/. Embryo!, exp. Morph. Vol. 64, pp. 149-168, 1981
Printed in Great Britain © Company of Biologists Limited 1981
\ 49
Calcium and spreading behaviour of amphibian
blastula and gastrula cells
By JACQUELINE L E B L A N C 1 AND IRVING BRICK 2
From the Department of Biology, New York University
SUMMARY
Cell locomotion involves several structural-functional activities: membrane extensibility,
microfilament regulation and adhesive interactions. There is evidence for Ca2f requirement
in all of these. Our data may clarify the role of Ca2+ in locomotion and adhesion. Morphologic and spreading responses of isolated blastula-late gastrula Rana pipiens germ layer cells
to varying molar concentrations of Ca2+: 0-Ca2+, Standard Ca2+ (Barth's A'solution), 1-5 x
and 20 x Std Ca2+ were viewed by S.E.M. after 1 h in culture. Ionic strength and pH were
constant. All cells showed quantitative relationships between Ca2+ concentration and surface
extensibility, projection formation and presumably adhesion, but with tissue- and stagespecific variations. Cells in Ca2+-free medium fail to adhere (50%), flatten or form surface
projections. Cells in media with increasing Ca2+ generally formed more numerous and
extensive surface projections, spread and adhered to a greater extent. In some cases there
were no quantitative differences in response between 1 -5 x and 20 x standard Ca2+. Cells in
suspension for 1 h in standard solution remained spherical, forming no projections. We infer
from these results that both Ca2+ and contact with a physical substratum, cell-cell or cellglass are required for mobilization of the various systems involved in locomotion and
adhesion. In addition, components of these systems are quantitatively activated by increased
availability of Ca2+.
INTRODUCTION
Cell locomotion requires participation of several structural-functional cell
activities. These include (1) plasmalemma extensibility, (2) organization and
regulation of a submembrane microfilament and microtubule apparatus to form
locomotor organelles, (3) cell periphery adhesive interactions with a substratum
and (4) energy availability. There is considerable evidence that Ca 2+ is involved
in the operation of all these activities (Weiss, 1970; Durham, 1974; Spooner,
1975). Experiments reported here provide data which may clarify some aspects
of Ca 2+ participation in cell movement and adhesion.
Cells move most frequently by forming projections of varied kinds which
1
Author's address: The College of Staten Island, The City University of New York,
Staten Island, New York, U.S.A.
2
Author's address: Department of Biology, New York University, 952 Brown Building,
Washington Square, New York, New York, 10003, U.S.A.
* The work reported here was submitted to the faculty of the Graduate School of Arts
and Science in partial fulfilment of the requirements for the degree of Docor of Philosophy
at New York University.
150
J. L E B L A N C AND I. BRICK
attach to a substratum. This has been seen in vivo in many embryos (Gustafson,
1964; Brick, Schaeffer, Schaeffer & Gennaro, 1974; Spiegelman & Bennett,
1974; Nakatsuji, 1976). Cell microprojections are also involved in the initial
process of intercellular association and in making focal adhesions in cell
attachment to a substratum (Abercrombie, Heaysman & Pegrum, 1971;
Izzard & Lochner, 1976; LeBlanc & Brick, 1981).
Microprojection formation is associated with microfllaments as observed in
many species (Spiegelman & Bennett, 1974; Nakatsuji, 1976). Concentrations of
cortical microfllaments are seen at sites of close apposition to the substratum
and at intercellular contacts (Abercrombie et ai, 1971; Heaysman & Pegrum,
1973). The relationships and deployment of microfllaments suggest that microfilament Ca2+-dependent contractile activity is involved in formation and
withdrawal of cell protrusions and probably related to cell extensibility and,
therefore, locomotion.
Cell periphery adhesive interactions are also related to Ca 2+ availability.
Ca2+-free medium aids embryo disaggregation (Jones & Elsdale, 1963), and
reaggregation of amphibian blastula and gastrula cells can occur by restoration
of Ca 2+ (Steinberg, 1962; Jones & Elsdale, 1963). Weiss, 1960, has shown that
Ca 2+ is essential for attachment of cells to glass and Taylor, 1961, showed that
Ca2+-free medium inhibits cell spreading. Curtis, 1962, suggested that Ca 2 +
decreases cell surface charge thus promoting adhesion. Schaeffer, 1972, showed
that Ca 2+ reduced electrokinetic mobility of various amphibian embryo cells.
We propose that Ca 2+ has other, indirect roles in cell adhesion by virtue of the
suggested relationship of Ca 2+ to cell projections and formation of cell-cell
contacts commonly achieved via cell projections.
Two categories of experiments are reported. In the first series Ca 2+ concentrations were varied for germ-layer cells of amphibian blastulae and gastrulae.
Our objectives were to determine (1) the role of Ca 2+ in cell extension over the
substratum, (2) the Ca 2+ requirement for initial adhesion and/or subsequent
projection formation in cell spreading and (3) if varying Ca 2+ concentration
would alter the form and number of projections extended onto the substratum.
LeBlanc & Brick (1981) have demonstrated stage and presumptive (Pr) tissuespecific variations in spreading and adhesive behaviour of Rana pipiens blastula
and gastrula cells. We have here used the same array of cells in order to evaluate
the role of Ca 2+ in the various cell behaviours.
The second series of experiments evaluated the substrate-projection relationship. Inasmuch as spreading cells invariably adhere and conform to their
substrata, the influence of such direct surface contacts on cell form must be
considerable (Elsdale & Bard, 1972). We considered two possibilities; (1) the
substratum might bring about a redistribution of projections already present
on the surface of a cell settling on the substratum, or (2) that a substratum
might be required for actual projection formation. To test these hypotheses,
cells from one Pr tissue type, at one stage were maintained in suspension under
Calcium and spreading behaviour of amphibian cells
151
the same environmental conditions as spreading cells (Le Blanc & Brick, 1981),
the only difference was lack of a substratum.
MATERIALS AND METHODS
Induced ovulation and fertilization of Rana pipiens were according to Rugh
(1962). Embryos were maintained at 13 °C in spring water. Blastula, early-midlate gastrula stages were used (St 9-11 1/2, Shumway, 1940).
Embryonic regions utilized were: (1) Blastula (St 9) Pr head endoderm
(Pr dorsal lip); Pr notochord; Pr inner neural ectoderm; Pr inner epidermal
ectoderm. (2)Early gastrula (St 10) Pr head endoderm (dorsal lip); Pr notochord;
Pr inner neural ectoderm; Pr inner epidermal ectoderm. (3) Mid-gastrula (St 11)
Pr head endoderm (leading edge of the invaginating fold); Pr notochord (dorsal
lip); Pr inner neural ectoderm; Pr inner epidermal ectoderm. (4) Late gastrula
(St 11 1 /2) Pr head endoderm (leading edge of the invaginating fold); Pr notochord (chordamesoderm midway between anterior and posterior ends); Pr
inner neural ectoderm; Pr inner epidermal ectoderm.
Methods for dissection, disaggregation and culturing have been previously
reported (LeBlanc & Brick, 1981). In the present experiments, all cells spread
for 60 min on glass under sterile conditions at 23-25 °C, the time required for
marked spreading to occur.
Modifications of the standard solution, Barth's X solution, were used for the
spreading studies (Barth & Barth, 1959), pH 7-5, ionic strength 0-09797. Barth's
solution is prepared in three parts, A, B, and C; parts B and C consist of buffers.
We, therefore, varied only part A in subsequent test solutions. Ionic strength
and pH were kept constant. Only part A with varied Ca 2+ concentrations is
listed.
Solution 1
Part A - Ca2+ free
NaCl
5-2809 g
KC1
00750 g
MgSO4.7H2O
0-2040 g
H2O to 500 ml
Solution 2
Part A - 1 -5 x molarityof Ca2+ in standard
NaCl
50845 g
KC1
00750 g
MgSO4.7H2O
0-2040 g
Ca(NO3)2.4H2O
01197 g
CaCl2.2H2O
00902 g
H2O to 500 ml.
Solution 3
Part A - 2 x molarity of Ca2+ in standard
NaCl
50191 g
KC1
00750g
MgSO4. 7H2O
0-2040 g
Ca(NO3)2.4H2O
01594 g
CaCI2.2H2O
01203 g
H2O to 500 ml
Solution 4
Part A - Ca2+-Mg2+-free
NaCl
5-4260 g
KC1
00750 g
H2O to 500 ml
When changes in Ca 2+ concentration in solutions 1-3, or Ca 2+ and Mg 2+ in
solution 4 were made, ionic strength was maintained by adjusting the con-
152
J. L E B L A N C AND I. BRICK
centration of NaCl. Solutions 1-4 were constructed to view the effect of Ca 2+
on cell spreading behaviour. Mg 2+ was not adjusted in solutions 1-3 because
it is a cofactor in membrane enzyme reactions and because it is a divalent
cation associated with cell adhesiveness. Any membrane changes observed
should be attributable specifically to Ca 2+ and not to a change in concentration
of polyvalent cations in general. NaCl was chosen to be adjusted because it is
readily diffusible. The largest change in NaCl molarity was in solution 4 where
an additional 00047 mole was added. In solutions 1-3, the largest change in
NaCl was 0-0022 mole.
Osmolality of the incubation media was measured on a Fiske osmatic automatic osmometer, model 130; the readings were as follows: Barth's X solution
= 171 milliosmoles; solution 1 (Ca2+-free) = 174 milliosmoles; solution 2
(1-5 x molarity of Ca2+) = 171 milliosmoles; solution 3 (2 x molarity of Ca2+) =
167 milliosmoles; solution 4 (Ca2+-Mg2+-free) = 177 milliosmoles.
Preparation of cells on coverslips for S.E.M. has been reported previously
(LeBlanc & Brick, 1981). Cells were examined in an AMR 1000 Scanning Electron Microscope at 20 kV.
For each Pr cell type at each stage and in the various media, at least two
cultures were made, and in most cases, more than two. In each culture, cells
from all areas on the substratum were observed, the number of examined cells
ranging from 20-60. Description of a Pr cell type at a particular stage and in a
particular medium will be characteristic of typical surface behaviour for those
cells, unless otherwise noted.
Cells for the suspension study were prepared as for the cell spreading observations. These cells, in Barth's X medium, were drawn up into a 0-575 mm
diameter polyethylene tubing fitted to a microinjector. The polyethylene tubing
was mounted on the base of a dissecting microscope so cells could be observed
during the 30 or 60 min suspension. The cells were manually manipulated back
and forth for 30 to 60 min, during which they remained positioned in the slug
of moving medium and were not observed to form contacts with each other or
Fig. 1. Pr inner neural ectoderm cell isolated from blastula and spreading on glass
for 1 h in medium without Ca2+. x 1999. Scale mark equals 50/im.
Fig. 2. Pr inner neural ectoderm cell isolated from blastula and spreading on glass
for 1 h in medium without Ca2+ and Mg2+. x 770. Scale mark equals 13 /*m.
Fig. 3. Pr inner epidermal ectoderm cell isolated from mid-gastrula and spreading
on glass for 1 h in medium without Ca2+. x2380. Scale mark equals 4-2 /im.
FIG. 4. Pr inner epidermal, ectoderm cell isolated from mid-gastrula and spreading
on glass for lh in medium without Ca2+and Mg2+. x 2856. Scale mark equals 3 5 /tm.
Fig. 5. Pr head endoderm cell isolated from late gastrula and spreading on glass for
1 h in medium without Ca2+ and Mg2+. x 1218. Scale mark equals 8-2 /im.
Fig. 6. Pr notochord cell isolated from late gastrula and spreading on glass for
1 h in medium without Ca2+. x 2404. Scale mark equals 4-
Calcium and spreading behaviour of amphibian cells
153
154
J. L E B L A N C AND I. BRICK
with the tubing. Cells were fixed immediately at the end of 30 min or 1 h,
placed on a flotronic membrane and dehydrated as previously reported (LeBlanc
& Brick, 1981) and examined by S.E.M.
RESULTS
(1) Attachment and spreading of cells in media lacking (1) Ca2+ and (2) Ca 2+ and
Mg2+: All Pr tissues at all stages studied attached to the substratum without
forming extensions or spreading (Figs. 1-6). Generally, about half of the cells
on a coverslip adhered. This was determined by adding fixative to the coverslip
while observing the cells with a dissecting microscope. The unattached surfaces
are virtually devoid of projections, even if these were usually present on the
freshly disaggregated cells, and surface infolding and delineation of underlying
cortical structures are very evident. The morphologic features displayed by
each cell type at each stage are uniform. There are no subpopulations as was the
case in several of the cell types in Standard Barth's X solution (LeBlanc &
Brick, 1981) and in media with increased calcium.
(2) Attachment and spreading of cells in media with 1-5 x and 2-0 x molarity of
Ca2+: With the exception of a few isolated cells, most cells adhere to the glass
in these media. All observations are summarized in Table 1. For ready comparison, the morphologic behaviour of each cell type at each stage in Standard
Barth's X solution (LeBlanc & Brick, 1981) is included in the table.
(A) Pr inner neural ectoderm: At most stages, the major process of extension
is the lamellipodium. Late blastula cells adhere and spread by forming lamellipodia which often extended from the entire periphery (Fig. 7). In media with
2 x Ca 2+ some cells assume an elongate U-shaped configuration with a broad
lamellipodium extending to the glass from one peripheral region (Fig. 8).
Early gastrula cells spread via broad membranous lamellipodia which often
projected filopodia to the glass similar to mid-gastrula cells in which the lamellipodia radiated filopodia or microspikes (Fig. 9). Some mid-gastrula cells,
Fig. 7. Pr inner neural ectoderm cell isolated from blastula and spreading on
glass for 1 h in medium with l-5x std. Ca2+. x 1800. Scale mark equals 5-6 ywm.
Fig. 8. Pr inner neural ectoderm cell isolated from blastula and spreading on glass
for 1 h in medium with 2 x std. Ca2+ x 960. Scale mark equals 10-4 /«m.
Fig. 9. Pr inner neural ectoderm cells isolated from mid-gastrula and spreading on
glass for 1 h in medium with 1 -5 x std. Ca2+ x 2400. Scale mark equals 4-2 /*m.
Fig. 10. Pr inner neural ectoderm cell isolated from mid-gastrula and spreading on
glass for 1 h in medium with 2 x std. Ca2+ x 3300. Scale mark equals 3 0 fim.
Fig. 11. Pr inner epidermal ectoderm cell isolated from blastula and spreading on
glass for 1 h in medium with 2 x std. Ca2+. x 1513. Scale mark equals 66 /tm.
Fig. 12. Pr inner epidermal ectodeim cell isolated from early gastrula and spreading
on glass for 1 h in medium with 1 -5 x Std. Ca2+. x 2400. Scale mark equals 4-2 fim.
Calcium and spreading behaviour of amphibian cells
1t
155
Solution
Mid-gastrula
Pr inner neural
ectoderm cells
1-5 x«
2+
*Std.
Cell morphology
Relatively spherical
Lamellipodia
(Fig. 12)
Relatively spherical
Lamellipodia
(Fig. 13)
Relatively spherical
or elongated
Relatively spherical
Relatively Elongated Relatively spherical Dumbell Relatively Relatively spherical
spherical
spherical
Filopodia Stem-like Filopodia with inter- Extended Long fine Stem with filopodia
vening webbing
periphery filopodia
& filopodia
Two populations
Two populations
Semi-flattened
Lamellipodia
Semi-flattened
Lamellipodia
Fairly wide filopodia
Stem-like with
encircling membrane and lamellipodia
with filopodia
Relatively spherical Relatively spherical
Varying width
Fine filopodia
filopodia
Relatively spherical Relatively spherical
Lamellipodia, often Fairly broad filopodia
(Fig. 21)
with filopodia
Relatively spherical Relatively spherical
Lamellipodia, often Fairly broad filopodia
with filopodia
(Fig. 22)
(Fig. 17)
Two populations
Lamellipodia
(Fig. 11)
Relatively spherical
Filopodia
Relatively spherical
Relatively spherical
Relatively spherical
Filopodia
Relatively spherical
Filopodia, numerous
& fairly wide
Relatively spherical
Pr notochord cells
Relatively spherical
Fine filopodia
Relatively spherical
Stem-like
Pr head
endoderm cells
Relatively spherical
Filopodia
Relatively spherical
Lamellipodia
Pr inner epidermal
ectoderm cells
1 -5 x std. Ca2+ Relatively Elongated Often elongated &
spherical
flattened
Spreading protrusions *Std.
Cell morphology
Cell morphology
2 x std. Ca
Spreading protrusions 2xstd. Ca2+
Cell morphology
Relatively spherical
Filopodia
Cell morphology
*Std.
Relatively spherical
Spreading protrusions *Std
Filopodia
2+
Cell morphology
1-5 x std. Ca Flattened
Lamellipoda
Spreading protrusions 1-5 x std. Ca2+
(Fig. 7)
Flattened, often U2+
Cell morphology
2 x std. Ca
shaped
Lamellipodia
2+
Spreading protrusions 2 x std. Ca
(Fig. 8)
Characteristics
Early gastrula Cell morphology
*Std.
Spreading protrusions *Std
Blastula
Stage
Table 1. One hour cell spreading studies
o
w
z
o
2
r
w
r
pn
Late-gastrula
2 x std. Ca 2+
*Std.
2+
2 x std. Ca2+
Spreading protrusions 2 x std. Ca
Cell morphology
Cell morphology
Often long convoluted Broad stem &
stem
lamellipodia
(Fig. 18)
•Results from LeBlanc & Brick, 1981
Relatively Elongated Flattened
spherical
Broad
Broad
Lamellipodia
stem
stem
(Fig. 16)
Broad stem with
Broad lamellipodia
extending membrane with filopodia
(Fig. 24)
Semi-flattened
Broad stem with
filopodia
Long stem
(Fig. 20)
Semi-Flattened
Relatively spherical
Often elongate
Relatively Elongated Elongated & flattened Dumbell Relatively Relatively spherical
spherical with ellongated end
spherical
Extended Long fine Broad stem
Filopodia Stem-like Lamellipodia &
periphery filopodia
filopodia
& filopodia
Two populations
Relatively Elongated Often elongated &
Relatively spherical Relatively spherical
spherical
flattened
or elongated
or elongated
Lamelli- Stem-or
Lamellipodia, often Often broad bulbous Broad stem &
podia
rope-like with tortuous folding stem
lamellipodia
(Fig. 10) (Fig. 15)
(Fig. 19)
(Fig. 23)
Two populations
Two populations
15xstd. Ca2+ Relatively Elongated Flattened
spherical
Spreading protrusions l-5xstd. Ca 2+ Broad
Broad
Lamellipodia
stem
stem
Two populations
Spreading protrusions *Std.
Cell morphology
Spreading protrusions 2 x std. Ca2+
Cell morphology
Spreading protrusions l-5xstd. Ca2+ Lamelli- Stem-like Lamellipodia
podia
(Fig. 9)
Two populations
158
J. L E B L A N C AND I. BRICK
similar to late gastrula cells, projected stem-like protrusions to the glass. In
2 x Ca 2+ a number of mid-gastrula cells exhibit branching, relatively broad,
'rope-like' extensions to the glass and to surfaces of neighbouring cells across an
intervening space (Fig. 10).
(B) Pr inner epidermal ectoderm: These cells spread and adhered via membranous lamellipodia at all stages (Figs. 11-16). Late blastula cells in 2 x Ca 2+
(Fig. 11) tend to have broader lamellipodia than when in 1-5 x Ca 2+ . Figure 14
shows an early gastrula cell projecting a relatively broad lamellipodium onto the
upper surface of an adjacent cell. Note the ruffling of portions of the periphery.
These cells can also project long filopodia across the upper surfaces of cells
which they are contacting. Mid-gastrula cells often exhibit an elongate morphology and tend to be markedly flattened against the glass. In 2 x Ca 2+ some
cells produced membranous extensions with a tortuous folding and component
regions resembling a lobopodium (Fig. 15). Projections to the substratum rarely
developed from the entire cell periphery. Most late gastrula cells bear filopodia
at the periphery of their lamellipodia (Fig. 16). There is also extensive spreading
of cells onto surfaces of neighbouring cells.
(C) Pr Head endoderm: Late blastula cells in 1-5 x Ca 2+ produce short stemlike adhesions, while in 2 x Ca 2+ they exhibit short stems, but with an encircling
membranous extension with peripheral filopodia radiating from the stem.
Early gastrula cells of the dorsal lip region in media with enhanced Ca 2+
extended membranous protrusions to the glass (Fig. 17) which may be relatively
flat and smooth, or undulating with filopodia extending from their periphery
(Fig. 17). Cells of the leading edge of the invaginating fold (mid-gastrula)
adhere to the glass via stem projections in these media. Cells either exhibit a
very elongate often convoluted stem (Fig. 18), or a broader, shorter bulbous
stem (Fig. 19), the latter more common in medium with 2 x Ca 2+ and the former
more common in medium with 1-5 x Ca2+. Late gastrula cells of the leading edge
of the invaginating fold exhibit long, stem-like adhesions in medium with
1-5 x Ca 2+ and broader stem adhesions with extending membranous protrusions
Fig. 13. Pr inner epidermal ectoderm cell isolated from early gastrula and spreading
on glass for 1 h in medium with 2 x std. Ca2+. x 2394. Scale mark equals 4-2 /im.
Fig. 14. Pr inner epidermal ectoderm cells isolated from early gastrula and spreading
on glass for 1 h in medium with 2 x std. Ca2+. x 2268. Scale mark equals 4-4 /um.
Fig. 15. Pr inner epidermal ectoderm cell isolated from mid-gastrula and spreading
on glass for 1 h in medium with 2 x std. Ca2+. x 5400. Scale mark equals 1 -9 /im.
Fig. 16. Pr inner epidermal ectoderm cell isolated from late gastrula and spreading
on glass for 1 h in medium with 2 x std. Ca2+. x 5280. Scale mark equals 1 -9 /*m.
Fig. 17. Pr head endoderm cell isolated from early gastrula and spreading on glass
for 1 h in medium with 2 x std. Ca2+. x 5400. Scale mark equals 1-9/tm.
Fig. 18. Pr head endoderm cells isolated from mid-gastrula and spreading on glass for
1 h in medium with 1-5 x std. Ca2+. x 600. Scale mark equals 16-7/mi.
Calcium and spreading behaviour of amphibian cells
ib
— 18
159
160
J. L E B L A N C AND I. BRICK
2+
in medium with 2 x Ca . Cells in 1-5 x Ca 2+ , with long convoluted projections
were seen adhering to the glass and also to the surface of other cells (Fig. 20).
(D) Pr notochord: These cells, at late blastula project filopodia to the glass in
medium with 1-5 x Ca2+, but in medium with 2 x Ca2+, although filopodia are
produced there are also points on the cell periphery with membranous projections. Early gastrula cells produce filopodia in both media and unattached
surfaces have numerous filopodia and bulbous microvilli-like projections
(Figs. 21, 22). The behaviour of these cells changes at mid-and late-gastrula.
At mid-gastrula, cells from the dorsal lip in both media produce broad stem
adhesions and lamellipodia, the latter are more common in medium with
2 x Ca 2+ and in this concentration, long, finger-like protrusions from the
unattached surfaces are present, Fig. 23. Cells taken from the chordamesoderm
mid-way along the anterior-posterior axis at late gastrula in l-5xCa 2 + , form
broad stem-adhesions with peripheral filopodia, while these cells in 2 x Ca 2+
form broad lamellipodia with peripheral radiating filopodia (Fig. 24).
(3) Suspension of dissociated Pr inner neural ectoderm cells, late gastrula, for
30 to 60 min: These experiments were done to determine whether surface
projections would be produced in the absence of contact with a substratum,
either living or non-living. Pr late gastrula inner neural ectoderm cells after
30 min or 1 h of suspension in Barth's X solution were spherical, with relatively
smooth surfaces (Fig. 25).
DISCUSSION
There are three noteworthy aspects of the data which clarify and provide
additional insight into the relationship of Ca 2+ to plasma membrane extensibility, surface projection formation, cell adhesion and spreading, at least for
amphibian embryo cells during gastrulation. These are (1) failure in Ca2+-free
medium of cells to adhere, flatten and form surface projections; (2) the enhanced
ability of these cells in media with additional Ca2+, compared to these cells in
Fig. 19. Pr head endoderm cell isolated from mid-gastrula and spreading on glass
for 1 h in medium with 2 x std. Ca2+ x 1164. Scale mark equals 8-6 /tm.
Fig. 20. Pr head endoderm cells isolated from late gastrula and spreading on glass
for 1 h in medium with 1-5 x std. Ca2+. x 1200. Scale mark equals 83 /<m.
Fig. 21. Pr notochord cell isolated from early gastrula and spreading on glass for
1 h in medium with 1-5 x std. Ca2+. x 1294. Scale mark equals 7-7 pm.
Fig. 22. Pr notochord cell isolated from early gastrula and spreading on glass for
1 h in medium with 2 x std. Ca2+. x 1176. Scale mark equals 8-5 /tm.
Fig. 23. Pr notochord cells isolated from mid-gastrula and spreading on glass for
1 h in medium with 2 x std. Ca2+. x 798. Scale mark equals 12-5 /im.
Fig. 24. Pr notochord cells isolated from late gastrula and spreading on glass for
1 h in medium with 2 x std. Ca2+ x 1356. Scale mark equals 7-
Calcium and spreading behaviour of amphibian cells
24
161
162
J.
LEBLANC
AND I. BRICK
P5
Fig. 25. Pr inner neural ectoderm cell isolated from late gastrula and maintained for
1 h in suspension in Barth's X solution. x2760. Scale mark equals 3-6 ,wm.
standard medium (LeBlanc & Brick, 1981), to form more numerous, more
extensive surface projections, increased spreading and presumably increased
adhesiveness; and (3) the requirement for a solid substratum, even in the presence of Ca2+, for plasma membrane extensibility and surface projection
formation.
There is a quantitative relationship between Ca 2+ availability and cell surface
morphologic expression. While there were some variations in the specific
features of surface extensibility and projection formation among the various
cell types in media with Ca 2+ and within each cell type in the two media with
increased calcium and the standard medium (LeBlanc & Brick, 1981), all cells
responded quantitatively with respect to Ca 2+ concentration. This is clearly
evident in the absence of spreading and projection formation in all cells in the
absence of Ca 2+ and the enhanced response in these respects by all cells in the
several Ca 2+ concentrations. Although some cell types, i.e. early and late
gastrula Pr inner neural ectoderm, early and late gastrula Pr inner epidermal
ectoderm, early gastrula Pr head endoderm and early gastrula Pr notochord cells
responded similarly in 1-5 x and 2xCa 2 + , others nevertheless, demonstrated
enhanced extensibility and projection formation in 2 x Ca 2+ compared to
l-5xCa 2+ , i.e. blastula and mid-gastrula Pr inner neural ectoderm, blastula
and mid-gastrula Pr inner epidermal ectoderm, blastula, mid- and late-gastrula
Pr head endoderm and blastula, mid- and late-gastrula Pr notochord cells.
We have shown that external Ca 2+ is not essential for initial adhesion of
amphibian embryo cells to glass. However, half the cells did not adhere in
contrast to cells in standard medium (LeBlanc & Brick, 1981) or in the media
with increased Ca 2+ in which almost all cells adhered. Ca 2+ or Mg 2+ effect on
initial adhesion of cells cannot be excluded since intracellular sources of these
ions may provide low, but sufficient concentrations. In both ion deficient
Calcium and spreading behaviour of amphibian cells
163
media cell surface morphologies were similar and the cells remained spherical
suggestive of a low degree of adhesiveness. These coinciding observations
suggest that these effects may be primarily due to absence of Ca2+.
The importance of Ca 2+ for cell-cell and cell-substratum adhesion has long
been recognized (Feldman, 1955; Steinberg, 1962; Jones & Elsdale, 1963).
Steinberg (1962) demonstrated that Ca 2+ binds to embryonic cell surfaces. Tissue
culture studies indicate that Ca 2+ is essential for attachment of cells to glass
(Weiss, 1960). Taylor (1961), however, did not find Ca 2+ essential forcell attachment to glass, but in media containing serum, omission of Ca 2+ resulted in
spreading inhibition.
All cells are probably negatively charged at physiological pH with many
cation-binding groups. Curtis (1962) postulated that Ca 2+ might decrease
negative surface charge of cells, and thus by decreasing repulsive forces allow
cells to come into close approximation where Van der Waals-London forces
might promote adhesion. Ca 2+ significantly reduces surface charge density of
various amphibian gastrula cells, presumably thereby promoting adhesion
(Schaeffer, 1972).
Cell spreading may be dependent on sequential formation of new adherent
plaques. Only focal points of adhesion may be produced in cell substratum
attachment and cell microprojections appear involved in early intercellular
association (Abercrombie et al., 1971; Izzard & Lochner, 1976). Abercrombie
et al. (1971) and Heaysman & Pegrum (1973) demonstrated concentrated
areas of microfilaments in cortical cytoplasm beneath adhesive plaques. Microfilaments are also located beneath cell-cell contacts (Heaysman & Pegrum,
1973); close appositions with concentrations of subplasmalemma microfilaments being formed within 20 sec of the first visible contact. This points to
extremely rapid mobilization of microfilament assembly. In fibroblasts, the
entire lamella region of spreading cells is filled with a meshwork of microfilaments which appear to insert on the cell membrane inner surface (Abercrombie
et al., 1971; Spooner, Yamada & Wessells, 1971; Di Pasquale, 1975). Spooner
etal. (1971), using Cytochalasin B, concluded that this network was indispensable
for locomotion, particularly in the extension phase (Luduena & Wessells, 1973).
It, therefore, seems a likely hypothesis that contractile activity of these microfilaments may be involved in protrusion formation and their withdrawal. There
is evidence that such cytoplasmic microfilaments may be f-actin, and therefore,
contractile (Pollard, 1972; Spooner, Ash, Wrenn, Frater & Wessells, 1973), and
apparently insert on the plasma-membrane (Pollard & Korn, 1973). Spooner
(1975) suggests microfilaments might be regulated by uptake or release of Ca2+.
The present study indicates that Ca 2+ levels affect surface morphology by
being related to protrusion formation. Microfilaments may be involved in cell
protrusion formation and the activity of microfilaments in turn regulated by
Ca 2+ . Evidence for the presence of microfilaments in protrusion is extensive
(Baker 1965; Betchaku & Trinkaus, 1974; Di Pasquale, 1975; Nakatsuji, 1976).
164
J. L E B L A N C AND I. BRICK
Ben-Shaul, Ophir, Cohen & Moscona (1977), from freeze-fracture studies on
chick embryonic retinal cells, observed pits frequently arranged as a rim at
bases of blebs or lobopodia. They suggest these might represent anchoring sites
for contractile structures, such as microfilaments and speculated that contraction of such a system with an accompanying cytoplasmic flow might result in
projection of small blebs or lobopodia.
There is evidence which suggests that cell membranes have Ca 2+ pumping
activity that may be analogous to that of sarcoplasmic reticulum (Perdue, 1971;
Hurwitz, Fitzpatrick, Debbas & Landon, 1973). Letourneau & Wessells (1974)
have speculated that Ca 2+ pumping may be a component of cell locomotion.
Huxley (1973) noted a number of motile cells, which contained an actin-like
protein frequently identified with filaments, and that many of the same cells
contained myosin-like proteins. He suggested that the actin filament-myosin
head assembly may be a basic motile mechanism. Durham (1974) suggested that
all nonmuscle movements, if they involve actin and myosin, are controlled by
Ca 2+ flows across the membranes, which, in turn, are determined by chemical
and electrical processes at those membranes.
It appears, therefore, that a submembranal contractile system may be involved
in cell extensibility and that it may be regulated by Ca2+. Without Ca 2+ in the
medium, in vitro cells in this study were not able to form protrusions. In
addition, even if disaggregated cells of a Pr tissue type at a particular stage
initially exhibited surface projections (LeBlanc & Brick, 1981) these cells in
Ca2+-free medium were almost completely devoid of projections. Ca2+-free
and Ca2+-Mg2+-free media could not maintain surface projections. It would
appear that a certain extracellular level of Ca 2+ may be required for maintaining
the structure of a surface projection.
Increases in Ca 2+ concentrations used in experimental media are well within
the physiological range of tolerance determined by Barth & Barth (1974) for
amphibian embryo cells. In media with 1-5 x Ca 2+ and 2-0 x Ca 2+ of standard
molarity the various Pr cells at the four stages, exhibited alterations in forms of
protrusions extended onto the surrounding glass; cellular projections were
visibly different from those formed in Barth's X solution (LeBlanc & Brick,
1981). In some cases there were obvious form differences in the cell extensions
between cells incubated in medium with l-5xCa 2 + and in medium with
2-0 x Ca2+. However, in other instances there were no detectable differences,
although there was always a change in spreading behaviour from that seen in
standard medium. There may well exist a maximum extracellular Ca 2+ concentration, dependent to a degree on Pr tissue type and stage, above which
there would be no further structural alterations in protrusions. In addition to
changes in protrusion form, in media with increased molar concentration of
Ca 2+ , there was also exhibited, in some instances, a change in distribution of
projections onto the substratum. Generally, limited areas of the periphery
became dominant regions of extension rather than the entire periphery.
Calcium and spreading behaviour of amphibian cells
165
Alterations in overall cellular morphology were also observed in certain tissue
types at a particular stage (e.g. mid-gastrula Pr notochord cells) in media
with increased Ca2+.
Cells from all Pr tissue types adhered in approximately the same frequency as
in Barth's X solution. This is in direct contrast to the frequency of adhesion in
media deficient in Ca2+. Both Pr neural and epidermal ectoderm, at each stage,
in solutions with increased Ca2+, generally exhibited membranous, lamellipodial extensions rather than filopodia. Lamellipodia in other cells exhibit a
microfilamentous network (Spooner et al., 1971; Luduena & Wessells, 1973;
Di Pasquale, 1975). This network is extensive and may involve more active
cellular mobilization than microfilaments in filopodia, with a higher Ca2+
dependency. A second possibility, which is suggested by the results of longer
incubation of the two Pr ectodermal tissues (LeBlanc & Brick, in preparation),
where after 1 h filopodia were observed, and after 5 h lamellipodia, is that the
spreading process may have been accelerated by the presence of increased
Ca 2+ , perhaps by a more rapid mobilization of underlying cortical structures,
such as microfilaments. Filopodia may be extended in greater numbers and
undergo fusion into a membranous structure during the 1 h period. Filopodia
which are coalescing and with intervening cytoplasmic webbing are not unusual.
In addition, ectodermal cells, in increased Ca2+, show more flattening and more
intercellular contacts.
Pr head endoderm and Pr notochord cells also exhibit alterations in protrusions related to extracellular Ca 2+ levels. These tend to be numerically increased
or thicker filopodia, extensive stem adhesions and lamellipodia. These cells in
increased Ca 2+ also can exhibit highly convoluted stem or fingerlike regions
which may be markedly elongated. This morphological alteration may require
extensive membrane and underlyingcortical structure rearrangements. These two
Pr tissues in increased Ca2+ also exhibit a higher incidence of intercellular contact.
Since spreading cells invariably adhere and conform to their substrate, the
influence of such direct surface contacts on cell form must be considerable.
North (1970) suggested that phagocytosis is basically the same as the spreading
of a cell on a surface; the cell on a flat surface is, in effect, attempting to phagocytose a sphere of infinite diameter. The membrane is responding to contact
with the external structure by the adjustment of its adhesiveness and possibly
the tension in an actomyosin network by utilizing a mechanism which may
involve Ca2+ (Durham 1974). Wolpert & Gingell (1968) argued that inducers of
endocytosis bring about a response by direct electrical effect on the membrane
potential difference, and Gingell (1970) showed how a reduction in membrane
potential would lead to entry of Ca 2+ and a resulting contractile response.
Contact of the cell membrane with a substratum may result in membrane
potential and conformational changes which result in an entry of Ca2+.
We theorized that a substratum, living or non-living, would influence the
initial mechanisms for protrusion of cell projections. We considered two
166
J. L E B L A N C AND I. BRICK
possibilities. First, that the surface of cells suspended in Barth's X solution for
30 min or 1 h would develop an even distribution of cellular projections, such as
filopodia. If this were the case, it was reasoned that the concentrated areas of
projections seen on the surface adjacent to the glass (LeBlanc & Brick, 1981)
were the result of redistribution of projections in response to contact with a substratum. The second alternative was that after 30 min or 1 h of suspension,
during which the cells were allowed no lasting contact with a substratum, living
or non-living, cells would be devoid of projections. They might, however, have
a few surface blebs. Small blebs have the appearance of 'empty' vesicles
emerging from the cell surface and may be the result of changes in membrane
fluidity only (Ben-Shaul et al., 1977), and, therefore, possibly do not require the
mobilization of such cellular structures as microfilaments.
Our observations fit the second possibility; cell surfaces of Pr inner neural
ectoderm cells were relatively smooth and devoid of projections. Areas of slight
folding and elevations observed are in no way comparable to the blebbing seen
in freshly disaggregated cells (LeBlanc & Brick, 1981). It may be, following
removal of contact with other cells in disaggregation, that cell membranes
experience an immediate fluidity change in Ca2+-Mg2+-free disaggregation
medium. Whereas after 30 min or 1 h in medium with both Ca 2+ and Mg 2+ and
a reduced pH (7-5) this is no longer evident. Holtfreter (1943) observed that a
pH of 9-6, or more, resulted in the entire surface of isolated amphibian embryo
cells breaking forth in a number of hyaline blisters which resembled rapidly
protruding and retracting 'bubbles'. In the present study, disaggregation
medium pH was 8-9. A substratum, living or non-living, appears required for
formation of protrusions, as well as a medium which, from our observations,
must contain Ca 2+ .
REFERENCES
M., HEAYSMAN, J. E. M. & PEGRUM, S. M. (1971). The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Expl Cell Res. 67,
359-367.
BAKER, P. C. (1965). Fine structure and morphogenetic movements in the gastrula of the
tree frog, Hyla regilla. J. Cell Biol. 24, 95-116.
BARTH, L. G. & BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula in
unconditioned medium. /. Embryol. exp. Morph. 7, 210-222.
BARTH, L. G. & BARTH, L. J. (1974). Ionic regulation of embryonic induction and cell
differentiation in Rana pipiens. Devi Biol. 39, 1-22.
BEN-SHAUL, Y., OPHIR, I., COHEN, E. & MOSCONA, A. A. (1977). SEM study of dissociated
embryonic cell surface activity. IITRI/SEM2, 29-36.
BETCHAKU, T. & TRINKAUS, J. P. (1974). An in situ study of surface expansion during Fundulus epiboly. / . Cell. Biol. 63, 24a.
BRICK, I., SCHAEFFER, B. E., SCHAEFFER, H. E. & GENNARO, J. F. JR (1974). Electrokinetic
properties and morphologic characteristics of amphibian gastrula cells. Ann. New York
Acad. Sci. 238, 390-407.
CURTIS, A. S. G. (1962). Cell contact and adhesion. Biol. Rev. 37, 82-129.
Di PASQUALE, A. (1975). The locomotory activity of epithelial cells in culture. Expl Cell Res.
94,191-215.
ABERCROMBIE,
Calcium and spreading behaviour of amphibian cells
167
A. C. H. (1974). A unified theory of the control of actin and myosin in nonmuscle
movements. Cell2,123-136.
ELSDALE, T. & BARD, J. (1972). Collagen substrata for studies on cell behaviour. /. Cell Biol.
54, 626-637.
FELDMAN, M. (1955V Dissociation and reaggregation of embryonic cells of Triturus alpestris.
J. Embryo!, exp. Morph. 3, 251-255.
GINGELL, D. (1970). Contractile responses at the surface of an amphibian egg. /. Embryol.
exp. Morph. 23, 583-609.
GUSTAFSON, T. (1964). The role and activities of pseudopodia during morphogenesis of the
sea urchin larva. In Primitive Motile Systems in Cell Biology (ed. R. D. Allen & N. Kamiya),
pp. 333-349. New York: Academic Press.
HEAYSMAN, J. E. M. & PEGRUM, S. M. (1973). Early contacts between fibroblasts. An ultrastructural study. Expl Cell Res. 78, 71-78.
HOLTFRETER, J. (1943). A study of the mechanics of gastrulation. Part I. /. exp. Zool. 94,
261-318.
HURWITZ, L., FITZPATRICK, D. F., DEBBAS, G. & LANDON, £. J. (1973). Localization of
Ca2+ pump activity in smooth muscle. Science 179, 384-386.
HUXLEY, H. E. (1973). Muscular contraction and cell motility. Nature 243, 445-449.
IZZARD, C. S. & LOCHNER, L. R. (1976). Cell-to-cell substrate contacts in living fibroblasts:
an intereference reflexion study with an evaluation of the technique. /. Cell Sci. 21, 129159.
JONES, K. W. & ELSDALE, T. R. (1963). The culture of small aggregates of amphibian embryonic
cells in vitro. J. Embryol. exp. Morph. 11, 135-154.
LEBLANC, J. & BRICK, I. (1981). Morphologic aspects of adhesion and spreading behaviour
of amphibian blastula and gastrula cells. /. Embryol. exp. Morph. 61, 145-163.
LETOURNEAU, P. C. & WESSELLS, N. K. (1974). Migratory cell locomotion veisus nerve axon
elongation. Differences based on the effects of lanthanum ion. J. Cell Biol. 61, 56-69.
LUDUENA, M. A. & WESSELLS, N. K. (1973). Cell locomotion, nerve elongation, and microfilaments. Devi Biol. 30, 427-440.
NAKATSUJI, N. (1976). Studies on the gastrulation of amphibian embryos: Ultrastructure of
the migratory cells of anurans. Wilhelm Roux. Archiv. devl Biol. 180, 229-240.
NORTH, R. J. (1970). Endocytosis. Seminars Hemat. 7, 161-171.
PERDUE, J. F. (1971). The isolation and characterization of plasma membranes from cultured
cells. III. The ATP-dependent accumulation of Ca2+ by chick embryo fibroblasts. /. biol.
Chem. 246, 6750-6759.
POLLARD, T. D. (1972). Progress in understanding amoeboid movement at the molecular
level. In The Biology of Amoeba (ed. K. W. Jeon), pp. 291-317. New York: Academic
Press.
POLLARD, T. D. & KORN, E. D. (1973). Electron microscopic identification of actin associated
with isolated amoeba plasma membranes. / . biol. Chem. 248, 448-450.
RUGH, R. (1962). Experimental Embryology. Minneapolis: Butgess Publishing Co.
SCHAEFFER, H. (1972). Cell electrophoresis of disaggregated amphibian gastrula cells: changes
in electrophoretic mobility in response to extracellular calcium and pH, and various chemical
agents. Doctoral Thesis, Department of Biology, New York University.
SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. Anat. Record 78,
139-148.
SPIEGELMAN, M. & BENNETT, D. (1974). Fine structural study of cell migration in the early
mesoderm of normal and mutant mouse embryos (T-locus: t9/t9). J. Embryol. exp. Morph.
32,723-738.
SPOONER, B. S. (1975). Mictofilaments, microtubules, and extracellular materials in morphogenesis. BioScience 25, 440-451.
SPOONER, B. S., ASH, J. F., WRENN, J. T., FRATER, R. & WESSELLS, N. K. (1973). Heavy
meromyosin binding to microfilaments involved in cell and morphogenetic movements.
Tissue Cell 5, 37-46.
SPOONER, B. S., YAMADA, K. M. & WESSELLS, N. K. (1971). Microfilaments and cell locomotion. /. Cell Biol. 49, 595-613.
DURHAM,
168
J.
LEBLANC
AND I. BRICK
M. S. (1962). Calcium complexing by embryonic cell surfaces: relation to intercellular adhesiveness. In Biological Interactions in Normal and Neoplastic Growth (ed.
M. J. Brennan & W. L. Simpson), pp. 127-140. Boston: Little, Brown and Co.
TAYLOR, A. C. (1961). Attachment and spreading of cells in culture. Expl Cell Res. Suppl. 8,
154-173.
WEISS, L. (1960). Studies on cellular adhesion in tissue culture. III. Some effects of calcium.
Expl Cell Res. 21, 71-77.
WEISS, L. (1970). Cell contact phenomena. In In Vitro: Advances in Tissue Culture (ed. C.
Waymouth), pp. 48-78. Baltimore: Williams and Wilkins.
WOLPERT, L. & GINGELL, D. (1968). Cell surface membrane and amoeboid movement.
Soc. Exp. Biol. Symp. 22, 169-198.
STEINBERG,
{Received 7 October 1980, revised 5 March 1981)