/. Embryol. exp. Morph. Vol. 60, pp. 201-234, 1980
Printed in Great Britain © Company of Biologists Limited 1980
201
The cellular basis of epiboly: An SEM study of
deep-cell rearrangement during gastrulation in
Xenopus laevis
By R. E. KELLER 1
From the Department of Biology, Yale University, New Haven,
Connecticut and Department of Biology, Indiana University
SUMMARY
Measurements of several indices of shape, contact, position and arrangement of deep cells
in the late blastula and gastrula were made from scanning electron micrographs of carefully
staged, fractured embryos in order to describe the cellular processes which account for the
increased area of the deep region of the gastrula during extension of the dorsal marginal zone
and epiboly of the animal region. At the onset of gastrulation, the deep cells of the dorsal
marginal zone become elongated, extend protrusions between one another along radii of the
embryo and interdigitate to form fewer layers of cells of greater area in a process of radial
interdigitation. When interdigitation is complete, the deep region consists of one layer of
columnar cells which then flatten and spread and thus account for additional increase in area
of the deep region. During epiboly of the animal region, interdigitation occurs and the number
of cell layers decreases without the changes in cell shape seen in the dorsal marginal zone.
These differences may be related to the anisotropy of expansion (extension and convergence)
in the dorsal marginal zone as opposed to uniform spreading in the animal region, or they
may reflect an active cell motility in the dorsal marginal zone as opposed to a passive behavior in the animal region. A cellular and mechanical model is presented in which active
(autonomous) spreading is brought about by active, force-producing interdigitation and
subsequent flattening of deep cells. A model of passive spreading (stretching) is also presented. These observations suggest experiments that would determine the relationship of
cell behavior to the mechanics of gastrulation.
INTRODUCTION
Embryologists have always been fascinated with the process of amphibian
gastrulation and have directed much effort toward discovering its secrets. Roux
(1894, 1896) ascribed the global rearrangements of gastrulation to the behavior
of individual cells which he had isolated and observed in a simple culture
medium. From tissue recombination experiments, Holtfreter (1939) concluded
that the differentiation of a hierarchy of cell type specific affinities governs
tissue behavior and thus the movements of gastrulation. On the basis of extensive observation of the structure of the gastrula and of the behavior of
1
Authors present address: Department of Zoology, University of California, Berkeley,
California 94720, U.S.A.
202
R. E. KELLER
individual cells and groups of cells in vitro. Holtfreter (1943 a, b, 1944) set forth
some ideas on cellular behavior and the mechanics of gastrulation. Schechtman
(1942) and others (see Spemann, 1938) did surgical rearrangement and explantation experiments which showed both a regional autonomy and a cooperation
on the part of various sectors of the gastrula. However, these studies did not
produce a comprehensive description of cellular behavior in vivo or a mechanism relating specific patterns of cell behavior to the generation of forces appropriate to produce the observed distortion of the gastrula. The opacity of the
amphibian embryo has prevented the direct observation in vivo of the cellular
activities that actually occur during gastrulation, and therefore they remain
unknown.
To establish the cellular mechanisms of gastrulation, it is necessary to answer
several questions. What are the paths of cell movement? What cellular activities
bring about these movements? How does cellular behavior generate the necessary
mechanical forces? How is this cellular behavior controlled?
A series of studies on gastrulation in the African clawed frog, Xenopus laevis,
has been directed at answering these questions. The paths of cell movements
were mapped by vital dye marking of both the superficial and the deep layers
of the embryo. Movements in both layers are similar in their major features to
those found in other amphibians (compare Vogt, 1929, Pasteels, 1942). The
animal region undergoes a nearly uniform expansion (epiboly). The dorsal
marginal zone (DMZ) spreads rapidly toward the blastopore (extension), and
concurrently it narrows (convergence). The lateral marginal zone (LMZ) and
the ventral marginal zone (VMZ) also spread toward the blastopore but to a
lesser degree. Both epiboly and extension consist of an increase in area; in the
latter case the increase is anisotropic and directed toward the blastopore (see
Keller, 1978). However, Xenopus differs fundamentally in that mesoderm is not
found on the surface of the embryo but lies entirely in the deep marginal zone
and is covered by a monolayer of suprablastoporal endodermal cells (see
Nieuwkoop & Florshutz, 1950; Keller, 1975, 1976). The superficial layer remains intact during gastrulation, which is in contrast to the results of work on
other amphibians (Vogt, 1929; but see L0vtrup, 1975). A quantitative analysis
of the apical area, number, and shape of superficial cells by time-lapse cinemicrography of living Xenopus gastrulae (Keller, 1978) showed that these cells
spread, divide, and undergo rearrangements and a temporary change in shape
which accounts for the change in area and for the distortion of the superficial
layer during epiboly, extension and convergence.
The activities of the deep cells could not be seen in the intact embryo, but a
scanning electron microscopic analysis of fractured gastrulae suggests what
their behavior might be. Regions of the gastrula undergoing epiboly and
extension become thinner and show a decrease in the number of layers of
deep cells (see Keller & Schoenwolf, 1977; Keller, unpublished data). There is
no evidence of cytolysis or cell death. Indeed, this is unlikely since the total
Cellular basis of epiboly in Xenopus
203
(a)
(by
(c)
mm
(d)
Fig. 1. Radial interdigitation of deep cells is proposed as the cellular mechanism of
spreading of the deep region of the Xenopus gastrula during epiboly and extension.
From the outside inward, three layers of cells are shown («): the superficial layer,
the inter deep layer, and the inner deep layer (shaded). Cells of the two deep layers
extend protrusions inward or outward, along radii of the embryo, and move between
one another (arrows, b); this process is called radial interdigitation (the cells move
along radii of the embryo). When interdigitation is complete, the inner deep layer
consists of one layer of elongated, columnar deep cells (c). These cells then flatten
and spread id). The process of deep-cell interdigitation and subsequent deep-cell
shortening and spreading increases the area occupied by the deep layer. Concurrently, superficial cells flatten and spread (see Keller, 1978). Cell division occurs
as well but has not been shown for clarity of illustration.
cellular volume increases slightly during gastrulation (Tuft, 1965). Time-lapse
cinemicrography shows that deep cells do not disappear from the deep layer by
moving outward into the superficial layer. They cannot migrate more deeply
into the interior because they are bounded internally by the interface, which is
a clearly defined discontinuity with the underlying involuted cells. Vital dye
mapping shows that a marked sector of the deep region expands as a contiguous
mass (Keller, 1976). These facts suggest that deep cells undergo a local migration inward or outward, between one another, along radii on the embryo, and
thus transform several layers of deep cells into one layer of greater area. Such
a process of radial interdigitation would account for the spreading of the deep
region during extension of the marginal zone and epiboly of the blastocoel roof
(Fig. 1; see the Discussion and Fig. 13, Keller, 1978).
In the present study, measurements of indices which quantitatively describe
the positions, the shapes, and the arrangements of the deep cells show that
radial interdigitation occurs and that it and the subsequent flattening of a single
204
R. E. KELLER
layer of deep cells provide the increased area necessary for epiboly and extension
of the deep layer. Furthermore, these data suggest a sequence of changes in deepcell shape and contact as a cellular mechanism of radial interdigitation. This
paper and an earlier one (Keller, 1978) provide a quantitative description of
cellular behavior of both the superficial and the deep cells during epiboly.
From these data, models relating local cellular behavior, a global system of
mechanical stresses, and the spreading movements of the deep and superficial
layers were generated.
MATERIALS AND METHODS
Procedures for fixation and scanning electron microscopy. Xenopus embryos
were obtained by standard mating procedures (see Keller, 1975) and dejellied
as described previously (Keller & Schoenwolf, 1977) or by cysteine hydrochloride treatment (1-75 g in 50 ml distilled water with 0-3 g Tris buffer, adjusted
to pH 7-8 with NaOH). They were then washed in 10% Steinberg or NiuTwitty solution, staged according to Nieuwkoop & Faber (1967), and fixed in
2-5 % glutaraldehyde in 0-1 M sodium cacodylate (pH 7-4-7-6) at room temperature. They were then fractured with a blunt knife as described previously
(Keller & Schoenwolf, 1977), dehydrated in ethanol, dried (liquid CO2 criticalpoint-drying), mounted on stubs with silver paste, coated with gold-palladium
(60:40) in a Polaron sputter-coater or a Denton vacuum evaporator, and photographed on Polaroid Type 55 film with an Etec Auto Scan electron microscope.
Magnification of electron micrographs, vertically and horizontally, was determined from micrographs of a grid, the dimensions of which were measured with
a light microscope fitted with a calibrated filar micrometer.
Anatomy and terminology of the embryo. The regions to be discussed in this
paper are found in the outer shell of the embryo (lightly shaded regions, Fig. 2).
As this outer shell expands and moves vegetally, its lower margin is involuted.
It is continuous with the involuted material (darkly shaded regions, Fig. 2) at the
blastoporal lip where involution occurs, and is separated from the involuted
material in all other regions by a clearly defined discontinuity, the interface (see
Fig. 2). The outer shell consists of a monolayered epithelium of superficial cells
and a deep region of one or more layers of non-epithelial deep cells (Fig. 3).
There are two types of deep cells. Inner deep cells are those deep cells that bound
the blastocoel or that form the interface with the involuted mesoderm (see
Figs. 2 and 3). Inter deep cells are all those deep cells that do not bound the
blastocoel or the interface (Fig. 3).
Definition and measurement of parameters of cell position, size, and arrangement. Indices that measure several relevant characteristics of superficial cells,
inter deep cells and inner deep cells were defined (Fig. 3) and measured in
scanning electron micrographs of known magnification (370-420). First, the
maximum extent of the superficial cells, the inter deep region, and the inner
deep cells in the direction perpendicular to the surface (along radii) of the
Cellular basis of epiboly in Xenopus
205
BR
DMZ
VMZ
DMZ
VMZ
Stage... 8
ANE
ANE
DMZ
VMZ
DMZ
VMZ
Stage...
Fig. 2. Two components can be distinguished in the Xenopus gastrula - an outer shell
(lightly shaded), which undergoes spreading or expansion toward the blastopore,
and an inner region (darkly shaded), which has undergone involution and is clearly
separated from the outer shell by a clearly defined discontinuity, the interface (IN)
(see Keller & Schoenwolf, 1977). This study describes changes in the morphology and
arrangement of cells in the following sectors of the outer shell of the embryo: BR,
blastocoel roof; DMZ, dorsal marginal zone; VMZ, ventral marginal zone; ANE,
anterior neural ectoderm. The diagrams are approximate representations of the
stages indicated, which include the late blastula (stages 8 and 9), the early gastrula
(stages 10-10£), the mid gastrula (stage 11), and the late gastrula (stage 11| and 12|).
embryo were measured and the positions of these points with respect to the
surface of the gastrula were recorded, averaged, and the mean plotted graphically as the layer boundary (Fig. 3). Only cells for which the inner and outer
boundaries could be clearly determined were included.. The superficial layer and
the inner deep layer are, by definition, monolayers; in contrast, the inner deep
region may consist of one or several layers or tiers of cells. In the latter case only
the inner-most or outer-most boundaries were recorded. Second, individual
superficial cells were chosen and the shortest route was traced to the interior,
jumping from cell-to-cell (Fig. 3). The mean number of cells passed through in
all micrographs of a given region and stage was recorded as the layer index.
Third, the number of inner deep cells was divided by the total number of deep
cells to give the per cent inner deep cells. Fourth, the number of inner deep cells
extending through the entire deep region and contacting the inner surfaces of the
14
EMB 60
206
R. E. KELLER
[Superficial
• Interdeep
Unnerdeep
Layer index
3. Percent inner deep cells: no. inner deep cells/no, inner + inter deep cells, x 100.
4. Percent inner-super cell contact: no. inner-super. Contacts (#)/no. inner deep
cells, x 100.
5. Percent inner-inter deep layer interdigitation: Inner-inter layer overlap (o)/total
deep layer thickness (b), x 100.
6. Height/length ratio {H/L).
7. Length/width ratio (£/ W).
8. Apical area of inner deep cells {A).
Fig. 3. A diagram of a typical midsagittal surface of a fractured embryo shows the
relationship of the superficial, inter deep and inner deep cells (see Materials and
Methods for definitions) and the various parameters of cell position, shape and
arrangement used in this study (No. 1 through No. 8). The layer boundaries (No. 1)
of the superficial (triangles), the inter deep region (squares), and the inner deep
layer (circles), are mean values of the positions of the inner and outer boundaries
(dotted lines) of large numbers of individual cells in each category. The layer index
(No. 2) is the mean number of steps from the superficial layer to the interface, taking
the shortest route and jumping from cell to cell as indicated by the example above.
Parameters Nos. 3, 4 and 5 are indices of cell position and contact and parameters Nos. 6, 7 and 8 are indices of cell shape. These are described briefly above
and in detail in the Materials and Methods.
superficial cells was divided by the total number of inner deep cells to give the
per cent inner-to-superficial contact. Fifth, the mean overlap of inter deep cells
and inner deep cells was calculated as a percentage of the^thickness of the entire
deep region and designated the per cent interdigitation (Fig. 3). Sixth, the maximum extent of cells along the animal-vegetal (meridian) lines of the embryo
was divided into their maximum extent along radii (normal to the surface) of
the embryo to give a height-length (h/l) ratio. Seventh, the length (as defined
above) of the inner apices of the inner deep cells was divided by their maximum
extent along latitude lines of the embryo to give a length-width (l/w) ratio. Note
that these ratios reflect both cell shape and orientation. Eighth, the inner apices
of the inner deep cells were traced and their areas determined with a planimeter,
corrected for magnification, averaged, and the mean apical area plotted graphically. The mean, the standard deviation, the standard error of the mean, and the
frequency distribution were determined for nearly all parameters for every stage
and region considered. Means (and in some cases errors) are presented graphically, and other data will be noted where they are important. Lastly, the mean
volume of inner deep cells was estimated from their apical area and height at
several stages of development. All measurements were of fixed and criticalpoint-dried material which shows some shrinkage (Keller & Schoenwolf, 1977).
Cellular basis ofepiboly in Xenopus
207
RESULTS
Dorsal marginal zone (DMZ). The changes in cell morphology and arrangement occurring in the DMZ of the late blastula and throughout gastrulation
are shown in a series of representative electron micrographs (Fig. 4) and by
quantitative data (Figs. 5 and 6).
At stage 8 the DMZ is about 125 /im thick and consists of a superficial cell
layer and an inner deep cell layer, each about 50 /im thick, and a slightly thicker
region of one and a half layers of inter deep cells (Fig. 6). By stage 9, the DMZ
as a whole has thinned slightly due to thinning of all three of its component
regions. The superficial cells, the inner deep cells, and probably the inter deep
cells as well, decrease in their linear dimensions following cell division. These
cells can be viewed as nearly cuboidal or as cylinders (roughly) with the height
equal to diameter, since their h/l ratios are about 1-0, and both the inner deep
cells and the superficial cells have a l/w ratio of about 1-0 (see page 212 and
Keller, 1978). Thus the one cycle of inner deep cell division taking place from
stage 8 to 9 (see Fig. 18) should reduce the linear dimensions of these cells by
21 %, assuming, correctly (Tuft, 1965), that cell growth does not occur. The
observed decrease in height is 20 %. Concurrently, the thickness of the superficial layer is reduced by 35%. Time-lapse cinemicrography (see Keller, 1978)
a population average of just under two anticlinal cell divisions occur in this
period, which would reduce the linear dimensions of these cells by about 36 %.
As a result of cell division there are more deep cells of smaller size and thus the
layer index has risen to 4-1, even though the DMZ has become thinner.
The events in the period from stage 9 to 10 appear to be in preparation for
the onset of gastrulation (Fig. 4). The DMZ thickens by nearly 20 /tm, all of it
due to an increased number of layers (layer index is 5-5). The mean cell size
decreases again; approximations of the inner deep cell volume indicate that these
cells have divided twice in this period (compare stages 8 and 10 in Fig. 4; see
Fig. 18). The mean h/l ratio of inner and inter deep cells has risen to 1-3 and
1 -4 (Fig. 6), and both ratios are significantly greater than that of the superficial
layer (non-parametric Wilcoxon Rank Sum test; P < 0-01).
In summary, stages 8 and 9 appear to comprise a preparatory phase in which
the number of cells and layers of cells increases as the mean cell volume decreases by division, and in which the DMZ reaches its maximum thickness.
The period from stage 10 to 10+ marks the beginning of gastrulation. It is
characterized by a rapid thinning of the DMZ and a rapid decrease in the
number of cell layers in the inter deep region (Fig. 5). Thus it has been designated the multiple-layer interdigitation phase. The layer index decreases from
5-5 to just above 3-0 (Fig. 6), which means that the inter deep region decreased
from 3-5 cell layers to just one. The mean h/l ratios of inner and inter deep cells
are significantly greater than 1-0 at stage 10 and continues to increase. For the
first time, a few (about 8 %) of the inner deep cells pass through the entire deep
14-2
208
R. E. KELLER
< •
10+
Cellular basis of epiboly in Xenopus
209
Fig. 4. Scanning electron micrographs of the dorsal marginal zone (DMZ) show
the cell morphology and arrangement typical of the blastula (stage 8) and gastrular
stages (stages 10+ to 12£). Note the absence of thinning, the increase in the
number of cell layers, and the decrease in the cell size from stage 8 to 10, followed
by a decrease in the number of cell layers and thinning from stage 10 onward. Note
also, that the deep cells become increasingly elongated from stage 10£ to 11 and
form columnar cells (stage 11). Then they shorten from stage 11 to H i . The
micrographs are reproduced here at about 270 x .
region and contact the superficial cells, and the per cent inter digitation of inner
and inter deep cells shows a parallel increase (Fig. 6). During these events the
DMZ expands and extends rapidly toward the blastopore (see Keller, 1978).
Since stage 9, important changes have occurred in the protrusions of the
cell bodies of deep cells. It will be useful to make a distinction between macroprotrusions and microprotrusions of the cell body. The definition of these terms
assumes that all cell shapes are modifications of a spherical cell body, which is,
in fact, usually polymorphic but approaches a spheroidal or cuboidal shape on
the average, much like those of the DMZ at stage 8 (Fig. 4). A large extension
that comprises a major part of the cell body will be called a macroprotrusion.
A small extension of either the cell body or of a macroprotrusion is called a
microprotrusion. These are nearly always attached distally to other cells (see
Fig. IOCQ.
At stage 8, the deep cells are polymorphic but generally cuboidal and show
only microprotrusions of the cell body, which serve as attachments to adjacent
cells (see stage 8, Fig. 4). Many deep cells of stage 9 and nearly all of them at
stage 10+ bear macroprotrusions which are attached distally to adjacent cells
by microprotrusions, usually filiform in shape (arrows, stage 10+ and 10^,
Dig. 4). By stage 10^, these macroprotrusions tend to be aligned along radii of
the embryo and thus contribute to the increased h/l ratio (Fig. 6). Macroprotrusions may be thought of as transitional structures in a process of changing
cell shape. For example, when a macroprotrusion reaches the size of the cell
210
R. E. KELLER
120
120
* ^ ^ - \ ^
/
^ - ^
\
\
Whole _
Super..
\
100
Inter
Inner _
\
80
60
- - — - / \
^
^
\
40
> 1
°
20
0
Time (h)
Stage (h)
-o
^
i
i
i
i
i
i
i
i
i
9
10
10 10+
i
i
11
i
i
12
13
l
l
14
104- i
Fig. 5. Above, the positions of the superficial layer, the inter deep layer, and the
inner deep layer of the dorsal marginal zone (DMZj are plotted from stage 8 through
stage 12i. Below, the total thickness and the thicknesses of the superficial layer, the
inter deep layer and the inner deep layer are plotted for the same stages. Each point
is a mean of an average of 54 measurements.
body, the cell would no longer be considered a spheroidal or cuboidal cell with
a macroprotrusion, but an elongate or columnar cell. At stage 10 + a few of the
deep cells, particularly the inner deep cells, are already columnar, and many
are by stage 10£.
The DMZ thins slowly by 15 % in the next period (stage 10 + to 10£). This is
not due to thinning of the individual cell layers but is accomplished by a slow
and partial interdigitation of inter and inner deep cells which produces an 18 %
per cent decrease in the thickness of the deep region (Fig. 5). The inter deep
Cellular basis of epiboly in Xenopus
211
I"
40
30
20
10
2-6
2-2
1-8
1-4
10
0-6
Time (h)
Stage
6
8
7
8
9
9
10
10 10+
11
12
104- 11
13
14
Fig. 6. Above, the layer index, the per cent inner deep cells, the per cent interdigitation
of inter and inner deep layers, and the per cent inner-to-superficial cell contact are
plotted for stages 8 through 12|. Below, the height/length {h/l) ratios for the superficial, inter and inner deep cells are plotted for the same stages. Each h/l data point
is a mean of an average of 61 measurements. Each layer index data point is a mean
of an average of five measurements from each of 44 specimens.
region thins by a reduction in the mean number of cell layers from 1-2 to 0-8
(the layer index decreases from 3-2 to 2-8). In spite of the overall thinning of the
deep region, both the inner and inter deep cells actually increase in height
relative to their diameter (increased h/l ratio; Fig. 8). The per cent interdigitation rises to nearly 30 % by stage 10^. Concurrently, there is an increase in the
per cent inner-to-superficial ceil contact (Fig. 6) such that 30 % of the inner
deep cells extend through the deep layer by stage 10-}. By stage 10^, most deep
cells, particuiarly the inner deep cells, are elongate, aligned along radii of the
embryo, and bear numerous terminal and lateral microprotrusions (see stage 10£,
Fig. 4).
To summarize, from stage 10+ to stage 10^ there is a slow decrease in the
212
R. E. KELLER
thickness of the DMZ due to partial interdigitation of a layer of inter deep cells
and a layer of inner deep cells. Concurrently, both cell types become progressively more elongate and columnar.
From stage 10^ to 11, the DMZ thins by about 20 /«n (29 %). About 6 /tm
of this is due to thinning of the superficial layer, and the rest is due to the rapid
completion of the interdigitation of inner and inter deep cells (Fig. 6). As a
result, the layer index decreases from 2-8 to 2-2, 88 % of the deep cells are inner
deep cells, and the inner-to-superficial cell contact reaches 90% by stage 11.
Concurrently, the h/l ratio increases to a maximum of 2-2 (Fig. 6), and the deep
region then consists of one layer of tall, columnar cells (stage 11, Fig. 4). This
and the previous period can be considered the two-layer interdigitation phase.
From stage 11 to 11^, these columnar inner deep cells undergo flattening and
spreading (compare stages 11 and 11^ in Fig. 4), and as a result the DMZ
decreases from 50 to 36 [im in thickness. The flattening is indicated by a decrease in the h/l ratio of the inner deep cells from 2-2 to 1-6 (Fig. 6) and by a
corresponding increase in apical area (see Fig. 8). By stage 11 \, the layer index has
reached 2-0 and the inter deep cells no longer exist, since nearly all have become
inner deep cells by interdigitation. From stage 11^ to 12£, the DMZ thins by
4 /.im, losing an average of about 2 /tm in each layer (Fig. 5), probably because of
continued flattening and spreading of cells, but this time in the superficial layer
as well as the deep layer. Thus at the end of gastrulation, the DMZ consists of
a superficial layer about 14 ptm thick and a deep layer about 21 /im thick (Fig. 5).
The period from stage 11 to 12^, characterized by shortening and flattening of
the columnar array of deep cells, is called the deep-cell spreading phase.
Change in deep-cell shape as related to extension, convergence, and interdigitation
Extension and convergence in the superficial layer occurs in part by a
temporary rise in l/w ratio of superficial cells (Keller, 1978). Perhaps deep cells
undergo a similar l/w change. Such a change should be reflected in the l/w
ratios of the inner apices of the inner deep cells of the DMZ, which are exposed
by digging out all involuted material from gastrulae fractured midsagittally
(Fig. la, b). They are closely packed, relatively flat, and have filiform protrusions extending across their neighbors (Fig. 7 c). On the basis of the data thus
far, the hypothesis that the mean l/w of these apices is equal to one could not be
rejected (P = 0-01). The same was true when the length and width axes were
rotated 45° to their proper orientation. Thus these polymorphic apices are isodiametric when considered as a population. This means that the increase in
h/l ratio during interdigitation is not due to decrease in length as the cells
become relatively wider; instead, their height increases at the equal expense of
length and width. Similarly, when the h/l ratio decreases during shortening and
spreading (stage 11 onward), the height is decreasing to the equal benefit of both
length and width.
Cellular basis of epiboly in Xenopus
213
These facts suggest that inner deep cells behave as cylinders in which the height
increases at the expense of apical area during the interdigitation phases, and
decreases to the benefit of apical area during the deep-cell spreading phase.
Indeed, if the expected apical areas of inner deep cells are calculated on the basis
of mean change in cell volume (Figs. 8, 18) and corrected for the observed
changes in h/l ratio (Fig. 6), they lie close to the values actually observed (Fig. 8).
An increased mean h/l ratio of inter and inner deep cells is associated with
interdigitation and a decreased h/l ratio is associated with their subsequent
spreading. But does the entire population or only part of it show these changes
in h/l ratiol The frequency distribution of h/l ratios of inner deep cells (Fig. 9)
is not obviously bimodal at any stage except perhaps stage 11, and thus it
appears that no large fraction of the population fails to show change in h/l ratio.
The increasing broadness of the distribution (Fig. 9) might be explained in
several ways. Each cell may undergo a single cycle of h/l increase and decrease
during gastrulation but with individual cells some degree out of phase with one
another. Also, some cells may show a large excursion of the h/l ratio and others
much less. Alternatively, individual cells may be passing rapidly through many
such cycles, again, out of phase with one another.
Cells undergoing division contribute to the lower h/l clases. Inner deep cells
undergo anticlinal divisions by rounding up (h/l of about 1-0), dividing to form
dumb-bells (h/l of less than 1-0), and finally forming pairs of rounded daughter
cells (see Fig. 22, Keller & Schoenwolf, 1977). A large number of these rounded
cells and dumb-bells are seen at stage 11. The relatively high frequency
of cells with h/l ratios of less than 1-0 at stage 11 (Fig. 9) may represent this
population of dividing cells but the change in cell size (see Fig. 4) suggests that
the majority of cells have divided earlier, somewhere between Stage 10£ and
11.
Relationship of protrusion formation, cell shape and interdigitation. During
interdigitation deep cells usually bear macroprotrusions which are aligned along
radii of the embryo (Fig. 4) and thus contribute to the increased h/l ratio. By
stage 10^, 51 % of deep cells have h/l ratios above 2-0 (Fig. 9) and these cells
either have large, oriented macroprotrusions or they are elongated and cylindrical
(Fig. 4). Many inter deep cells of the DMZ have a rounded cell body with a
long, gently tapered macroprotrusion extending between adjacent cells in the direction of interdigitation (see No. 1, Fig. 10c, d). Others have shorter, more
blunt macroprotrusions of the cell body, also insinuated between cells to the inside (see No. 2 in Fig. 10a, b and e). Macroprotrusions of inter deep cells lying
adjacent to the superficial layer often extend inward to the interface and form
a small apex there (see No. 3 in Fig. 10a, e). Microprotrusions of several types
are found on macroprotrusions, as well as on the cell bodies, and these connect
the cells to one another. There are terminal filiform (Fig. 10 a, e), lateral filiform
(Fig. \0a, c, d and e), terminal lamelliform (Fig. 10b), and lateral lamelliform
(Fig. 10b) types of microprotrusions. Some of these microprotrusions may be
214
R. E. KELLER
' K
Cellular basis of epiboly in Xenopus
4
215
•
2-6
O
II
K U
o
X
14
10
10
12
Time (h)
14
Fig. 8. The change in h/l ratio of inner deep cells is related to change in the area of
the inner deep cell apices. As the h/l ratio increases (from 8 to 12-2 h), the observed
apical area decreases and this decrease is greater than that expected, given that these
cells show the volume changes in Fig. 18 and that they are roughly cylindrical with
a height/diameter (h/d) ratio of 10 (since these cells are isodiametric (see page 212),
length, as measured in this study, should approximate the diameter and therefore
(h/d) and (.h/l) are used interchangeably). After 12-2 h, the (h/l) ratio decreases
toward 1 0 and the apical area increases and approaches that value expected from cell
volume calculations, assuming the cells to be cylindrical with {h/d) equal to 1-0. If
the cells are assumed to be cylindrical and cell volume data is used to calculate apical
area, with adjustment for the observed change in (h/l), Ihe calculated apical areas
approximate those actually observed (compare' observed' and' cyh, h/d adj.'). These
data show that the inner deep cells begin as cylinders with height about equal to
diameter, change to cylinders with height greater than diameter, and back again to
near the original shape (see drawings at the top of the Figure). Each data point is a
mean of an average of 460 measurements of apical area of individual cells.
FIGURE 7
Fig. 7. Low power electron micrographs of stage-11 (a) and - H i (b) gastrulae from
which most of the involuted mesoderm and endoderm has been removed, show the
exposed inner apices of the inner deep cells. These apices were photographed by
tilting the specimen about 90 degrees, such that the electron beam passes just above
the opposite rim and normal to the surface of the inner apices of the inner deep cells
in the following regions; DMZ, dorsal marginal zone; VMZ, ventral marginal zone;
BR, blastocoelroof; ANE, anterior neural ectoderm. DL indicates the dorsal lip and
IM shows some involuted mesodermal cells. At higher magnification, the inner
deep cell apices (c) of the dorsal marginal zone of stage 11 appear to be nearly flat,
polymorphic in shape, and connected to one another by filiform protrusions extending across the apices of adjacent cells (pointers). Magnifications are: (a) 41 x ,
bar = 100/mi; (b) 54x, bar = 100/*m; (c) 288 x, bar == 10/mi.
216
R. E. KELLER
12-5
Fig. 9. Frequency distributions of cell height/length (h/l) ratios are shown for seven
stages of development. After the onset of gastrulation (stage 10 + ), the frequency
distribution broadens and shifts to the higher values of (h/l), until stage 11, when it
begins to move downward.
retraction fibres. Superficial and deep cells are connected to one another by
microprotrusions extending from both cell types (Fig. \0a, e). In rare cases, all
in the blastocoel roof, a macroprotrusion of a superficial cell extends through
the deep layer and forms an apex bounding the blastocoel (Fig. 10/). The inner
apices of the inner deep cells are connected by marginal filiform protrusions
which extend, often a considerable distance, across the apices of adjacent cells
(Fig. 7 c). These are found without noticeable variation throughout the DMZ,
VMZ, ANE and BR regions of the gastrula.
FIGURE
10
Fig. 10. Several types of protrusions are found on the superficial, inter and inner
deep cells. Small protrusions (microprotrusions) of several types are found. These are
lateral filiform protrusions (//) found extending laterally from superficial (c), inter
(dand e) and inner (a, c and e) deep cells, and lateral lammeliform protrusions (11 in
d). Terminal filiform protrusions (//) and terminal lammeliform protrusions (tl) are
found on the large blunt or tapered extensions (macroprotrusions) of the cell body of
inter or inner deep cells (a, b and e). Some cells have a rounded cell body with long,
tapered, lanceolate macroprotrusions extending between cells deep or superficial to
it (cells No. 1 in c and d). Others are more blunt (cells No. 2 in a, b, and e). Some
extend the full depth of the deep layer and have a small apex (sa) on the interface
(cell no. 3 in c and e). Some superficial cells have protrusions extending deeply
into the deep layer (cell No. 4 in a) and in rare cases they may extend through the
deep layer and form an apex on the interface (pointer i n / ) .
Cellular basis of epiboly in Xenopus
(c)
217
(f)
218
R. E. KELLER
The formation of protrusions oriented along radii of the embryo may reflect
an active locomotion of cells between one another in this direction. Cells may
actively migrate between one another by using a cyclical locomotory process
involving the extension of a macroprotrusion, which attaches distally by microprotrusions, to adjacent cells, and shortening of the macroprotrusion, thus
pulling the cell body toward it. There is no direct evidence for this, but
preliminary evidence on the behaviour of cells in an in vitro model system
suggests that active interdigitation does, in fact, occur. The entire outer shell of
the gastrula (shaded region in Fig. 2) is removed and pinned with glass needles,
inside up, to a bed of Permoplast in a culture dish of modified Steinbergs
solution. Clumps of cells are then added to this surface and their behavior
recorded with time-lapse cinemicrography, followed by fixation and scanning
electron microscopy. Deep cells of aggregates from regions thought to be
undergoing active interdigitation do, in fact, migrate between the native
inner deep cells on which they are placed and form a smooth pavement of polygonal apices indistinguishable from those of the native cells (see Fig. 7 c). In
contrast, cells which have already undergone involution and which do not
normally interdigitate further, remain as a clump sitting on the inner apices
of the inner deep cells. Some of the marginal cells of the clump will migrate a
short distance but they show no tendency to move between the cells serving a
substratum.
The range and continuity of the h/l frequency distributions, and the appearance of a continuous morphological series from a rounded cell body, to a rounded
cell body bearing an increasingly larger, often oriented, macroprotrusion, and
finally an elongate or columnar cell, suggest that, in addition to first serving as
locomotory structures, the formation of oriented macroprotrusions also serves
as a step in deep-cell elongation and thus the formation of the columnar array
of deep cells at stage 11 (Fig. 4).
Anterior neural ectoderm. Through stage 10^, a large region on the dorsal
side of the embryo appears to be nearly homogeneous, but from stage 11 onFig. 11. An overview of equivalent regions of the dorsal side of the gastrula at stage
10-| (above) and 12£ (below) shows the changes in cell size, shape and arrangement
which have taken place between these two stages. As the dorsal marginal zone
becomes thinner, it extends to the right, toward the blastopore, where the mesoderm
of the deep region undergoes involution in the mesodermal involution zone (MIZ).
The mesodermal cells then move to the left, toward the animal pole, along the inner
apices of the inner deep cells, forming the interface (INT). By stage 12|, the DMZ
has thinned and the inner deep cells are cuboidal. In contrast, the ANE has not
thinned, complete interdigitation has not occurred, and the inner deep cells are
relatively tall. The inner deep cell apices of the DMZ are large (mean of 309 ± 27 /im2)
and those of the ANE relatively small (115 ± 12 /*m2) (see inserts above). Most involuted mesoderm has been removed from the second specimen, but a few involuted
mesodermal cells (IM) underlying the ANE remain. Magnifications are 128 (above)
and 122 (below).
Cellular basis of epiboly in Xenopus
Stage 10 "2
219
220
R. E. KELLER
60
.a 80
100
120
6 0
' 50
100
4-0
80
60
30
40
2-0
20
10
Time (h)
Stage
6
8
8
9
9
10
10
10+
11 12
13
lOi-11 11-f
14
12
Fig. 12. Above, the positions of the superficial layer, the inter deep cell layer, and
the inner deep cell layer of the anterior neural ectoderm (ANE) are plotted from
stage 8 through 12£. Below, the layer index, the per cent inner deep cells, the per
cent interdigitation, and the per cent of inner-to-superficial cell contact are plotted for
the same stages. Each data point for position is a mean of an average of 50 measurements. Each data point for the layer index is a mean of five determinations from
each of an average of 30 specimens.
ward, a region high in the dorsal sector, the prospective anterior neural ectoderm
(ANE), remains thick while the region closer to the blastopore (the DMZ) thins
(Fig. 11). The layer index and the total thickness of each region were compared
statistically at stage 10, 10|, 11, and \\\, to determine at what stage the hypothesis that the two regions were identical with respect to these parameters would
be rejected and thus indicating separate treatment of the two regions. These
parameters were chosen because they showed a nearly normal frequency distribution. Both parameters were significantly different between the two regions at
but not before stage 11, according to the Student's t test (P < 0-01) and thus
at stage 11 and later the two regions were treated separately (see Figs. 5 and 12).
Cellular basis of epiboly in Xenopus
221
3-4
30
Super.
Inter
Inner
2-6
^2-2
1-8
1-4
10
0-6
Time (h)
Stage
6
8
9
10
10
10+
11
12
13
lOf 11 14-
14
12}-
Fig. 13. The height/length (h/l) of the superficial, inter deep and inner deep cells
of the anterior neural ectoderm (ANE) are plotted for stages 8 through 12$. Each
data point is a mean of an average of 46 measurements.
At stage 11, the ANE remains 15 /im thicker than the DMZ; the layer index
remains higher (2-7 versus 2-2); the percent interdigitation and percent inner-tosuperficial cell contact remain less than 30% compared to 90% and 100%
respectively, for the DMZ (compare Figs. 6 and 13). The h/l ratios of inner and
inter deep cells rise to 3-6 and 2-5 respectively, by stage 1\\ and decreases only
slightly by stage \2\ (Fig. 13). At the end of gastrulation the ANE is 64 /im in
thickness, or about double that of the DMZ. Thus in the ANE, deep-cell elongation occurs but, unlike the situation in the DMZ (Fig. 5), interdigitation does
not follow (Fig. 12).
Ventral marginal zone. The ventral marginal zone (VMZ) could not be distinquished from the DMZ until stage 10 (Fig. 14) and therefore the data are
combined and the plots of all parameters are identical for the DMZ and VMZ
to stage 10. At the onset of gastrulation, the VMZ shows similarities in appearance to the DMZ (compare Fig. 4 with 13 and Figs. 5 and 6 with 15) but thinning
is slower in the VMZ. This is to be expected since the extension and involution
of the ventral sector is delayed relative to the DMZ (see Keller, 1976). Also,
the VMZ shows an earlier but less pronounced rise in h/l ratio of the inner deep
cells, and no rise at all among the inter deep cells (Fig. 15). Indeed, only the
low VMZ (the region from which quantitative data was taken, Fig. 15) seems
to behave like the DMZ in showing an increased h/l ratio of inner deep cells.
At or after stage 10-^-, the higher part of the VMZ (nearer the animal pole)
behaves like the blastocoel roof (see Figs. 16 and 17); quantitative measurements
from micrographs taken in the high VMZ are similar to those from the BR.
For example, the mean total thicknesses for the low VMZ, the high VMZ, and
15
EMB 60
222
R. E. KELLER
10
fir &
Fig. 14. Scanning electron micrographs of the ventral marginal zone (VMZ) of the
stages indicated (at the lower right) show the change of cell morphology and
arrangement through the gastrula stages (stage 10+ through 12£). The micrographs
are reproduced at about 280 x .
the BR at stage 10^ are 76, 48 and 43 /tm respectively. The correspondingly
layer indices are 2-9, 2-2, and 2-1.
Blastocoel roof. The changes in cell size, shape and arrangement in the course
of epiboly of the blastocoel roof (BR) are shown in electronmicrographs (Fig. 16)
and by quantitative data (Fig. 17). There are two periods of thinning of the
blastocoel roof. The first, of 53%, occurs from stage 8 to stage 10+ and is
distributed between all three layers. These changes are probably due to three
rounds of cell division from stage 8 to 10 + (Fig. 18), and these alone would reduce
the linear dimensions of the cells by 45 %. Since the cells remained cuboidal or
Cellular basis of epiboly in Xenopus
8
9
9
10
10
10+
11
10J
12
11
13
ni
14
12
223
hr.
Stage
Fig. 15. Above, the positions of the superficial, the inter deep and the inner deep layers
of the ventral marginal zone are plotted for stages 8 through 12\. The center, the
layer index, the per cent inner deep cells, the per cent interdigitation, and the per cent
inner-to-superficial cell contact is plotted for the same stages. Below, the (h/l) ratios
of superficial, inter deep and inner deep cells are plotted. Each data point for
position is a mean of an average of 27 measurements, and each data point for the
(/;//) ratio is a mean of an average of 30 measurements. Layer index data points are
means of an average of five determinations from each of 26 specimens.
approximately cylindrical with height and diameter equal (Fig. 17), change in
cell shape probably has little effect on layer thickness in this period.
Concurrent with the decrease in their linear dimensions, complete interdigitation of inner and inter deep cells occurs by stage 10 + . In the BR, the
tendency of cell divisions in the pregastrular period to increase the number of
cell layers is more than countered by thinning, and the number of cell layers
actually decreases slightly (Fig. 17). In contrast, the DMZ shows no permanent
thinning prior to the onset of gastrulation, and therefore cell division increases
15-2
224
R. E. KELLER
11
•T ^
?
".* > . /
v
•
, •/
Fig. 16. Scanning electron micrographs of the blastocel roof at the stages indicated
show the change in morphology and arrangement of cells in the blastula (stages 8
and 9) and in the gastrula (stages 10 through 11£). Magnification is about 285 x .
Cellular basis of epiboly in Xenopus
E
225
Thi ckness
40
60
80
100
30
2-6
•
idex
2-2
1-8
14
14
§ 10
0-6
•
Time (h)
Stage
^—- Y 8 ^
r
6
8
i
i
7
•
1
i
8
9
9
10
10
i0+
1
1
1
,
,
1
11
12
13
104 11 114
14
12
Fig. 17. Above, the positions of the superficial layer, the inter deep layer, and the
inner deep layer are plotted for stages 8 through 12£. In the center, the layer index,
the per cent inner deep cells, the per cent interdigitation, and the per cent inner-to-
superficial cell contact are plotted for the same stages. Below the (h/l) ratios of the
superficial, inter deep and inner deep cells are plotted for stages 8 through 12i.
Each data point for cell position is a mean of an average of 37 measurements. Each
(h/l) data point is a mean of an average of 36 measurements. Layer index data points
are means of an average of five determinations from each of 14 specimens.
the number of layers. This is consistent with the spreading behavior of the two
regions. Spreading, and presumably thinning, occurs throughout the blastula
and gastrula stages in the blastocoel roof, but occurs only after the onset of
gastrulation in the DMZ (see fig. 3, Keller, 1978). Indeed, it is likely that spreading of the deep region vegetally during the blastula stages actually supplies cells
to the deep marginal zone (see Discussion, page 232).
The /;// ratio of inner and inter deep cells of the BR does not increase to the
extent found in the DMZ (compare Figs. 6 and 17), and the decrease in the
h/l ratio after stage lO? brings these values to well below 1-0. This may be
related to major differences between the DMZ and the BR in the mechanics and
behavior of cell spreading (see Discussion, page 231). In fact, there is a rapid
226
R. E. KELLER
decrease in h/l ratio of inner deep cells of the BR between stage 11 and 11-^
(Fig. 17), which may be related to events in the ANE. Above (toward the
animal pole) and perhaps to the sides of the thickened ANE at stage 11-^-, there
is a region of the BR which shows very thin, flattened inner deep cells (Fig. 16).
The margins of the inner apices of these cells seem to be overlapping one another
in the direction of the thickened ANE. This may indicate a shifting and spreading of cells toward the ANE as the cells there become columnar and take up
less area.
Relationship between layer indices and interdigitation
The layer index and the per cent inner deep cells were chosen as related but
independently evaluated criteria of thickness in terms of cell layers, and they
serve as checks on one another. The latter should equal one, divided by the
layer index minus one. Using this relationship, the mean values of the layer
index for a given region and stage was used to calculate the expected value of the
per cent inner deep cells and this value was compared to the observed per cent
inner deep cells. The correlation coefficient between the expected values
and the observed mean values is 0-98 for the BR and DMZ and 0-96 for the
VMZ, and the slopes are 1-04, 0*99 and 0-91 respectively when expected values
(ordinate) are plotted against observed values (abscissa) (data not shown). It is
also obvious from the graphed data that there is a rather sensitive inverse
relationship between the layer index and the per cent inner deep cells (see Figs. 6,
12, 15 and 17). The per cent inter digitation and the per cent inner-to-superficial
cell contact show coordinate behavior from stage 10 +onward (Figs. 6, 12, and
15) in the DMZ and at all stages in the BR (Fig. 17). At these stages two layers
of deep cells interdigitate to form one layer. More inner deep cells contact
superficial cells, and increasing numbers of inter deep cells, already in contact
with superficial cells, become inner deep cells. Thus as the layer index approaches
2-0, the per cent inter digitation, the per cent inner deep cells, and the per cent
inner-to-superficial cell contact approach 100 as expected (Figs. 6, 15 and 17).
The coordinate behavior of these parameters suggest that they accurately
reflect the positions and the layering of deep cells.
Cell division. Since the apical area and the height of inner deep cells are known
it is possible to calculate, approximately, the volume of inner deep cells. Since
it is known that the total cell volume increases very little through gastrulation
(see Tuft, 1965), any reduction in mean cell volume is due to division. Thus, the
number and times of cell division cycles for all major sectors of the embryo can
be estimated from change in mean cell volume. The minimum mean volume is
reached at stage 11^ (12-8 h) for all regions. Working backwards to the points
at which this minimum volume is doubled, it is estimated that the inner deep
cells divide once during gastrulation, twice between 8 and 9-5 h, and once
before that, for a total of four rounds of division in the period covered in this
study. Inner deep cells tend to divide anticlinally during gastrulation, forming
Cellular basis of epiboly in Xenopus
227
dumb-bells or pairs of rounded cells lying next to one another (see figure 22,
Keller & Schoenwolf, 1977). Superficial cells also divide anticlinally during this
period (Keller, 1978). It is not yet clear whether all divisions during gastrulation
are of this orientation and what role this might have in spreading.
DISCUSSION
A cellular model for spreading of the DMZ. On the basis of the quantitative
data in Figs. 5 and 6 cellular behavior can be related to the increase in area
during extension of the marginal zone {\9d). This model accounts for increase
in area alone. The mechanism of directing this areal increase toward the blasopore will not be considered and for simplicity I have shown only the events from
the three-layered stage (stage 10 + ) onward. First the inner and inter deep cells
interdigitate by extending protrusions between one another, along radii (and
perpendicular to the surface) of the embryo, and concurrently they become
columnar in shape. As interdigitation proceeds the mean number of cell layers
decreases. As the inter and inner deep cells wedge between one another, the deep
region becomes thinner and occupies greater area. When interdigitation is
complete, there is only one layer of columnar deep cells. These cells then flatten
and occupy more area, which results in additional expansion of the deep region.
Concurrent with the expansion of the deep region the superficial layer expands
by flattening, spreading, and division of the superficial cells (see Keller, 1978).
The" same process of interdigitation occurs during stage 10, but involves the
interdigitation of multiple layers of inter deep cells with one another and with
inner deep cells. A similar sequence of events occurs in the low VMZ and presumably in the lateral marginal zone as well, but at progressively later stages
and perhaps with less change in the h/l ratio. In some respects this model may
apply to the blastocoel roof, but in this case there is no marked increase in
//// ratio of deep cells and the cellular activities involved may be different.
It is appealing to think of the increase in h/l ratio as being related to the extension of protrusions of inner and inter deep cells between one another and
thus to the process of interdigitation, but h/l ratio is not always associated with
interdigitation and reduction in the number of layers of cells. The anterior
neural ectoderm shows such an increase without interdigitation, and all other
sectors of the gastrula show interdigitation and decrease in the number of cell
layers, but only the DMZ shows a large increase in the h/l ratio of deep cells.
In the DMZ interdigitation might be an active process requiring an increased
h/l ratio, but in all other sectors it might be a passive process (see page 231)
which does not require such an increase. Alternatively, the larger increase in the
DMZ may be associated with the extension and convergence movements which
are unique to this region. But extension and convergence would be expected to
involve changes in the length and width dimensions, as is the case in the superficial layer (Keller, 1978), rather than changes in height. In fact, deep cells
228
R. E. KELLER
10
0-8
0-6
0-4
1 -M-2-3
0-2
10
Time (h)
12
14
Fig. 18. Estimates of mean inner deep cell volume were calculated from the mean
apical area and the mean height for the blastocoel roof (BR), the dorsal marginal
zone (DMZ) and the ventral marginal zone VMZ. The volume at any time (Vt) was
divided by the volume at 6 h (V6) and this parameter of relative volume (Vt/V6) is
plotted against age in hours (h). From the changes in volume it is estimated that about
four cell divisions take place over the period studied - three in the late blastula stage
and one in the course of gastrulation. The minimum cell volumes at stage 11$
(12-2 h) are 3800, 4200, 4800/tm3 for BR, DMZ and VMZ respectively.
FIGURE
19
Fig. 19. A model of cellular behavior during active spreading of the marginal zone
was constructed from interpretation of the quantitative data presented in this
study. It consists of radial interdigitation of several layers of deep cells to form one
layer of tall, columnar cells. These then shorten and flatten. Both the interdigitation phase and the shortening phase result in spreading of the deep region.
A mechanical model of active spreading (b), correlated with the cellular model (a),
was constructed from observations in the literature and from current experiments.
The superficial layer and the deep layer are connected in a bilayer system and each is
represented by a series of elements. The superficial layer is assumed to have no
intrinsic capacity to spread but is under tension and tends to shrink (arrows pointed
toward one another). The deep layer is assumed to be under compression due to areal
expansion (arrows pointed away from one another) brought about by active interdigitation and shortening of deep cells (a). As a result of these forces in each layer,
the bilayer system curves outward, opposite the curvature of the embryo. The entire
outer shell of the embryo can be represented by a bilayer system of this type which
has been forced into the normal curvature of the embryo by attachment at the margins
of the blastopore to the involuted material (shaded in c). Such a spherical shell, consisting of a bilayer of superficial tension-deep compression is mechanically stable;
this can be demonstrated by cutting a hole in a rubber raquet ball and turning it
inside-out (d). Small cuts in the surface gape open, which indicates that the superficial layer is under tension and therefore the deep layer must be under compression.
For contrast, a model of cell rearrangement during passive spreading is shown (e,f).
Tension in the superficial layer would stretch it (e); the superficial cells would expand
and flatten; deep cells would then rearrange to occupy the increased area on the
inner surface of the superficial epithelium (f).
229
Cellular basis of epiboly in Xenopus
• I—I)—II—li—i!—I
I—II—II—II—II—I
•
I—IhHI—IhHI—I
(a)
(c)
(e)
(d)
230
R. E. KELLER
appear to be isodiametric and thus extension and convergence probably occurs
solely by lateral interdigitation similar to that seen in the superficial layer
(Keller, 1978), rather than by change in cell shape.
Is extension of the DMZ or epiboly in general an active process! In order to
make a mechanical model which relates this cellular behavior to the production
of mechanical forces sufficient to bring about the increase in area during epiboly
and extension, it is important to know whether the cell behavior in a given
region is an active, intrinsic property of the cell which generates force, or if it
is a passive response to forces generated elsewhere in the embryo. For example,
the extension of the DMZ might be autonomous and due to local cell behavior,
or it might be due to passive stretching in response to tension generated by
constriction of the circumblastoporal region or by involution. The grafting and
isolation experiments of Spemann (1931; see page 101, Spemann, 1938) and
Holtfreter's observations on exogastrulation (1933) suggest that spreading of the
animal half and extension of the marginal zone are autonomous processes
arising from forces generated by local cell behavior. But it is not clear whether
the observed marginal zone extension is postgastrular notochord extension or
the earlier gastrular extension (see Holtfreter, 1944). Also, there has been no
quantitative comparison of DMZ extension in situ with involution proceeding
and in situ with involution blocked. Schechtman (1942) observed elongation in
the absence of involution but this may have been notochord elongation. Expansion of the ectoderm in isolated ventral halves (Spemann, 1931) or in exogastrulae (Holtfreter, 1933) was presumed from the folded appearances of the
specimens and was not documented quantitatively. Time-lapse films of exogastrulae show that the ectoderm becomes corrugated by rapid constrictions of
the apices of superficial cells and by the appearance of holes in the epithelium,
perhaps as the result of powerful contractions of the cell apices (Keller, unpublished data). Thus, it is shrinkage, not expansion, that generates the folded
morphology. Holtfreter's observation that dorsal marginal zone explanted onto
glass or a field of endoderm will elongate and that this process is dependent on
the original cellular arrangement remaining intact (Holtfreter, 1944), is the
strongest evidence for active extension. Until supporting evidence is available,
though, it is prudent to consider both active and passive models of spreading.
A mechanical model for active expansion. Assuming that expansion is active,
it is unlikely that this is due to active expansion of the superficial layer. When the
superficial epithelium is cut, the wound margins gape open, indicating that it is
under tension. The fact that the apices of superficial cells of the extending
DMZ show an increase in l/w ratio followed by a decrease as the cells rearrange
suggests that this region behaves as a viscoelastic system which is stretched,
under tension (see Keller, 1978). These cells have machinery for producing
tension. Superficial cells contain circumapical bands of microfilaments which
bind heavy meromyosin and are undoubtedly actin (Perry, 1975). Apparently,
the cortical microfilament system can be made to contract by several conditions
Cellular basis of epiboly in Xenopus
231
which raise the intracellular free calcium levels (Gingell, 1970). If EGTA is
injected into the egg, the cortex relaxes, bulges outward, and finally bursts
(Baker & Warner, 1972). These observations suggest that the cortical region of
the egg and, at later stages, the apices of the superficial cells contain a calciumregulated contractile system which is maintained under tension. If the superficial layer of the Xenopus gastrula is under tension and yet epiboly or extension
is an active or intrinsic property, the force for spreading must arise in the deep
region, probably from the active interdigitation and subsequent shortening of
deep cells (Fig. \9d). In this model, the outer shell of the gastrula would consist
of a bilayer system - a superficial layer under tension and an attached deep
region with a tendency to expand forcefully (Fig. 19b). Spreading of the deep
region would be resisted by tension in the superficial layer and outward curling
of the bilayer would result. However, if the bilayer is constrained by attachment
of its margins to the involuted material at the rim of the blastopore, and if the
resistance to compression in the deep region is greater than the tension in the
superficial layer, spreading of the entire bilayer would result from active expansion of the deep region (Fig. 19 c). Such a bilayer system is mechanically
stable. This can be demonstrated by cutting a hole in a rubber raquet ball and
turning it inside-out such that the superficial component is under tension and the
deep component is under compression (Fig. 19 d).
There is considerable evidence in support of the bilayer model described
above. It is a common observation that when pieces of the superficial-deep
bilayer of the gastrula are excised, their immediate behavior is to curl to the
outside, opposite the curvature of the embryo, suggesting that the deep layer is
under compression and that the superficial layer is under tension. Second, cuts
made in the superficial layer gape open, again suggesting tension. Third, the
evidence presented in this paper shows that the proposed method of spreading
of the deep region does occur. Fourth, preliminary evidence (see last section of
the Results) shows that excised deep cells of the marginal zone prior to involution will actively interdigitate between cells of the inner surface of the blastocoel
roof or the marginal zone. Furthermore, there is apparently a behavioral
change upon involution, for cells which have involuted do not interdigitate
between the inner deep cells of the uninvoluted marginal zone but instead use
their apices as a substratum for migration (see Nakatsuji, 1974, \915a, b, and
Schoenwolf, 1977). This behavioural change from an interdigitating to a
migrating cell type is correlated with the morphological differences between
preinvolution and involuted cells, and is the basis for the formation of the
interface between the two cell types (see Keller & Schoenwolf, 1977).
A mechanical model for passive expansion. In contrast, a passive model of
expansion would hold that the superficial epithelium is stretched by tension
generated at the margin of the blastopore and that the superficial cells passively
spread (Fig. 19e). As they do so the deep cells would actively rearrange themselves to occupy the increased area available to them but would not forcibly
232
R. E. KELLER
interdigitate or generate a spreading force. In this model, the entire shell of
uninvoluted material would be pulled down over the involuted material by a
dynamic locus of circumblastoporal constriction or perhaps by involution itself.
Differences in thickness and areal spreading between the deep and the superficial layers. The total cell volume remains nearly constant during gastrulation
(see Tuft, 1965), and the extracellular spaces in the regions undergoing epiboly
extension do not appear to change, since cells in these regions seem to be closely
packed at all stages. Therefore an inverse relationship should exist between the
thickness and the amount of areal expansion in a given region. Overall, this
expected inverse relationship is found in the superficial layer from the early
gastrula stage onward, but the amount of thinning in the deep region is much
greater than that in the superficial layer, implying that spreading in the deep
region should far exceed that of the superficial layer. As calculated from its
decrease in thickness, the area of the deep region of the DMZ should increase
by a factor of 2-56 from the early gastrula (stage 10 + ) to the late gastrula (stage
12^), whereas actual expansion of the superficial layer of this region is only by
a factor of 1-95 (Keller, 1978). The same figures for the deep region of the
blastocoel roof from stage 10+ to 11£ are 1-79 for expected and 1-2 for actual
expansion. The thinning, and therefore spreading, of the deep region is most
pronounced in the period from stage 10 to 10 + , in which the deep region must
expand by a factor of 1-85 in a short time. Where does this newly generated
area go? Undoubtedly it is fed into the involuting mesodermal cell stream,
which forms early in Xenopus (see Nieuwkoop & Florshiitz, 1950; Keller, 1976),
and it probably also contributes to the rapid extension of the dorsal marginal
zone vegetally during this period (see Keller, 1978).
Since the deep region expands more rapidly than the superficial layer it must
move vegetally faster. Indeed, the upper border of the prospective mesoderm
in the deep region lies above the corresponding upper boundary of the suprablastoporal endoderm in the early gastrula but coincides with it in the neurula
(Keller, 1976). The great expansion of the deep marginal zone in the early
gastrula stage suggests that its upper boundary lies much higher in the blastula
at stage 8 or 9, than indicated on maps of the early gastrula (Keller, 1976).
Experimental cellular analysis of amphibian gastrulation. Since the behavior
of superficial cells can be followed by time-lapse cinemicrography and at least
some aspects of deep cell behaviour can be studied by the methods described
here, an experimental analysis of gastrulation movements at the cellular
level is in order. First, it is important to determine whether cell behavior
in both the deep and the superficial layers is regionally determined and autonomous, or if it is passive and dictated by systemic mechanical forces. This
question can be answered unambiguously by grafting the superficial layer, the
deep layer, or both from one region of the gastrula to another and determining
with time-lapse cinemicrography and SEM whether cells of the grafted patch
undergo spreading and rearrangement as they would have in their original
Cellular basis of epiboly in Xenopus
233
location or adopt the pattern characteristic of the region into which they were
grafted. Secondly, experiments must be done to map the mechanical stresses in
the embryo and relate this to cellular behavior. Beloussov, Dorfman &
Cherdantzev (1975) mapped mechanical stresses by recording the behavior
of freshly isolated fragments of the gastrula. Additional work of this type must
be done in order to understand the forces that actually bring about distortion
of the embryo. Thirdly, the social behavior of cells with different or changing
morphogenetic properties should be studied in vitro. Fourthly, these cells
should be examined at the ultrastructural and molecular levels for differentiation
of cytoskeletal, junctional, and cell surface components which might relate to
these differences in morphogenetic behavior. It is important that these last two
studies be directed toward cell populations with specific functions, such as preinvolution deep cells and deep cells after they have involuted, rather than
traditionally important geographic regions such as the 'dorsal lip' which contains both these and probably more types of cells.
1 wish to thank Professors R. Briggs and J. P. Trinkaus for their support and suggestions,
Dr W.-T. Chen, Dr G. Radice, and M. Shure for helpful discussions, Dr John Gerhart for
reading the manuscript, and Dorothy Barone and Patricia White for technical assistance.
This work was supported by NTH grant No. USPHS-HD-07137 to J. P. Trinkaus and
No. R01 GM 05850-21 to R. W. Briggs, and by American Cancer Society Fellowship
No. PF 1174 to the author.
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(Received 10 December 1979, revised 6 May 1980)