/. Embryol. exp. Morph. Vol. 46, pp. 5-20, 1978
Printed in Great Britain © Company of Biologists Limited 1978
The role of the cell surface
in the migration of primordial germ cells in early
chick embryos: effects of concanavalin A
By H. LEE1, N. KARASANY1 1 AND R. G. NAGELE, JR 1
From the Department of Biology, Rutgers University, Camden, N.J.
SUMMARY
Effects of concanavalin A (Con A) on the morphology and migration of primordial germ
cells (PGCs) in stage-6 to -12 chick embryos were investigated. Con A, at a sublethal dose
(10 /tg/ml), inhibited migration of PGCs from the germinal crescent area to other parts of the
embryo. Affected PGCs were more rounded without the usual cytoplasmic extensions, but
the integrity of other structures was unaffected. Nearly identical results were obtained with
another lectin, wheat germ agglutinin (10/ig/ml). Histochemistry using Con A-horseradish peroxidase revealed that PGCs in control embryos had a thin, rather uniform layer
of extracellular coat material (ECM). Con A appeared to alter the distribution of ECM on
PGCs, i.e. some parts of the cell surface were devoid of any detectable ECM, while others
had small, scattered patches of ECM. Con A effects were alleviated by a-methyl-D-mannoside.
Overall results of the present study indicated that the observed inhibition of PGC migration
in early chick embryos is a consequence of Con A-induced alterations of cell surface properties.
INTRODUCTION
Since the periodic acid-Schiff (PAS) reaction was first applied to identify
primordial germ cells (PGCs) in the chick embryo (Meyer, 1960), their origin
and distribution have been extensively studied by Clawson & Domm (1969),
Meyer (1964) and Fujimoto, Ukeshima & Kiyofuji (1976). The presence of
abundant glycogen in the cytoplasm is considered the most distinguishing
feature of PGCs. In addition, the large size relative to surrounding cells and
fragmented nucleoli are useful for identification. PGCs of the chick are first
detectable in the hypoblast of the germinal crescent area at the primitive streak
stage of development. As development proceeds, PGCs separate from the
hypoblast, enter the blood vessels, and are later incorporated into the gonad.
Fujimoto et al. (1976) have suggested that PGCs enter the blood vessels by
amoeboid movements. Most of them eventually reach the germinal ridges, but
some go astray and are found in various parts of the embryo, where they later
degenerate.
1
Authors' address: Department of Biology, Rutgers University, Camden, New Jersey
08102, U.S.A.
6
H. LEE, N. KARASANYI AND R. G. NAGELE, JR
Recently, much attention has been given to the mechanism of cell migration.
This process is believed to be a result of membrane activities which involve
interactions between cell surface macromolecules and submembranous contractile microfilaments (Edelman, 1976; Nicolson, 1976). One of the most
extensively used probes for studying cell surfaces is concanavalin A (Con A).
Con A, a globular protein isolated from the Jack Bean, has been shown to bind
to specific cell surface carbohydrate residues (Goldstein, Hollerman & Merrick,
1965). The most interesting property of this plant lectin is its ability to agglutinate embryonic and transformed cells, but not their normal adult counterparts
(Rapin & Burger, 1974). Con A also inhibits closure of the chick neural tube
(Lee, 1976), migration of murine ascites tumor cells (Friberg, Cochran &
Golub, 1971), and amphibian epidermal cells (Donaldson & Mason, 1977). In
this study, we investigated effects of Con A on PGCs in early chick embryos
with emphasis on the role of the cell surface in their migration.
MATERIALS AND METHODS
Fertile White Leghorn eggs were incubated at 37-5 °C to obtain embryos at
stages 6-12 of development (Hamburger & Hamilton, 1951). These embryos
were chosen because the migration of PGCs from the germinal crescent area
towards posterior embryonic regions usually begins at stage 6 and is completed
by stage 12 (Fujimoto et al. 1976). Unless otherwise stated, stage-6 or -7 embryos
were explanted by New's (1955) technique and cultured on 1 ml of thin albumin
( = nutrient medium) with or without 10/^g/ml Con A (Sigma Chem. Co.,
St Louis, Mo.). At various intervals during incubation, some embryos were fixed
and processed for either electron microscopy or histochemistry as described
below. Others were fixed in Bouin's fluid, embedded in paraffin, serially sectioned
at 8 jam, and stained with Delafield's hematoxylin and eosin.
Electron microscopy
Embryos were fixed in half-strength Karnovsky's (1965) fixative for 2 h at
room temperature. After a thorough washing in 0-08 M cacodylate buffer
(pH 7-2), embryos were postfixed for 1 h in 1 % osmium tetroxide. After an
additional brief rinse in buffer, the embryos were washed in three changes of
maleate buffer (pH 5-0) over a period of 30 min. They were then stained en bloc
with 1 % uranyl acetate in maleate buffer for 1-5 h at room temperature, briefly
rinsed in maleate buffer, dehydrated in a graded ethanol series, embedded in
Spurr's (1969) resin, and sectioned. Thick sections (0-5-1-0 /tm) for light
microscopy were stained with toluidine blue. Thin sections (silver/pale gold)
were contrasted with aqueous uranyl acetate and lead citrate and examined
with a JEM 100B electron microscope.
Migration of primordial germ cells
7
Histochemical technique for identification of PGCs
The PAS reaction described by Meyer (1960) with minor modifications was
used to identify PGCs. Briefly, embryos were fixed in Rossman's fluid (Fujimoto
et al. 1976) for 3-12 h, followed by several rinses in 9 5 % ethanol until they
were clear of the fixative. Some were placed on slides and coated with 2 %
celloidin (North Carolina Biological Supply Co., Burlington, N.C.). Others
were dehydrated in two changes of absolute ethanol, double embedded in
celloidin and paraffin, serially sectioned at 10 jmn, mounted on slides, and coated
with a thin layer of 2 % celloidin. Both whole embryos and sections were treated
with 0-5 % periodic acid for 2 h and stained with Schiff's reagent for 30-60 min
at room temperature. This was followed by three changes of 0-5 % sodium
metabisulfite (5 min each) and a 10 min wash in tap water. Specimens were
dehydrated through a graded ethanol series, cleared in xylol, and mounted in
Permount.
Ultrastructural visualization of Con A binding sites
The procedure described by Bernhard & Avrameas (1971) with minor
modifications was used. Embryos were fixed for 1 h in half-strength Karnovsky's
(1965) fixative, thoroughly rinsed in cacodylate buffer, and treated for 1 h with
100/Ag/ml Con A. Following a rinse in cacodylate buffer, they were treated
for 1 h with 1 mg/ml horseradish peroxidase (HRP). After an additional brief
rinse, the diaminobenzidine (DAB) reaction was carried out at room temperature
for 30 min. Embryos were postfixed for 30 min in half-strength Karnovsky's
(1965) fixative, followed by 1 % osmium tetroxide for 1 h. They were then
dehydrated in a graded ethanol series and embedded in Spurr's (1969) resin.
Sections (silver/pale gold) were contrasted with uranyl acetate and lead citrate
and examined with a JEM 100B electron microscope.
RESULTS
Morphology and distribution of PGCs in stage-6 to -12 embryos
PGCs were readily recognizable as relatively large spherical cells possessing
PAS-positive material ( = glycogen) (Fig. 1 A). The size of PGCs ranged from
12 to 20 /im in diameter and remained unchanged during their migration from
the germinal crescent area towards posterior parts of the embryo. While in the
blood vessels, they appeared smaller (diameter = 9-16 /im) and more rounded.
The number of PGCs varied considerably among embryos of the same developmental stage due largely to individual variations and preparative procedures
(Meyer, 1964; Clawson & Domm, 1969; Fujimoto et al. 1976). Thus, in all
cases the distribution of PGCs in various parts of the embryo is expressed in
terms of the percentage of the total number.
Stage-6 to -7 embryos. There were 150-250 PGCs in each embryo with a mean
H. LEE, N. KARASANYI AND R. G. NAGELE, JR
B
Fig. 1. (A) Portion of germinal crescent area of stage-6 embryo showing PGCs
(arrows) revealed by PAS reaction. AO, area opaca; AP, area pellucida. Scale
line = 40/*m. (B) Transverse section through germinal crescent area of stage-6
embryo. Section was stained with toluidine blue. Note most PGCs (arrows) are
located in space between epiblast (EP) and hypoblast (HP). Scale line = 40 /tm.
of 219. Over 90% of the PGCs were found in the germinal crescent area and
others had migrated to the area pellucida just anterior to the head-fold. No
PGCs were encountered posterior to the primitive streak in any of the 24
embryos examined. In the germinal crescent area, 65-72% of the PGCs had
already separated from the hypoblast and were found in the space between the
epiblast and hypoblast (Fig. IB).
Electron microscopy revealed that PGCs, especially those found in the region
just anterior to the head-fold, had finger-like extensions (filopodia) which
projected from larger and more blunt membrane undulations (Fig. 2 A). Subsurface microfilaments were occasionally observed within these cytoplasmic
extensions. In many cases it was difficult to determine the leading edges of
migrating PGCs because (1) extensions projecting from PGCs were not as
conspicuous as those described in other cell types (Revel, 1974) and (2) the
direction of their migration often did not coincide with the plane of a transverse
section through the embryo. Favorable sections did show, however, that the
number of protrusions was greatest on the portion of the cell surface opposite
the eccentrically located nucleus. The distribution of glycogen granules varied
among PGCs. Yolk, PAS-negative lipids, and mitochondria were abundant
(Fig. 2B). The Golgi complex was well developed and typically showed small
vesicles which appeared to be budding from the ends of stacked crescentric
saccules (Fig. 2C). The nucleus was spherical to ovoid (average diameter =
8 /im) and eccentrically located. The nucleolus was fragmented and appeared
as several electron-opaque masses (Fig. 2B).
Stage-8 to -12 embryos. In stage-8 embryos, the number of PGCs ranged
from 132 to 286 (average = 226) - only a slight increase (P > 0-05) from that
observed in stage-6 to -7 embryos. The distribution of PGCs was nearly identical
to that in stage-6 to -7 embryos, except that 6-9 % had migrated to the area
Migration of primordial germ cells
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K^'S?;
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N
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Fig. 2. (A) Surface morphology of migrating PGC found just anterior to head-fold
ofstage-6 embryo. Notefinefinger-likefilopodia(F)projecting from membrane undulations. N, Nucleus. Scale line = 0-8 /im. (B) PGC found in germinal crescent area
of stage-6 embryo showing lipid (L), mitochondria (M), nucleolus (NU), and yolk
(Y). Scale line = 1 1 /*m. (C) Portion of PGC found in germinal crescent area of
stage-6 embryo showing well-developed Golgi complex (GC) and rod-shaped
mitochondria (M). N, nucleus. Scale line = 0-33 /*m.
10
H. LEE, N. KARASANYI AND R. G. NAGELE, JR
A. /
Fig. 3. (A) Embryo explanted at stage 7 and cultured for 14 h on medium containing
10 /*g/ml Con A. Morphological features of this embryo are comparable to those of
corresponding controls (cf. B) except that closure of brain is incomplete. Scale
line = 0-78 mm. (B) Embryo explanted at stage 7 and cultured for 14 h on plain
medium ( = thin albumin). Scale line = 0-78 mm.
lateral to the developing brain. No PGCs were ever observed in forming blood
islands. As development proceeded, migration of PGCs into more posterior
embryonic regions continued. By stages 9-10, some (4-7 %) had penetrated or
were enclosed by the blood vessels. The general morphology of PGCs remained
unchanged up to stage 10 of development.
In stage-12 embryos most PGCs were found in the blood vessels and were
circulating within the embryo. The number of PGCs ranged from 182 to 436
(average = 281). This great variation in number was due primarily to difficulties
in counting PGCs in the blood vessels (Meyer, 1964). The intravascular PGCs
[circulating PGCs of Clawson & Domm (1969)] assumed a more spherical shape
and thus appeared somewhat smaller (diameter = 9-16/^m) than those in
younger embryos (diameter = 12-20 jum). PGCs were easily distinguishable
from blood corpuscles because the former were larger, possessed considerable
amounts of yolk and PAS-negative lipids, and displayed cytoplasmic granules
with an intense PAS-positive reaction.
B
Migration of primordial germ cells
11
Table 1. Effects of different concentrations of Con A on the development of
embryos explanted at stage 6 or 7 and cultured for 14 h: by this time, about 86 %
of the control embryos had reached stage 10 of development
No. and % (in parentheses) of embryos
Con A
Gag/ml)
No. of
embryos
Little or no
development
Very
Abnormal
Abnormal
Normal
0
(Control)
5
10
15
20
30
42
0 (00)
0 (00)
3 (71)
39 (92-9)
34
48
34
32
28
0 (00)
0 (00)
3 (8-8)
3 (9-4)
4 (14-3)
0 (00)
0 (00)
6(17-6)
7(21-9)
7 (25-0)
3 (8-8)
13 (27-1)
13 (38-2)
12 (37-5)
12 (42-9)
31 (91-2)
35 (72-9)
12 (35-3)
10(31-3)
5 (17-9)
Table 2. Gross morphological features of control and 10 pig I ml
Con A-treated embryos shown in Table 1
Group
Control
No. of embryos explanted
% of embryos showing
Brain and neural tube
Partially open and/or
irregularly folded
Normal
Somites
No. of somite pairs ^ 8
No. of somite pairs > 9
Heart
Small and/or nonpulsatile
Pulsatile
Blood islands
42
Treated
48
7-1
27-1
92-9
. 72-9
14-3
85-7
29-2
70-8
190
810
1000
22-9
77-1
1000
Effects of Con A on morphology and migration of PGCs during stages 6-10 of
development
A series of experiments was carried out in which 176 embryos were explanted
at stage 6 or 7 and cultured on medium containing different concentrations of
Con A. Control embryos were treated in the same manner except that Con A
was not used. At various intervals, embryos were examined on a warm stage
(37-38 °C) under a dissecting microscope and grouped into four categories:
(1) little or no development - embryos at the same stage of development as when
explanted, (2) very abnormal - severely malformed embryos, (3) abnormal
embryos with one or more discernible abnormalities (Fig. 3 A), and (4) normal
(Fig. 3B). Results obtained after 14 h of incubation are summarized in Table 1.
12
H. LEE, N. KARASANYI AND R. G. NAGELE. JR
Fig. 4. PGCs found in area just anterior to developing brain of embryo explanted
at stage 6 and cultured for 6 h on medium containing 10 /*g/ml Con A. Note relatively
smooth cell surface (cf. Fig. 2A). Distribution and morphology of cell organelles are
comparable to those shown in Fig. 2B. ER, Endoplasmic reticulum; L, lipid;
M, mitochondria; N, nucleus; Y, yolk. Scale line = 0-8 fim.
Concentrations of 20 /^g/ml or higher inhibited morphogenesis of over 65 % of
the embryos. In contrast, 5 /£g/ml Con A had no apparent effect. Among all
the concentrations of Con A tested, 10 ^g/ml appeared to be most appropriate
for the purpose of the present study because it was sufficient to inhibit migration
of PGCs, but was still low enough to allow normal morphogenesis except for
closure of the neural tube and, to a lesser degree, the formation of somites
(Table 2). Blastodermal expansion, heart development, and blood island formation were not obviously affected (Fig. 3 A). Thus, Con A at a concentration
of 10 jLtg/ml was used in subsequent experiments.
Fifty-four stage-6 to -7 embryos were explanted and cultured on medium
containing 10 /^g/ml Con A. At various intervals, randomly selected embryos
were fixed and processed for electron microscopy or histochemistry using the
PAS reaction.
Con A had no obvious effect on the morphology of PGCs in seven embryos
examined at 2 h of incubation. By 4 h, over 92% of the PGCs (compared to
75-82% in controls) remained in the germinal crescent area in 8 out of the 14
Migration of primordial germ cells
13
(57.1 %) embryos. The number of PGCs ranged from 124 to 291 (average =
219) and was comparable to that in corresponding controls.
After 6-8 h of incubation, 9 out of the 16 (56-3 %) embryos showed signs of
neural fold fusion in the region corresponding to the future midbrain or anterior
portion of the hindbrain, a characteristic feature of the stage-8+ embryo
(Hamburger & Hamilton, 1951). The number of PGCs ranged from 131 to
294 with a mean of 234 (P > 0-05 compared to controls.) In all embryos,
78-84 % of the PGCs remained in the germinal crescent area, while others
were typically restricted to the area pellucida anterior to the developing brain.
Affected PGCs had relatively smooth surfaces without the usual cytoplasmic
extensions, but the integrity of other structures was unaffected (Fig. 4).
After 12-14 h of incubation, the neural tube in 5 out of the 17 (29-4 %) embryos
exhibited varying degrees of openness. The number of PGCs ranged from 192
to 332 (average = 276). There was no correlation between the number of PGCs
and general condition of the embryo. However, 56-62 % of the PGCs were
found in the area vasculosa in or near the germinal crescent area and 32-48 %
were clustered in the area pellucida just anterior to the brain. The remainder
were occasionally encountered in the somite or more posterior embryonic
regions.
We tested whether or not the observed Con A effects were consequences of
an alteration of culture medium. The medium, which produced the adverse
effects described above, was reused. Of the 24 embryos grown for 14 h on this
'used' medium, only two (8-3%) exhibited incomplete neural tube closure. In
all cases the morphology and distribution of PGCs were unaffected.
Additional experiments were carried out to see if the inhibitory effects of
Con A were permanent or could be alleviated by subsequent treatment with or
simultaneous presence of its inhibitor, a-methyl-D-mannoside (a-MM) (Sigma
Chem. Co., St Louis, Mo.). In the first series, 24 stage-6 to -7 embryos were
exposed to 10 /tg/ml Con A at 37-5 °C for 4 h, the treatment time found to be
required for producing microscopically observable Con A effects on PGCs.
After this treatment the embryos were subcultured for 6 h on medium containing
0-05 M aM-M. Of these embryos, 22 (91-7%) exhibited morphological features
typical of stage-8+ or -9 embryos and two (8-3 %) were abnormal. In all cases
the morphology and distribution of PGCs were unaffected. In the second
series, 24 stage-6 to -7 embryos were grown for 6-8 h on medium containing
both 10/ig/ml Con A and 0-05 M a-MM. Of these embryos, only one (4-2%)
appeared to be adversely affected and showed failure of neural-fold fusion at
the future hindbrain region. The morphology and distribution of PGCs were
again nearly identical to those of untreated controls.
E M B 46
14
H. LEE, N. KARASANYI AND R. G. NAGELE, JR
h
Fig. 5. Embryo explanted at stage 7 and cultured for 14 h on medium containing
10 yKg/ml WGA. Morphological features of this embryo resemble those of 10 /*g/ml
Con A-treated embiyos (cf. Fig. 3 A). Scale line = 0-78 mm.
Effects of wheat-germ agglutinin on morphology and migration of PGCs during
stages 6-10 of development
The effects of wheat-germ agglutinin (WGA) (Sigma Chem. Co., St Louis,
Mo.) were compared with those of Con A. Thirty-six stage-6 to -7 embryos
were cultured on thin albumin containing 10 ^g/ml WGA. At various intervals,
embryos were examined under a dissecting microscope to determine the degree
of development. Randomly selected embryos were fixed and processed for
electron microscopy or histochemistry using the PAS reaction. Effects produced
by WGA (10^g/ml) were almost indistinguishable from those of Con A
(10^g/ml). Both lectins inhibited PGC migration and neural tube closure
(Fig. 5), but Con A was more effective. WGA also had a 'smoothing' effect on
the surfaces of migrating PGCs, but this effect was not apparent until after 6 h
of treatment (compared to 4 h in Con A-treated cells).
The inhibitory effects of WGA were reversible. Twenty-four stage-6 to -7
embryos were pretreated with WGA (10 /Wg/ml) for 6 h. This was followed by
subculturing for 4 h on medium containing its inhibitor, 0-05 M N-acetyl-
Migration of primordial germ cells
Fig. 6. (A) Surface of PGC found in region just anterior to developing brain of
stage-8 embryo. Note thin and rather uniform layer of ECM as revealed by lectin
probe labelling. Scale line = 0-2 /tm. (B) Surface of PGC found in region just
anterior to developing brain of embryo explanted at stage 7 and cultured for about
6 h on medium containing 10/tg/ml Con A. ECM is not visible after lectin probe
labelling. Cell surface is smooth compared to that shown in (A). Scale line =
0-25 /tm. (C) Surface of PGC found in region just anterior to developing brain of
embryo explanted at stage 7 and cultured for about 6 h on medium containing
10 /*g/ml Con A. Small patches of ECM are scattered on cell surface. Scale line =
0-21 fim.
15
16
H. LEE, N. KARASANYI AND R. G. NAGELE, JR
glucosamine (NAGA) (Sigma Chem. Co., St Louis, Mo.). Only one (4-2%)
showed incomplete neural tube closure. Also, 22 of the 24 (91-7 %) stage-6 to -7
embryos cultured for 8-10 h on medium containing both WGA (10/tg/ml)
and NAGA (0-05 M) were normal. In all cases the distribution of PGCs was
comparable to that of untreated controls.
Effects of Con A on extracellular coat material {ECM) of PGCs
Twenty-four embryos were explanted at stage 6 or 7 and cultured on medium
with or without 10 /^g/ml Con A until the majority of controls had advanced
to stage 9 of development. Randomly selected embryos were processed by the
lectin probe method ( = HRP labelling). The following description is based on
the observations of those PGCs found in the region just anterior to the developing brain.
Control series. All PGCs had a thin, rather uniform layer of electron-dense
material (equivalent to ECM) on their surfaces (Fig. 6 A). The specificity of
the label was demonstrated by the absence of electron-dense material on the
surface of PGCs when the specimens, prefixed in Karnovsky's fixative, were
incubated with Con A in the presence of a-MM (0-02 M). Also neither DAB
alone nor DAB and peroxidase together was able to yield positive results.
Experimental series. Treatment of stage-6 to -7 embryos with 10 fig/m\ Con A,
in addition to the effect on surface topography discussed previously, caused an
alteration in the amount and distribution of ECM. HRP labelling showed that
in some sections the surface of PGCs was devoid of any detectable ECM
(Fig. 6B). In others, small patches of ECM were scattered on the cell surface
(Fig. 6C).
DISCUSSION
The present study was confined to the morphology, distribution, and migration
of PGCs in chick embryos during stages 6-12 of development. In stage-6
embryos, as previously described by Clawson & Domm (1969) and Fujimoto
et ah (1976), most PGCs were found in the germinal crescent area. By stage 8
the number of PGCs had slightly increased (P > 0-05) and their migration
towards posterior embryonic regions became apparent. In stage-9 to -10
embryos, some PGCs had either penetrated or were enclosed by the forming
blood vessels. The number of PGCs in the blood vessels progressively increased
until stage 12 of development. Fujimoto et ah (1976) have suggested that PGCs
enter the blood vessels by amoeboid movement. Similar movements might also
account for the migration of PGCs from the germinal crescent area into posterior
parts of the embryo because (1) they display short, yet conspicuous, cytoplasmic
extensions (lamellapodia and filopodia) and (2) the direction of their migration
appears to be independent of the general pattern of morphogenetic movements
during stages 6-12 of development (Rosenquist, 1966). While in the blood
vessels, PGCs appear smaller, a result of their more spherical shape, suggesting
Migration of primordial germ cells
17
that they are not actively migrating in the blood vessels, but are being passively
transported by the circulating blood.
As shown in Fig. 2 A, migrating PGCs often possessed finger-like extensions
(filopodia) which projected from larger and more blunt undulations. These
structures resembled the membrane ruffles and lamellapodia often seen at the
leading edges of migrating cells (Revel, 1974). Revel (1974) has suggested that
the properties of the substratum along which the cells migrate are among the
major factors affecting the direction of migration and that membrane ruffles
may be involved in searching for an appropriate substratum. Attachment to the
substratum and development of tension in these cytoplasmic extensions are
steps which appear to be essential to movement in many cell types. For instance, migrating chick mesodermal cells with prominent ruffles were found to
follow the diffuse fibrous stroma beneath the epiblast during early morphogenesis (Revel, 1974). Whether or not the substratum participates in directional
guidance for PGC migration remains to be answered.
This study showed that treatment of early chick embryos with Con A caused,
among other things, inhibition of PGC migration. In fact, Con A has been
shown to inhibit the migration of many other cell types such as murine ascites
tumor cells (Friberg et al. 1971) and amphibian epidermal cells (Donaldson &
Mason, 1977). The concentration of Con A (10 /*g/ml) used in the present study
was too low to cause any apparent cytotoxic effects. However, affected PGCs
often became more rounded and rarely showed cytoplasmic extensions. This
'smoothing' effect mediated by Con A on the surface of PGCs was not observed
until after at least 2 h of treatment. This time period may be a consequence of
the viscosity of thin albumin ( = nutrient medium) which hinders the diffusion
of Con A and thus delays its occupation of the minimum number of binding
sites necessary for producing observable morphological effects. At the same
time, even if saturation of Con A-binding sites is rapid, Con A-mediated
alterations in cell surface morphology may require some time to be of sufficient
magnitude as to become microscopically detectable. In addition, the Con A
effect was blocked by simultaneous presence of a-MM. Similar results were
obtained with another lectin, WGA and its inhibitor, NAGA. These findings
suggest that observed inhibition of PGC migration is a result of binding of
lectins to cell surface carbohydrate residues. Temmink, Collard, Roosien
& Van Den Bosch (1976) reported that incubation of human lymphoblasts
and lymphocytes with agglutinating concentrations of Con A or WGA
resulted in a 'smoothing down' of the cell surface. Since profiles of cytoplasmic
extensions could be seen in sections of these cells, they attributed this effect to
a covering over and sticking down of microvilli with the lectin. However, in
the case of Con A-treated PGCs, cytoplasmic extensions appeared to be
retracted and their surfaces exhibited only gentle undulations. Our observations
seem to be in line with those of Brown & Revel (1976) who reported that Con A
interfered with the ruffling activity of LA-9 cells. Since the formation of ruffles
18
H. LEE, N. KARASANYI AND R. G. NAGELE, JR
at the leading edges of cells has been shown to be requisite for their movement
(Abercrombie, Heaysman & Pegrum, 1972), Con A inhibits PGC migration,
at least in part, by interfering with ruffling activity. The precise nature of the
molecular events involved in this 'smoothing' effect remains unclear. However,
Rothman & Lenard (1977) have recently proposed that all ectoproteins must
necessarily be capable of interacting with submembranous microfilaments and
microtubules. These cytoskeletal elements have been implicated in the formation
of cytoplasmic extensions through their ability to interact with plasma membrane glycoproteins including Con A receptors (Edelman, 1976; Nicolson,
1976). If this is the case, the binding of Con A to surface receptors may restrict
their lateral mobility, thus increasing the rigidity of the cell surface and preven ting
the formation of membrane protrusions. Con A is known to bind to PGCs, to
other tissues of the chick embryo, and perhaps to the ovalbumin and ovomucoid
present in our culture medium. A possibility existed that the observed effect of
Con A on PGCs was a secondary one which resulted from an alteration of the
medium. This was found to be unlikely because (1) the same medium (initially
containing 10 /*g/ml Con A) could not be used twice to produce noticeable
effects on PGCs, (2) similar results could be obtained with WGA, and (3) the
Con A effect was eliminated by the presence of a competing sugar, a-MM. It
could also be argued that the low concentration of Con A (10 /*g/ml) used in
this study still has some effect on morphogenesis and this may secondarily affect
migration of PGCs. This is considered unlikely because, as pointed out above,
the direction of their migration appears to be independent of the general pattern
of morphogenetic movements.
Our histochemical studies showed that Con A altered the distribution of
ECM on PGCs. PGCs of untreated controls had a thin, rather uniform layer
of ECM on their surfaces (Fig. 6 A). By contrast, in Con A-treated cells, some
parts of the cell surface were devoid of any detectable ECM (Fig. 6B), while
other parts had small, scattered patches of ECM (Fig. 6C). Although this
configuration is possibly artifactual, it is observed only in Con A-treated cells
and repeatedly. Taylor, Duffus, Raff & DePetris (1971) reported that Con A
could cross-link and redistribute membrane receptors, leading to patching and
capping. That patched receptors move from the active edges of cells has been
demonstrated by many investigators (e.g. Abercrombie et al. 1971; Brown &
Revel, 1976; Albertini, Berlin & Oliver, 1977) and this movement is believed to
involve submembranous microfilaments (Edelman, 1976; Nicolson, 1976).
These cytoskeletal elements have long been thought to participate in cell
locomotion through their transient assemblies in strategic areas of the cell.
Thus if microfilaments are preoccupied with patch and cap formation as in the
case of Con A-treated cells, they are effectively removed from the prospective
leading edge of the cells and are unavailable for ruffling activity as discussed
above. Under these conditions, cells would be unable to move. The extensive
shifting of patched receptors to other areas of the cell may also provide the
Migration of primordial germ cells
19
driving forces for the observed redistribution of ECM in Con A-treated cells.
Overall results of the present study indicate that the observed inhibition of
migration of PGCs in early chick embryos is a consequence of Con A-induced
alterations of cell surface properties.
This study was supported by grants from the Research Council and the Charles and
Johanna Busch Memorial Fund of Rutgers University.
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(Received 21 June 1977, revised 3 March 1978)