AMER. ZOOL., 13:989-1006 (1973).
Microtubules and Microfilaments in Amphibian Neurulation
BETH BURNSIDE
Department of Anatomy, University of Pennsylvania, Philadelphia, Pennsylvania
19104
SYNOPSIS: In the salamander embryo, the morphogenetic movements of neurulation
are correlated with two cell shape changes in the neural epithelium: elongation and
apical constriction of the columnar neural plate cells. Cells first elongate to form the
flat open neural plate and then constrict apically as the plate rolls up to form the
neural tube. Evidence is presented that these cell shape changes are intrinsic to the
cells themselves and that they play a causal role in the morphogenetic movements.
Neural plate cells contain numerous microtubules oriented parallel to the axis of
elongation. These microtubules are critical to the elongation process. Possible mechanisms for microtubule function in cell elongation are considered. During apical
constriction the cells contain bundles of microfilaments which encircle the cell apex
in purse-string fashion. Evidence is presented which suggests that microfilament
bundles play an active role in apical constriction, and that this localized contraction
is produced by filament sliding.
Early morphogenesis is primarily accomplished by movements of embryonic epithelia: spreading, thickening, or folding
of coherent cell sheets. Though many of
these processes have been described and
mapped since the early phases of experimental embryology, the actual mechanisms
of epithelial movements are only beginning to be understood. In the discussion to
follow, I will use neurulation in the salamander embryo as a model system for an
examination of the mechanisms of epithelial morphogenesis. Neurulation consists of the morphogenetic movements
which generate the neural plate and the
neural tube. These two processes involve
placode formation and invagination respectively (thickening and folding) of simple
columnar epithelia, and are representative
of many morphogenetic movements that
shape organ primordia in all embryos.
The data to follow indicate that the morphogenetic movements of neural plate and
neural tube formation can be directly corThis work was supported in part by U.S. Public
Health
Service Research Grants HD00725,
HD00143, 6M0046, and HD06326, and in part by
Pre- and Post-doctoral fellowships GM30828.
Gratitude is extended to Doctors E. D. Hay, K.
R. Porter, A. G. Jacobson, J. P. Revel, J. R. McIntosh, and J. G. Hollyfield for their helpful support and stimulating discussions of this work, and
to John Woolsey for his artistic assistance.
989
related with changes in shape of the constituent cells of the neural epithelium.
Furthermore, since changes in cell shape
are intrinsic to the cells themselves, it
seems appropriate to postulate that they
play a causal role in the observed morphogenetic movements. Finally, these active
cell shape changes, elongation in neural
plate formation and apical constriction in
neural tube formation, appear to be dependent on the activities of intracellular
cytoskeletal and contractile elements, cytoplasmic microtubules and microfilaments.
I will conclude by examining some of the
current hypotheses for explaining how microtubules and microfilaments contribute
to cell elongation and localized contraction
respectively, and by considering their applicability to salamander neurulation.
NEURAL PLATE MORPHOGENESIS AND
DIFFERENTIAL CELL ELONGATION
Mapping and histological studies with
Taricha torosa embryos have revealed a
direct correlation between the morphogenetic movements of neural plate formation and differential cell elongation in the
neural epithelium (Burnside and Jacobson,
1968).
The T. torosa embryo exhibits several
attributes which facilitate a mapping
990
BETH BURNSIDE
Stage 15
Stage 13
B
Pathways of
Cell Displacement
FIG. 1. Maps of the morphogenetic movements of
neural plate formation. The movements of spots of
pigment were followed in time lapse films starting from the intersections of superimposed coordinate grid at stage 13, as seen in A. Tracings of
these pathways of movement between stages 13
and 15 are indicated in B. The endpoints of these
pathways were connected to generate the deformed
coordinate grid seen in C. Comparing coordinate
grids from stages 13 and 15 allows one to determine relative changes in surface area for different
regions of the neural plate. (Redrawn from Burnside and Jacobson, 1968.)
study of cell movements during neural
plate formation. A flat open neural plate
forms on the dorsal surface of the embryo
and can be time-lapse filmed in normal
orientation. The neural plate remains a
simple columnar epithelium throughout
neurulation and has a variegated pigmentation pattern with spots of pigment restricted to single cells or small groups of
cells. It is, therefore, possible to trace cell
movements during neural plate formation
by following spots of pigment on time-lapse
films of the dorsal surface of the embryo.
By choosing spots of pigment located at
the intersections of a superimposed coordinate grid to follow, one can simultaneously measure both cell displacement and local changes in surface area of different regions of the neural plate. (Fig. 1). Changes
in local surface area are illustrated by comparing the original coordinate grid to a
deformed coordinate grid which is generated by connecting the end points of cell
displacement pathways. For example, regions of the anterior plate decrease as much
as 70% in surface area while areas of the
central posterior regions increase only
slightly.
It is also possible to compare the cells
in a particular region of the coordinate
map before and after neural plate formation. This is accomplished by relating the
coordinate maps to serial sections of embryos at the two stages and comparing
equivalent regions. Thus, cell displacements during neural plate formation are
taken into account. At the end of gastrulation, all the columnar cells of the neural
epithelium are approximately the same
height. By the open neural plate stage,
however, there has occurred a differential
elongation of neural plate cells, the degree
of elongation in a particular region being
directly correlated with the reduction in
surface area of that region as observed on
the coordinate map (Fig. 2). If one measures cell diameters in histological sections
and used these diameters to calculate apical surface area changes of individual cells
in a specific region of the neural plate,
one finds that these cellular apical surface area changes correlate very closely
with surface area changes for that region
as measured from the coordinate maps.
Since cell volume remains relatively constant (see Fig. 2), it is clear that changes
991
NEURULA MICROTUBULES AND MICROFILAMENTS
Stage 13
Area of Free
Apical Surface
Area A Measured
From Map
Volume
Stage 15
245 p
-46%
14,200.p3
(-3.5%)
13,700 p3
FIG. 2. Schematic illustration of the correlation
between the decrease in area of a specific region
of the neural plate and the degree of elongation
of cells in that region. When cells from a particular region of the neural plate (indicated by
shaded area) are measured before and after elongation, one finds that the change in apical free
surface area of the individual cells corresponds to
the change in surface area in that region measured from the coordinate maps. Height and diameter were measured for 20 cells each from serial
sections of embryos at stage 13 and 15, then apical
surface area and volume were calculated by assuming the cells were cylindrical. A second region not illustrated was compared for the two
stages; change in apical free surface area was
—31% for individual cells as compared to —27%
for that region in the coordinate map. Since the
volume of the cells remains relatively constant in
both cases, change in surface area is clearly produced by a redistribution of the cell mass into a
longer cell. Thus, the degree of elongation is directly related to surface area changes.
in apical surface area of the cells result
from a redistribution of the mass of the
cell from a low to high columnar form.
Thus, the change in apical surface area of
the cell is directly related to the degree of
elongation of the cell.
Furthermore, the mapping studies demonstrate that the change in surface area
is directly related to the displacement of
cells observed during neural plate formation. Consequently, the patterns of cell
displacement in morphogenesis of the neural plate are directly related to localized
differential elongation of the constituent
cells of the neural epithelium.
It is not difficult to visualize how local-
992
BETH BURNSIDE
ized differential elongation could contribute to the observed cell displacement.
Since the cells are bound to one another
in a coherent cell sheet, a decrease in the
apical surface area of a given cell will
necessarily displace its neighbors. One
might expect that an appropriate gradient
of cell elongation (with its concomitant
shrinkage of the cell apex) might be invoked to explain the deformations of the
coordinate map observed during neural
plate formation. Such a pattern of gradients in cell elongation has in fact been
found by Jacobson and Gordon (unpublished). Using computer simulations of
shrinkage patterns combined with certain
restrictions to account for an interaction
between the neural plate at the midline
and the underlying notochord, these workers have been able to generate deformations of a coordinate map equivalent to
those observed in the embryo mapping
studies.
These observations do not, of course,
demonstrate that cell elongation causes the
morphogenetic process. This conclusion is
supported by two observations which indicate that the capacity to elongate is intrinsic to the cells themselves. First, isolated neural plates undergo normal morphogenetic movement when unattached to
a substrate, suggesting that the movements
do not result from locomotion across the
underlying tissues. Second, and more conclusive, is Holtfreter's observation (1947)
that single isolated cells from the salamander neural plate retain their columnar
shape and continue to elongate in culture.
Since the capacity to elongate is intrinsic
to the cells and does not represent a passive accommodation to external forces, and
since differential elongation is directly related to the observed cell displacement,
it seems logical to conclude that cell elongation plays a causal role in the morphogenetic movements of neural plate formation.
MECHANISMS OF ACTIVE CELL SHAPE CHANGES
DURING NEURULATION
During neurulation, cells of the sala-
mander ectoderm undergo three shape
changes (Fig. 3). At the end of gastrulation, all the ectodermal cells are indistinguishable from one another and form a
simple low columnar epithelium (stage 1213 according to Twitty and Bodenstein in
Rugh, 1962). During neurulation, cells of
the presumptive neural epithelium first (1)
elongate dramatically to form the placodelike neural plate (stage 13 to 15), and then
(2) become flask-shaped by apical constriction as the plate rolls up to form the neural tube (stage 15 to 20). Presumptive epidermal cells, on the other hand, (3) flatten throughout neurulation and eventually form a squamous epithelium. Two of
these processes, cell elongation and localized contraction (here apical constriction)
appear to be fundamental processes by
which cytomorphogenesis is accomplished
in a large number of systems examined
(see Burnside, 1971, for review).
Elongation and localized contraction
have been increasingly associated with activities of intracellular microtubules and
cytoplasmic filaments respectively. Microtubules are almost always observed oriented parallel to the long axis of asymmetric cells or cell processes, and in most cases
disruption of microtubules with various
inhibitors result in the concomitant disappearance of cell asymmetry. Associations of
50-70 A cytoplasmic filaments (termed microfilaments) with localized contraction is
derived from a variety of observations. Appearance of microfilaments in many cases
is temporally and spatially correlated with
localized contraction, and furthermore, microfilaments have been found oriented so
as to be able to cause observed contraction phenomena. In a number of cases microfilaments have been shown to be biochemically identical to muscle actin, and
in many more cases microfilaments have
been shown to bind heavy meromysin (a
fragment of muscle myosin) to form arrowhead complexes identical to those
formed with muscle actin. Thicker filaments (80-100 A in diameter) have also
been described in a number of cell types,
but their function is not well understood.
Since they do not appear to be temporally
NEURI.'LA MlCROTL'Bl'LES AND MlCROFILAMENTS
993
STAGE 19
FIC. 3. Schematic illustration of three cell shape
neural epithelial cells first elongate to form the
changes exhibited by salamander ectoderm cells
neural plate (stage 15) and then constrict apicalduring neurulatioii. At the end of gastrulation
ly as the plate rolls up to form the neural tube
(stage 12), the entire ectoderm is a uniform sim(stage 19). Epidermal cells flatten throughout neuple columnar epithelium. During neurulatioii, rulation. (From Burnside, 1971.)
or spatially correlated with cytoplasmic
contraction it has been suggested that they
play a structural role (see Burnside, 1971).
In the present study, electron microscopic examination of urodele ectodermal cells
during neurulation has revealed a consistent pattern of occurrence and orientation
of microtubules and cytoplasmic filaments.
Elongating neural plate cells contain numerous microtubules aligned parallel to
the axis of elongation (paraxial microtubules) and bundles of microfilaments
which encircle the cell apex like a purse
string (Fig. 4, 5, 6). Flattening presumptive epidermal cells on the other hand,
contain randomly arranged microtubules
and relatively few microfilaments in the
apical end of the cell. Instead, the cells
contain thicker 100 A filaments which resemble tonofilaments of differentiated uro-
994
BETH BURNSIDE
dele epidermal cells (Fig. 7, 8). Since it
seems unlikely that these 100 A filaments
play an active role in epidermal cell shape
determination they will not be considered
further in this discussion.
Microtubules and cell elongation
The elongation process in the neural
>!r.te is clearly dependent on the presence
of intact microtubules. Concentrations of
colchicine, vinblastine, and colcemid which
disrupt microtubules also block cell elongation in isolated neural plates (Fig. 9).
These observations, therefore, corroborate
:he conclusion implied by paraxial alignment that microtubules play an active role
in cell elongation. Similar dependence of
elongation on microtubules has been demonstrated for a number of types of elongating cells (see Burnside, 1971). Nevertheless the actual mechanism by which microtubules function in cell elongation is not
understood.
In considering possible mechanisms for
microtubule function in cell elongation,
it is helpful to examine three hypotheses
modified from theories of microtubule
function in spindle elongation and cytoplasmic transport. These hypotheses are
diagrammed in Figure 10 and may be
briefly described as follows: (1) force may
be generated by elongation of the microtubules themselves by addition of subunits
(similar to the dynamic equilibrium model
for spindle elongation of Inoue and Sato,
1967); (2) force against the apical and basal
ends of the cell may be generated by the
active sliding of an apical set of microtubules against a basal set to decrease overlap (similar to the sliding filament model
10p
FIG. 4. Schematic illustration of orientation of
microtubules and microfilaments in elongating
neural plate cells. Numerous microtubules are
aligned parallel to the cells' long axis and circumferential bundles of micronlaments encircle the
cell apex in purse-string fashion. These bundles
of micronlaments form specific attachments with
desmosomal filaments. It is not yet clear whether
microtubules extend the full length of the cell as
illustrated here, but this interpretation is consistent with reported observations. There is no significant difference between average numbers of
microtubules at different apico-basal levels of the
cells (see Fig. 11).
NEURULA MICROTUBULES AND MICROFILAMENTS
995
FIG. 5. Electron micrograph from a section cut
perpendicular to the long axis of a stage 15 neural
plate cell. Paraxial microtubules are seen in circular cross-section. The uniformity of these crosssections illustrates the extent of alignment of microtubules in these cells. Note that in a some
microtubules lie close enough to neighboring mi-
crotubules to interact by microtubule bridges (arrows) and that in b many microtubules are found
in close proximity to mitochondria (arrows).
Mitochondria (m) are also aligned parallel to the
long axis and seen in cross section. Both X 58,000.
(Fig. 5a from Burnside, 1971.)
for spindle elongation of Mclntosh et al.,
1969); (3) microtubules may deplete the
apical and extend the basal end of the cell
by a basally directed transport of cytoplasmic elements (similar to models for axoplasmic flow and spermatid elongation)
(Kreutzberg, 1969; Fawcett et al., 1971).
According to the first hypothesis (dynamic equilibrium model), microtubules
would elongate by addition of subunits
from an available pool and thereby exert
a push on the apical and basal ends of
the cell. This model in its simplest form
would predict that microtubules extend
the full length of the cell or can form
lateral associations with one another which
are stronger than the forces generated by
elongating the microtubule. It would also
predict that there would be an increase in
the amount of microtubule protein in the
polymerized state during the elongation
process.
The second hypothesis (sliding filament
model) suggests that force may be produced by the active sliding of microtubules
against one another. Sliding would be generated by means of intertubular bridges analogous to those between actin and myosin
filaments in the sliding filament model of
skeletal muscle contraction. In this case,
however, active sliding would produce decreased overlap and a pushing force, whereas in muscle the active sliding increases
overlap and exerts a pulling force. In its
simplest form this hypothesis would predict an apical and basal set of microtubules which overlap in the central region
of the cell. Overlap would be decreased
by sliding during elongation. This model
would not necessarily require an increase
in the amount of microtubule protein in
the polymerized state or the existence of
996
FIG. 6.
parallel
stage 15
are seen
BETH BURNSIDE
Electron micrograph from a section cut
to and just below the free surface of a
neural plate cell. Microfilament bundles
coursing near die lateral cell membranes
and forming specific attachments with desmosome
(d) filaments. Microfilaments also appear to be
associated with the lateral cell membranes (arrows) . X 66,000. (From Burnside, 1971.)
NEURULA MICROTUBULES AND MICROFILAMENTS
997
FIG. 7. Schematic illustrations of the flattening
cells of the epidermis. Microfilaments in the apical
region of the cell have been replaced by discrete
bundles of thicker 100 A filaments which appear
to course the cell from desmosome to desmosome.
Microtubules appear to be randomly oriented
throughout the cell.
FIG. 8. Electron micrograph of an epidermal cell
in a stage 15 embryo illustrating bundles of 100 A
filaments (arrows). These filaments are indis-
tinguishable from tonofilaments of larval epidermal cells. X36,000. (From Bumside, 1971.)
BETH BURNSIDE
998
r
FIG. 9. Light micrographs illustrating the effect
of colchicine on cell elongation in isolated neural
plates. Neural plates (np) in cross section are
shown in a immediately after explanting at stage
15, and in b and c after four hours in culture: b,
in the presence of 1(HM colchicine in Holtfreter's
solution, or c, in normal Holtfreter's solution. Cells
in the colchicine treated neural plate have failed
to elongate. X120.
a pool of available microtubule precursors. One would expect to find at least
some of the microtubules to be close
enough together to interact by means of
microtubule bridges (Hepler et al., 1970).
The third hypothesis (basally directed
transport) would specify that microtubules
generate a force, not by themselves pushing against the apical and basal ends of
the cell, but by creating a basally directed
flow of cytoplasm. In most epithelia, the
basal end of the cell is probably more distensible since it lacks the junctional complexes and microfilamentous "cortex"
found in the apical end. Cytoplasmic flow
along the microtubule might conceivably
be produced either (1) by means of microtubule bridges to paniculate cytoplasmic
elements (Jarlfors and Smith, 1969), or (2)
by means of a pumping motion created by
waves passing down the microtubule (Roth
et al., 1970). In nerve axons, synaptic vesicles connected to microtubules by bridges
have also been observed (Jarlfors and Smith,
1970). The existence of wave motions
along microtubules is more difficult to demonstrate by conventional techniques, but
has not been ruled out (Roth et al., 1970).
The basally directed transport hypothesis
would predict that microtubules either extend the length of the cell or form relatively stable lateral associations since microtubules would have to be prevented
from moving toward the apex in order to
produce a basalward movement of cytoplasm. Microtubules would then have to
lengthen as the cell elongates, but in this
case microtubule elongation itself would
not be exerting a force. In the case of
bridge action for transport, one might expect to see close associations of cytoplasmic constituents and microtubules if not
actual bridges between them. •
As a first step in considering the applicability of these hypotheses of neural plate
cell elongation, counts have been made of
microtubules in neural plate cells before
and after elongation and in cells which
have elongated to a different extent (Burnside, 1971). Because of the paraxial alignment of microtubules, it is possible to
count circular cross sections of micro-
NEURULA MICROTL'BULES AND MICROFILAMENTS
tubules in sections cut at right angles to
the long axis of the neural plate cell (Fig.
5). But before counts from cells in different parts of the neural plate or different
embryos could be compared, it was necessary to ascertain whether any section
through a neural plate cell yielded a number of microtubules per cell representative
for all apico-basal levels of that cell. When
three separate apico-basal levels of a small
group of neural plate cells were compared,
no significant differences between mean
numbers of microtubules per cell at each
level were observed (P > 0.5) (Fig. 11).
Therefore, it was assumed that numbers of
microtubules per cell counted from random sections of individual cells would be
representative for those cells.
A comparison of cells in equivalent regions of the neural plate before and after
elongation (Fig. 11) indicates that the average number of microtubules per cell decreases as the cell elongates. The number
of microtubules per cell decreases significantly from 163 at stage 13 to 108 at stage
15 (0.01 < P), while the cell elongates
from 50 ^ to 94 ^ in length. Interestingly,
if one computes the total cumulative
length of microtubules per cell at each
stage, by multiplying numbers of microtubules per cell by cell length, one finds
that total cumulative length of microtubule remains approximately constant
(9800 n at stage 13 to 10,100 /* at stage 15).
Thus, in spite of the decrease in numbers
of microtubules per cell during elongation,
there is conservation of the total cumulative length of microtubules and correspondingly, the total amount of microtubule protein in the polymerized state.
When cells from two regions of the stage
15 neural plate which have elongated to
different extent are compared (Fig. 11),
one finds no significant difference (P >
0.1) in average numbers of microtubules
per cell between cells which have elongated to nearly twice their original height
(88%) and cells which have elongated
much less (47%). It is, therefore, clear that
the total cumulative length of microtubules in the longer cell (108 microtubules per cell x ^ c = 10,100 p) is
999
greater than that in the shorter cell (86
microtubules per cell X 74 p = 6364 p).
Thus, the degree of elongation appears to
be more closely related to the total cumulative length of microtubules than to the
number of microtubules per cell in cross
section.
Now let us consider whether these observations are consistent with any of the hypotheses for microtubule function in cell
elongation which have been described
above. Although it is not yet possible to
unequivocally eliminate any of the three
hypotheses, these observations set forth a
number of requirements for which the different proposed mechanisms must account.
It is, therefore, possible to begin to restrict
the applicability of the proposed mechanisms and point to limitations of the
theories in explaining neural plate cell
elongation.
First, the simplest form of the dynamic
equilibrium model, elongation of microtubules themselves by addition of subunits
to pre-existing microtubules, does not seem
applicable because of the decrease in numbers of microtubules and constant total
cumulative length of microtubules per cell.
Rather, one would have to propose a more
complicated mechanism involving extension of some microtubules at the expense
of others that are broken down. This more
complicated mechanism is, of course, still
theoretically possible.
The second hypothesis, sliding of polarized microtubules to decrease overlap, is
consistent with these observations. The
constant total cumulative length of microtubules and the decrease in numbers of microtubules per cell are consistent with overlapping sets of microtubules. Since the cell
elongates to nearly twice its length, complete overlap of microtubule sets before
elongation would be reduced by sliding to
almost no overlap in the elongated cell.
The same sets of microtubules would be
present before and after elongation, hence
no change in total cumulative length of
microtubules would be observed. The decrease in numbers of microtubules per cell
could result from counting mainly overlapped regions at stage 13 and mainly non-
1000
BETH BURNSIDE
1 Elongation of Microtubules by
Addition of Subunits
2 Sliding of Overlapping Sets
of Microtubules
3 Basally Directed Transport of
Cytoplasmic Constiuents Along
Microtubules
Idol
NEURULA MICROTUBULES AND MlCROFILAMENTS
Stoge 13
Mean Number of
Microtubules = 163±8
Anterior
Neural Plate
Stage 15
Posterior
Neural Plate
1
Total Cumulative
Length = 9800 u
Mean Number of
Microtubules O08 1 5
Mean Number of
Microtubules =86±12
Total Cumulative
Length5 6340 p
Total Cumulative
Length = 10,100 u
FIG. 11. Comparison of numbers of paraxial mi- pared before and after elongation (stage 13 and
crotubules counted in cross sections of neural plate stage 15) , one finds a significant decrease in avcells. Before it was possible to compare numbers erage numbers of microtubules per cell as the cell
of microtubules per cell for cells from different elongates (P < 0.01) . Nevertheless there is a conembryos or different regions of the neural plate, servation of total cumulative length of microtubules (number microtubules per cell X cell
it was necessary to ascertain whether any section
of a cell gave representative counts for that cell. height) . Counts of microtubules in cells from two
Since the average numbers of microtubules per regions of the same neural plate which have eloncell for three apico-basal regions of stage 15 neural gated to a different extent do not differ signifiplate cells did not differ significantly (P < 0.5) , cantly (P > 0.1) . The total cumulative length of
it was assumed that any section through a cell microtubules, however, is less in cells which have
would yield representative counts. When cells from elongated less (Burnside, 1971).
equivalent regions of the neural plate are com-
overlapped regions at stage 15.
There is another observation, however,
which is not consistent with this hypothesis. If the decrease in numbers of microtubules per cell results from counting
overlapped portions of microtubules at
stage 13 and nonoverlapped portions at
stage 15, one would expect to see a decrease
in numbers of microtubules lying close
enough together to interact by microtubule bridges (see Hepler et al., 1970).
Counts to determine percentages of microtubules with neighbors closer than 600 A
indicate that the fractions of microtubules
with close neighbors remain almost exactly
the same at both stages (Burnside, 1971).
Furthermore, one might expect to see a
higher percentage of microtubules close
neighbors in the central as opposed to the
apical or basal regions of the cells. This
relationship was not observed. Rather, the
percent of microtubules with close neigh-
FIG. 10. Schematic illustration of three possible
mechanisms of microtubule function in cell elongation. In 1, force is generated by elongation of
the microtubule themselves by addition of subunits; in 2, force is generated by the active slid-
ing of an apical and basal set of microtubules to
decrease overlap; and in 3, force is generated by
directed transport of cytoplasmic constituents
along microtubules; thereby depleting the apical
and extending the basal end of the cells.
1002
BETH BURNSIDE
10
20
30
Microtubule
Density
(Number /p 2 of Cell Cross Section)
FIG. 12. A scatter diagram illustrating the relationship between percentage of microtubules having close neighbors and the density of microtubules
in the cell cross section. There is a significant correlation between percent microtubules with close
neighbors and microtubule density (P < 0.01).
The equation for the regression is Y = 29.3 -f1.0X. The regression coefficient is 0.685; with 20
degrees of freedom, this value is significantly different from zero (P < 0.01).
bors appears to be directly correlated with
the density of microtubules in the cell
cross section (Fig. 12). These cells undergo
interkinetic nuclear migration, with the
nucleus migrating to the basal end of the
cell to synthesize DNA and to the apical
end to undergo mitosis, so that the cross
sectional area of these cells is highly variable. Nevertheless the number of microtubules observed in cross section remains
relatively constant. Not surprisingly, more
microtubules with close neighbors are observed when these microtubules are constricted into a smaller area. One further
problem with the sliding hypothesis is that
after the open neural plate stages, cells
continue to elongate during neural tube
formation—so that finally cells have elongated to almost three times their original
length (see Fig. 3). This degree of elongation is incompatible with the sliding of
two overlapping sets of microtubules.
At first glance, the results from microtubule counts neither support nor rule out
the transport hypothesis of cell elongation.
If, however, one assumes that for purposes
of transport a particular volume of cytoplasm is acted upon by a certain increment
of microtubule length, then these observations are consistent with this hypothesis.
Since the volume of the cell remains constant, the relationship of cytoplasmic volume to increment of microtubule length is
maintained by the constant total cumulative length of microtubules as the cell elongates. This maintenance of total cumulative length in an elongating cell necessarily requires a decrease in numbers of microtubules per cell in cross section. Furthermore, this hypothesis may explain the
smaller total cumulative length of microtubules in cells which elongate less. Since
the volume of the two cell types are equal,
one might expect that decreasing the ratio
of microtubule length to cytoplasmic volume might reduce the efficiency of transport and, hence, reduce the extent of elongation. In keeping with this hypothesis, it
is common to see cytoplasmic constituents
in close proximity to microtubules, for example, mitochondria may lie closely parallel to microtubules over a large portion of
their length (Fig. 13). Mitochondria are
generally found oriented parallel to the
axis of elongation in neural plate cells
(see Figs. 5b and 13). This orientation may
represent alignment with cytoplasmic flow.
Neural plate cells also contain numerous
microtubules oriented parallel to and just
below the apical surface: these microtubules could perhaps further stabilize the
apical ends of the cell and provide a dense
apical "cortex" to counteract the retrograde push on the microtubules produced
by basal ly directed flow along them.
Although none of the three hypotheses
can be unequivocally ruled out, I favor
the directed transport hypothesis as best
fitting the available data described above.
The directed transport hypothesis is also
attractive since it obviates the necessity of
proposing completely separate mechanisms
for cytoskeletal and transport functions of
microtubules. A second and stronger advantage of this hypothesis is that it is more
easily tested than the other two, since
NEURULA MICROTUBULES AND MICROFILAMENTS
basally directed transport should be detectable in isolated neural plate cells which
elongate in culture (Holtfreter, 1947). Experiments examining this possibility are
1003
in progress.
Microfilaments and apical constriction
Several observations suggest that microfilaments play an active role in apical constriction in neural plate cells. Microfilaments are arranged in circumferential
bundles around the cell apex and, therefore, would be appropriately oriented to
produce a purse string-like apical constriction. Bundles of microfilaments are attenuated and lost in presumptive epidermal
cells whose apices are enlarging as the cell
flattens. In neural epithelial cells, microfilaments become pronounced at precisely the time of apical constriction, and
bundles of microfilaments grow thicker as
the cell apex constricts. It seems unlikely
that this increase in thickness represents
passive accumulation since microfilaments
remain relatively straight and parallel
within the bundles as though under tension.
Further temporal and spatial correlation
of microfilaments with localized contraction in neural plate cells comes from experiments in which micronlament formation and contraction are experimentally induced to occur atypically at the basal ends
of the cells (Burnside, 1972). If early neural plates are explanted into a simple salt
solution with the basal surface exposed to
the medium, bundles of microfilaments are
formed at the new free surface and the
basal ends of the cells constrict. This basal
constriction curls the whole epithelium toward the basal surface (Fig. 14). Normally,
bundles of microfilaments are present only
at the apical ends of neural plate cells.
Within a few minutes after explantation,
however, new bundles of microfilaments
appear at their basal ends. These microfilament bundles grow thicker and the basalFIG. 13- Electron micrograph of longitudinal section of neural plate cell. Note that mitochondria
(m) and microtubules are oriented parallel to the
long axis. Note also that one mitochondrion is
closely associated with a nearby microtubule for
a large portion of its length (arows) and that the
distance between the mitochondrion and the microtubule is relatively constant over this distance.
X 38,000.
1004
BETH BURNSIDE
2 - 5 MIN
30 MIN
5 HR
FIG. 14. Camera lucida drawing of neural plates
explanted with and without underlying tissues.
The region of this neural plate with exposed basal
surface begins to curl immediately after dissection
and continues to curl basalward until it has folded
back upon itself and the basal surface is no longer
exposed.
ward curling continues until the epithelium has folded back upon itself and the
basal surface is no longer exposed. At this
time bundles of filaments are much reduced. Thus, in addition to the normal
correlation of apical microfilaments with
apical invagination of the neural tube,
concomitant microfilament formation and
contraction can be experimentally produced in the basal ends of the cells.
Cytochalasin B experiments also suggest
a correlation between microfilaments and
apical constriction in neural plate cells
(Burnside, 1972, and unpublished). Without describing the vehement controversy
as to whether cytochalasin B interacts directly or specifically with microfilaments
(and it seems likely that it does neither),
I will assert that when cytochalasin both
disrupts microfilaments and simultaneously blocks an observed cytoplasmic contraction associated with those filaments, this
result jjrovides further circumstantial correlation of the observed contraction with
the observed microfilaments. This result
does not prove that microfilaments cause
the contraction any more than other circumstantial correlations prove causality.
Cytochalasin B at low concentrations
(less than 5 mg/ml) blocks apical constriction in isolated neural plates without disrupting the integrity of the neural epithelium. In these neural plate cells, the
apical microfilament bundles no longer
contain detectable linear filaments but are
seen as the dense granular masses typical
of other studies of cytochalasin effects on
microfilaments (Wessells et al., 1970).
When basal microfilament formation is induced in explanted plates in the presence
of cytochalasin, granular masses rather
than microfilament bundles are found in
the basal ends of the cells. The basal ends
of the cells fail to constrict and the plate
remains flat (Burnside, 1972). Thus, cytochalasin simultaneously abolishes localized
contraction and disrupts the integrity of
microfilaments in neural plate cells during
normal apical constriction and also during
experimentally induced basal constriction.
Though none of the above observations
demonstrate that microfilaments cause
apical constriction (there is no proof that
microfilaments cause cytoplasmic contraction in any system), the hypothesis that
microfilaments do play a causal role in cytoplasmic contraction is clearly the most
likely interpretation of these results and
other published observations of microfilament behavior. Therefore, I will provisionally assume that microfilaments do play
an active role in apical constriction and in
the following discussion examine some possible mechanisms of microfilament function.
At present there are no physiological examples of cytoplasmic contraction being
mediated by shortening of individual filaments. On the other hand, abundant evidence for sliding of filamentous structures
in contractile processes has accumulated
from studies of skeletal muscle (see Young,
1969), smooth muscle (e.g., Sanger and Hill,
1972), and fiagellar axonemes (Summers
NEURULA MICROTUBULES AND MICROFILAMENTS
Stage 13
1005
Stage 19
Diameter of Cell A p e x "
0.32
0.17M
Thickness of
f
Microfilament Bundle^
Stage 13
Stage 19
Cross Sectional Area 2.3xlO6A2
of Filament Bundle
Maximum Number of
800
Filaments Per Bundle
Circumference
59.6 u
of Cell
Total Length of
Filaments Per Cell 47,600 u
8.4xlO 6 A 2
FIG. 15. Calculations o£ total length of microfilaments in the apical circumferential bundles before and after constriction. Diameters of apical
microfilamcnt bundles were measured from electron micrographs for stages 13 and 19 (immediately after gastrulation and at neural tube closure).
Diameters of the apices of cells were determined
from thick plastic sections. Cross-sectional area of
the microfilament bundles was calculated by assuming that the bundles were circular in crosssection. This area was divided by the cross-sectional area of a single 60 A microfilament to estimate total numbers of filaments that could be
2,960
18.7M
55,600 M
present, assuming maximal packing. Cell circumference was estimated by assuming the cells were
round at the apex. Since the microfilament bundles
encircle the apex, the circumference of the cell
apex was multiplied by the total number of filaments in the bundle to estimate the total length
of filaments per cell. According to these calculations the total length of filaments remains relatively constant as the cell apex constricts, therefore
suggesting that the decrease in circumference and
the increase in thickness of the bundle results
from an increased interdigitation of the original
complement of filaments. (From Burnside, 1971).
1006
BETH BURNSIDE
and Gibbons, 1972). Consequently, it is
cogent to look for filaments and for evidence of sliding when considering models
for the mechanism of non-muscular cytoplasmic contraction.
The increase in thickness of microfilament bundles during neural plate cell
apical constriction lends support to the hypothesis that sliding of filaments plays a
role in non-muscular cytoplasmic contraction (Burnside, 1971). First the circumferential microfilament bundles increase in
thickness during apical constriction while
the constituent microfilaments remain relatively straight and parallel within the
bundles, as though subjected to tension. The
filaments themselves retain a constant
diameter. Furthermore, when one estimates
the total length of microfilaments in the
bundles before and after apical constriction (Fig. 15) one finds that there is little
change in the total length of filaments
while the cell apex constricts. This observation suggests that the increase in thickness
of the bundles and the decrease in circumference result from increased interdigitation
of the original complement of filaments.
The nature of the motive force for sliding of microfilaments within the circular
bundles is still a matter of conjecture. Many
questions remain to be answered. What generates the sliding force? Are there myosin
filaments which have not been preserved
in fixation? Could monomeric myosin mediate sliding of actin filaments as suggested by
Pollard and Korn (1972) for Acanthamoeba? Do the desmosmal attachments of microfilaments act like Z-bands, so that the interaction of filaments from neighboring desmosomes resembles contraction of a sarcomere? These and many other aspects of
microfilament behavior and biochemistry
are only in their earliest stages of clarification.
In summary, we have seen that, in the
salamander embryo, macroscopically visible
morphogenetic movements can be correlated
on the cellular level with active changes in
cell shape, and that these changes in cell
shape are to some extent dependent on intracellular cytoskeletal and contractile organelles, microtubules and microfilaments.
The actual mechanisms by which microtubules and micronlaments mediate cell
shape change are not yet clear, but in view
of the intense current interest in their
morphology, their biochemistry, and the
nature of their interactions, it seems safe to
predict that a clearer understanding of cytomorphogenesis will soon be forthcoming.
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