AMER. ZOOL., 24:649-655 (1984)
Migration-Directing Liquid Properties of
Embryonic Amphibian Tissues1
GRAYSON S. DAVIS
Department of Biology, Valparaiso University,
Valparaiso, Indiana 46383
SYNOPSIS. Deep ectoderm, mesoderm and endoderm excised from gastrulating amphibian embryos spontaneously undergo liquid-like movements in organ culture. Cell populations of these tissues on nonadhesive substrata will round up into spheres, spread over
one another and segregate (sort out) from one another just as immiscible liquid droplets
do. In ordinary liquids, movements like these are controlled by surface tensions; perhaps
surface tensions also control the similar movements of liquid-like tissues. One necessary
condition for tissue surface tension analysis is that the tissue must be able (just as ordinary
liquids are able) to spontaneously relax internal stretching forces (shear stresses). When
cellular aggregates of the germ layers were deformed by gentle compression between
parallel glass plates, cells within the aggregates were initially stretched. However, the cells
soon returned to their original undistorted shapes. Thus, cell stretching forces were
gradually relaxed by cell rearrangements. The in vitro spreading movements of the deep
germ layers imply that the surface tension of ectoderm should be greater than the surface
tension of mesoderm which should be greater than the surface tension of endoderm.
Quantitative measurements of tissue surface tensions made by parallel plate compression
confirm precisely that relationship. Furthermore, the surface tensions of these tissues
remain constant regardless of the amount of aggregate flattening—another necessary
condition for valid surface tension measurements. These results demonstrate that amphibian deep germ layers possess fundamental liquid properties which are sufficient to direct
their liquid-like rearrangements in organ culture. Furthermore, I also report that one of
these properties, surface tension, displays a preliminary correlation with density of cell
surface charge (assessed by electrophoretic mobility) and with the onset of in vivo mesodermal involution.
INTRODUCTION
A morphogenetic event can be analyzed
at many different levels. One can attempt
to determine which genes are active, which
proteins are being synthesized, which cell
organelles are functioning and which cells
are growing, dividing, changing shape or
moving. However, information about
microscopic events may not be sufficient
for a complete explanation of morphogenesis. Often, a macroscopic analysis of the
movement-generating mechanism is
required. This paper reviews a macroscopic analysis of certain in vitro tissue
movements (which often resemble normal
morphogenesis) and then examines certain
microscopic events which could produce
those movements.
According to Newton's laws, all move' From the Symposium on Castrulation presented
at the Annual Meeting of the American Society of
Zoologists, 27-30 December 1982, at Louisville, Kentucky.
ments which occur where friction is present must be the result of unbalanced forces.
Therefore, for every tissue movement
which we observe, we should be able to
identify an unbalanced force which acts to
make those tissues move. This kind of
physical analysis has been especially fruitful for certain embryonic tissues which display liquid-like movements in organ culture. When irregularly-shaped explants of
these tissues are cultured on nonadhesive
substrata (agar, e.g.), the tissue fragments
will round up to form spheres (Holtfreter,
1939). When two such spheres composed
of different liquid-like tissues are touched
together, they will fuse as one tissue spreads
about the other in a characteristic envelopment pattern (Holtfreter, 1939). When
two different, liquid-like tissues are disaggregated into single cells, and the cells are
mixed and reaggregated, the cell types will
sort out from one another, eventually
forming the same stable configuration
attained by fragment fusion (Townes and
Holtfreter, 1955).
649
650
GRAYSON S. DAVIS
In ordinary liquids, movements like these
are controlled by the surface tensions of
liquid interfaces (Adamson, 1967). Perhaps surface tensions can also direct similar
movements of liquid-like tissues. The control mechanism of such movements is significant because the final arrangement
attained by some tissue combinations
resembles complex in vivo morphology.
Townes and Holtfreter (1955), for example, found that certain tissue fragments or
even dissociated cells from an early
amphibian neurula could autonomously
rearrange to form miniature replicas of late
amphibian neurulae. However, these
authors suggested that the different cell
types of the embryo were able to segregate
themselves by cell-specific adhesion and by
chemotaxis. Noting that the time course of
such cellular rearrangements is very similar to types of liquid behavior controlled
by intermolecular adhesions, but quite different from that expected from chemotaxis, Steinberg proposed that rounding up,
aggregate fusion and cell sorting could be
explained by differential cellular adhesion
alone (Steinberg, 1962a, b, 1963). To define
strength of adhesion in physical terms,
Steinberg (1964) referred to "works of
adhesion" (the amounts of reversible work
performed in forming or separating a unit
area of adhesion). Unfortunately, because
of the difficulties in measuring the changes
in free energy and in cell contact area,
works of adhesion have never been quantified for any tissues (Steinberg, 1964).
Phillips (1969) has pointed out that, for
liquid substances, surface tensions are
physically equivalent to works to adhesion
and offer the further advantage of being
measurable for living tissues. A macroscopic analysis of liquid-like tissue movements could address the following questions. Do those embryonic amphibian
tissues which behave like liquids actually
possess fundamental liquid properties
which make surface tension analysis appropriate? If so, are their surface tensions of
the right strengths to direct tissues in their
in vitro (and perhaps in vivo) migrations?
What microscopic cell properties contribute to tissue surface tensions?
FIG. 1. Diagram by Holtfreter (1943) of cells dissected from a morula. In contrast to the deep cells,
the outermost cells possess a more heavily pigmented,
nonadhesive, exteriorly-directed surface.
FUNDAMENTAL LIQUID PROPERTIES
OF THE DEEP TISSUES
In addition to the usual distinctions
between germ layers, Holtfreter found that
the surface cells of the amphibian embryo
are significantly different from the deep
cells (Holtfreter, 1943) (Fig. 1). He proposed that the exterior cells form a "surface coat" because their more heavily pigmented, exteriorly-directed surfaces were
nonadhesive to other cells. During gastrulation, cells of the surface coat do exchange
neighbors laterally, but they do not sink
into the deep layers, nor do deep cells come
out onto the surface (Keller, 1978).
It is important to remember that liquidlike behavior is a property of the deep tissues but may not be characteristic of coated
cells. In all of our experiments with midgastrula Rana pipiens embryos, we peeled
away and discarded the coated surface cells
and then dissected out the underlying deep
or uncoated tissues. In these experiments,
deep ectoderm, mesoderm and endoderm
excised from embryos exhibited fundamental liquid behavior. First, when excised
pieces of these tissues were cultured on a
nonadhesive substratum, they rounded up
like liquids, adopting nearly spherical
shapes in 4-5 hr. Scanning electron micrographs showed that the shapes of the cells
inside these tissues were nearly isodiametric both before and after rounding up
LIQUIDITY OF EMBRYONIC AMPHIBIAN TISSUES
(Davis and Phillips, submitted for publication). This implies that rounding up proceeds by a process of cell rearrangement
rather than by shape changes of immobile
cells.
Ordinary liquid droplets round up
because their subunits can move with
respect to one another while trying to maximize their adhesions to each other (Symon,
1971). Furthermore, this type of subunit
redistribution permits liquids to relax
internal shear stresses which would otherwise accompany overall shape changes.
Do cells of liquid-like tissues also display
this second fundamental liquid property?
To find out, we gently flattened spherical
aggregates of deep ectoderm, mesoderm
and endoderm between two parallel, agarcoated coverslips and fixed them at various
times during compression so that the shapes
of their interior cells could be observed
with the scanning electron microscope
(Phillips and Davis, 1978). The interior cells
of uncompressed aggregates are loosely
packed and rounded (Fig. 2A). When the
aggregate's overall shape is changed by
compression, internal stresses are initially
created which stretch all the cells (Fig. 2B).
Stretched cells then gradually rearrange
themselves to relieve those internal stresses.
By the end of fifteen minutes of compression, no orientated cell stretching was
apparent in the aggregate's interior, even
though the aggregate remained under constant compression (Fig. 2C). Although the
cells were initially stretched (like the subunits of an elastic solid), they gradually
rearranged themselves (like the coherent,
mobile subunits of a viscous liquid) to relax
stretching forces inside the aggregate.
Substances which display this type of shortterm solid behavior and long-term viscous
liquid behavior are appropriately termed
elasticoviscous liquids (Phillips et al., 1977;
Phillips and Steinberg, 1978).
A third liquid property is demonstrated
by the fusion behavior of deep ectoderm,
mesoderm and endoderm aggregates.
When two such aggregates touch, one
spreads around the other in a tissue-specific pattern of envelopment. Deep ectoderm always goes to the inside, deep
651
FIG. 2. Diagram of the shapes of cells within initially
round aggregates before and after differing durations
of constant compression. A. Before compression, interior cells are isodiametric. B. During initial compression, the overall shape change experienced by the
aggregate creates internal stresses which stretch all
the cells. C. However, after continued compression,
the stretched cells are able to rearrange themselves
and thereby relax the stress. Cells gradually return
to their original undistorted shapes, even though the
aggregate remains under constant compression.
endoderm always goes to the outside, and
deep mesoderm is intermediate since it surrounds ectoderm and is itself surrounded
by endoderm (Phillips and Davis, 1978). In
a similar experiment, Steinberg and Kelland (1967) excised chunks of tissue from
the sides of amphibian gastrulae. Each tissue fragment contained coated ectoderm,
deep ectoderm, deep mesoderm and deep
endoderm. If the surface layer of coated
ectoderm cells was peeled away, the
remaining deep layers rounded up into a
single sphere and rearranged such that
endoderm was outermost, mesoderm in the
middle and ectoderm innermost. This
result is not unexpected given the behavior
of fused pairs of aggregates, but it is the
opposite of the gastrula's normal germ layer
652
GRAYSON S. DAVIS
FIG. 3. Diagram of an experiment by Steinberg and
Kelland (1967). A piece of tissue containing coated
ectoderm (black), deep ectoderm (dark gray), deep
mesoderm (medium gray) and deep endoderm (light
gray) was excised from the flank of a gastrula. When
this fragment was allowed to round up in organ culture, the tissues rearranged to form the normal germ
layer architecture.
'AO
organization. Moreover, this inside-out
architecture raises the question of what, if
anything, liquid behavior in general and
fragment fusion in particular have to do
with normal, in vivo morphogenesis. However, Steinberg and Kelland also found that
if the excised fragment was allowed to
round up with the coated ectoderm cells
in place, that sheet of coated cells seemed
to be held at the exterior by its nonadhesive surface. As the coat spread over the
surface of the aggregate, the deep tissues
rearranged to form the normal germ layer
architecture (Fig. 3). This observation suggests that when coated ectoderm is present, liquid-like fusion behavior of the deep
germ layers is sufficient to direct tissue
explants to form the normal germ layer
configuration (Phillips and Davis, 1978).
SURFACE TENSION AND AGGREGATE
FUSION BEHAVIOR
But what of the fusion behavior of the
deep tissues? What does the "insidedness"
hierarchy of ectoderm > mesoderm >
endoderm imply about the liquid properties of those tissues? When two ordinary
liquid droplets fuse, the one which goes to
the inside has the higher surface tension
(Fig. 4). By analogy, that tissue which goes
inside in a fragment fusion should have the
higher tissue surface tension. The aggregate fusion behavior of deep ectoderm,
mesoderm and endoderm predicts that the
FIG. 4. Diagram of two similarly-sized, immiscible
liquid droplets (A and B) fusing in medium O. Their
final arrangement is determined by the surface tensions (<T'S) of the AO, BO and AB interfaces. If B
surrounds A, then <jAO > <rBO. The surface tension of
the AB interface determines the degree, but not the
direction, of envelopment.
surface tensions, er's, will be in the following sequence:
crECT > o-MES > crEND
(1)
How can the surface tension of living
aggregates be measured to see if this prediction is valid?
Of course, surface tension is that force
which promotes droplet rounding up and
opposes increases in surface area. To measure tissue surface tension then, one can
deform initially spherical aggregates with
a known force, wait for them to reach
mechanical equilibrium and monitor the
increase in aggregate surface area. Figure
5 is a diagram of a device which facilitates
such measurements. Here an aggregate is
gently compressed between two parallel,
agar-coated glass plates. The top plate is
immobile and the bottom plate is attached
to one end of a flexible quartz fiber. The
opposite end of the fiber is held by a simple
micromanipulator. The compressing force
applied to the aggregate can be increased
by turning a dial on the micromanipulator.
LIQUIDITY OF EMBRYONIC AMPHIBIAN TISSUES
FIG. 5. Diagram of a device for compressing cellular
aggregates. An aggregate (a), surrounded by medium,
is compressed between two agar-coated glass plates.
The lower plate (gc) is attached to one end of a flexible
quartz fiber (f). The other end of the fiber is held by
a simple micromanipulator. Turning the dial (d) will
increase or decrease the force applied to the aggregate and the flexure of the fiber. Since the bending
constant of the flexible quartz fiber has been previously determined, measurement of the fiber's degree
of deflection will permit a calculation of the force
applied to the aggregate. The shape of the aggregate
may be monitored by side view photographs.
If the applied force is increased, the aggregate will be flattened more and the quartz
fiber will be bent more. Since the fiber has
been calibrated, a simple measurement of
its degree of deflection will permit a calculation of the force (F) applied to the
aggregate. The deforming pressure (AP)
experienced by the aggregate is equal to
the force applied divided by the area of
contact between the aggregate and glass
plate. The sides of the aggregate can be
described by two radii of curvature. The
horizontal radius (parallel to the compressing plates) is called R,, and the vertical
radius (perpendicular to the compressing
plates) is called R2.
These variables can be measured from
side view photographs of aggregates
undergoing compression. Their values may
then be substituted into the Young-Laplace
equation (Adamson, 1967),
(2)
653
to compute the value of surface tension (a).
This method gives the correct value for
the surface tension of water (i.e., air-water
interface) when small air bubbles are compressed between the plates (Davis and Phillips, submitted for publication). Furthermore, the surface tension value obtained
is constant regardless of the degree of
compression. All liquid substances must
have area-invariant surface tension. By
contrast, the surface tension of a solid substance always increases as the degree of
compression increases (Zemansky, 1957).
Therefore, if the liquid behavior of deep
ectoderm, mesoderm and endoderm is
directed by their surface tensions, then
their germ layer surface tensions must not
only satisfy equation (1), they must also be
area-invariant.
Measurements of deep ectoderm, lateral
mesoderm and endoderm excised from
mid-gastrula Rana pipiens embryos do satisfy equation (1) (Table 1). The probability
that any two of these values are equal is
less than 0.03. Furthermore, these values
are area-invariant, which could not be true
if these tissues were solid-like. Deep ectoderm, mesoderm and endoderm do resemble liquids. They have classically defined
(area-invariant) surface tensions, and furthermore, the values of their surface tensions are appropriate to direct their
observed rounding up, sorting out and
fusion behavior.
PRELIMINARY CORRELATIONS OF
SURFACE TENSION, SURFACE CHARGE
AND MESODERMAL INVOLUTION
Tissue surface tension measurements
have provided quantitative data supporting the above macroscopic analysis of liquid-tissue behavior. Those same measurements may now be used to help identify
TABLE 1. Area-invariant surface tension values (in ergs / centimeter2 ± standard errors) of tissues excised from midyolk plug stage Rana pipiens.
Tis!
Ectoderm
Mesoderm
Endoderm
Number of cases
Surface tension
Probability that surface tension
values are inconsistent with the
predicted sequence
2.92 ± 0.82
0.64 ± 0.09
0.39 ± 0.03
mes: P < 0.025
ect
ect :£ end: P < 0.018
mes £ end: P < 0.021
654
GRAYSON S. DAVIS
those microscopic (e.g., ultrastructural)
properties of cells which produce tissue
specific germ layer surface tensions. Those
cell characteristics which are shown to correlate with surface tension would then be
implicated in the control of liquid-like tissue movements.
Some authors have proposed that negative charges on the surfaces of cells produce electrostatic forces which may reduce
cell-cell adhesiveness by preventing the
close approach of cell surfaces (Pethica,
1961; Garrod and Gingell, 1970; Lee,
1972). If this is true, then tissues with
greater densities of surface charge would
be less adhesive and so have lower surface
tensions. Therefore, according to equation
(1), the surface charge of deep ectoderm
should be lower than that of deep mesoderm which should be lower than that of
deep endoderm. In fact, measurements of
cell surface charge (using the rates of electrophoretic mobility of germ layer cells)
found precisely this relationship (Schaeffer
et al., 1973a). Furthermore, there is a correlation between surface charge and adhesion. Deep tissues in organ culture will dissociate if the pH of the surrounding
medium is increased (Townes and Holtfreter, 1955). Schaeffer e/aZ. (19736) found
a corresponding increase in cell electrophoretic mobilities as the pH increased.
This is consistent with the proposed inverse
relationship between surface tension and
surface charge.
Changes in surface charge also correlate
with certain morphogenetic movements of
gastrulating germ layers. At the blastula
stage, dorsal mesoderm possesses an electrophoretic mobility very similar to that of
blastula ectoderm. However, at the early
gastrula stage, shortly before the involution of dorsal mesoderm, that tissue's electrophoretic mobility decreases to became
intermediate between ectoderm and
endoderm (Schaeffer et al., 1973a), suggesting that this surface charge relationship, and therefore the surface tension
relationship in equation (1) may be developed in preparation for mesodermal involution. Is there a corresponding change in
the surface tension of blastula mesoderm
(from ectoderm-like to intermediate
between ectoderm and mesoderm)?
To answer this question, we have made
surface tension measurements of several
tissues excised from early gastrula stage
embryos (Davis and Phillips, in preparation). Preliminary results for dorsal mesoderm indicate that, after its involution, this
tissue may no longer possess a liquid-like
(area-invariant) surface tension. This nonliquid behavior makes dorsal mesoderm an
inappropriate tissue to test for a correlation between surface tension change and
morphogenesis. However, lateral and ventral mesoderm excised from the early gastrula did possess area-invariant surface tensions. Lateral mesoderm, which was nearly
ready to involute, had a surface tension
somewhat less than that of ectoderm of the
same stage. Ventral mesoderm, which
would not begin to involute for several
hours, had a surface tension greater than
that of ectoderm. The surface tension of
lateral mesoderm was significantly lower
than the surface tension of ventral mesoderm, suggesting that the surface tension
of mesoderm does decrease in preparation
for its involution. Further analysis and
additional experiments are now underway
to examine this correlation more carefully.
An evaluation of the covariance between
the surface tension of a tissue and the surface charge of its cells could permit an
assessment of the potential role of cell surface charge as a primary determinant of
the morphogenesis of liquid-like tissues.
Moreover, quantitative measurements of
surface tension provide a criterion to
appraise the contributions of other ultrastructural properties to the control of liquid-like tissue flow.
ACKNOWLEDGMENTS
I am grateful to Dr. Herbert M. Phillips
for his help and guidance and to Ms. Sandra Mitchell and Ms. Margaret MacQueen
for their expert technical assistance. This
research was supported by N.S.F. grants
GB-40041 and PCM 78-0593 to H.M.P.,
N.I.H. traineeship HD00430 and a Valparaiso University Research Fellowship to
G. S. D.
LIQUIDITY OF EMBRYONIC AMPHIBIAN TISSUES
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