jf. Cell Sci. 68, 49-67 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
49
ECTODERMAL FRAGMENTS FROM NORMAL FROG
GASTRULAE CONDITION SUBSTRATA TO SUPPORT
NORMAL AND HYBRID MESODERMAL CELL
MIGRATION IN VITRO
NORIO NAKATSUJI AND KURT E. JOHNSON*
Department of Anatomy, The George Washington University Medical Center,
Washington, D.C. 20037, U.SA.
SUMMARY
Using time-lapse cinemicrography and scanning electron microscopy, we have shown that normal
Rana embryos and gastrulating hybrid embryos have extracellular fibrils on the inner surface of the
ectodermal layer. These fibrils are absent prior to gastrulation and appear in increasing numbers
during gastrulation. They can also be deposited in vitro where they condition substrata in such a way
that normal presumptive mesodermal cells placed on them show extensive attachment and unoriented cell movement. These fibrils are also present in some arrested hybrid embryos, but in reduced
numbers, or are lacking in other arrested hybrid embryos. Explanted ectodermal fragments from
arrested hybrid embryos fail both to condition culture substrata by the deposition of fibrils and to
promote cell attachment and translocation. In contrast, ectodermal fragments from normal embryos
can condition culture substrata so as to promote moderate cell attachment and, for one particular
gamete combination, even cell translocation of presumptive mesodermal cells taken from arrested
hybrid embryos. These results provide new evidence to support the hypothesis that extracellular
fibrils represent a system that promotes mesodermal cell migration in amphibian embryos. Differences in the fibrillar system in urodele and anuran embryos are discussed in relation to fundamental differences in the mode of mesodermal cell migration in these two classes of Amphibia.
INTRODUCTION
One of the most interesting problems in studies of gastrulation in amphibian embryos is the mechanism of directed cell locomotion of prospective mesodermal cells
along the inner aspect of the roof of the blastocoel (Nakatsuji, 1983). The roof of the
blastocoel is a primitive epithelium in amphibian gastrulae. This epithelium is not
bounded in the gastrula stage by a basement membrane containing many layers of
collagen fibres, proteoglycans and glycoproteins in a dense meshwork. Instead, it is
underlain by a gossamer network of extracellular fibrils with a diameter of
approximately 0-1 pun. The extracellular fibrils have been observed in several species
of urodele gastrulae (Nakatsuji, Gould & Johnson, 1982; Nakatsuji, 1983; Nakatsuji
& Johnson, 1983a) and several species of anuran gastrulae (Nakatsuji & Johnson,
19836), includingXenopus laevis and two species of normal Rana embryos (Rana
pipiens and R. sylvatica). In an earlier study, we reported an absence of extracellular
fibrils in Rana gastrulae (Nakatsuji & Johnson, 19836) but we now know that this
•Author for correspondence.
50
N. Nakatsuji and K. E. Johnson
report was partially in error. With improvements in fixation methods, we have now
shown that extracellular fibrils on the basal surface of the blastocoelic epithelium also
occur in R. pipiens and R. sylvatica embryos. This improvement in fibril fixation has
allowed us to make tests of the hypothesis that extracellular fibrils promote cell
locomotion in vivo and in vitro.
In the present study, we have made use of large numbers of normal and hybrid
embryos by collecting normal embryos or by fertilizing the eggs olR. pipiens with the
sperm of several different species of Rana. In these interspecific hybrid embryos, two
kinds of developmental events most often follow, if fertilization and cleavage are
initiated normally (Moore, 1955). In one, formed by fertilizing the eggs of R. pipiens
with the sperm of a closely related but distinct species, R. palustris, gastrulation and
subsequent development occur in a completely normal fashion, although at a rate
slightly slower than the rate of gastrulation in normal control embryos. In all other
gamete combinations, fertilization and cleavage occur normally with a normal rate,
but an abrupt developmental arrest occurs at the onset of gastrulation. These arrested
embryos fail to progress through the gastrula stages and subsequently die between 1
day and 7 days after arrest. The time of death after arrest at the early gastrula stage
is highly variable for different gamete combinations but is an invariant and consistent
feature of any given combination. For example, arrested hybrid embryos formed by
fertilizing the eggs of R. pipiens with the sperm of R. catesbeiana die 1 day after arrest
(Johnson, 1976). In contrast, arrested hybrid embryos formed by fertilizing the eggs
of R. pipiens with the sperm of R. sylvatica or R. temporaria die 7 days after arrest
and undergo considerable cytodifferentiation before arrest (Johnson, 1971; Johnson
& Adelman, 1984).
It is convenient to use shorthand notation to designate different kinds of gamete
combinations: normal R. pipiens embryos will be called R. pip. Normal Rana sylvatica embryos will be called R. syl; R. pipiens eggs fertilized with R. palustris sperm
will be called pal; R. pipiens eggs fertilized by R. sylvatica sperm will be called syl;
R. pipiens eggs fertilized by R. temporaria sperm will be called temp; R. pipiens eggs
fertilized by R. catesbeiana sperm will be called cat; R. pipiens eggs fertilized by R.
clamitans sperm will be called clam.
We have discovered that extracellular fibrils are absent in blastulae and increase in
amount during gastrulation in R. pip, R. syl embryos and pal embryos. Explanted
fragments of the roof of the blastocoel of all three gastrulating embryos will deposit
extracellular fibrils onto plastic substrata under appropriate conditions in vitro. Substrata conditioned in this manner by fragments from either normal embryos or pal
embryos will subsequently support the attachment and locomotion of presumptive
mesodermal cells from either homologous normal embryos or from heterologous
normal embryos (Table 1). We have also found that extracellular fibrils are generally
absent in arrested hybrid blastulae. They either fail to appear at all after arrest or
appear in small numbers, depending upon the particular gamete combinations under
study. Explanted fragments from arrested hybrid embryos have few if any fibrils and
they deposit few fibrils on plastic substrata in vitro. Furthermore, when seeded with
presumptive mesodermal cells from either normal or hybrid embryos, substrata
Normal frog ectoderm allows cell movement in vitro
51
Table 1. Conditioning effects of normal and hybrid ectodermal layers on attachment
and locomotion of mesodermal cells
Conditioning effects*
Ectoderm
layer
Mesoderm
cells
R.pip
R.pip
R.pip
f
Mean velocity!
++
13
1
0
4- 1 ± 1-2/im/min
cat
7
6
1
1-8 ± 0-6fxm/min
cat
cat
R.pip
cat
0
0
4
0
0
4
R.pip
temp
4
4
0
R.pip
temp
R. pip
temp
0
0
2
0
2
4
ND
ND
R. pip
pal
pal
pal
R. pip
pal
4
4
4
0
0
0
0
0
0
ND
ND
R.pip
syl
syl
R. pip
clam
clam
syl
R. pip
syl
0
0
0
4
0
0
0
1
4
ND
ND
ND
da
R.pip
clam
0
0
0
1
6
0
0
1
3
ND
ND
ND
(« = 84)
(n = 53)
NDt
ND
0- 6 ± 0-2/un/min
(« = 43)
3- 5 ± 1-1 ^m/min
(» = 26)
Conditioning effects were scored by counting the attached cells in a conditioned area.
/
f Mean velocity is calculated from time-lapsefilmsof the conditioned area with good cell attachment (+ + ).
J ND indicates that the mean velocity was not determined.
'conditioned' by fragments from fibril-deficient arrested hybrid embryos support
neither strong attachment nor cell locomotion. These results give further support to
the hypothesis that oriented fibrils promote oriented movement of presumptive
mesodermal cells by contact guidance.
MATERIALS AND METHODS
Embryos
R. pipiens females and various Rana male species (R. painstris, R. sylvatica, R. temporaria, R.
catesbeiana and R. clamitans) were obtained from Nasco (Fort Atkinson, Wisconsin) and C. D.
Sullivan (Nashville, Tenn.). R. sylvatica embryos from natural spawnings were collected in the
field in Fairfax County, Virginia. They were stored in plastic boxes at 4°C in tap water supplemented with antibiotics and salts (Johnson & Adelman, 1981). Females of R. pipiens were ovulated by
pituitary injection (Rugh, 1962). Fertilization was carried out by stripping eggs into sperm suspensions made from macerated testes from various Rana species males in 10% Steinberg's solution
52
N. Nakatsuji and K. E. Johnson
(SS). Fertilization for temp embryos has been described previously (Johnson & Adelman, 1984).
Developmental stages of normal embryos were determined according to Shumway (1940). Jelly
coats were first removed manually with Dumont no. 5 forceps, and the remaining thin layer of jelly
was dissolved by incubating eggs for lOmin in 0-7% sodium thioglycolate in 50% SS (pH8-6),
followed by 10 rinses in 10% SS.
Media
The composition of the medium used in this study has been published in detail previously
(Nakatsuji & Johnson, 1982). Briefly, the basic salt solution is a modified Steam's solution (MSS)
(Stearns & Kostellow, 1958) buffered with 5mM-HEPES (Sigma). Mesodermal cells were
dissociated in 0-02 M-sodium citrate (Feldman, 1955) in Ca2"1"-, Mg^-free MSS. For explants of the
ectodermal layer, we used the culture medium used in the previous study (Nakatsuji & Johnson,
1982), with a pH of 8'0, a Ca2+ concentration of 110/IM, and containing 0 - 5 % bovine serum
albumin (Sigma). At this Ca2+ concentration, ectodermal fragments adhere to the plastic substratum and remain flat, as in the case of conditioning by Ambystoma gastrula ectoderm layers
(Nakatsuji & Johnson, 1983a). The dissociated mesodermal cells were cultured in the same culture
medium with a Ca2+ concentration of 100 fXM.
Conditioning of substrata and culture of dissociated cells
Procedures for conditioning culture substrata have been described in detail (Nakatsuji & Johnson,
1983a). Rectangular pieces of the ectodermal cell layer were cut from the dorsal side of early
gastrulae (stage 11; Shumway, 1940) of normal or hybrid embryos, in MSS, using fine forceps and
hair loops. The whole blastocoelic roof with a round shape (instead of the rectangular pieces) was
used for some of the conditioning experiments in which the conditioning effect was compared, using
normal and hybrid conditioning fragments and cells, without examination of the alignment of the
cell trails along the blastopore-animal pole axis.
The ectodermal pieces from normal and hybrid embryos were explanted in plastic tissue-culture
dishes (35 mm diameter, Falcon Plastics, no. 3001) with their inner basal surface covered by fibrils
(in the case of normal embryos and pal hybrid embryos) resting on the dish surface. Two explants
were placed in a dish, and cultured for 4 h at 22-24 °C. For the examination with scanning electron
microscopy, the explants were cultured on plastic coverslips for tissue culture (Thermanox tissue
coverslips, Miles Laboratories) placed in plastic Petri dishes. The margin and blastopore-animal
pole axis, in the case of rectangular pieces of the explant, were marked on the dish or coverslip
surface by scratching with the tip of Dumont no. 5 forceps.
Explants were removed by gently flushing them away with a Pasteur pipette, from a direction
perpendicular to the blastopore— animal pole axis in the case of rectangular pieces, to avoid possible
artificial stress alignment of the fibrils along this axis. The conditioned surface was rinsed with two
changes of the culture medium, and seeded with the dissociated mesodermal cells from middle
gastrulae (stage 12) of either normal or hybrid embryos using a Pasteur pipette. When the ectoderm
and mesoderm from normal embryos are used for the experiment, the cells on the conditioned area
start to attach and locomote in 20min. There was no cell attachment outside the conditioned area.
The large size of amphibian embryonic cells allows direct counting of the attached cells using a
higher magnification (50X) of the dissecting microscope. Unattached cells are spherical and displace
with the slightest agitation of the dish. Attached cells were flattened and bipolar or polygonal with
lamellar substratum adhesions at the corners of stretched cells. The attached cells were counted and
scored on each conditioned area. Time-lapse 16 mm films (Kodak, Plus-X Reversal) were taken with
20 X or 10 X phase-contrast objective lenses at 8-s intervals, controlled by a Nikon cine-autotimer
CFMA for 1 h or longer within 1-3 h after the seeding of cells.
The coverslips were fixed for scanning electron microscopy by transferring them into a fixation
solution of 2-5% glutaraldehyde in 0 - 0 5 M - P I P E S (Sigma) buffer (pH7-3) supplemented with
5 mM-CaCU. The same buffer with Ca 2+ was used for the post-fixation with 1 % OsCU, followed
by dehydration through an ethanol series, critical-point drying through liquid CO2, and sputter
coating with gold-palladium. The samples were examined with a JEOL JSM35 scanning electron
microscope. Whole embryos were fixed and processed in the same manner, except that they were
cut open in the prefixation solution with a razor blade.
Normal frog ectoderm allows cell movement in vitro
53
Analysis of the films and cell trails
The method of drawing cell trails from films was described in detail previously (Nakatsuji &
Johnson, 1982). Briefly, we projected the film at 4-min intervals and marked the centre of the cell
body. For the further analysis of the cell trails, we used an image-analysis system with a microcomputer (Graphics Tablet connected to Apple II Plus Computer), and a computer program that
we developed and described previously (Nakatsuji & Johnson, 1983a). It gives the alignment
parameterR of each cell trail, as well as its length. The/? value is log 2r, wherer = 'ZAy/'LAx and
Ay is the vertical component of a vector tangential to a point on a cell trail and Ax is the horizontal
component of a vector tangential to a point on a cell trail. A random, unaligned cell trail would give
an R value of zero, while alignment along the blastopore-animal pole axis of the ectoderm explant
that conditioned the surface, or perpendicular to that axis, would give positive, or negative, values
of R, respectively. For example, an R value of +1-0 means that the cell has made twice as much
displacement along the blastopore—animal pole axis as perpendicular to that axis. An R value of — 1 -0
means the opposite. An R value of 0 means random alignment or no net translocation. (See Nakatsuji
& Johnson, 1983a for details.)
RESULTS
Observations on fibrils in vivo
In an earlier publication, we reported that extracellular fibrils were extremely
sparse on the inner surface of the roof of the blastocoel in R. pipiens gastrulae. We have
subsequently made dramatic improvements in our ability to fix extracellular fibrils in
R. pipiens gastrulae and hybrid embryos thanks largely to helpful suggestions received
from Drs J. LeBlanc and I. Brick of New York University. Preliminary experiments
with fixation conditions revealed that the Ca2+ concentration infixativesolutions was
critical. Using the PIPES buffer described above with 1 mM-Ca2+ added, there is a
modest improvement in fibril preservation although few fibrils are observed in most
cases. If the Ca2+ concentration is increased to 5 ITIM, there is a dramatic improvement
in fibril fixation. Further increases in Ca2+ concentration to lOmin do not improve
fibril fixation, as judged by the frequency of fibrils encountered in the scanning
electron microscope (SEM). Fibrils are either very sparse or entirely absent in stage
8 and stage 9 (Shumway, 1940) R. pip blastulae (Fig. 1A). Fibrils are also extremely
sparse in normal R. syl embryos and in gastrulating interspecific hybrid pal in stage
8 and 9 blastulae. We have examined a number of other hybrid embryos at these stages
as well and have found that there are few if any fibrils present prior to the onset of
gastrulation in controls or at gastrular arrest in arrested hybrid embryos.
At the onset of gastrulation (stage 10), there is a sudden dramatic increase in the
number of extracellular fibrils lining the inner aspect of the roof of the blastocoel in
both normal R. pip (Fig. 1B) and R. syl embryos and in the gastrulation hybrid pal as
well. As gastrulation proceeds through stage 11 and 12, there is a further increase in
the number of fibrils in both kinds of normal embryos (Fig. lc) and in thepal hybrid.
These changes, however, are not seen in arrested hybrid embryos. Two basic results
were obtained in this study. (1) For the temp, syl and clam arrested hybrids there are
a few fibrils present at stages 10, 11 and 12 (Fig. 2 B - D ) . It is important to realize here
that these arrested hybrid embryos do not undergo gastrulation and consequently
N. Nakatsujt and K. E. Johnson
Fig. 1
Normal frog ectoderm allows cell movement in vitro
55
never progress beyond stage 10. When we describe an arrested hybrid embryo as stage
11 or stage 12, we mean that the control normal embryos for that cross have reached
stage 11 and 12, respectively. (2) The second result we obtained, using cat embryos,
was quite different. In these arrested hybrids, essentially no fibrils are formed at any
stage after arrest at stage 10 (Fig. 2A). Some typical results from our studies so far are
shown in Figs 1 and 2.
Conditioning experiments in vitro
We have performed a large number of experiments in which we have conditioned
artificial substrata with explanted fragments of the roof of the blastocoel from normal
embryos, gastrulating hybrid embryos and arrested hybrid embryos. We have seeded
such conditioned substrata with dissociated presumptive mesodermal cells from normal embryos, gastrulating hybrid embryos and arrested hybrid embryos. Three
interesting results were obtained from these studies. First, we were able to show that
explanted ectodermal fragments from R. pip and R. syl normal embryos promote the
attachment and rapid translocation of homologous presumptive mesodermal cells
(Fig. 3A) (Table 1). For example, R. pip conditioned substrata support translocation
at a rate of 4-1 fim/m\n ± 1-2/xm/min S.D. (n = 84) for R. pip cells. We have also
measured heterologous combinations of ectoderm and mesodermal cells. R. pip
ectoderm also conditions for R. syl mesodermal cells with extensive cell attachment
and cell translocation at a rate of 3-3 ^m/min ± 0-8/im/min S.D. (n = 7). Ambystoma
maculatum ectoderm has a modest conditioning effect for R. pip cells, with cell
attachment and cell translocation at a rate of 2-3 ^m/min ± 0'5 /xm/min S.D. (n = 9).
A. maculatum ectoderm can also condition for A', laevis mesodermal cells (Nakatsuji
& Johnson, 1983a). Second, we found that ectoderm from R. pip would condition
substrata such that even cells from arrested hybrid embryos would show moderate
attachment and even substantial rates of translocation. R. pip conditioned substrata
support translocation at a rate of 1 -8^m/min ± 0-6/im/min S.D. (n = 53) for cai cells
(Fig. 3c) (Table 1) and 0 - 6^m/min ± 0-2/im/min for temp cells. In other gamete
Fig. 1. Scanning electron micrographs of the inner aspect of the roof of the blastocoel in
stage 9 (A) , stage 10 (B) and stage 11 (c) R. pipiens gastrulae and a stage 11 (D) cat arrested
gastrula. Fibrils are present in B and c but lacking in A and D. X5400; bar, 2/im.
Fig. 2. Scanning electron micrographs of the inner aspect of the roof of the blastocoel in
a stage 11 (A) cat arrested gastrula. X5400; a stage 12 (B) syl arrested gastrula. X6000; a
stage 11 (c) clam arrested gastrula; and a stage 12 (D), temp arrested gastrula. X6000. All
micrographs lack fibrils. Bar, 2f.im.
Fig. 3. Photographs from time-lapse films of plastic Petri dishes conditioned by ectodermal explants and seeded with presumptive mesodermal cells, A-C are R. pipiens ectoderm
conditioning for R. pipiens cells (A, B) and cat (c) cells, D is cat ectoderm conditioning for
cat cells. I n A and c, cells are attached by lamellipodia (arrows). No cell attachment is seen
in B because this is a region of the plastic dish outside the boundaries of the conditioning
explant. No cell attachment is shown in D because cat ectoderm does not condition for cat
cells nor does it condition for R. pipiens or other hybrids (results are shown in Table 1).
The rounded cells do form active filopodia that can be seen in B and D (arrows) protruding
into the surrounding medium. X300; bar, 50fun.
N. Nakatsuji and K. E. Johnson
Fig. 2. For legend see p. 55.
Normal frog ectoderm allows cell movement in vitro
Fig. 3. For legend see p. 55.
57
58
N. Nakatsuji and K. E. Johnson
Fig. 4. Trajectories for the centre of the cell bodies of mesodermal cells during 1 h of
movement. The solid circles show the starting points, and open circles show ending points.
In A, c and D R. pipiens ectoderm conditioning for R. pipiens (A) mesodermal cells; cat
(c) mesodermal cells; and temp (D) mesodermal cells. In B, pal ectoderm conditioning for
pal mesodermal cell. Bar, 100/im.
Normal frog ectoderm allows cell movement in vitro
59
combinations, attachment and translocation was slight (Table 1). Some examples of
normal and hybrid cells attached to conditioned substrata are shown in Fig. 3. In all
of the cultures that we examined, conditioning effects were limited to the area of the
culture covered by the explanted ectodermal fragment. In areas of the culture dish
outside the boundaries of the conditioning fragment, there was no cell attachment and
no translocation for all combinations of conditioning ectoderm and presumptive
mesodermal cells (Fig. 3B, Fig. 9). Third, we found that ectoderm from arrested
hybrid embryos had no conditioning effects (Fig. 3D).
Time-lapse film analysis of cell translocation and cell orientation
We have made a detailed study of cell translocation of normal and hybrid cells from
time-lapse films (Fig. 4). We have found that normal cells show substantial rates of
translocation (Fig. 4 and Table 1). In some preparations, we explanted rectangular
fragments of the roof of the blastocoel where the long axis of the rectangle was parallel
to the animal pole—blastopore axis of the gastrula. We then measured the orientation
of the cell trail (R value) with respect to the animal pole—blastopore axis. When we
conditioned substrata with R. pip ectoderm and seeded them with R. pip mesodermal
cells, we found that the cell trails were extensive but only weakly oriented with respect
to the animal pole—blastopore axis (R = 0-\Z) (Fig. 5) and not polarized (Fig. 6).
7-
-2
-1
+1
+2
Fig. 5. Histogram showing ilic distribution of cell trails according to their R values. A
positive R value indicates cell locomotion parallel to the animal pole-blastopore axis of a
rectangular conditioning ectodermal explant. This histogram represents data from 84 R.
pipiens mesodermal cells on a surface conditioned by R. pipiens ectoderm. The
distribution is centred around a 0 value for R and is symmetrical but appears to be slightly
shifted towards the right with a mean and standard deviation of 0-12 ±0-72. The null
hypothesis that the population mean n^O is rejected with a level of significance of
0-05 < P < 0-10 (statistic, z = 1-56).
60
N. Nakatsuji and K. E. Johnson
AP
o
o8
°
o
o
oodb
fco d ( c0
cP
o
oo
oo°
°o
°
o °o
o . o <
0
Fig. 6. Distribution of the end-points of the cell trails when the start-points are superimposed on the origin of the co-ordinate, and the direction toward the animal pole (AP)
is adjusted to they-axis. All cell trails were traced for 1 h. There is no preferential orientation of end-points toward the animal pole. Forty-one cells have positive y values and 42
cells have negative values. One cell has a zero y value. These trails belong to R. pipiens
mesodermal cell9 migrating on a substratum conditioned by R. pipiens ectoderm. Bar,
100 [m\.
When we conditioned substrata with R. pip ectoderm and used arrested hybrid test
cells, we found that cat cells showed moderate translocation that was substantially less
than when we used R.pip test cells; and that temp cells showed very little movement
at all (Fig. 4 and Table 1).
Fig. 7. Scanning electron micrographs of cultures where R. pipiens explants conditioned
forR. pipiens (A, B) mesodermal cells; cat (c) mesodermal cells; and temp (D) mesodermal
cells. In A, the scratch made in the plastic substratum indicates the boundary of the
conditioning explant. X 36; bar, 400 /im. I n B , there is a higher-power view of five attached
cells with lamellipodia (arrows; from the left of centre in A. X320; bar, 100 ^m. In c and
D, there is extensive attachment with many lamellipodia (arrows) for cat (c) and temp cells
(D). X540; bar, 50fun.
Normal frog ectoderm allows cell movement in vitro
Fig. 7
61
N. Nakatsuji and K. E. Johnson
Scanning electron microscopic observations
We found thati?. pip ectodermal fragments deposit fibrils and bits of cellular debris
on plastic substrata after a 4-h conditioning period. Some of this debris appears to be
the remains of small filopodia or retraction fibres that sheared off when conditioning
fragments were washed away by a stream of culture medium expelled from a Pasteur
pipette. The deposited fibrils, which formed anastomosing networks, were not as
prominent on conditioned substrata as they were in intact fragments of embryos.
When R. pip presumptive mesodermal cells were seeded on substrata conditioned by
R. pip ectoderm, the test cells were flattened considerably and formed broad fan-like
lamellipodia (Figs 7A— B, 8A) and were extensively elongated, assuming a distinct
fusiform shape, presumably due to traction forces exerted by lamellipodia. In many
instances, long filopodia were observed preceding these lamellipodia (see Trinkaus &
Erickson, 1983). Cells from arrested hybrid embryos seeded on substrata conditioned
by R. pip explants do not attach as extensively as cells from normal embryos. In the
scanning electron microscope, hybrid cells are not nearly as flattened as normal cells
(Fig. 7c, D) . Instead, they remain more or less rounded up in culture and form smaller
Fig. 8. Scanning electron micrograph of a culture where R. pipiens explants conditioned
iorR. pipiens mesodermal cells. In A, a cell has a large lamellipodium with several filopodia
(arrows) projecting from it. X1200; bar, 10/im. In B, filopodia are closely associated with
fibrils and attached globules (arrows). X6000; bar, 2/im.
Normal frog ectoderm allows cell movement in vitro
63
Fig. 9. Scanning electron micrograph of a culture where a R. pipiens explant conditioned
for cat cells. These cells are outside the boundaries of the conditioning explant and remain
rounded up without the typical lamellipodium formation shown on portions of the substratum underneath the conditioning explant. However, they have filopodia and lamellalike protrusions (arrow) on the cell surface. These protrusions show active movement in
time-lapse films. These cells should be compared with the more flattened cells shown in
Figs 3c and 7c. X720; bar, 20 nm.
numbers of filopodia and associated lamellipodia. Attached hybrid cells are also not
as fusiform as cells from normal embryos on conditioned substrata. Perhaps hybrid
lamellipodia are not as capable of exerting traction forces or do not adhere as firmly
as normal cells. We have examined normal and hybrid cells in cultures in which
substrata had been conditioned but cells had been deposited outside the conditioned
area. In these areas, both normal and hybrid cells remain rounded up due to a lack
of substratum adhesion (Figs 3B and 9).
DISCUSSION
The present results show that normal embryos and gastrulating hybrid embryos have
extracellular fibrils in vivo. These fibrils are absent prior to gastrulation and appear in
increasing numbers during gastrulation. Furthermore, thesefibrilscan be deposited in
vitro, where they condition substrata so that presumptive mesodermal cells seeded on
them can show extensive but unoriented and unpolarized cell movement. These fibrils
64
N. Nakatsuji and K. E. Johnson
are present in reduced numbers in some arrested hybrid embryos and are altogether
lacking in other arrested hybrid embryos. Explanted ectodermal fragments from
arrested hybrid embryos fail to deposit fibrils and to promote cell attachment and
translocation. Finally, we have shown that ectodermal fragments from normal embryos can condition culture substrata in such a way as to promote moderate cell
attachment, and in one instance even cell translocation of presumptive mesodermal
cells taken from an arrested hybrid embryo (cat).
It appears now that extracellular fibrils on the inner aspect of the roof of the
blastocoel of amphibian gastrulae are widely distributed among both the urodeles and
anurans. We have already reported the existence of such fibrils in three species of
urodeles, A. maculatum, A. mexicanum and Cynopspyrrhogaster (Nakatsuji, Gould
& Johnson, 1982; Nakatsuji & Johnson, 19836). The fibrils in A. maculatum are
significantly oriented along the animal pole-blastopore axis in vivo (Nakatsuji et al.
1982). They are also deposited in an oriented fashion in vitro and can promote
oriented cell locomotion along the animal pole-blastopore axis of conditioning fragments. Furthermore, this oriented locomotion shows a slight polarization and cells
move preferentially on them toward the animal pole rather than toward the blastopore,
under certain conditions (Nakatsuji, 1983; Nakatsuji & Johnson, 1983a). We also
reported previously the existence of fibrils in one anuran species, namely A", laevis
(Nakatsuji & Johnson, 19836); and now, since we have improved our fixation
procedure, we find fibrils in two other species of anurans, R. pip and R. syl. Fibrils
are also present in an interspecific hybrid that undergoes gastrulation (pal). Interspecific arrested hybrid embryos, however, have far fewer fibrils than normal embryos. It is safe to conclude that extracellular fibrils on the inner surface of the roof
of the blastocoel in gastrulae are common features of both urodeles and anurans.
There is a striking difference in the number of fibrils in vivo in urodeles and
anurans; the former invariably show many more fibrils. The number of fibrils
deposited in vitro when urodele conditioning fragments are used is also considerably
greater. The fibrils in urodeles are also more oriented, both in vivo and in vitro. The
migrating mesodermal cells in urodeles often move as isolated cells or small cell
clusters, as they migrate across the inner aspect of the roof of the blastocoel. In
contrast, anuran cells form compact masses rather than loosely arranged groups of
cells. We suggest that migrating mesodermal cells in both urodeles and anurans use
these fibrils as a contact guidance system. Perhaps the cells in urodeles require a more
formal fibrillar orientation mechanism because they are loosely arranged and thus
require more oriented extracellular fibrils to guide them towards the animal pole. In
contrast, perhaps the migrating mesodermal cells in anurans do not require such an
organized system of extracellular fibrils because they form a more compact mass of
cells.
The significance of our observations for the control of cell migration in amphibian
embryos may be appreciated better if one considers the behaviour of an isolated
migrating mesodermal cell in a urodele embryo. Once such a cell broke away from the
pack of other migrating mesodermal cells, hypothetically it would loose contact inhibition of cell migration as an orienting mechanism. Once this orienting mechanism
Normal frog ectoderm allows cell movement in vitro
65
was lost, the cell would be forced to migrate at random so that the direction back
towards the blastopore would be the same as the direction away from the blastopore.
If there were a contact guidance system within the embryo, such as the oriented and
polarized extracellular fibrils that we have described (Nakatsuji, 1983; Nakatsuji et
al. 1982; Nakatsuji & Johnson, 1983a,b), then even an isolated cell would still have
orienting cues in its environment and would continue to move directionally, i.e. away
from the blastopore towards the animal pole. Such a system would have a greater
efficiency than a system where only contact inhibition of locomotion was in effect. In
contrast, in anuran embryos, the migrating mesodermal cells have never been observed to have loose cells where they have lost their contacts with neighbouring migrating
mesodermal cells. In this kind of a system, the direction back towards the blastopore
might be prohibited by other migrating mesodermal cells coming from behind by
contact inhibition. Isolated presumptive mesodermal cells iromR.pip gastrulae show
strong contact inhibition of lamellipodial activity and cell locomotion when they are
studied in isolation in vitro (Johnson, 1976) and, most probably, they also exhibit this
behaviour in vivo. Unfortunately, we are unable to make a definitive test of this
hypothesis due to the opacity of amphibian embryos and the consequent impossibility
of a direct analysis of cell movement in vivo.
The differences between normal and hybrid embryos with respect to the system of
extracellular fibrils provides further evidence for the importance of this fibrillar system in promoting cell migration. When extracellular fibrils are present in vivo, either
in normal embryos or in interspecific hybrid embryos where gastrulation does in fact
occur, mesodermal cell migration is extensive but unoriented. These fibrils are also
deposited in vitro and promote cell attachment and translocation. When extracellular
fibrils are reduced in number or absent in vivo in arrested hybrid embryos, mesodermal cell migration fails to occur. Explants from arrested hybrid embryos do not
deposit fibrils in vitro and there is no promotion of cell attachment and translocation.
Finally, the inability of cells from arrested hybrid embryos to attach and translocate
in vitro can be partially restored by explants from normal embryos, which deposit
fibrils. This represents the first evidence that behavioural abnormalities of hybrid
cells can be corrected by factors derived from normal embryos and provides further
substantial evidence that these extracellular fibrils promote cell adhesion and cell
movement. It seems possible that the mechanism of this effect lies in the ability of
extracellular fibrils to serve as an attachment site for filopodia, thereby promoting the
formation of lamellipodia, which are attachment and locomotory organelles of many
cells in vivo and in vitro (Trinkaus, 1976).
At the present time, we have little information concerning the chemical nature of
these fibrillar materials. Closely related experimental studies in amphibia, however,
suggest that these fibrils contain fibronectin. Boucaut & Darribere (1983a, b) have
found that gastrulae of Pleurodeles waltlii have an abundance of fibronectin lining the
inner aspect of the roof of the blastocoel but much less in the same location at the
blastula stage. Our earlier work (Nakatsujiet al. 1982; Nakatsuji & Johnson, 1983a,b)
shows that much fibrillar material lines the inner surface of the roof of the blastocoel
of urodele embryos but that these fibrils are sparse or absent prior to the onset of
66
N. Nakatsuji and K. E. Johnson
gastrulation. Gualandris, Rouge & Duprat (1983) have also shown a network of fine
anastomosing fibrils lining the inner aspect of the roof of the blastocoel in P. waltlii
embryos that were stained by fluorescent soy bean agglutin, a lectin with specificity
for ./V-acetyl-a-D-galactosamine and galactose. The similarity in the appearance and
pattern of these fibrillar networks between P. waltlii and other urodeles (Nakatsuji et
al. 1982; Nakatsuji & Johnson, 19836) is striking.
Oriented fibrils of fibronectin presumably would have precisely the properties
postulated for the fibrils in this study; namely, promotion of cell attachment and cell
locomotion. Heasman et al. (1981) have shown that primordial germ cells from X.
laevis embryos migrate up the dorsal mesentery of the gut along a domain that is rich
in fibronectin. Furthermore, they were able to show that explanted dorsal mesentery.
cells will deposit fibronectin in vitro, and promote elongation and migration of seeded
primordial germ cells along lines parallel to the orientation of stretched mesentery
cells and associated oriented fibrils of fibronectin. Normal R. pip embryos also show
increases in synthesis of extracellular glycoconjugates during gastrulation (Johnson,
1977a-c, 1978), whereas arrested hybrid embryos show defects in extracellular
glycoconjugate synthesis. In the future we plan to study fibronectin distribution in
normal and hybrid embryos to determine whether the extracellular fibrils described
in the present work contain fibronectin, laminin, proteoglycan, or some combination
of similar basement membrane components.
We thank Drs Albert K. Harris and J. P. Trinkaus for their helpful suggestions on a draft of this
manuscript. This research was supported by NIH grant HD 13419 to K.E.J.
REFERENCES
BOUCAUT, J.-C. & DARRIBERE, T. (1983a). Presence of fibronectin during early embryogenesis in
amphibian Pleuwdeles waltlii. Cell Differ. 12, 77-83.
BOUCAUT, J.-C. & DARRIBERE, T. (19836) Fibronectin in early amphibian embryos. Migrating
mesodermal cells contact fibronectin established prior to gastrulation. Cell Tiss. Res. 234,
135-145.
FELDMAN, M. (1955). Dissociation and reaggregation of embryonic cells of Triturus alpestris. J.
Embryol. exp. Morph. 3, 251-255.
GUALANDRIS, L., Roucfi, P. & DUPRAT, A. M. (1983). Membrane changes in neural target cells
studied with fluorescent lectin probes. J. Embryol. exp. Morph. 77, 183-200.
HEASMAN, J., HYNES, R. O., SWAP, A. P., THOMAN, V. & WYLIE, C. C. (1981). Primordial germ
cells oiXenopus embryos: The role of fibronectin in their adhesion during migration. Cell 27,
437-447.
JOHNSON, K. E. (1971). A biochemical and cytological investigation of differentiation in the
interspecific hybrid amphibian embryo Rana pipines $ X Rana sylvatica C?.J. exp. Zool. 177,
191-206.
JOHNSON, K. E. (1976). Ruffling and locomotion in /fana/>i/»e;ts gastrula cells. Expl CellRes. 101,
71-77.
JOHNSON, K. E. (1977a). Extracellular matrix synthesis in blastula and gastrula stages of normal
and hybrid frog embryos. I. Toluidine Blue and lanthanum staining. J'. Cell Sci. 25, 313-322.
JOHNSON, K. E. (19776). Extracellular matrix synthesis in blastula and gastrula stages of normal
and hybrid frog embryos. II. Autoradiographic observations on the sites of synthesis and mode
of transport of galactose- and glucosamine-labelled materials. J. Cell Sci. 25, 323-334.
JOHNSON, K. E. (1977C). Extracellular matrix synthesis in blastula and gastrula stages of normal
and hybrid frog embryos. I l l . Characterization of galactose- and glucosamine-labelled materials.
J. Cell Sci. 25, 335-356.
Normal frog ectoderm allows cell movement in vitro
67
JOHNSON, K. E. (1978). Extracellular matrix synthesis in blastula and gastrula stages of normal and
hybrid frog embryos. IV. Biochemical and autoradiographic observations on fucose-, glucose-,
and mannose-labelled materials. J. Cell Set. 32, 109-136.
JOHNSON, K. E. & ADELMAN, M. R. (1981). Circus movements in dissociated cells in normal and
hybrid frog embryos. J. Cell Sci. 49, 20S-216.
JOHNSON, K. E. & ADELMAN, M. R. (1984). Circus movements in dissociated cells in normal and
two new hybrid frog embryos. J . Cell Sci. 68, 69-82.
MOORE, J. A. (1955). Abnormal combinations of nuclear and cytoplasmic systems in frogs and
toads. Adv. Genet. 1, 139-182.
NAKATSUJI, N. (1983). Cell locomotion and contact guidance in amphibian gastrulation. .Am. Zool.
(in press).
NAKATSUJI, N., GOULD, A. C. & JOHNSON, K. E. (1982). Movement and guidance of migrating
mesodermal cells in Ambystoma maculatum gastrulae. J. Cell Sci. 56, 207-?,?,?,.
NAKATSUJI, N. & JOHNSON, K. E. (1982). Cell locomotion in vitro by Xenopus laevis gastrula
mesodermal cells. CellMotil. 2, 149-161.
NAKATSUJI, N. & JOHNSON, K. E. (1983a). Conditioning of a culture substratum by the ectodermal layer promotes attachment and oriented locomotion by amphibian gastrula mesodermal cells.
jf. Cell Sci. 59, 43-60.
NAKATSUJI, N. & JOHNSON, K. E. (19836). Comparative study of extracellular fibrils on the
ectodermal layer in gastrulae of five amphibian species. J. Cell Sci. 59, 61-70.
RUGH, R. (1962). Experimental Embryology, 3rd edn. Minneapolis: Burgess Pub. Co.
SHUMWAY, W. (1940). Stages in the normal development of Ranapipiens. Anal. Rec. 78, 139-147.
STEARNS, R. N. & KOSTELLOW, A. B. (1958). Enzyme induction in dissociated cells. In The
Chemical Basis ofDevelopment (ed. W. D. McElroy & B. Glass), pp. 448-457. Baltimore: Johns
Hopkins University Press.
TRINKAUS, J. P. (1976). On the mechanism of metazoan cell movements. In The Cell Surface in
Animal Embryogenesis and Development (ed. G. Poste & G. L. Nicholson), pp. 225-329.
Amsterdam: North-Holland.
TRINKAUS, J. P. & ERICKSON, C. A. (1983). Protrusive activity, mode and rate of locomotion, and
pattern of adhesion of Fundulus deep cells during gastrulation. J . exp. Zool. 228, 41-70.
(Received 9 November 1983 -Accepted 14 December 1983)
Note added in proof
Recentlly, Boucaute/a/. {Nature, 307, 364—367, 1984) have prepared antibodies to Ambystoma
mexicanum fibronectin. Fluorescent anti-fibronectin antibodies bind to a fibrillar network on the
inner aspect of the roof of the blastocoel of A. mexicanum gastrulae. When transplantation experiments were performed so that the inner surface of the roof of the blastocoel projected outwards
towards the vitelline membrane, mesodermal cell migration failed to occur over the patch of transplanted tissue. Finally, these authors showed that injection of monovalent Fab1 fragments of antifibronectin antibodies prevent gastrulation but not neurulation.