Download Liquid-Tissue Mechanics in Amphibian Gastrulation: Germ

AMER. ZOOL., 18:81-93 (1978).
Liquid-Tissue Mechanics in Amphibian Gastrulation: Germ-Layer Assembly
in Rana Pipiens
HERBER'I M. PHILLIPS
Department of Anatomy, Wayne State University School of Medicine, Detroit, Michigan
48201
AND
GRAYSOX S. DAVIS
Department of Biology, University of Virginia, Charlottesville, Virginia 22901
SYNOPSIS During amphibian gastrulation, migrating subsurface germ layers may flow past
one another like immiscible viscous liquids in response to tissue surface tension forces. We
describe here two physical tests for liquid-tissue morphogenesis in cultured aggregates of
subsurface ectoderm (E), mesoderm (M) and endoderm (N) excised from mid-yolk-plug
Rana pipiens gastrulae. (i) Liquids are coherent substances in which subunits can slip past
one another to relax internal shear stresses. We find, in cross-sections of cell aggregates
fixed during compression, that cells within flattened aggregates are intially deformed, but
do, as predicted, gradually reassume their original, undistorted shapes, (ii) Surface
tensions (y's) govern ordinary liquid-droplet spreading; e.g., if equal-sized droplets A and
B fuse in medium 0, B spreads around A when yA0 > yao- When pairs of subsurface
aggregates are cultured together, N surrounds M and E, and M surrounds E. To see if yE0
>
"YMO > TNO. we flatten aggregates with quartz fibers calibrated to measure the force of
compression. As predicted, under the same flattening force, E aggregates are rounder
than M aggregates, which are rounder than N aggregates. Furthermore, a second surface
tension relationship can account for the autonomous involution of M between E and N;
and these surface tension relationships can also explain the inversion of E, M and N by
coated ectoderm to produce normal gastrular germ-layer arrangements. We conclude
that, combined with active cell shape changes in solid-like surface cell layers and also with
autonomous elongation of dorsal lip mesoderm, tissue surface tension control of liquidtissue flow in subsurface germ layers is a key morphogenetic mechanism in amphibian
gastrulation which might be regulated by changes in intercellular adhesiveness.
IXTRODL'CTIOX
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partment of Biology, by NSF grant GBg40041 to R a t h e r , t h a n attempting to inventory the
H.M.P. G.S.D. is supported by NIH traineeship myriad changes in differentiating cell surHD00430.
faces, we have sought to characterize the
81
82
HERBERT M. PHILLIPS AND GRAYSON S. DAVIS
mesoderm and endoderm in late blastula
and gastrula stages by cell size (smaller,
intermediate and larger, respectively) and
color (dark gray, light gray and white,
respectively). We have not attempted to
determine the precise fates of these cell
populations, and simply avoid using cells
wherever the above morphological differences are not perfectly distinct under
the dissecting microscope. However, the
three cell populations so defined lie precisely in the positions, both in blastula and
gastrula stages, assigned to presumptive
ectoderm, mesoderm and endoderm by
Vogt (1929) in his dye-marking experiments on Rana esculenta and Ranafusca.
As Holtfreter (1943a,6; 1944) did, we
can peel away with dissecting needles a
continuous, single-cell layer from the surface of the late blastula, and from external,
archenteric and blastopore lip surfaces in
gastrula stages, thus uncovering multilayered subsurface populations of presumptive ectoderm, mesoderm and endoderm. Under the dissecting microscope,
subsurface cells of each type appear to be
adhering uniformly, although sometimes
quite loosely, to other cells on all sides. By
contrast, the outer faces of the surface cell
populations appear to be shiny—"coated"
in Holtfreter's terminology. We find, as he
did (op. cit.), that the shiny sides of these
surface cells are non-adhesive with respect
to blastula and gastrula cells of all types.
(By contrast, their "uncoated" lateral and
basal sides do adhere to other uncoated
ment, certain embryonic tissues may become cell surfaces.)
temporarily converted into liquid-like cell masses
We observe, as Holtfreter did, that durwhich flow from one configuration into another ing gastrulation shiny surface ectoderm
guided by tissue surface tension forces. While plus underlying subsurface ectoderm
those tests involve only artificial combina- gradually spread over the entire surface of
tions of morphogenetically remote chick the embryo. In late (mid-yolk-plug—
cell populations, this hypothesis may also Shumway [1940] stage 11+) gastrulae,
be applied to contiguous, migrating popu- after dissecting away surface ectoderm, we
lations of subsurface ectoderm, mesoderm routinely obtain samples' of subsurface ecand endoderm in amphibian gastrulation. toderm from the anterior hemisphere of the
embryo opposite the receding yolk plug
(including all of the roof of the now greatly
LOCATING AND DISSECTING COATED AND SLBdiminished
blastocoel) by peeling it off of
SLRFACE GERM LAYERS
underlying mesoderm and endoderm with
In our analysis of tissue rearrangements a hairloop. We also confirm Holtfreter's
in Northern Rana pipiens embryos, we op- conclusion that in these late gastrula stages
erationally define presumptive ectoderm, coated endoderm cells cover the plug and
morphogenetic forces immediately responsible for tissue movements, avoiding,
insofar as is possible, artifacts due to in
vitro tissue manipulation.
The monumental studies of Holtfreter
(1939; 1943a,6; 1944) and of Townes and
Holtfreter (1955) demonstrate that small
groups of cells excised from amphibian
gastrulae and neurulae and placed in
organ culture can move autonomously i.e.,
independently of the rest of the embryo, to
form miniature copies of in vivo embryonic
structures. Therefore, these germ-layer
migrations are evidently driven by intrinsic
morphogenetic forces originating within
the tissues themselves. Moreover, these
forces seem to operate normally, and thus
can be reliably examined, in in vitro culture
conditions.
Steinberg (1963, 1964, 1970, 1975) has
pointed out that the tissue-spreading and
cell-sorting movements observed in vitro in
amphibian germ-layer (as well as laterstage embryonic chick) tissue masses are
analogous to rounding-up, droplet coalescence and the break-up of emulsions
into homogeneous phases in systems of
immiscible liquids. The intrinsic forces
governing these latter processes are the
surface tensions at immiscible liquid interfaces. Steinberg and I and co-workers
(Phillips, 1969, and in preparation; Phillips and Steinberg, 1969, 1978, and in
preparation; Phillips et al., 1973, 1977a;
Phillips et al., 1977ft) have been refining and
testing the hypothesis that, during develop-
LIQUID-TISSUE MECHANICS IN GASTRULATION
the inner lip of the blastopore, and continue inward to line the floor, sides and
anterior roof of the archenteron. We dissect subsurface endoderm out of the region
underneath the coated endoderm lining
the floor of the archenteron. Similarly,
coated mesoderm covers the outer lip of
the blastopore, and dorsal coated
mesoderm may provide the lining for the
posterior roof of the early archenteron.
(According to Vogt [1929] and Mayer
[1931], in anurans this dorsally involuted
surface mesoderm soon becomes displaced
by coated endoderm migrating up to line
the entire archenteron roof. Others argue
that all surface cells which in the anuran
blastula are located above subsurface
mesoderm will actually become part of
subsequent endodermal structures [Keller,
1975].) We note that the surface cells lining
the outer lip of the blastopore (they cover
subsurface layers of involuting mesoderm
cells and thus up to this stage would be
classified as "coated mesoderm" cells on
morphological grounds) seem to lose their
shiny coats once they have moved a few cell
diameters forward into the embryo away
from the outer margin of the lip. In late
gastrula stages, after dissecting away surface (coated) ectoderm cells and then peeling off one multilayered group of subsurface cells (subsurface ectoderm in anterior
regions, yet-to-be-involuted subsurface
medoserm in posterior regions), we can
peel a several-cell-thick layer of lateral
(and, if desired, ventral) involuted subsurface mesoderm off of the underlying endoderm. Moreover, if we avoid freshly
involuted mesoderm close to the blastopore, our mesoderm aggregates have no
shiny, coated cells and will adhere on all
sides to other subsurface cell aggregates.
(For reasons to be explained below, we
avoid using subsurface dorsal mesoderm in
our tests for tissue liquidity.)
83
microfilaments evidently produce active
cell shape changes which cause tissue thickening and bending. Similar cell shape
changes have been observed in coated amphibian germ layers—e.g., bottle-cell formation in surface cells at the blastopore lip
(see Holtfreter, 1943a,6; Baker, 1965;
Perry and Waddington, 1966). Therefore
this intracellular force-generating mechanism may be responsible for external, archenteric and blastopore lip morphologies during amphibian gastrulation.
It requires, however, that tissues behave
like deformable solids. Cells must be
tightly bound to one another to prevent
cell shuffling movements that would disrupt coordinated tissue deformations.
While useful in thin-tissue morphogenesis,
this prohibition of cell shuffling movements would cause severe cell distortions
in subsurface, thick-tissue masses during
gastrulation (Fig. 1). In fact, coordinated
cell shape changes in gastrulating germ
layers seem to be confined to surface
layers; cells in subsurface layers appear to
maintain relatively undistorted shapes as
they migrate (e.g., Holtfreter, 19436;
Baker, 1965; Nakatzuji, 1975). We have
therefore been exploring the hypothesis
that subsurface germ layers instead flow as
liquid-like masses during gastrulation.
Also, since subsurface tissue movements
seem to be governed by intrinsic morphogenetic forces (see above), we have
been examining the further possibility that
subsurface germ-layer flow is directed by
tissue surface tension forces.
TESTS FOR CELL SLIPPAGE PERMITTING LIQUIDTISSUE FLOW
A liquid is defined (Symon, 1971) as a
cohering substance in which internal shear
stresses are spontaneously dissipated as the
subunits of which it is composed slip past
one another. Consider two models for
originally spherical cell aggregates in cul"SOLID" AND "LIQUID" TISSUES IN AMPHIBIAN
ture medium that are deformed by being
CASTRULATION
compressed between parallel glass
In single-cell-thick epithelia in a variety coverslips (Fig. 2). In the elastic-solid
of embryonic systems (Wessels et al., 1971; model, cell slippage movements are prohibBurnside, 1973; Spooner, 1973; Karfun- ited. Therefore, initial horizontal stretchkel, 1974), cytoplasmic microtubules and ing of interior cells should continue as long
HERBERT M. PHILLIPS AND GRAYSON S. DAVIS
(A)
N
(B)
/
FIG. 1. In anuran amphibian gastrulae, thick prospective germ layers migrate past one another as
cohering, multilayered cell masses, as schematically
diagrammed here. A, A blastula with monolayered
surface and multilayered subsurface ectoderm (E and
E), mesoderm (M — but see text — and M) and endoderm (N and N) will form a trilayered gastrula. B,
If strong cross-linking were to prevent cell slippage,
these solid-tissue redistributions would result in
major cell distortions (although not necessarily in the
directions shown simply for illustrative purposes
here). C, In fact, most subsurface cells are not markedly distorted during gastrulation.
as the aggregate remains compressed. By
contrast, in the viscous-liquid model,
aggregates would behave like slow-flowing
liquids composed of elastic subunits (cells).
Thus, during prolonged aggregate compression, initially stretched cells should be
free to slip past one another and return to
their original, undistorted shapes, even
though the aggregate would remain flattened.
To test for cell slippage in deformed cell
aggregates, we allowed excised fragments
of subsurface ectoderm, mesoderm or endoderm to round up for several hours in
Steinberg's medium (Hamburger, 1962) in
dishes coated with agar (Colab Ionagar
#2). We compressed each one, immersed
in medium, between parallel glass surfaces
in a miniature vise. We fixed aggregates
during compression by gently pipetting
LIQUID-TISSUE MECHANICS IN GASTRULATION
A. Elasticsolid
model
85
B. Viscous
liquid
model
I. Before compression
II. Initial compression
I. Continued compression
FIG. 2. Liquids are cohering substances in which
internal shear forces are relaxed by subunit slippage
movements. A, Elastic-solid model for compressed
aggregates: cell stretching but no cell slippage. B,
Viscous-liquid model (i.e., elasticoviscous-liquid
model — see Phillips and Steinberg, 1978): initial cell
stretching, then slow cell slippage.
HERBERT M. PHILLIPS AND GRAYSON S. DAVIS
2% formalin in culture medium under the
coverslip. Fifteen minutes later the
coverslips were removed and aggregates
were prepared for scanning electron microscopy by standard techniques. After
critical-point drying, we broke compressed
aggregates in planes perpendicular to
their flattened sides to observe cell shapes
in cross-section in the scanning electron
microscope.
Most of our experiments to date have
been performed on subsurface endoderm
aggregates (18 samples), although the few
samples of subsurface ectoderm and
mesoderm which we have examined have
yielded similar results. Loosely packed, initially rounded cells in uncompressed
aggregates (Fig. 3A) become markedly flattened during the first minute of compression (Fig. 3B). After five minutes of compression, interior cells are still quite flattened, although surface cells may begin to
return to their original rounded shapes
(Fig. 3C). However, after fifteen minutes
of compression, virtually all cell stretching
disappears as shear stresses become relaxed, leaving rounded cells within these
flattened aggregates in their original undistorted shapes (Fig. 3D).
These observations are consistent with
predictions derived from the viscousliquid model for cell aggregates and clearly
contradict those derived from the elasticsolid model. Similar cell slippage movements occur in centrifugally deformed
embryonic chick liver aggregates (Phillips
et.al., 1977a) and in whole, intact chick
limb buds compressed with tiny surgical
clamps (Daggy and Phillips, in preparation). However, cell slippage in these
later-staged embryonic chick tissues seems
to require at least several hours, which is
consistent with the slower rate of
rounding-up of chick aggregates (1-2 days)
compared to amphibian germ-layer
aggregates (2-6 hours).
By liquid-tissue flow we mean changes in
the configurations of cohering cell masses
that leave cells undistorted thereafter.
Therefore, the spontaneous relaxation of
internal shear forces reflected in these cell
shape changes is the sole physical prerequisite for liquid-tissue flow. Tissue
0 1 MM
FIG. 3. Scanning electron micrographs of subsurface N aggregates fixed before compression (A), and
during compression after one (B), five (C) and 15
minutes (D), and then broken to reveal interior cell
shapes in cross-section. (Variations in compressed
aggregate heights are due to differing distances set
between the rigid compression plates in our miniature clamping vise.)
liquidity is in principle compatible with, yet
need not depend upon, active cell motility.
Cells in liquid tissues could dissipate shear
forces by self-propelled translocations or
LIQUID-TISSUE MECHANICS IN GASTRULATION
87
by passive slippage movements or both.
Also, tissue liquidity, of course, neither
assumes nor requires that individual cells
must be liquid droplets, any more than
individual molecules in ordinary liquids
need themselves be liquids.
other [Phillips, 1969, and in preparation].
The correlation between completeenvelopment
configurations
and
aggregate-medium surface tensions has
been confirmed for embryonic chick cell
aggregates in previous aggregatecentrifugation experiments [op. cit.; Phillips
and Steinberg, 1969, and in preparation;
TESTS FOR SURFACE TENSION CONTROL OF TISSUE
et al, 19776J.
Phillips
FLOW
Holtfreter (1944) and Townes and
Surface tension is the force within (tan- Holtfreter (1955) reported that, in fused
gent to) a liquid interface which opposes pairs of fragments of gastrula and neurula
any increase in its surface area, if cell subsurface germ layers, endoderm tends to
aggregates behave like liquid droplets, surround ectoderm and mesoderm. Steinthen, in the absence of external deforming berg and Kelland (1967) confirmed these
forces, their behavior should be deter- findings and also reported that subsurface
mined simply by tissue surface tensions. mesoderm tends to surround subsurface
For example, two immiscible but mutually ectoderm. We have obtained similar readhering droplets A and B immersed in sults in our laboratory (Phillips, Mitchell,
medium O form three liquid-liquid inter- MacQueen and Davis, in preparation). Acfaces, A-O, B-O and A-B, with surface cordingly, if these subsurface germ-layer
tensions yA0, yBn a n d TAB respectively (Fig. movements are determined by tissue sur4). When pairs of similarly-sized droplets face tensions, as predicated above, then
with approximately spherical interfaces the surface tensions of subsurface eccoalesce, the surrounding droplet will have toderm (E), mesoderm (M) and endoderm
a lower surface tension with respect to the (N) in medium (O) should fall into the
medium than does the surrounded drop- sequence
let: i.e., (Fig. 4), if droplet B spreads around
droplet A, yB0 is less than yA0 (Phillips, 1969,
and in preparation; Torza and Mason,
1969). (Special cases of this relationship
for coalescing droplets of greatly differing
size are discussed in Heintzelman, Phillips
and Davis fin preparation]. However, this
relationship holds regardless of dropletsize if one droplet completely envelops the
TEO > 7MO > TNO-
(1)
To test this prediction, we have again
resorted to aggregate compression. However, in this case, one compressing glass
chip is mounted on the end of a flexible
quartz fiber (Fig. 5). Using preweighed bits
of aluminum foil, we determine the bending constant of the fiber. If we then observe how much the fiber bends during
compression, we can calculate how much
upward force is flattening the aggregate.
Once internal cell stretching within a deformed aggregate has been dissipated by
cell slippage, its surface tension is the sole
force resisting deformation. Thus, the
greater that surface tension, the rounder
should be the shape of the aggregate
under a given force of compression. In
FIG. 4. For comparably-sized immiscible droplets A
and B fusing with approximately spherical interfaces principle, quantitative measurements of
in medium 0, spreading configurations are deter- droplet-medium surface tensions can be
mined by the three surface tensions yA0, yB0 and yAB. determined by this technique (e.g., YoneIf B spreads around A (rather than A around B), yB0 da, 1964, but see Hiramoto, 1967; Phillips,
< yA0. (yAB determines immiscibility, and in this case
affects the extent of spreading but not the direction of in preparation).
envelopment.)
In practice, we allow freshly excised
HERBERT M. PHILLIPS AND GRAYSON S. DAVIS
88
///7/ffA
V////II
0
FIG. 5. An aggregate (A) in culture medium (O) is
compressed between two glass chips, one of which
(GC) is glued to a quartz fiber (F) which can be raised
or lowered at its other end. The greater the surface
tension at the A-O interface, the rounder will be the
equilibrium shape of the compressed aggregate. (Preliminary analysis of aggregate shapes suggests that
these aggregates form small contact angles with the
compressing glass surfaces.)
samples of subsurface ectoderm, mesoderm and endoderm from stage 11 +
Rana pipiens gastrulae to round up in
organ culture 2-8 hours. Initially round
aggregates are placed on the moveable
glass chip, which is then raised until the
aggregate becomes flattened against the
coverslip mounted on the roof of the
chamber. Within ten minutes after the
final turn of our compression dial, these
aggregates adopt stable ("equilibrium")
shapes which remain constant for at least
another 20 minutes thereafter. (By this
time, of course, internal stretching forces
have been dissipated by cell slippage — see
Fig. 3).
In qualitative terms, our results reveal
that, when flattened by the same compressing force, subsurface ectoderm aggregates
adopt much rounder equilibrium shapes
than subsurface mesoderm aggregates,
which adopt slighdy rounder shapes than
subsurface endoderm aggregates (Fig. 6).
(The height-to-width ratios of mesoderm
and endoderm aggregates compressed by
the same force partially overlap, but preliminary estimates of their aggregate-medium surface tensions are nevertheless statistically different.) Precise quantitative
determinations of aggregate-medium
surface tensions are still in progress. However, our preliminary results are consistent
with relation (1) above and thus tentatively
confirm the predicted correlation between
aggregate behavior and germ-layer surface tensions.
TISSUE-TISSUE SURFACE TENSIONS AND MESODERMAL INVOLUTION
When subsurface samples of all three
germ layers are combined in triplet-fusion
and cell-sorting experiments, endoderm
surrounds mesoderm, which surrounds ectoderm (Townes and Holtfreter, 1955).
The mechanism by which coated germlayer tissues invert this configuration to
form the normal germ-layer arrangement
is discussed below. Here we wish to point
out that a second surface tension relationship, in addition to (1) above, is required to
explain triplet-fusion behavior. The
spreading of mesoderm between ectoderm
and endoderm implies (Fig. 7) that
7 EN > 7 EM +
(2)
E
M
N
0.1mm
FIG. 6. Representative profiles of subsurface aggregates flattened by equal compressing forces until they
adopted stable shapes (see text and Fig. 5). As predicted, aggregate resistance to expansion of surface
area falls into the sequence E > M > N.
89
LIQUID-TISSUE MECHANICS IN GASTRULATION
FIG. 7. When all three subsurface tissues fuse, forming inverse germ-layer configurations, M spreads
between E and N. In a liquid-tissue model, the surface
tensions driving this process can be represented by
vectors drawn tangent to the E-M, E-N and M-N
interfaces. It is evident by inspection that the point at
which all three meet will move to the right in the
above diagram when y^M + ^ N <1YEN- This relationship would account for the spontaneous flow of M
into E-N interfaces observed in in vitro experiments
and for autonomous involution of lateroventral
mesoderm during gastrulation in vivo.
that is, the E-N interface resists expansion
more than the E-M and M-N interfaces
combined. We are only now attempting to
make the tissue-tissue surface tension measurements required to test this relationship
directly. However, tissue behavior, both in
vitro and in vivo, suggests that the flow of
subsurface mesoderm between subsurface
ectoderm and endoderm consistent with
this relationship is a major force in amphibian gastrulation.
For example, Holtfreter (1944) described an experiment (Fig. 8) in which
Lehmann removed the coated and subsurface ectoderm from a small area in the
ventral region of a gastrula, thereby exposing underlying subsurface endoderm. Into
that wound he placed a piece of dorsal lip
mesoderm, only part of which was covered
by coated mesoderm. Subsurface mesoderm fused with the exposed subsurface
endoderm; but in addition, at the edge of
the wound, the subsurface mesoderm went
on to spread between subsurface ectoderm
and endoderm. This movement dragged
the coated mesoderm cells on the exterior
surface of the aggregate inward to form an
archenteron-like cavity. Of course, such
transplanted dorsal lips go on to induce
secondary axes in the- host embryos
(Spemann and Mangold, 1924).
Schechtman (1942) mechanically isolated various portions of the blastopore lip
in intact Hyla regilla gastrulae using cellophane inserts, and found that lateral and
ventral mesoderm involute autonomously,
but dorsal mesoderm involutes only when
connected with lateral mesoderm. Recall
that in early gastrulae, while bottle cell
formation in surface mesodermal layers
can produce some initial blastopore lip
invagination, there is no dorsal subsurface
ectoderm-endoderm interface into which
the subsurface mesoderm can then spread.
By contrast lateral and ventral lip
mesoderm is confronted with such an E-N
interface. Given the capacity of dorsal lip
mesoderm to spread in such an interface
(as demonstrated by Lehmann's experiment described above), Schechtman's results support the hypothesis that mesodermal involution in vivo is driven by the
flow of subsurface lateroventral mesoderm
M
B.
M N
\
M
M N E E
FIG. 8. Diagram by Holtfreter (1944) of experiments by Lehmann (our labels added). When implanted into a wound in the ventral ectoderm of a
host embryo, a graft of dorsal lip mesoderm spontaneously flows into the subsurface E-N interface.
The coated surface of the graft forms an
archenteron-like cavity, and can give rise by induction
to an entire secondary axis. Tissues here are labeled
as in Figure 1.
90
HERBERT M. PHILLIPS AND GRAYSON S. DAVIS
into the E-N interface. We propose that
this autonomous tissue flow is driven by
intrinsic tissue-tissue surface tension forces
according to relation (2) above.
GERM-LAYER INVERSION BY COATED ECTODERM
Given surface tension relations (1) and
(2) for subsurface tissues, and given the
additional fact that the shiny side of coated
ectoderm is non-adhesive towards other
cells, one can explain how the presence of
coated ectoderm would cause subsurface
germ layers to form their normal in vivo
arrangement (ectoderm surrounding
mesoderm surrounding endoderm). In
qualitative terms, "non-adhesive" in this
case means, of course, that the coated sides
of cells adhere more strongly to the culture
medium than to other cells. This would
explain observations by Holtfreter (1943a,
1944) and Townes and Holtfreter (1955)
that in tissue-spreading and cell-sorting
experiments coated cells usually tend to
collect at and spread over the surfaces of
cell aggregates.
The following argument, diagrammed
in Figure 9, first formulated by Steinberg
and Kelland (1967), is restated here in
terms of tissue surface tensions, (i) Once
on the outside of an aggregate (or embryo), coated ectoderm forms an wncoated
ectodermal boundary facing the subsurface cell populations which it has enveloped, (ii) Since their surface tensions
cause germ-layers to be immiscible (e.g., as
demonstrated graphically by their cellsorting behavior), subsurface ectoderm
will adhere to "its own kind," and thus will
displace subsurface mesoderm and endoderm along the inner (uncoated, adhesive) boundary of the surface layer of
coated ectoderm, (iii) Tissue-tissue surface
tenions (relation (2) and Fig. 7 above)
causes subsurface mesoderm to spread
into the E-N interface, (iv) This displaces
subsurface endoderm to the most interior
position in this tissue system.
Steinberg and Kelland (op. cit.) performed a direct behavioral test of this
explanation in their "sandwich experiments" (Fig. 10). They simply excised
whole chunks of tissue from the sides of
FIG. 9. Our diagram of the sequence of events
proposed by Steinberg and Kelland (1967) as a
mechanism by which coated ectoderm could assemble
subsurface germ layers into their normal gastrular
arrangement (see text). This mechanism requires
only surface tension relations (1) and (2) plus the
observed fact that the coated faces of surface ectoderm cells are non-adhesive.
Rana pipiens gastrulae at sufficiently late
stages so that lateral mesoderm had already spread between ectoderm and endoderm. Each excised fragment then contained portions of all three subsurface
germ layers. On some fragments Steinberg
and Xelland left the surface layer of coated
ectoderm intact; from others they peeled it
LIQUID-TISSUE MECHANICS IN GASTRULATION
A. With coated ectoderm
-E
M
N
B. Without coated ectoderm
M
N
FIG. 10. Our diagram of the "sandwich" experiments by Steinberg and Kelland (1967), which were
designed to test the effect of removing coated ectoderm from samples of subsurface E, M and N. As
they predicted, excised pieces of lateral tissue from
late gastrulae (containing all three subsurface germ
layers) in organ culture form (A) the normal arrangement when coated ectoderm has been left intact
and (B) an inverted arrangement when coated ectoderm has been removed from otherwise identical
tissue samples.
away. Organ culture fragments with
coated ectoderm formed round, multilayered aggregates with the normal gastrular germ-layer arrangement. In otherwise identical fragments with the coated
ectoderm removed, the resulting configurations of subsurface germ layers were
reversed. This demonstrates that coated
ectoderm is the sole additional factor required to cause subsurface germ layers to
adopt their proper orientations during in
vitro tissue spreading. Holtfreter (1944)
and Townes and Holtfreter (1955) obtained similar results in various combinations of subsurface germ layers with and
without coated ectoderm.
This explanation further implies that, in
the absence of coated ectoderm, the normal distribution of subsurface germ layers
should be unstable. An early observation
by Holtfreter (1944) provides an elegant in
vivo confirmation of this prediction. He
placed a whole, intact late-gastrula in dissociating medium. After many coated ectoderm cells had fallen off the embryo, but
91
before further cell dissociation could proceed, he returned the embryo to normal
culture medium. At first the endoderm
from the archenteron began to emerge
from the blastopore and spread over the
denuded, now largely uncoated surface of
the embryo. However, the remaining
coated ectoderm soon spread out to cover
the rest of the surface of the embryo, then
halted the advance of the endoderm and
eventually forced it to recede back to its
interior position! Thus, in the normal embryo, coated ectoderm may confine endoderm to its proper internal location as
gastrulation proceeds. (By means of this
analysis one can also account for the envelopment of mesoderm by endoderm in
the absence of ectoderm in vivo, e.g., as
Holtfreter [1933] observed in whole embryos following exogastrulau'on.)
CONCLUSION
Liquid-tissue flow is clearly not the sole
morphogenetic mechanism operating in
amphibian gastrulation. For example, active cell shape changes evidently cause
elastic-solid deformations in surface cell
layers which initiate blastopore lip imagination and maintain the external contours
of the lip during gastrulation (see above).
Another apparently solid-like forcegenerating movement is the spontaneous
elongation of dorsal lip mesoderm, which
Schechtman (1942) described in Hyla, and
which one of us also confirmed in Rana
pipiens (Phillips, in preparation). For
example, excised pieces of dorsal lip
mesoderm in organ culture will at first
round up like aggregates of lateroventral
lip mesoderm. Unlike the latter, however,
a dorsal lip aggregate (with or without
coated mesoderm) will then form a
cylindrically-shaped protrusion several cell
diameters in thickness and many cell
diameters long—an obvious departure
from a simple minimum-surface-area
configuration characteristic of isolated
liquid droplets.
The significance of this dorsal lip
mesodermal elongation is shown by
Schechtman's experiments {op. cit.) in
which he mechanically isolated dorsal lip
92
HERBERT M. PHILLIPS AND GRAYSON S. DAVIS
mesoderm or removed it entirely from
otherwise intact, whole embryos. (As
explained above, when mechanically isolated from lateral lip mesoderm, dorsal
lip mesoderm does not involute. However, it does still elongate, so dorsal lip
elongation occurs independently of its
own involution.) In the absence of normally involuted dorsal lip mesoderm,
Schechtman obtained "ring" embryos,
which were greatly compressed in an
anterior-posterior direction, but which still
exhibited involution of mesoderm along
the lateral and ventral lips of the blastopore. Thus, dorsal mesoderm elongation,
while not required for lateroventral
mesodermal involution, is evidently necessary for the maintenance of overall embryonic shape (and, in addition, he found,
for dorsal convergence of involuting
mesoderm and for subsequent closure of
the blastopore over the yolk plug). While
the mechanism of dorsal mesoderm elongation remains entirely unknown (this occurs long before the notochordal elongation due to cell vacuolization described by
Mookerjierfa/. [1953]), suffice it to say that
it is clearly a significant force in amphibian
gastrulation which is evidently different
from and independent of subsurface
germ-layer flow.
While recognizing the need for several
different force-generating mechanisms to
combine in producing amphibian gastrulation, we nevertheless wish to emphasize
the importance of liquid-tissue flow as a
major factor in this process, not only for
mesodermal involution (discussed above),
but perhaps also for ectodermal epiboly
and for the anterior spreading of subsurface endoderm on the inner margin of
blastocoel-roof ectoderm. (This seals off
the blastocoel and creates the subsurface
E-N interface into which involuting
mesoderm then flows.) Amphibian gastrulation has been a subject of scientific investigation for nearly a century, but has to this
day remained a mechanically puzzling
process. We suggest that this has been in
large part due to a failure to appreciate
conceptually and to explore experimentally the mechanism of liquid-tissue flow
governed by tissue surface tensions as a
potential explanation for subsurface
germ-layer movements. (Credit should be
given, however, to Holtfreter [1943a,6;
1944] and to Rhumbler [quoted therein]
for preliminary explorations in this direction, as well as to Steinberg and Kelland
[1967] and Steinberg [1970] for partial
formulation of this hypothesis.) We are
now investigating this mechanism in other
thick-tissue morphogenetic processes,
specifically limb bud formation in chick
embryos (Daggy and Phillips, in preparation; Heintzelman et al., in preparation).
Physical studies of morphogenetic
movements at the cell-and-tissue level of
analysis are not only essential in providing
a complete mechanical explanation of
these phenomena, but may also prove to
be useful in molecular approaches to these
problems. For example, in liquid-tissue
systems, those molecular interactions
which can be shown to affect tissue surface
tensions would be morphogenetically significant in the regulation of tissue flow;
those which do not—interesting as they
may be for other reasons — would in this
context be morphogenetically irrelevant.
Moreover, regulation of intercellular
adhesiveness, e.g., via changes in cell-cell
bonding strengths and/or patterns (Steinberg, 1963, 1964, 1970, 1975; Phillips,
1969, and in preparation), constitutes the
simplest, although not the only, means of
generating and modulating the tissue surface tensions which direct liquid-tissue
flow.
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