/ . Embryol. exp. Morph. Vol. 60, pp. 163-171, 1980
Printed in Great Britain © Company of Biologists Limited 1980
153
Cell surface properties of amphibian embryonic
cells
By MOTOKO MATSUDA 1
From the Biological Institute, Faculty of Science, Nagoya University
SUMMARY
Gastrula and neurula embryos of Cynopspyrrhogaster were dissociated into cell suspensions
with ethylenediaminetetraacetic acid (EDTA), trypsin, and alkali solution. The cells were
cultured in Niu-Twitty's balanced salt solution and their aggregates were examined histologically. The results indicated that the capacity of amphibian embryonic cells for aggregate
formation, sorting out, and notochord differentiation was not suppressed by EDTA and
alkali treatments. Trypsin treatment, however, virtually suppressed the cell's capacity for
aggregate formation. When aggregate formation resulted from trypsin-dissociated cells, the
aggregates showed the sorting out and no differentiation of notochord.
INTRODUCTION
The cell surface plays an important role in embryonic development. Changes
in celt surface properties, resulting in the formation of stronger intercellular
adhesion, are necessary for embryonic morphogenesis (Gustafson & Wolpert,
1967; Ede & Agerbak, 1968; Johnson, 1970; Bellairs, Curtis & Sanders, 1978).
Layer specificities and tissue specificities exist upon embryonic cell surfaces
(Moscona & Moscona, 1952; Townes & Holtfreter, 1955; Moscona, 1968;
Steinberg, 1970; Gottlieb, Rock & Glaser, 1976; Marchase, Vosbeck & Roth,
1976; Tickle, Goodman & Wolpert 1978). Embryonic cells also possess lectin
receptors, cell recognizing substances, and specific cell ligands on their surfaces
(Goldschneider & Moscona, 1972; KJeinschuster & Moscona, 1972; O'Dell,
Tencer, Monroy & Brachet, 1974; Moscona, 1974; Krach, Green, Nicolson &
Oppenheimer, 1974; Hausman & Moscona, 1976; McClay, Chambers &
Warren, \911a\ McClay, Gooding & Fransen, 19776; Shur, 1977a, b\ Morriss
& Solursh, 1978; Nosek, 1978; Ben-Shaul, Hausman & Moscona, 1979;
Boucant et al. 1978; Hausman & Moscona, 1979). These data suggest that the
embryonic cell surface has a role in cellular adhesiveness during morphogenesis
and in embryonic cell differentiation.
Patricolo (1967) indicated that EDTA-dissociated amphibian gastrula cells
reaggregated but did not differentiate. The dorsal lip of the amphibian gastrula,
however, differentiated into chorda cells (Townes & Holtfreter, 1955; Jones &
1
Author's address: Biological Institute, Faculty of Science, Nagoya University, Nagoya
464, Japan.
164
M. MATSUDA
Elsdale, 1963). These data suggest that changes of embryonic cell surface
properties cause abnormal cell aggregation and inhibition of cell differentiation.
To investigate the cell surface properties in relation to embryonic cell differentiation, dissociated amphibian embryonic cells were cultured and examined for
aggregate formation, cellular sorting out, and notochord differentiation.
MATERIAL AND METHODS
Cynops pyrrhogaster embryos were obtained according to Matsuda & Oya
(1977). Early gastrula (st. 11), middle gastrula (st. 13b), early neurula (st. 15),
and neurula (st. 18) embryos were used. Embryo stages were determined from
Okada & Ichikawa (1947).
Embryos were sterilized in 70 % alcohol for 50 sec. Chorions and vitellin
membranes were removed with iridectomy scissors and watchmaker's forceps.
The individual embryo was placed in 2 ml of dissociating medium in a Linbro
semimicrotray microwell (FB-16-24-TC), whose bottom was coated with 1 %
agar, and incubated for 30 min at 25 °C. After the treatment, the dissociating
medium was replaced with Ca 2+ - and Mg2+-free Niu-Twitty's balanced salt
solution (CMF) and the embryo was dissociated into a cell suspension by gentle
pipetting. Trypsin (0-4%; Difco 1:250) in CMF, EDTA ( 1 0 ~ 3 M ; Katayama
Chemical Co.), and alkali solution (CMF of pH 9-8 justified with KOH) were
used as the dissociating media. Dispersed cells were given one minute to settle
out of the suspension. The medium was removed and replaced five times with
Niu-Twitty's balanced salt solution (BSS; Flickinger, 1949). Dissociated cells of
one embryo were cultured in BSS in a well, which was rotated on a gyratory
shaker at 70 rev./min at 20-23 °C. As controls, st. -11, -13b, -15, and -18 embryos,
which were dechorioned, were cultured in the same manner as described above.
When the control embryos developed at sts. 27-29, the cultures were fixed for
3-5 h in 10 % neutral formalin, rinsed in running tap water, dehydrated, and
embedded in paraffin. These paraffin materials were sectioned at 10 fim and
stained with picronigrosin (one part of 1 % nigrosin solution and nine parts of
saturated picric acid) for 2 h at room temperature.
RESULTS
Recovery of the dissociated cells. Dissociated middle gastrula cells are shown
in Fig. 1. Yolk platelets derived from broken cells are evident. Recovery of these
cells after the dissociation is summarized in Table 1. Our results showed that
almost all cells of intact embryos were recovered after EDTA and alkali dissociations, (see Suzuki, Kuwabara & Kuwana, 1976) but only half the cells were
recovered after trypsin dissociation.
Viability of the dissociated cells. Results of viability tests of dissociated cells
are summarized in Table 2. Viability of dissociated cells with each treatment at
Embryonic cell surface properties
Fig. 1. Dissociated middle gastrula cells. A, EDTA-dissociated cells; B, alkalidissociated cells; C, trypsin-dissociated cells. (Phase contrast.)
165
166
M. MATSUDA
Table 1. Number of dissociated cells recovered from one embryo
Stages
11
13b
15
18
EDTA-dissociated
cells
26 000 ±800*
56000 ±3600
64800 ±2800
73 600 ±1600
Trypsin-dissociated
cells
14 700 ±1400
30 800 ±2800
26000 ±800
3 5 600 ±4000
Alkali-dissociated
cells
20 800 ±2400
51600 ±2000
60 800 ±1600
74 800 ±2800
* ± Standard deviation.
Table 2. Viability of dissociated amphibian embryonic cells
Stages
EDTA-dissociated
cells
11
13b
15
18
87-3 ±4-89%*
87-2±4-36
89-7±4-13
92-8 ±6-23
Trypsin-dissociated
cells
Alkali-dissociated
cells
95-4 ±1-90
95-8 ±1-49
98-7 ±0-52
97-8 ±1-60
77-7 ±7-30
76-8 ±4-45
90-7 ±5-43
89-4 ±8-67
± Standard deviation.
Table 3. Formation of aggregates from dissociated cells
Stages
EDTA-dissociated
cells
Trypsin-dissociated
cells
Alkali-dissociated
cells
•S)
«©
«(D
"©
0/
/o
11
13b
15
18
-g)
each developmental stage was over 85 % except for the alkali-dissociated
gastrula cells.
Formation of aggregates. The results of aggregate formation from the dissociated cells are summarized in Table 3. Dissociated cells usually formed one aggregate per well. In the cultures of EDTA-dissociated and alkali-dissociated cells,
most cells joined the aggregate, while only a few hundred remained on the agar
and failed to participate. The majority of these cells did not exclude trypan blue.
Embryonic cell surface properties
167
Table 4. Histological observation of aggregates from dissociated amphibian
embryonic cells
Aggregates which showed the
sorting out
11
13b
15
13
14
22
24
11
18
26
22
21
11
14
22
24
11
18
26
22
21
10
4
11
1
0
0
11
1
1
10
0
21
3
0
2
0
10
3
5
23
8
19
11
14
22
24
0
18
26
22
21
1
18
EDTA
Alkali
EDTA
Alkali
Trypsin
EDTA
Alkali
EDTA
Alkali
to to
Stages
No. of aggregates
which
No. of
- differentiated
Type of
notochord
dissociation aggregates Number Type A TypeB TypeC
In the cultures of trypsin-dissociated cells, hardly any aggregates were formed
although the majority of individual cells excluded trypan blue.
Histological observation of the aggregates. Histological observations of the
aggregates are summarized in Table 4. All the aggregates exhibited smooth
spherical forms.
Two out of 13 aggregates, which were formed by EDTA-dissociated early
gastrula cells, showed m ixtures of endodermal, ectoderrnal, and mesodermal cells.
Differentiated notochord was not observed in these two aggregates. Ten out of
the remaining 11 aggregates showed central cell masses which were formed by
ectodermal and mesodermal cells, easily distinguished from the surrounding
endodermal cells by differences in cellular shape and size. Differentiated notochords were observed in these cell masses (Fig. 2). The pattern of this cell
arrangement was termed pattern A. One out of the 11 aggregates did not form
the central cell mass constituted by ectodermal and mesodermal cells. These
cells remained close to the surface of the aggregate, differentiating into notochords, surrounding mesenchymes, and epidermis at the periphery, endodermal
cells centrally. This cell arrangement was termed pattern C (Fig. 3). The cell
arrangement of the control embryos (Fig. 4) resembled pattern C. Twenty-two
aggregates from EDTA-dissociated middle gastrula cells were examined. The
sorting out of endodermal cells and ectodermal and mesodermal cells occurred
in all aggregates. Eleven out of 22 aggregates showed pattern A. In the remaining
aggregates, ectodermal and mesodermal cells were distributed in the central
portion, embedded in endodermal cells, but these cells accumulated and reached
the surface (Fig. 5). This arrangement was termed pattern B. As the developmental stage of donor embryos proceeded, the proportion of pattern B and C
aggregates to total aggregates became larger.
The sorting out of endodermal cells and ectodermal and mesodermal cells
168
M. MATSUDA
0-25 mm
0-25 mm
0-25 mm
0-25 mm
6 '
Fig. 2. Section of aggregate formed by EDTA-dissociated early gastrula cells
(pattern A). Notochord indicated by arrow.
Fig. 3. Section of aggregate formed by alkali-dissociated middle gastrula cells
(pattern C). Notochords indicated by arrows.
Fig. 4. Section of control embryo. Early neurula embryo was cultured and fixed
at st. 28.
Fig. 5. Section of aggregate formed by EDTA-dissociated middle gastrula cells
(pattern B). Notochords indicated by arrows.
Fig. 6. Section of aggregate formed by trypsin-dissociated middle gastrula cells.
The section shows sorting out of endodermal and other cells. No significant differentiation is observed.
Embryonic cell surface properties
169
occurred in all aggregates formed by alkali-dissociated cells. The majority of
aggregates showed pattern C. Differentiated notochords were observed in all
aggregates.
Eleven aggregates of trypsin-dissociated middle gastrula cells were observed.
All aggregates showed segregation of endodermal cells and other cells. These
segregation patterns resembled pattern C. Histological examination, however,
did not demonstrate any significant differentiation (Fig. 6).
DISCUSSION
In this experiment, the differentiation of notochords was shown within the
aggregates of EDTA-dissociated early gastrula cells, although Patricolo (1967)
reported that the aggregates of dissociated early gastrula cells did not demonstrate any significant differentiation. Jones & Elsdale (1963) showed that EDTAdissociated dorsal-lip cells from a gastrula of Triton alpestris differentiated into
chorda cells in in vitro culture. The difference between our results and those of
Patricolo (1967) probably derive from differences in cell-dissociating procedures
and culture methods.
Formation of aggregates from trypsin-dissociated cells resulted in fewer and
smaller aggregates. Differentiated notochord was not observed in these aggregates. The small volume of aggregates was caused by the decrease in the number
of reaggregated cells. Moscona & Moscona (1967) commented that trypsin
dissociation of chick embryonic cells was superior to EDTA dissociation with
regard to cell dissociation, cell recovery, and cell differentiation. The results of
this experiment, however, showed that trypsin dissociation of amphibian
embryonic cells reduced recovery of cells, formation of aggregates, and differentiation of cells, compared to EDTA and alkali dissociations.
The results of the present experiment also indicated that the capacity for
aggregate formation, sorting out, and notochord differentiation was not
suppressed in amphibian embryonic cells by EDTA and alkali treatments.
Trypsin treatment, however, virtually suppressed the capacity of cells for
aggregate formation. These data suggested that trypsin-sensitive component(s)
for cell reaggregation existed upon embryonic cell surfaces. Trypsin-sensitive
cell-surface component was required for the aggregation of embryonic neural
retina cells and for the adhesion of those cells and optic tectum (McClay et al.
1911b; Hausman & Moscona, 1979; Ben-Shaul et al. 1979). When aggregate
formation resulted from trypsin-dissociated cells, sorting out of endodermal
cells and other cells occurred but notochord differentiation was not observed.
These results indicated that the capacity for cellular sorting out was not suppressed by trypsin treatment, whereas that of notochord differentiation was
suppressed. Chorda cells differentiated autonomously from dorsal lip cells
(Jones & Elsdale, 1963). The cell surface of presumptive notochordal cells is
possibly changed by trypsin treatment, and these cells may not aggregate
170
M. MATSUDA
(including the cells dissociated on agar or those dying) and/or may not differentiate.
REFERENCES
BELLAIRS, R., CURTIS, A. S. G. & SANDERS, E. J. (1978). Cell adhesiveness and embryonic
differentiation. J. Embryol. exp. Morph. 46,207-213.
BEN-SHAUL, Y., HAUSMAN, R. E. & MOSCONA, A. A. (1979). Visualization of a cell surface
glycoprotein, the retina cognin, on embryonic cells by immuno-latex labelling and scanning
electron microscopy. DevlBiol. 72,89-101.
BOUCANT, J. C , BERNARD, B., AUBERY, M., BOURRILLION, R. & HONILLION, CH. (1979).
Concanavalin A binding to amphibian embryo and effect on morphogenesis. /. Embryol.
exp. Morph. 51,63-72.
EDE, D. A. & AGERBAK, G. S. (1968). Cell adhesion and movement in relation to the developing limb pattern in normal and talpid3 mutant chick embryo. J. Embryol. exp. Morph. 20,
81-100.
FLICKTNGER, R. E. (1949). A study of the metabolism of the amphibian neural crest cells
during their migration and pigmentation in vitro. J. exp. Zoo/. 112, 465—484.
GOLDSCHNEIDER, I. & MOSCONA, A. A. (1972). Tissue-specific cell surface antigens on embryonic cells. /. CellBiol. 53,435-449.
GOTTLIEB, D. 1., ROCK, K. & GLASER, L. (1976). A gradient of adhesive specificity in developing avianretina.Proc. natn. Acad. Sci., U.S.A. 73,410-414.
GUSTAFSON, T. & WOLPERT, L. (1967). Cellular movement and contact in sea urchin morphogenesis. DevlBiol. 42,442-498.
HAUSMAN, R. E. & MOSCONA, A. A. (1976). Isolation of retina-specific cell-aggregating factor
from membranes of embryonic neural retina tissue. Proc. natn. Acad. Sci., U.S.A. 73;
3594-3598.
HAUSMAN, R. E. & MOSCONA, A. A. (1979). Immunologic detection of retina cognin on the
surface of embryonic cells. Expl Cell Res. 119,191-204.
JOHNSON, K. E. (1970). The role of changes in cell contact behaviour in amphibian gastrulation. /. exp. Zool. 175,391-428.
JONES, K. W. & ELSDALE, T. R. (1963). The culture of small aggregates of amphibian embryonic cells in vitro. J. Embryol. exp. Morph. 1 1 , 1 3 5 - 1 5 4 .
KRACH, S. W., GREEN, A., NICOLSON, G. & OPPENHEIMER,
S. (1974). Cell surface changes
occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins. Expl Cell Res. 84,191-198.
KLEINSCHUSTER, S. J. & MOSCONA, A. A. (1972). Interactions of embryonic and fetal neural
retina cells with carbohydrate binding phyto-agglutinins: cell surface changes with differentiation. Expl Cell Res. 70,397-410.
MARCHASE, R. B., VOSBECK, K. & ROTH, S. (1976). Intercellular adhesive specificity. Biochem.
Biophys. Acta 457,385-416.
MATSUDA, M. & OYA, T. (1977). Induced oviposition in the newt Cynops pyrrhogaster by
subcutaneous injection of human chorionic gonadotropin. Zool. Mag. 86, 44-47.
MCCLAY, D. R., CHAMBERS, A. F. & WARREN, R. W. (1977). Specificity of cell-cell interactions in sea urchin embryos. Appearance of new cell-surface determinants at gastrulation.
DevlBiol. 56,343-355.
MCCLAY, D. R., GOODING, L. R. & FRANSEN, M. E. (1977). A requirement for trypsinsensitive cell-surface components for cell-cell interactions of embryonic neural retinal cells.
J. CellBiol. 75,56-66.
MORRISS, G. M. & SOLURSH, M. (1978). Regional differences in mesenchymal cell morphology
and glycosaminoglycans in early neural-fold stage rat embryo. /. Embryol. exp. Morph. 46,
37-52.
MOSCONA, A. A. (1968). Cell aggregation: Properties of specific cell-ligands and their role in
the formation of multicellular systems. Devi Biol. 18, 250-277.
Embryonic cell surface properties
111
A. A. (1974). Surface specification of embryonic cells: lectin receptors, cell
recognition and specific cell ligands. In The Cell Surface in Development (ed. A. A. Moscona), p. 67. New York: Wiley.
MOSCONA, A. A. & MOSCONA, H. (1952). Dissociation and aggregation of cells from organ
rudiments of the early chick embryo. /. Anat. 86,283-301.
MOSCONA, A. A. & MOSCONA, M. H. (1967). Comparison of aggregation of embryonic cells
dissociated with trypsin or versene. Expl Cell Res. 45,239-243.
NOSEK, J. (1978). Changes in the cell surface coat during the development of Xenopus laevis
embryo; detected by lectins. Wilhelm Roux1 Arch, devl Biol. 184, 181-193.
O'DELL, D. S., TENCER, R., MONROY, A. & BRACHET, J. (1974).. The pattern of concanavalin
A-binding sites during the early development of Xenopus laevis. Cell Differ. 3, 193-198.
OKADA, K. & ICHIKAWA, M. (1947). Normal table of Triturus pyrrhogaster (BOIE). Ann.
Rept. ExptlMorphol. (Tokyo) 3,1-6.
PATRICOLO, E. (1967). Differentiation of aggregated embryonic cells of amphibians (Discoglossuspictus). ActaEmbryol. exp. 10,75-100.
SHUR, B. D. (1977a). Cell-surface glycosyltransferase in gastrulating chick embryos. I.
Temporally and spatially specific patterns of four endogeneous glycosyltransferase activities. Devi Biol. 58,23-39.
SHUR, B. D. (19776). Cell-surface glycosyltransferase in gastrulating chick embryos. II.
Biochemical evidence for a surface localization of endogeneous glycosyltransferase activities. Devi Biol. 58,40-55.
STEINBERG, M. S. (1970). Does differential adhesion govern self-assembly processes in
histogenesis? Equilibrium configurations and the emergence of a hierarchy among populations of embryonic cells. /. exp. Zool. 173,395-434.
SUZUKI, A., KUWABARA, Y. & KUWANA, T. (1976). Analysis of cell proliferation during early
embryogenesis. Develop., Growth and Differ. 18,447-455.
TICKLE, C , GOODMAN, M. & WOLPERT, L. (1978). Cell contacts and sorting out in vitro: the
behaviour of some embryonic tissues implanted into the developing chick wing. /. Embryol.
exp. Morph. 48,225-238.
TOWNES, P. L. & HOLTFRETER, J. (1955). Directed movements and selective adhesion of
embryonic amphibian cells. /. exp. Zool. 128,53-120.
MOSCONA,
{Received 6 November 1979, revised 28 April 1980)