Effect of oxygen concentration on morphogenesis of
cranial neural folds and neural crest in cultured rat embryos. J. Embryol. exp. Morph. 54,
17-35.
MORRISS, G. M. & SOLURSH, M. (1978). The role of primary mesenchyme in normal and abnormal
morphogenesis of mammalian neural folds. Zoon 6, 33-38.
MORRISS-KAY, G. M. (1981). Growth and development of pattern in the cranial neural epithelium
of rat embryos during neurulation. J. Embryol. exp. Morph. 65 (Supplement), 225-241.
NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. L. (1976a). Improved development of head-fold
rat embryos in culture resulting from low oxygen and modifications of the culture serum.
J. Reprod. Fert. 48, 219-222.
NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. L. (19766). Comparison of growth in vitro and in
vivo of post-implantation rat embryos. /. Embryol. exp. Morph. 36, 133-144.
PATTEN, B. M. (1948). Embryology of the Pig. Philadelphia: Blakiston Co.
SCHLIWA, M. (1982). Action of cytochalasin D on cytoskeletal networks./. Cell Biol. 92, 79-91.
SHEPARD, T. H. & GREENAWAY, J. C. (1977). Teratogenicity of cytochalasin D in the mouse.
Teratology 16, 131-136.
SPOONER, B. S. (1974). Morphogenesis of vertebrate organs. In Concepts of Development (ed. J.
Lash & J. R. Whittaker), pp. 213-240. Stamford: Sinauer Assoc.
SPOONER, B. S. (1978). Cytochalasins as probes in selected morphogenetic processes. In
Cytochalasins: Biochemical and Cell Biological Aspects, (ed. S. W. Tanenbaum). Amsterdam:
Elsevier/North Holland Biomedical Press.
STEELE, C. E. & NEW, D. A. T. (1974). Serum variants causing the formation of double hearts
and other abnormalities in explanted rat embryos. /. Embryol. exp. Morph. 31, 707-719.
TANNENBAUM, J. (1978). Approaches to the molecular biology of cytochalasin action. In
Cytochalasins: Biochemical and Cell Biological Aspects, (ed. S. W. Tanenbaum). Amsterdam:
Elsevier/North Holland Biomedical Press.
TANNENBAUM, J., TANENBAUM, S. W. & GODMAN, G. C. (1977). The binding sites of cytochalasin
D. II. Their relationship to hexose transport and to cytochalasin B. /. Cell Physiol. 91,
239-248.
WEBSTER, W. & LANGMAN, J. (1978). The effect of cytochalasin B on the neuroepithelial cells of
the mouse embryo. Am. J. Anat. 152, 209-222.
MIRANDA,
WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR, E. L.,
WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmental
processes. Science 171, 135-143.
M. J. (1980). The effects of cytochalasins on the ultrastructure of neurulating hamster
embryos in vivo. Teratology 22, 59-69.
WILEY,
(Accepted 1 April 1985)