/ . Embryo!. exp. Morph. Vol. 57, pp. 95-106, 1980
Printed in Great Britain © Company of Biologists Limited 1980
95
Changes in lectin-mediated agglutinability during
primary embryonic induction in the
amphibian embryo
By FRANCISCO D. BARBIERI, 1 SARA S. SANCHEZ 1
AND ENRIQUE J. DEL PINO 1
From the Institute de Biologia del Desarrollo,
Universidad Nacional de Tucumdn and
Consejo Nacional de Investigaciones Cientificas y Tecnicas,
San Miguel de Tucumdn, R. Argentina
SUMMARY
The present study was undertaken to investigate structural alterations at the surface of
presumptive neural cells after primary embryonic induction. For this purpose, plant lectinmediated agglutinability of dissociated cells from the epiblast of Bufo arenarum gastrulae
was tested. Two fragments of epiblast were excised from the same mid-gastrula: one from
the dorsal side of the egg, making contact with the invaginating chordamesoblast and
assumed to be composed of determined cells and the other from the ventral region of the
egg, facing the blastocoele cavity and assumed to be composed of undetermined cells. Cells
of the pooled fragments were dissociated in calcium-free Holtfreter's solution with potassium
oxalate and incubated in the presence of different concentrations of phytohemagglutinin
and concanavalin A. Epiblast cells overlying the archenteron roof are less agglutinated with
both lectins than undetermined cells. On the other hand, when egg fragments were removed
from the dorsal and ventral regions of early gastrulae before the archenteron was formed,
no significant difference in lectin-mediated agglutinability was observed, even after having
been cultured in vitro in absence of inducing tissue. These results suggest that the target
of the inducing signal generated in the mesoblast is likely to be located on the surface of
epiblast cells. Additional experiments showed that cells pretreated with colchicine, cytochalasin B or colchicine and cytochalasin B simultaneously exhibit no significant variation
in agglutinability, suggesting that the cytoskeleton was not be involved in the cell surface
alteration here described.
Treatment of whole embryos or sandwich explants with concanavalin A or phytohemagglutinin has no effect on neural tube formation, suggesting that the carbohydratecontaining binding sites for these lectins are not involved in primary embryonic induction.
Changes in cell agglutinability described in this paper are to be interpreted thus as a secondary
expression of structural alterations in the cell surface concomitant with neural determination.
INTRODUCTION
In the amphibian gastrula, cells underlying the dorsal lip of the blastopore
are capable of inducing the differentiation of the central nervous system. As
1
Authors' address: Instituto de Biologia del Desarrollo, Chacabuco 461, 4000 S.M. de
Tucuman, R. Argentina.
7-2
96
F. D. BARBIERI AND OTHERS
cells are invaginated through the blastoporal cleft, contact is established
between the two components of the primary inducing system: the organizer
cells of the archenteron roof and the reacting cells of the overlying epiblast.
Since the discovery of primary induction by Spemann & Mangold (1924), an
impressive amount of work has been devoted to clarifying its mechanism of
action, especially as regards the chemical nature of the signal transferred from
the organizer to the presumptive neural area. Very little attention has been
paid, however, to the immediate changes triggered by the inducer in the
reacting cells.
A first approach to establish whether primary induction is accompanied by
a modification of the surface of epiblast cells has now been afforded by the
use of plant lectins. These proteins which bind to specific saccharides (see
Sharon & Lis, 1972) have been widely used to investigate the nature and
functional significance of carbohydrate-containing cell surface receptor sites
on a wide variety of eukariotic cell types (Nicolson, 1974). The experimental
design used in the present study essentially consisted of measuring agglutination
with phytohemagglutinin and concanavalin A of dissociated neuroectoblast
presumptive cells before and after contact has been established between these
cells and the invaginating cells of the archenteron roof. To the best of our
knowledge the results obtained show for the first time that the epiblast cell
surface is affected during neural induction. In addition, the effect of these
lectins on induction has been assayed in order to establish whether their
carbohydrate-binding sites are involved in this process.
MATERIALS AND METHODS
Embryos
Females of the toad Bufo arenarum were injected with a fresh suspension
of homoplastic hypophysis and kept at 23 °C. About 12 h after injection,
animals were pithed, eggs were removed from the ovisacs, fertilized in vitro,
and allowed to develop in Holtfreter's solution (Holtfreter, 1931) to the
required stage.
Surgery
When the eggs had reached the desired stage, egg envelopes were removed
with sharpened forceps. Specific areas of the embryo were dissected with
tungsten needles and hair loops in either standard Holtfreter's solution (for
agglutination assays) or Barth's solution (Barth & Barth, 1959) containing
50 /tg/ml penicillin and 30 /tg/ml dihydrostreptomycin (for explant experiments).
Agglutination assays
About twenty fragments excised from the embryos or explants cultivated
in vitro were pooled in fresh Holtfreter's solution and then transferred to the
Cell surface changes during neural induction
97
dissociation medium contained in dishes covered with agar purified according
to Clausen (1969). In order to minimize cell breakage, three different dissociating media were previously tested: (a) calcium-free Holtfreter's with
0-034 M potassium oxalate added (Feldman, 1955); (b) Ca-free Holtfreter's with
15mg/100mlEDTA (Schaeffer, Schaeffer & Brick, 1973) at pH7-5; and (c)
Ca-free Ringer's containing 1 mM Tris buffer at pH 7-5. Cell breakage was
estimated by the presence of free yolk platelets at the bottom of the container
and the formation of a viscous material to which cells stick. Cell breakage
occurred with all dissociation procedures but was least when embryo fragments
were incubated in Holtfreter-oxalate solution. After washing three times in
this same solution, fragments were incubated undisturbed at room temperature
until most of the cells were spontaneously dissociated (45-60 min).
For agglutination assays, dissociated cells were carefully drawn into a
Spemann micropipette and transferred to 100 ji\ of Holtfreter-oxalate containing either phytohemagglutinin (PHA) (Difco) or concanavalin A (Con A)
(Sigma, Type IV) at the concentrations indicated in each experiment. Attempts
to transfer a predetermined number of cells proved to be detrimental to cell
integrity due to the large size and fragility of amphibian gastrula cells. Since
the release of intracellular materials is likely to affect cell agglutinability, it
was judged preferable to work with an undetermined number of cells kept as
far as possible constant for each experiment. As an additional precaution to
prevent artifactual results, the cell number and the volume of dissociating
solution transferred were very small as compared to the volume of the agglutination medium. The test was performed within a plastic ring mounted on
agar-coated glass slides. Control cells were incubated in the absence of lectins,
in PHA with 0-1 M JV-acetyl-D-galactosamine (Sigma, Grade III) and Con A
plus 0-1 M a-methyl-D-mannoside (Sigma, Grade III). After gentle shaking,
cells were left undisturbed for 30 min at room temperature. Plates were then
shaken to disperse non-agglutinated cells and scored under a microscope. To
determine the percentage of agglutinated cells, the number of clusters of more
than five cells and dispersed cells were counted in three different microscopic
fields (Noonan & Burger, 1973), at x200 magnification.
To assess the effect of colchicine and cytochalasin B on cell agglutinability,
agglutination assays were performed on cells incubated for 30 min in the
presence of these inhibitors prior to agglutination. Colchicine was dissolved
in Holtfreter-oxalate and cytochalasin B in the same solution containing 5%
dimethyl sulphoxide (Sigma, Grade I).
Effect of lectins on induction
Three procedures were used to test whether lectins inhibit primary induction.
(1) When the blastoporal groove had just formed (stage 10, according to Del
Conte & Sirlin (1951)), a slit was opened at the ventral side of manually
dejellied eggs, allowing a free communication between the blastocoele and
98
.
F. D. BARBIERI AND OTHERS
A
B
Fig. 1. (A) Section of an egg fixed at the stage when dissection was carried out.
(B) Diagrammatic representation of egg dissection to obtain the determined
(hatched pattern) and undetermined (dotted pattern) portions of epiblast. Scale
bar is 200 fim.
the medium. Embryos were then incubated in Barth's solution containing the
lectin to be tested, either for 20 h and then transferred to standard Barth's, or
continuously in the presence of the lectin. Control operated embryos were
cultured for the same period in Barth's solution. (2) Lectins dissolved in
Barth's solution were injected into the blastocoele with glass micropipettes
controlled by a Leitz micromanipulator as described elsewhere (Manes &
Barbieri, 1976), and eggs were allowed to develop in Barth's solution. Control
embryos were injected with Barth's solution. (3) The inducer tissue (dorsal
lip of the blastopore) removed from an early gastrula was enveloped in a piece
of ventral epiblast of the same age following the 'sandwich method' of Holtfreter
(Holtfreter, 1933). Immediately after removal both pieces were washed in
lectin solution, each dorsal lip enveloped in the epiblast, and the explants
incubated for 5-7 h at room temperature (about 20 °C). The explants were
finally washed and cultivated in Barth's solution.
RESULTS
Agglutination of presumptive neural cells prior to and after ectoblast-mesoblast
interaction
In order to assess whether there is any significant change in lectin-mediated
agglutinability of cells from the presumptive neuroectoblast in contact or not
with the invaginating chordamesoblast, mid-gastrulae (stage 11) were dissected
as indicated in Fig. 1. At this stage the advancing Bufo arenarum archenteron
Cell surface changes during neural induction
99
is about halfway (Fig. 1 A), and it is possible to dissect the presumptive neural
plate free of contaminating cells. The roof of the blastocoele was cut out
in such a way that a rectangular flap extending from the ventral side of the
embryo to the blastopore, where it remained attached, was obtained. The flap
was then folded over the blastopore so to expose its inner portion. The area
free of mesoblast and endoblast cells was cut out first (these fragments will be
referred to as undetermined epiblast) (Fig. 1B). The remaining portion of the
flap was then separated from the archenteric roof. At this stage, this could be
done with relative ease by interposing a hair loop between both cell layers
from the surface of section towards the blastopore. Once detached from the
roof of the archenteron, the epiblast layer was cut out free with a needle.
Based on the assumption that the inducing signal has already been transferred
from the archenteric roof to this territory (see Deuchar, 1970), it will be
referred to as determined epiblast (Fig. IB). Fragments of embryos were pooled
and the cells, after disaggregation, were tested for agglutinability with PHA
and Con A as described in Materials and Methods. Cell breakage was negligible
as assessed by the presence of yolk platelets in the medium and, as was expected
on the basis of their morphological similarity, cells removed from the dorsal
and ventral side of the embryo were apparently damaged to the same extent.
The results obtained have shown that cells derived from both regions are
agglutinated with PHA at relatively low concentrations, and with Con A to
a lesser extent. As shown in Table 1 and Fig. 2, the agglutination response to
both lectins was generally lower for the determined epiblast than the undetermined epiblast. In all the experiments agglutination by PHA and Con A
was completely inhibited by JV-acetyl-D-galactosamine and a-methyl-D-manoside, respectively.
Agglutination of undetermined epiblast cells of the dorsal and ventral region of
the embryo
To establish whether regional differences in cell agglutinability exist regardless
of induction further experiments were undertaken. PHA-mediated agglutinability of epiblast cells excised from the dorsal and ventral regions of the embryo
at the onset of gastrulation was compared. Just after the blastoporal lip was
formed (stage 10), two rectangular fragments of about the same size were
dissected free from each embryo: one from the dorsal region at the side where
the blastopore was located, and the other from the opposite ventral region
of the embryo. Care was taken to exclude the animal pole and the marginal
region. Cells of the pooled fragments were dissociated and assayed for agglutination with PHA. As shown in Table 2, no significant differences are apparent
in cells from the dorsal and ventral areas.
To investigate whether the relative extent of dorsal cell agglutination might
be reduced in the course of gastrulation independently of neural induction, a
control experiment with epiblast fragments cultured in vitro in the absence of
100
F. D. BARBIERI AND OTHERS
Table 1. Lectin-mediated agglutination of undetermined and
determined presumptive neuroectoblast cells
Lectin
Concentration
(/tg/ml)
—
PHA
10
25
50
150
50 + 0-1 MiV-acetylD-galactosamine
Con A
50
150
250
50 + 0-1 Ma-methylD-mannoside
Undetermined Determined
epiblast
epiblast
0
0
17
36
59
83
55
80
91
92
100
0
6
8
25
17
27
10
40
6
31
14
89
67
90
92
100
100
97
100
100
0
0
12
18
85
65
100
82
100
100
20
47
85
85
73
87
90
0
0
7
0
46
15
36
63
92
90
0
0
0
0
0
0
Individual results are given, expressed as percentage of agglutinated cells according to
the procedure described in Materials and Methods.
an inducing stimulus was carried out. Dorsal and ventral fragments were
dissected from an early gastrula as in the preceding experiment and kept in
Holtfreter's solution up to the time non-operated embryos reached stage 11
(6 h at about 20 °C). By this time, explants appeared curled but not completely
closed. When PHA-induced agglutination of dissociated cells from dorsal and
ventral explants was compared, no difference was evident (Table 3).
Cell surface changes during neural induction
101
Fig. 2. Agglutination by Con A of dissociated epiblast cells from Bufo arenarum
gastrula. (A) Dispersed cells from the undetermined epiblast. (B) Same cells
incubated with Con A (50 /ig/ml). (C) Dispersed cells from the determined epiblast.
(D) Determined cells in the presence of Con A (50/ig/ml). Scale bar is 200 /*m.
102
F. D. BARBIERI AND OTHERS
Table 2. PHA-mediated agglutination of dissociated cells from the dorsal
and ventral regions of the embryo at the onset of gastrulation
PHA
(/*g/ml)
Dorsal epiblast Ventral epiblast
10
25
36
44
40
70
100
100
44
50
50
60
60
100
The results are given as in Table 1.
Table 3. PHA-mediated agglutination of dissociated cells from explants of
the dorsal and ventral regions of the embryo cultivated in vitro
PHA
(/ig/ml)
Dorsal
epiblast
Ventral
epiblast
10
20
40
50
60
70
90
93
20
30
45
55
88
85
92
25
The results are given as in Table 1.
Effect of colchicine and cytochalasin B on lectin-induced agglutination
To test the influence of colchicine and cytochalasin B on cell agglutinability,
experiments were performed with dissociated cells derived from the determined
and undetermined epiblast. Typical data are presented in Table 4, which
compares the agglutination response of both cell types in the presence or
absence of drugs. Using either Con A or PHA, no significant variation in
agglutinability was found when inhibitors-treated cell suspensions were compared
to controls.
Lectin effects on primary induction
The experiments described above suggest that surface glycoproteins binding
to Con A and PHA might be involved in the mechanism of primary induction.
Therefore, as the possibility arises that these lectins exert an inhibitory effect
on the induction process, an additional series of experiments was undertaken
to test this hypothesis. As was pointed out in Materials and Methods, lectin
influence on induction was assayed on the whole embryo as well as on explanted
pieces of combined inductor and undetermined epiblast.
In the first series, three microinjection experiments were conducted with
Cell surface changes during neural induction
103
Table 4. Effect of inhibitors on ledin-induced agglutination
of epiblast cells
PHA (/tg/ml)
Con A (/tg/ml)
250
A
50
25
A.
1
Additions
None
Colchicine
10"6M
10"4M
Cytochalasin B (30 ^g/ml)
Colchicine (10"6 M) +cytochalasin B
(30/ig/ml)
D
U
D
U
D
55
80
10
40
90
100
20
85
100
—
90
50
80
90
—
92
—
88
0
50
40
—
50
—
44
88
100
100
58
100
100
100
17
88
90
17
90
52
90
—
92
—
—
97
—
85
—
72
—
—
87
—
72
u
Abbreviations: U, undetermined cells; D, determined cells.
The results are given as in Table 1.
eggs from different batches. In each experiment, ten early gastrulae (stage 10)
were injected with 90 nl of a solution of PHA (5000 /*g/ml) and 11-90 nl of
a solution of Con A (1000-5000 /tg/ml). Under these treatment conditions, the
progress of morphogenesis appeared undisturbed. In injected eggs the neural
folds formed and fused at the same time as in Barth's-injected or uninjected
control eggs. Abnormalities did appear after 3 days, as lectins appear to
exert an inhibitory effect on hatching. Embryos assumed a tightly coiled
position within the vitelline envelope. When the envelope of these animals
was disrupted by means of sharpened forceps, apparently normal larvae were
obtained.
In the second series, gastrulae with an opened blastocoele (30 embryos)
were incubated in the presence of either 100 /tg/ml Con A or 50 /^g/ml PHA.
As compared to control gastrulae developing in Barth's solution, lectin-treated
gastrulae exhibited a slight retardation in the progress of blastopore invagination. In other ways, the development was exactly the same as in lectininjected eggs.
Three dorsal lip-epiblast sandwich experiments were carried out with embryos
from different clutches, each one consisting of ten lectin-treated and ten control
samples. Experimental explants were treated with either 50 ^g/ml PHA (two
experiments) or 50 fig/m\ Con A (one experiment). As in the above described
experiments, no significant differences were found between lectin-treated and
control explants. Adhesion between the combined fragments appeared unaffected, and in all the specimens the dorsal lip was completely enveloped by
the epiblast sheet. Active cilia were uniformly present over the surface of the
104
F. D. BARBIERI AND OTHERS
Fig. 3. Histological section of a sandwich explant pretreated with Con A 950 /*g/ml)
showing a typical neural tube, after 4 days. Scale bar is 200/tm.
explant and often streamers of mucus were seen. Examination of histological
sections showed also a quite uniform picture. Typical neural tissue was consistently observed in the vicinity of the implant (Fig. 3). Fragments of Bufo
arenarwn epiblast cultured without the associated blasoporal lip developed
into epidermis, melanophores and ciliated cells, but no autoneuralization was
observed under present conditions.
DISCUSSION
The present findings show that epiblast cells of Bufo arenarum gastrula
possess surface receptors for PHA and Con A capable of participating in an
agglutination reaction. This suggests that surface carbohydrates of the disaggregated cells contain 7V-acetyl-D-galactosamine and a-methyl-D-glucose or
D-mannose-like residues. Results presented here also show that dissociated
cells exhibit a higher agglutinability with PHA than with Con A, irrespective
of their location in the embryo. In spite of the difficulty in quantifying the
extent of cell agglutination in experiments with amphibian embryonic cells
and the high variability of absolute values, consistent relative results were
obtained in different experiments.
Under these experimental conditions, there is no difference in agglutinability
between dissociated epiblast cells from the dorsal and ventral regions of the
early gastrula. In agreement with this observation O'Dell, Tencer, Monroy &
Brachet (1974) have reported that fluorescent Con A does not exhibit any
Cell surface changes during neural induction
105
differential binding to the dorsal and ventral surface of Xenopus embryo.
Similar results have been obtained with fluorescent wheat germ and soybean
agglutinins (Tencer, according to Brachet, 1977). However, when epiblast cells
are underlaid by the invaginating archenteron roof, a decrease in agglutinability
both with PHA and Con A occurs. Whether this surface alteration is causally
related to the inducing action of the primary organizer remains an open
question. Although present data do not allow us to clear up this point, the
view is favoured by indirect evidence. The results of the agglutination assays
carried out with explanted dorsal and ventral epiblast fragments indicate that
we are not dealing with an autonomous regional change. It is further supported
by the close topographical and chronological correlation existing between the
cell surface alteration manifested by lectin-induced agglutination and the
establishment of epiblast-chordamesoblast contact.
An interesting possibility suggested by present results is that we are probably
dealing with an early response to the inducing stimulus, i.e. ascribable to the
period of cell determination. In fact, taking into account the time elapsed
between the onset of gastrulation (stage 10) and the moment when the agglutination change was detected (stage 11), the modification of the cell surface
structure is expected to have occurred in about 6 h at 20 °C. In addition, this
time period closely correlates with the increase in RNA synthesis ascribable
to induction as reported by Brahma (1966). The author observed that when
explants of epiblast cells of Triturus pyrrhogaster gastrulae are cultivated in
combination with the organizer, an enhanced incorporation of [3H]uridine in
the nuclei is detected 6 h after the beginning of contact with the mesoblast.
The fact, also shown in the present report, that PHA and Con A do not
interfere with neural induction in the toad, suggests that lectin receptors are
not directly concerned in this process. The change in cell agglutinability, if
relevant to primary induction, would be a secondary expression of the cell
surface modification determined by the inducing signal probably due, as suggested for other cell systems (see Nicolson, 1974), to the mobility of lectin
receptors. In this particular case, however, receptor mobility does not seem
to depend on the peripheral cytoskeleton composed of microtubules and
microfilaments (see Nicolson, 1976). In fact, under present conditions, colchicine
and cytochalasin B used either separately or together did not affect the agglutinability of embryonic Bufo arenarum cells.
Incidentally, other effects of lectins on the gastrula of the toad have been
observed. PHA and Con A at the higher concentrations tested (50 and 100 /^g/ml
respectively) retard morphogenesis as reported for Amblystoma maculatum
(Moran, 1974). An interesting observation was that at lower concentrations
(microinjection experiments) both lectins lead to a complete arrest of hatching
without any apparent interference with normal embryogenesis. This effect may
be due to a block of the lytic of the hatching enzyme as a result of lectin
binding to carbohydrates of the fertilization envelope.
106
F. D. BARBIERI AND OTHERS
Appreciation is expressed to Mr Eduardo Rothe for his assistance in the preparation of
the manuscript, to Mr Jose Greco for technical assistance and Mr Hugo Gomez for the
typing. Francisco D. Barbieri and Sara S. Sanchez are members of the Carrera del Investigador Cientifico, Consejo Nacional de Investigaciones Cientificas y Tecnicas (R. Argentina).
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{Received 18 July 1979, revised 5 December 1979)