Download An ECM substratum allows mouse mesodermai cells

Development 100, 587-598 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
587
An ECM substratum allows mouse mesodermai cells isolated from the
primitive streak to exhibit motility similar to that inside the embryo and
reveals a deficiency in the 7/7 mutant cells
KOICHIRO HASHIMOTO1, HIROKAZU FUJIMOTO2 and NORIO NAKATSUJI1
'Division of Developmental Biology, Meiji Institute of Health Science, 540 Naruda, Odawara, Kanagawa 250, Japan
Laboratory of Cell Biology, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan
2
Summary
The mesodermai cell layer is created by ingression
and migration of the cells from the primitive streak
region in mouse embryos on day 7 of pregnancy. In
order to study the mechanisms of mesodermai cell
migration during development, the mesodermai cells
isolated from the primitive streak were cultured on
various substrata, and cell behaviour and motility
were analysed with a time-lapse video system. The
mesodermai cells on the surface of extracellular
matrix (ECM)-coated dishes (ECM produced by bovine corneal endothelial cells) showed extensive migration at a mean rate of approx. SOjumh"1. They
also showed frequent cell division and exhibited contact paralysis of lamellipodia and contact inhibition of
movement. On plastic or glass surfaces, however, the
mesodermai cells became more flattened and less
motile (approx. 20—30jUmh~'). Cell shape and mean
rate of movement on the ECM were very similar to
those in situ, as investigated in a previous study
(Nakatsuji, Snow & Wylie, 1986). Therefore, this
culture condition could provide a useful experimental
system for analysing the cellular basis of normal
Introduction
There has been a rapid increase in our knowledge of
the locomotion of tissue cells in vitro (Porter &
Fitzsimons, 1973; Trinkaus, 1976; Bellairs, Curtis &
Dunn, 1982). Although most tissue cells in adult
animals retain a potential ability for active migration,
such motility is mainly displayed during embryonic
development, where directional movement of cells
takes place to build the complex organization of
tissues and organs (Trinkaus, 1984; Armstrong,
1985). However, we know relatively little about the
and abnormal morphogenetic movements in mouse
embryos.
Employing such a culture system, we studied
motility of the mesodermai cells from embryos homozygous for Brachyury (T) mutation, which are lethal
at the midgestation stage in utero. Histological observations have suggested that anomalous morphogenesis
of the T/T embryos may be brought about by defects
in migration of the mesodermai cells derived from the
primitive streak. When mesodermai cells from the
primitive streak of the T/T mutant embryos on days
8-9 were cultured on the ECM substratum, mean
rate of cell migration was significantly reduced compared to cells from normal embryos. Results support
the idea of retarded migration by the mutant mesodermai cells as an important factor causing abnormalities in morphogenesis.
Key words: extracellular matrix, cell motility,
gastrulation, mouse embryo, T mutation, primitive
streak, mesoderm, time-lapse video recording.
movement of embryonic cells involved in morphogenetic movements, because of our poor understanding of the nature of microenvironmental cues to
which cells are responsive during the course of their
movements (Trinkaus, 1976).
The study of events occurring inside multicellular
masses is much more difficult than the study of
isolated individual cells in vitro. In the case of opaque
embryos such as those of amphibians and birds, it is
not possible to look at moving cells in vivo. Morphological studies of fixed samples have provided information about the static features of cells presumed to
588
K. Hashimoto, H. Fujimoto and N. Nakatsuji
be moving. On the other hand, attempts to observe
cell movements in vivo have been successful in
the case of the relatively transparent embryos such as
those of sea urchin (Dan & Okazaki, 1956; Gustafson
& Kinnander, 1956), nematode (Deppe et al. 1978)
and fishes (Trinkaus, 1973, 1985; Eisen, Myers &
Westerfield, 1986). Even in such successful studies,
however, it has been difficult to manipulate the cells'
environment in an attempt to learn more about the
control of morphogenetic cell movements. Therefore, as an alternative, we have examined behaviour
of cells isolated from embryos in order to obtain
additional information on cell movement. Such in
vitro study allows a detailed examination of individual
embryonic cells and the interactions among them
under precisely controlled environmental conditions.
In these studies, it is most important to find culture
conditions that permit embryonic cells to exhibit a
similar motility in vitro to that in vivo (Nakatsuji &
Johnson,1982).
In recent years, there have been important advances in the analysis of the various morphogenetic
cell migrations, such as the neural crest cells (Le
Dourain, 1984; Thiery, Duband & Tucker, 1985a;
Thiery, Boucaut & Yamada, 19856), primordial germ
cells (Heasman et al. 1981; Heasman & Wylie, 1983;
England, 1983), amphibian mesodermal cells (Nakatsuji, 1984; Thiery et al. 19856) or sea urchin primary
mesenchymal cells (Katow, 1986). From these
studies, it has become apparent that extracellular
matrix (ECM), such as the basal lamina, plays a key
role in control of embryonic cell migration by providing substrata for cell attachment and movement.
We still know little about gastrulation movement in
mammals (for a review see Bellairs, 1986). Although
early mouse embryos are relatively transparent because of the small amount of yolk, they are not
accessible in situ due to the enveloping uterine and
decidua tissues. Based on examination of fixed materials, two conflicting hypotheses on the formation of
the mesodermal cell layer have been introduced. One
hypothesis is that the mesodermal cells migrate actively away from the primitive streak in both anterior
and antimesometrial directions (Batten & Haar,
1979; Tarn & Meier, 1982; Franke etal. 1982; Franke,
Grund, Jackson & Illmensee, 1983; for a review see
Beddington, 1983). The other is that the mesodermal
cells do not migrate away from the primitive streak,
but that they only appear to do so because of a shift in
the relative positions of the embryonic ectoderm
layer and of the primitive streak during rapid growth
of the embryo (Poelmann, 1981). Recently, Nakatsuji
et al. (1986) attempted to look directly at the migration of the mesodermal cells inside the whole
embryo using whole embryo culture and time-lapse
cinemicrography with Nomarski differential interference contrast (DIC) optics. They showed that the
mesodermal cells are motile and migrate actively
away fom the primitive streak in both anterior and
antimesometrial directions at a mean rate of approx.
1
Mutations that cause developmental defects in
early embryogenesis, such as T/t complex on
chromosome 17 of the mouse, provide useful tools in
the search for mechanisms of mammalian morphogenesis (Gluecksohn-Waelsch & Erickson, 1970;
Bennett, 1975; Sherman & Wudl, 1977). Brachyury
(T) mutation is lethal at the midgestation stage in
homozygotes, where it severely affects the axial
organization of the posterior trunk up to, but not
including, the forelimb buds on day 9 (Chesley,
1935). It also retards extension of the allantois,
inhibiting development of the complete umbilical
blood circulation (Gluecksohn-Schoenheimer, 1944).
Histologically, the chordamesoderm in the posterior
region of the T/ T embryo develops extensive tubular
structures instead of the notochord and somites by
day 9 (Spiegelman, 1976; Fujimoto & Yanagisawa,
1979). Griineberg (1958) proposed that anomalies of
the notochord may be involved in the massive abnormalities of the posterior trunk region in the T/T
embryos as a part of a more general disturbance of
the primitive streak. Indeed, the head process, derived from the cephalad extremity of the primitive
streak, is defective and the prenotochordal cells are
fewer in the archenteron area (Fujimoto & Yanagisawa, 1983). It is known that T/T embryos have a
bulky primitive streak at the middle of day 8 or the
later stages (Spiegelman, 1976). Morphometric analyses of histological sections of day-6 or -7 embryos
suggest that cells derived from the epiblast layer of
T/T embryos are stuck in the primitive streak region
(Yanagisawa, Fujimoto & Urushibara, 1981). These
morphological studies have suggested that anomalous
morphogenesis of T/T embryos is caused by lesions in
the migration of cells invaginated from the primitive
streak. Due to these effects in morphogenesis, the
T/T mutant serves as an excellent probe to study
mechanisms of mesodermal cell migration during
development.
In the present study, we examined the behaviour of
mesodermal cells isolated from the primitive-streakstage mouse embryos. We have compared three kinds
of substrata for cell culture in order to find culture
conditions that allow mesodermal cells to move in a
similar fashion to that in vivo. We found that dishes
coated with ECM produced by endothelial cells
provide substrata on which the mesodermal cells
exhibit behaviour most similar to that inside embryos
in terms of cell shape, rate of movement and cell
division. Employing such culture conditions, we
ECM and motility of mouse mesodermal cells
examined the effect of T mutation on the motility of
mesodermal cells by comparing the behaviour of
individual cells isolated from the T/T embryos with
that of those from normal embryos. Results directly
prove the T/T mutant mesodermal cells to be retarded in their migration ability.
Materials and methods
Mice
Randomly bred ICR (Crj:CD-l) strain mice were purchased from Charles River in Japan and kept at a normal
(7 a.m.-7 p.m. light) or reversed (4p.m.-4 a.m. light)
light/dark cycle. Heterozygous mice (T/+) were originally
supplied by Dr D. Bennett at the Sloan-Kettering Institute
for Cancer Research. Mice homozygous for the Robertsonian translocation Rb (4,17) 13 hub (hereafter designated
RblS) were originally supplied by the late Dr A. Gropp,
Medizinische Hochschule, Liibeck. They were in the 25th
and the 8-10th inbreeding generation, respectively, at the
time of the present study. The T/+ males were mated to
Rbl31Rbl3 females to produce short-tailed F] progeny
carrying the T mutation and the Rbl3 translocation (designated T/Rbl3). These F] mice were maintained under a
reversed light cycle.
Embryos
A male was mated with three to five females during the
dark period. When the vaginal plug was detected at the
beginning of the light period, the midpoint of the preceding
dark period was designated as day 0 of pregnancy. Embryos
used in this study were obtained from ICR females of day
6-5,7, 7-5, 8 or 9 of pregnancy and T/Rbl3 females of day 8
or 9 of pregnancy mated with T/Rbl3 males.
Pregnant females were killed by cervical dislocation.
Uteri were dissected out and transferred to Petri dishes
containing PB-1 solution (Whittingham & Wales, 1969).
Embryos were freed from the decidua tissue using fine
forceps under a binocular stereomicroscope.
T/T embryos derived from T/Rbl3 intercrosses were
distinguished from their normal littermates according to the
criteria of morphological abnormalities in the posterior
body and the allantois. The normal littermates, T/Rbl3 and
Rbl3/Rbl3, were used as controls. Effectiveness of the
morphological identification of T/ T embryos was confirmed
by karyotypic analysis of the cultured embryonic cells; cells
derived from morphologically identified 7/7homozygotes
contained no Robertsonian translocated chromosomes and
those from the normal embryos contained one or two
translocated chromosomes.
Isolation of the mesoderm cell layer
The mesoderm cell layer was isolated from the primitivestreak-stage embryo (egg-cylinder stage) obtained from
females of day 6-5 to 7-5 of pregnancy with sterile microsurgery and enzyme treatment, as described by Snow (1978a).
Briefly, the embryonic portion was dissected from the egg
cylinder and a single cut was made along the primitive
streak with a tungsten needle. The tissue fragment was
589
transferred into a mixture of 0-5% trypsin (Difco 1:250)
and 2-5% pancreatin (Sigma) in Ca2+,Mg2+-free phosphate-buffered saline (PBS-) for 5 to 15min at 4°C.
Enzyme digestion was stopped by rinsing twice with PBS—
supplemented with 2% calf serum. Afterwards, the fragment was gently agitated with a flame-polished micropipette. The mesoderm cell layer was carefully separated
from both the embryonic ectoderm and the primitive
endoderm layers with a pair of sharpened tungsten needles.
Using a stereomicroscope at higher magnifications, we
could distinguish the mesoderm layer, which appeared as a
loosely connected thin cell sheet with spaces present
between cells, from the much thicker cohesive cell sheet of
the embryonic ectoderm and the primitive endoderm,
which quickly shrank into a rounded-up cell mass. After
rinsing twice with culture medium, fragments of the mesodermal cell sheets were torn into small pieces and explanted
in culture dishes.
In the case of 8- to 9-day-old embryos, the mesodermal
cells were isolated from the primitive streak region occupying the caudal portion of the embryo, using the same
microsurgery and enzyme digestion.
Culture conditions
Three kinds of substrata were used in this study. Extracellular matrix (ECM)-coated dishes were purchased from
International Bio-Technologies Ltd (Jerusalem, Israel).
The dish surface had been coated with basement membrane
ECM produced by endothelial cells. The others were
Falcon plastic tissue culture dishes (Falcon 3001) and glass
coverslips placed in Falcon plastic Petri dishes (Falcon
1008). Small pieces of the mesoderm cell layer isolated from
each embryo were transferred into one of these dishes
containing Dulbecco's modified minimum essential medium
(DMEM) supplemented with glucose (final, 4-5gI"1) and
20 % fetal calf serum (Gibco). They were incubated at 37°C
with an atmosphere of 5 % CO2 and 95 % air.
Time-lapse video recording and analysis
Cell movement was recorded using a time-lapse video
system (Professional Editing Recorder BR-8600, JVC controlled by Time-lapse Video Controller SIV, Sankei,
Tokyo) equipped with a video camera (C1965, Hamamatsu
Photonics K. K., or CTC-2100, Ikegami Tsushin Co. Ltd),
which was connected to an inverted microscope (Nikon
Diaphoto-TMD or Olympus IMT-2) equipped with a
heated (37°C) box using xlO or x20 phase-contrast objective lenses. Single recording for 0 1 s was repeated at 30s
intervals.
Video recording during the period from 12 to 24 h after
the start of culture was analysed with a video digitizer
(For.A, Tokyo, Model FVW-300) connected to a personal
computer (NEC PC-9801). Rate of cell migration was
obtained by tracing the centre of the nucleus of each cell
every 30 min for the total of 5 h. When a cell entered mitotic
division during tracing, one of the daughter cells was chosen
for the succeeding tracing. When a cell left the field, its
tracing was stopped and discarded from the data. Movement of approximately 25 to 100 cells was analysed from
one video recording.
590
K. Hashimoto, H. Fujimoto and N. Nakatsuji
Results
Cell behaviour
Explants of the mesodermal cell sheet attached to the
bottom of an ECM-coated dish within l h of culture.
Attached cell masses continued to change their
shapes due to cell dislocation and cell division accompanied by vigorous blebbing within the cell mass.
Peripheral cells protruded cell processes onto the dish
surface. Shortly, marginal cells began to migrate
individually away from the periphery of the cell mass
(Fig. 1A). The whole cell masses became gradually
flattened as outgrowth proceeded. At the beginning
of outgrowth, individual cells migrated radially away
from the cell mass. Once fully dispersed, they moved
about apparently randomly on the substratum. The
outgrowth was completed within 12h of culture,
leaving no cell aggregate at the centre (Fig. IB).
Fig. 1. Phase-contrast micrographs of the mesoderm (A-C) or embryonic ectoderm (D) explants on the surface of
ECM-coated dishes. (A) Start of outgrowth after a few hours of culture. (B) Cells have completed their outgrowth and
dispersion by 12 h of culture. (C) A higher magnification view of the mesodermal cells during their extensive migration.
(D) The embryonic ectoderm cells remain as a cohesive cell sheet even after one day of culture. Marginal cells
frequently extend very thin large lamella (arrows). Bars, 100/.im.
ECM and motility of mouse mesodermal cells
Dispersed mesodermal cells were mostly bipolar or
unipolar, although some multipolar cells were observed (Fig. 1C). Each cell migrated in the direction
of an active lamellipodium. When lamellipodia of
migrating cells made contact with each other, the
lamellipodium stopped advancing immediately and
retreated locally at the site of contact, thus exhibiting
contact paralysis and contact inhibition of movement.
The lamellipodium at the opposite end of the cell next
became active and eventually the two cells moved
away from each other. Mesodermal cells obtained
from the primitive-streak-stage embryos and more
advanced embryos (day 8-9) exhibited almost the
same cell behaviour on the ECM-coated surface.
In the case of plastic tissue-culture dishes or glass
coverslips, attachment of the mesodermal cell mass to
the substratum required much more time than attachment to the ECM-coated dishes. Moreover, only a
minority of the explants attached. Although attached
cell masses became gradually flattened, few cells
migrated away from the periphery of the cell masses.
Most cells kept close contact with each other and
remained as a cell sheet (Fig. 2). Mesodermal cells on
plastic or glass surfaces took more circular and
591
flattened shapes than on the ECM-coated surface.
Each cell formed a large ruffling lamellipodium and
moved slowly toward that direction. Nuclei showed
frequent movement within the cytoplasm. This movement was more conspicuous in the cells on a glass
surface than on a plastic surface and much less so on
the ECM-coated surface. The cells showed contact
paralysis on plastic or glass surfaces as well.
Explants of the primitive endoderm layer did not
attach to any substrata. Those of the embryonic
ectoderm attached to the ECM-coated surface and
less frequently to the plastic or glass surfaces. The
explants flattened to become a relatively thick epithelial cell sheet on the ECM-coated surface, but
individual cells almost never moved away from the
explant, even after one day of culture (Fig. ID).
Cell motility
Table 1 shows the mean rate and standard deviation
(S.D.) of movement by mesodermal cells cultured on
the ECM-coated, plastic-culture-dish or glass surfaces. On the ECM-coated surface, mesodermal cells
obtained from the primitive-streak-stage embryos
moved at a mean rate of approx. 50 /im h~'. Mesodermal cells from the 8- or 9-day-old embryos were found
to move at the same rate as younger cells. On the
other hand, the mean rate on plastic or glass surfaces
was uniformly lower (approx. 20-30jumh"1), regardless of the age of embryos from which mesodermal
cells were isolated. The difference in the rate of
movement between the ECM-coated surface and
plastic or glass surfaces was found to be significant at
the level of P = 0-005 by Student's f-test. However,
there is no significant difference in the rate between
the plastic and glass surface.
Table 1. Motility (mean rate and S.D.) (\imh~1) of
the mesodermal cells isolated from ICR (CD-I) mouse
embryos on ECM, plastic or glass surfaces
Age of
embryo
(days)
6-5-7-5
8
9
A.
Fig. 2. (A) A phase-contrast micrograph showing
mesodermal explants cultured for one day on the surface
of a plastic culture dish. (B) A higher magnification view.
Bars, 100 pm.
Surface
ECM-coated
plastic
glass
51-7 ±19-3*
(n = 167)
50-8 ±9-7*
(n = 21)
48-0 + 22-2*
(n = 57)
26-6 ± 9 - 1 "
(n = 47)
29-0 ± 9 - 3 "
(n = 29)
22-7 ±7-3**
(n = 27)
22-4 ±9-8
(n = 24)
18-8 ±9-8
(n = 40)
19-4 ±7-2
(n = 26)
n Cell number measured.
* Significantly different from 'plastic' or 'glass' at the level of
P<0-005 (Student's (-test).
** Not significantly different from 'glass'.
592
K. Hashimoto, H. Fujimoto and N. Nakatsuji
Fig. 3 shows histograms illustrating distribution of
the rates of cell movement on the ECM-coated or
plastic surfaces. One remarkable feature is that the
distribution on the ECM-coated surface is broader
than that on the plastic surface. In most cases,
approx. 10% of cells moved faster than 80/imh" 1 ,
and approx. 30% of cells moved faster than
60/imh" 1 on the ECM-coated surface. The distribution on the plastic surface is shifted to the lower
rate range and there are no cells moving faster than
60/xmrr1.
Fig. 4 shows trajectories of the cell nuclei on the
ECM-coated, plastic or glass surfaces during a 5h
culture period. It is apparent that cell trails on the
ECM-coated surface are longer than those on the
plastic or glass surfaces. In most cases, they make
apparently random zigzag lines. At the periphery of
outgrowth, however, the direction of movement has a
tendency to move radially away from the centre of
explant. Cell trails on the glass or plastic surfaces are
relatively circular due to movement of the nuclei
within the cytoplasm. Nuclear movement within the
cytoplasm occurred even when the whole cell did not
translocate on plastic or glass surfaces. Therefore, the
L-
j
56-6
10-
5 -
^
V
1
• u
0
20
40
100
60
120
26-6
Fig. 4. Trajectories of the cell nuclei on the ECM-coated
(A), plastic (B) or glass (C) surfaces for 5 h traced from
the time-lapse video recording. Cell trails on A are much
longer than those in B or C. Bars, 100^m.
10-
5-
real rate of cell movement on these substrata seems to
be lower than the calculated values shown in Table 1.
I
20
40
60
80
100
Rate of movement
Fig. 3. Histograms showing distribution of the rates of
cell movement on the ECM-coated (A) or plastic (B)
surfaces. Total cell number is 49 (A) and 47 (B). The
mean values are shown by arrows.
Cell division
Mesodermal cells on the ECM-coated surface showed
frequent cell division accompanied by vigorous blebbing and rounding up of the cell body. Table 2 shows
the frequency of cell division observed during 5 h of
the video recording. The frequency is much lower on
ECM and motility of mouse mesodermal cells
593
Table 2. Frequency (per 100 cells) of cell division by cell shape, generally with only one active lamellipodium, by 30 to 60 min from the first sign of rounding
the mesodermal cells during 5 h culture on ECM,
up and blebbing.
plastic or glass surfaces
Surface
Age of
embryo
(days)
6-5-7-5
ECM-coated
plastic
glass
(no. of cell divisions/total cell no. examined)
130
(130/999)
0-9
(4/463)
1-5
(1/67)
the plastic or glass surfaces than on the ECM-coated
surface.
20 examples of cell division on the ECM-coated
surface were analysed to obtain the time course.
Vigorous blebbing started around the cell body as
soon as the cell body became rounded from more
angular shapes and the cell processes started to
detach from the substratum. Cytokinesis generally
started 20 to 30 min after rounding up of the cell body.
Blebbing resumed immediately before the completion of cytokinesis. The two daughter cells moved
rapidly away from each other and resumed normal
T mutant cells
No apparent differences were observed in cell morphology between explants taken from T/T and normal embryos (Fig. 5). The mesodermal cells showed
extensive migration with a spindle or stellar shape on
the surface of ECM-coated dishes. In contrast, on
plastic or glass surfaces, the cells took more flattened
shapes without extensive outgrowth from the
explants.
Table 3 shows the mean and standard deviation of
the rate of cell movement analysed from time-lapse
video recording. There was no significant difference
between T/T cells and control cells on plastic or glass
substrata, and the mean rate was uniformly low
(approx. 20,umh~1). On the ECM-coated surface,
however, mesodermal cells from normal embryos
moved faster (approx. 50/imh" 1 on day 8, and
approx. 40fimh"1 on day 9) than the cells from T/T
embryos (approx. 40,1ml h" 1 on day 8, and approx.
25 ftmh"' on day 9). Statistical analyses by Student's
Fig. 5. Phase-contrast micrographs of the mesodermal cells on the ECM-coated dish surface, after they completed the
outgrowth from small pieces of the mesodermal cell layer isolated from the primitive streak region. (A) Control cells;
(B) T/T cells. Bars, 50^m.
594
K. Hashimoto, H. Fujimoto and N. Nakatsuji
Table 3. Motility (mean rate and S.D.; \xmh ') of the mesodermal cells isolated from T / T and normal embryos
on ECM, plastic or glass substrata
Surface
Age of
embryo
Normal
T/T
ECM-coated
8
52-8 ± 22-6
(n = 34)
9
41-3 ±17-0
(n = 203)
* , ***
8
39-4 ±11-0
(n = 24)
9
25-5 ±11-6
(n = 301)
plastic
glass
21-8 ±6-6
(n = 24)
****
21-0 + 9-4
(n = 24)
****
24-3 ±8-4
(n = 25)
****
20-6 ±6-1
(n = 35)
16-9 ±4-9
(n = 23)
22-7 ±10-0
(n = 23)
n Cell number measured.
* Significantly different from 'plastic' or 'glass', P< 0-005 (Student's Mest for difference).
** Not significantly different from 'ECM'.
*** Significantly different from 'T/T', P< 0-005.
" " N o t significantly different from 'T/T'.
t-test showed these differences to be significant at the
level of P= 0-005.
Fig. 6 shows histograms illustrating distribution of
the rate of cell movement by the T/T and control
mesodermal cells of day-9 embryos. The distribution
is apparently shifted to the lower cell speed range in
the T/T cell group. One remarkable feature is that
approximately 10% of cells moved rapidly (faster
than 80(imh~l) in the control group, while there
were no such fast-moving cells among the T/T mutant
cell population. Such fast-moving cells were always
present among control cells, but never found in the
T/T cells when distribution of five groups each of the
T/T and control cells were examined.
Discussion
48-3
10-
5-
II I
20
40
60
100
B
26-2
U
10-
Behaviour of mesodermal cells in vivo and in vitro
Motility of mesodermal cells inside embryos was
examined by using whole embryo culture and DIC
optics (Nakatsuji et al. 1986). It showed that the
mesodermal cells migrated away from the primitive
streak at an average rate of 46Jumh~1 within threedimensional spaces in the early primitive-streak-stage
embryos. They moved slowly in a crowded area, but
more rapidly in areas of lower population density of
mesodermal cells and with larger cell-free spaces
(Nakatsuji et al. 1986). They took spindle, but not
flattened, cell shapes (Spiegelman & Bennett, 1974;
Spiegelman, 1976; Batten & Haar, 1979; Poelmann,
1981; Franke et al. 1982, 1983; Nakatsuji et al. 1986).
In the present study, we calculated the mean rate of
5-
I
20
lllui
40
60
80
Rate of movement (fim h" 1 )
100
Fig. 6. Histograms showing distribution of the rate of cell
movement analysed from time-lapse video recording. 50
mesodermal cells were analysed in cultures of explants
from a normal (A) and T/T (B) embryos on day 9. Mean
values are shown with arrows.
ECM and motility of mouse mesodermal cells
cell movement in vitro by mesodermal cells from the
primitive-streak-stage embryos to be 52Jumh~1 on
the ECM-coated surface, whereas it was much lower
on plastic or glass surfaces. Cell shape was bipolar or
stellar on the ECM-coated surface, but it was more
flattened on plastic or glass surfaces. These results
reveal a resemblance between the mesodermal cells
on the ECM-coated surface and those within an
embryo during gastrulation. Moreover, the mesodermal cells exhibited contact inhibition of movement in
the present study. It may contribute to the directional
migration of the mesodermal cells away from the
primitive streak and their slowdown in a crowded
area observed in vivo (Nakatsuji et al. 1986).
Cell divisions accompanied by extensive blebbing
were frequently observed while mesodermal cells
were migrating away from the primitive streak within
an embryo (Nakatsuji et al. 1986). We also observed
cell division by the mesodermal cells accompanied by
vigorous blebbing during outgrowth from an explant
on the ECM-coated surface. Such cell division occurred very frequently among the mesodermal cells
cultured on the ECM-coated surface, but much less
frequently on plastic or glass surfaces. Snow (1977,
I978a,b) has shown that the mesodermal cells in the
primitive-streak-stage embryos are rapidly proliferating (estimated cell cycle time, 14-22h). The time
course of cell division observed here in vitro is also
similar to that in vivo (Nakatsuji et al. 1986). These
results show that the behaviour of mesodermal cells
on the ECM-coated surface is much more similar to
that inside embryos than on plastic or glass surfaces.
ECM as the substratum for cell migration
In the primitive-streak-stage mouse embryo, mesodermal cells migrate away from the primitive streak
through an extracellular space between the embryonic ectoderm and primitive endoderm layers (Nakatsuji et al. 1986). Their cell processes attach to the
outer (basal) surface of the ectoderm layer, the inner
(basal) surface of the endoderm layer or adjacent
mesodermal cells (Spiegelman & Bennett, 1974; Spiegelman, 1976; Batten & Haar, 1979; Poelmann, 1981;.
Tarn & Meier, 1982; Franke et al. 1982, 1983; Nakatsuji etal. 1986).
Transmission electron microscopic studies have
revealed that the embryonic ectoderm layer has a
basal lamina which is almost continuous except for
the primitive-streak region (Batten & Haar, 1979;
Franke et al. 1982, 1983). The primitive endoderm
layer has no continuous basal lamina or lamina-like
structure. However, fragments of fuzzy amorphous
material (Franke et al. 1983; K. Hashimoto & N.
Nakatsuji, unpublished observation), which can be
stained with tannic acid (Herken & Barrach, 1985),
were found near the basal surface of the primitive
595
endoderm layer. Immunohistochemical studies of
mouse embryos (Adamson & Ayers, 1979; Wartiovaara, Leivo & Vaheri, 1979; Leivo, Vaheri, Timpl &
Wartiovaara, 1980; Leivo, 1983; Dziadek & Timpl,
1985; Herken & Barrach, 1985) have shown the
presence of laminin, fibronectin, type IV collagen and
heparan sulphate proteoglycan at the basal lamina of
the embryonic ectoderm layer or near the basal
surface of the primitive endoderm layer. Therefore, it
is very likely that the migrating mesodermal cells use
the ECM containing these molecules as the substratum for cell attachment and movement, in a
manner similar to the mesodermal cells of amphibian
gastrulae (Nakatsuji, Gould & Johnson, 1982; Boucaut & Darribere, 1983; Boucaut, Darribere, Boulekbache & Thiery, 1984; Nakatsuji, 1984; Nakatsuji,
Smolira & Wylie, 1985; Darribere, 1983; Boucaut,
Darribere, Boulekbache, Shi & Boucaut, 1985),
chick gastrulae (Critchley, England, Wakely &
Hynes, 1979) and sea urchin gastrulae (Katow, 1986).
ECM of the ECM-coated dish used in this study
was produced by bovine corneal endothelial cells
(personal communication from the distributor,
Funakoshi Pharmaceutical Co. Ltd, Tokyo). Immunohistochemical analyses reveal that main components of such basement membrane ECM are type
III and IV collagen, fibronectin and laminin (Gospodarowicz et al. 1979; Gospodarowicz & Tauber, 1980;
Vlodavsky, Lui & Gospodarowicz, 1980). The present study showed that the behaviour of mesodermal
cells on the ECM-coated surface is very similar to that
in vivo. Therefore, the ECM seems to contain adequate components necessary for the attachment and
movement by mesodermal cells, although it is produced by cells unrelated to those of the mouse
embryo.
An in vitro system for analysing abnormal
morphogenesis
Flint & Ede (1982) analysed the behaviour of mesodermal cells from the mouse amputated mutant using
an in vitro system. Morris (1973, 1975) observed the
effects of vitamin A on the behaviour in vitro of
mesodermal cells isolated from the primitive-streakstage rat embryos. In these studies, however, they
used glass or plastic surfaces as the culture substrata.
Use of an ECM-coated substratum might give further
insight into effects of the mutations or teratogens on
the cell behaviour. In the present study, we showed
that the mesodermal cells from the T/T mutant
embryos had a reduced rate of motility on the ECMcoated surface, compared to the cells from normal
embryos, but such a difference was not detectable in
the culture conditions using plastic or glass substrata.
Studies on viability of the T/T embryos have shown
that the metabolic activity and cell division continue
596
K. Hashimoto, H. Fujlmoto and N. Nakatsuji
beyond day 10 (Yanagisawa & Fujimoto, 1977a).
There is no statistical difference in the average
generation time of cells for normal and T/T embryos
on day 8 (Yanagisawa, Fujimoto & Urushibara,
1981). Although the mitotic activity at the posterior
end of the mutant embryo on day 9 is lower than that
in the anterior region (Yanagisawa & Fujimoto,
1977a), those tissues have potentiality to develop into
teratomas composed of fully differentiated tissues
when grafted into ectopic sites (Fujimoto & Yanagisawa, 1979). Thus, the reduced rate of movement in
the T/T cells could not be attributed to general
degeneracy.
The retardation of cell movement can be caused by
many factors. One of them is a change in the
cytoskeletal system, which might be affected by the
mutation as suggested in the case of f mutation
(Spiegelman & Bennett, 1974). Embryos homozygous for the t9 mutation show defective primitive
streak differentiation similar to that of the T/T
embryo, but at the earlier stages. No reduction in the
microfilaments or microtubules, however, has been
detected in the T/T embryonic cells (Spiegelman,
1976).
Another likely factor is a change in the cell surface
molecules related to cell adhesion. Shur (1982)
showed that cell surface glycosyltransferase activities,
which have been inferred to play a role in cell
migration (Shur, \911a,b), were different in normal
and 7/T mesenchyme cells. Yanagisawa & Fujimoto
{1911b) reported that reaggregation kinetics of the
dissociated embryonic cells was different in the T/T
cells and normal cells, suggesting some changes in the
cell surface properties. Similar differences are found
between normal and talpid3 mesenchyme cells in the
wing bud of the fowl (Ede & Agerbak, 1968; Ede &
Flint, 1975a). The talpid3 mutation also affects cell
motility in vitro (Ede & Flint, 19756).
One interesting finding concerning the role of ECM
is that in the T/ T embryos it is greatly decreased in all
areas when they are observed with scanning electron
microscopy (Jacobs-Cohen, Spiegelman & Bennett,
1983). Altered matrix molecules might result in faulty
organization of ECM and abnormal cell migration.
However, the results presented here suggest that
anomalous cell migration is caused by the intrinsic
character of the T/T mutant cells.
We thank Izumi Fuketa and Masako Nagatomo for
excellent technical assistance, and Misae Hirayama for
looking after the mouse mutant stocks. Karyotyping was
carried out by Hirofumi Suemori.
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{Accepted 22 April 1987)