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DEVELOPMENTAL
BIOLOGY
107,66-74
(1985)
Three Cell Recognition Changes Accompany the Ingression
of Sea Urchin Primary Mesenchyme Cells
RACHEL
Department
D. FINK AND DAVID R. MCCLAY’
of Zoology, Duke University,
Received February
Durham,
North
23, 1984 accepted in revised fwm
Carolina
2~06
July 27, 1984
At gastrulation
the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular
hyaline
layer and with neighboring
blastomeres as they pass through the basal lamina and enter the blastocoel. This
delamination
process was examined using a cell-binding
assay to follow changes in affinities between mesenchyme
cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage
micromeres (the
precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage
embryos
were used in conjunction
with micromeres raised in culture to intermediate
ages. The micromeres exhibited an
affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly,
micromeres had
an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell
affinity.
Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding
over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina
preparations,
there was an increase in affinity that was associated with developmental
time. Presumptive ectoderm
and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal
laminar components identified fibronectin
as one molecule for which the wandering
primary
mesenchyme cells
acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of
the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated
with the basal lamina and extracellular
matrix that lines the blastocoel.
@J 19x5 Academic PRSS. hc.
The environment of the primary mesenchyme cells
changes as they move from the wall of the blastula
into the blastocoel. They lose contact with the extracellular hyaline layer and with neighboring cells. The
cells ingress through the basal lamina, and begin a
series of migrations on the internal face of that structure. Okazaki (1960) observed the movements of the
primary mesenchyme cells after ingression and showed
that they follow a distinct pattern prior to and during
spicule formation. It was later demonstrated that
isolated micromeres follow the same behavioral patterns in culture as their in vivo counterparts, including
production of spicules (Okazaki, 1975).
We have studied the cell surface properties of micromeres and their descendents using cells from two
sources. Cells cultured in vitro provide a highly enriched
source of mesodermal cells that can be harvested at
any precise time during development, though it is
necessary to show that cultured cells behave as their
counterparts in viva. Micromeres and primary mesenthyme cells were also isolated and studied directly
from embryos. The use of these uncultured cells allowed
us to measure recognition properties at the two ends
of the behavioral spectrum, while the use of cells in
vitro
permitted us to examine recognition changes at
the precise time of primary mesenchyme cell ingression.
INTRODUCTION
The ingression of primary mesenchyme cells is the
earliest recognizable morphogenetic event of gastrulation in the sea urchin embryo. Several detailed studies
have documented the sequence of events in primary
mesenchyme migration. For example, Gustafson and
Kinnander’s time-lapse microcinematographic study of
sea urchin development focused on the extensive migrations of the primary mesenchyme cells (1956), and
demonstrated the sudden change in active motility of
these cells at ingression. Examinations of ingressing
primary mesenchyme cells by electron microscopy revealed a dramatic alignment of microtubules as the
cells changed shape (Gibbons et aL, 1969). More recently,
Katow and Solursh (1979) have studied the ultrastructure of these movements, and described the fibrous
nature of blastocoelic material that may serve as a
substrate for cell movement. These descriptive studies
form the necessary framework for functional studies
on the basis of morphogenetic movements. This paper
examines several cell recognition properties to determine their contributions to mesenchyme cell ingression
and migration.
’ To whom all correspondence
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66
FINK AND MCCLAY
Sea Urchin
Our working hypothesis is based on a model proposed
by Gustafson and Wolpert (1967) which suggested that
changes in cell-cell and cell-matrix
adhesions are
components of the morphogenetic movements of primary mesenchyme cells. To test this hypothesis we
have used a sensitive cell binding assay to measure
the affinity of sea urchin primary mesenchyme cells
for other embryonic components. We have previously
reported a developmentally regulated loss in affinity
for hyalin by postingressive mesenchyme cells at a
time corresponding to their normal release from the
hyaline layer (McClay and Fink, 1982). This report
shows that there are additional developmentally regulated changes in the adhesive behavior of mesenchyme
cells with nonmesodermal cells and with isolated basal
laminar components.
MATERIALS
AND
METHODS
Animals and Gametes
Arbaha punctulata were obtained from the Duke
University Marine Laboratory and Strongylocentrotus
purpuratus were purchased from West Coast Suppliers.
Gametes were shed by injection with 0.5 M KCl. Eggs
were fertilized with dilute sperm suspensions, and
fertilization membranes removed with P-aminobenzoic
acid (Paba, 10 mM) (McClay and Fink, 1982). Embryos
were grown in seawater filtered through a 0.45-pm
pore-size filter (Millipore) (MSW) containing penicillin
(l-2 pg/ml) and streptomycin sulfate (l-2 pg/ml)
(Sigma), or in calcium-free seawater (CF) through the
16-cell stage, at 15°C (Strongylocentrotus) or at 2224°C (Arbacia).
Cell Culture and Dissociation
Mesenchymal cells were isolated by two methods. In
order to obtain mesenchyme cells prior to complete
ingression, 16-cell-stage micromeres were isolated after
the method of Okazaki (1975). Briefly, fertilized eggs
were placed into CF after sperm addition and fertilization membrane removal. The embryos were grown to
the 16-cell stage in CF, then they were gently dissociated by swirling and pipetting. The dissociated embryos were layered on a discontinuous sucrose gradient
(4%,8% in CF), and the micromeres collected from the
4-8% interface. Micromeres were grown in MSW supplemented with 3% horse serum and antibiotics. Mesenchymal differentiation was monitored microscopically, and ingressive stage and wandering stage cultured
mesenchyme cells were harvested at the appropriate
times by gentle pipetting. Alternatively, mesenchyme
cells were isolated from mesenchyme blastula stage
embryos after the methods of McClay and Marchase
(1979) and Harkey and Whiteley (1981). This isolation
Cell Recognition
Changes
67
involves dissociating mesenchyme blastulae in a CF:
hyaline extraction medium (HEM) mixture, which
gently removes the noningressed blastomeres. The resulting basal laminar bags contain isolated primary
mesenchyme cells. These are gently sheared and the
cells collected by centrifugation.
Embryos at the blastula stage and beyond were
dissociated by treatment for l-5 min with HEM: 0.3
M glycine, 0.3 M NaCl, 10 mM KCl, 10 mM MgS04, 10
mM Tris, 2 mM EGTA (ethylene glycol bis@aminoethyl ether)-NJ’-tetraacetic
acid), pH 8.0 (McClay and
Fink, 1982). The hyaline layer was solubilized by HEM
and the embryos were easily dissociated by gentle
pipetting. Single-cell suspensions were separated from
undissociated clumps by pouring through 28-pm Nitex
mesh (Tetko). Cell suspensions were washed with MSW
and were found to exclude trypan blue (greater than
90% of a suspension) and, as a second test of cell
viability, the cells aggregated well in rotary culture.
Ectoderm cells were separated from endoderm cells by
procedures described previously (McClay and Marchase,
1979).
Basal Lamina Isolation
Basal lamina components were isolated as intact
basal laminar bags from embryos at the midblastula
stage and beyond. Embryos were lysed in 10 mM
NaHC03, 0.01% Triton X-100 (Wessel et aL, 1984). Bags
were collected and washed by centrifugation, and then
treated with DNase (30 pg/ml) (Sigma) to remove
trapped nuclei. Alternatively, embryos were dissociated
with HEM and the basal lamina bags collected by
gentle centrifugation (McClay and Marchase, 1979;
Harkey and Whiteley, 1981). In both cases the isolated
bags were sheared into fragments. Several experiments
used the following basal laminar components: fibronectin (Sigma), laminin (gift of H. Kleinman), collagen
(mixture of types I-IV, Sigma), chondroitin sulfate
(Sigma), and hyaluronic acid (Sigma).
Cell-Binding Assay
The cell-binding assay used has been described previously (McClay et al. 1981; McClay and Fink, 1982).
Briefly, proteins or cell monolayers are adsorbed onto
the bottom of polyvinyl chloride flat-well microtiter
plates. Aliquots of radiolabeled probe cells are then
added to each well and the plates sealed. The probe
cells are gently centrifuged into contact with the
substrate. The microtiter plates are then inverted, and
can be centrifuged in this inverted position to add a
dislodgement force of greater than lg. The microtiter
plates are then quick-frozen at -7O”C, and the bottoms
of the wells (containing the substrate layer and any
68
DEVELOPMENTALBIOLOGY
radiolabeled probe cells that remained bound during
the treatment) are clipped off for liquid scintillation
counting.
Three different substrates were used in the binding
assay. (1) Hyalin, the major protein component of the
hyaline layer, was purified from early cleavage stage
embryos (McClay and Fink, 1982) and directly adsorbed
onto the microtiter wells (lo-20 pg/well). (2) Fragments
of complete basal laminae were centrifuged onto the
microtiter wells (approximately 30 pg protein/well).
Purified components of basal lamina were adsorbed
directly onto the microtiter wells (lo-20 pg/well). Protein content of the adsorbed substrates was measured
by a miniaturized Bradford assay. (3) Cell monolayers
were generated by treating the microtiter plates with
a 21 solution of poly+lysine (2 mg/ml): glutaraldehyde
(3% in MSW), followed by extensive washings with
MSW. An aliquot of a single-cell suspension was then
added to each well (9 X lo5 cells/well), and the microtiter plates were centrifuged at 200~ to force the cells
onto the bottom of the well. Confluency of monolayers
was determined by microscopic observation.
A series of controls on the bioassay showed that the
cells remained intact (there was no radioactive material
released into the supernatant, and the ratio between
lz51-surface labeled: 3H-internally labeled cells did not
change after cells were removed from the substrate).
Monolayers were not released from the wells as determined by initial controls in which the monolayer cells
were radiolabeled. The substrate itself could be blanked
by washing a well with 10% fetal calf serum in MSW
to assure that cells bound to monolayers or matrix
materials rather than any free surface that might
exist on the well bottom (8% background binding
resulted if cells were exposed to an entire well surface
treated with 10% fetal calf serum; these same cells
bound at a level of 95100% to wells treated with polyL-lysine alone).
Micromeres, cultured mesenchyme cells, and newly
isolated mesenchyme cells were radiolabeled for use
as probe cells in the binding assays. To radiolabel
micromeres, fertilized eggs were placed in [3H]leucine
(1 &i/ml) in CF until the 16-cell stage (approx 3 hr
for Arbacia, 5 hr for Strongylocentrotus). [3H&eucine
(1 &i/ml) was added to cultured mesenchyme cells or
to morula stage embryos 4 hr before use as probe cells.
Labeled cells were washed extensively in MSW, and
added to the microtiter wells on ice in a total volume
of 0.3 ml. The same number of cells (approx 105; O.Ol0.05 cpm/cell) were then added to each well and the
plates sealed. Probe cells were centrifuged onto the
substrates at lo-20g. After 15 min incubation on ice,
the plates were inverted and centrifuged while in the
inverted position to add a dislodging force greater
VOLUME107,1985
than lg to the cells (McClay et al., 1981). Controls each
day included blanked wells for which the cells had no
affinity (3-8% background binding).
To estimate the strength of adhesion, test cells were
centrifuged away from a substrate at varying centrifugal forces. A plot of percentage binding versus relative
centrifugal force (RCF) applied was obtained for each
cell type on each of the different substrates. These
graphs were used to estimate the centrifugal force
necessary to remove 50% of the test cells from the
substrate at the different developmental stages (McClay
et aL, 1981). This centrifugal dislodgement force (Fn)
was calculated from the equation
FD =
(pm11
-
Pmedium)
X
Ke11 X RCF
in which Peel1is the specific density of the cell (determined for echinoderm cells to be 1.256 g/cm3); Pmedium
is the specific density of the medium (MSW, 1.024
g/cm3); and V,,, is the cell volume. A diameter of 16
pm was used for l&cell-stage micromeres, and 10 pm
for wandering mesenchyme cells. This calculation considers size difference between cells of different ages.
In some cases, the maximum binding achieved at a
removal force of lg was lower than 50%. For these
interactions, FD was estimated for the lg binding
value.
Mono&ma1 Immunojluorescence
Several primary mesenchyme cell-specific monoclonal
antibodies have been generated (McClay et aL, 1983)
and were used in this study. The monoclonal antibodies
were raised against Lytechinus variegates gastrula
membranes (McClay et aL, 1983; Wessel et aL, 1984).
These antibodies were used in indirect immunofluorescent assays on fixed embryonic tissues. To determine
the staining pattern in whole embryos, embryos were
fixed in Bouin’s fixative, dehydrated in an ethanol
series, embedded in paraffin, and serially sectioned at
5 pm (Wessel et ah, 1984). The mounted sections were
incubated with monoclonal supernatant in a humid
chamber. The sections were rinsed in phosphate-buffered saline containing 0.05% Tween-20 (PBS-Tw20),
and incubated with fluorescein isothiocyanate (FIT(?)conjugated rabbit anti-mouse Ig. The sections were
then rinsed with PBS-Tw20, converslips were added,
and they were viewed under a Leitz fluorescent microscope equipped with epiluminescence. For cultured
mesenchyme cells, cells were raised on glass coverslips.
At the appropriate time the cells were fixed in cold
acetone for 10 min, and air dried. Cells were then
stained with the same series of monoclonal antibody
followed by FITC-conjugated secondary antibody as
described above, and viewed under fluorescent light.
FINK
AND MCCLAY
Sea Urchin Cell Recognition Changes
Controls included sections without the monoclonal but
with the secondary antibody, substitution of myeloma
supernatant for antibody, and omitting the FITC secondary antibody to screen for autofluorescence. Each
of these was negative. A positive control using a
polyclonal anti-sea urchin serum and other monoclonals
with different specificities (McClay et a& 1983) was
included in most experiments to assure specificity of
the mesenchyme antibody.
RESULTS
Development
of Micromeres
in Culture
Cultured cells were used in the cell binding experiments to identify and determine the timing of changes
in cell recognition properties. In order to use such cells
it was necessary to establish the degree to which they
mimicked growth and differentiation in vivo. Sixteencell-stage micromeres were isolated and raised in culture, and exhibited the same morphological and behavioral patterns seen and previously described for cells
in vivo (Okazaki, 1975). Newly isolated micromeres
underwent a number of divisions, forming small clusters of cells. At the precise time of mesenchyme
69
ingression in control embryos, cultured mesenchyme
cells began pulsatory movements, extended transitory
pseudopodial cell processes, and became motile on the
culture dish. At the time corresponding to midgastrula
stage, cultured mesenchyme cells sent out filopodia
which overlapped and fused to form syncytial spicule
envelopes, within which mineralization occurred. Small
CaC03 crystal rudiments appeared, and the spicule
grew in vitro as the mesenchyme cells moved away
from the initial crystal, causing elongation. Thus, cells
cultured in vitro exhibited the same behavioral changes
as the primary mesenchyme cells in vivo.
To further assess the development of primary mesenchyme cells in vitro we examined the expression of
cell surface markers. Monoclonal antibodies against
primary mesenchyme cells were screened (McClay et
al, 1983; Wessel et al, 1984). In sectioned whole embryos, no fluorescent staining was seen until the mesenchyme blastula stage when primary mesenchyme
cells expressed the antigen (Fig. 1). As development
continued, the staining remained specific for primary
mesenchyme cells and for the envelope covering the
spicules they secreted.
When these antibodies were used to monitor cell
FIG. 1. Monoclonal
antibody staining in viwo and in vitro. Indirect immunofluorescence
using a monoclonal antibody specific for primary
mesenchyme cells shows that at the precise time of antigen appearance in viva, the cultured cells begin to show strong fluorescent staining.
At mesenchyme blastula stage, the antibody recognized mesenchyme cells that had completed ingression (a, b). This fluorescence remains
specific for primary mesenchyme cells and their products during development. At pluteus stage, the antigen binds to primary mesenchyme
cells and to the larval spicules (c, d). In vitro cells first show fluorescent staining when they become migratory
(e, f). This corresponds to
the mesenchyme blastula stage in viva. In the culture system, the antibody continues to recognize the mesenchyme cells and the envelope
surrounding the spicule (g, h). Scale bars: (a) 15 pm; (c) 20 pm; (e) 8 pm; (g) 12 pm.
70
DEVELOPMENTALBIOLOGY
surface antigens on cultured mesenchyme cells, the
cells became fluorescent as they became migratory. At
that time, which corresponds to ingression in viva,
cells began to react strongly with the antibodies (Fig.
1). Throughout spicule development in vitro, the staining pattern was identical to that in whole embryos.
The coincidental
appearance of these mesodermalspecific antigens on cultured mesenchyme cells suggests
that their cell surface is comparable to this cell type
in viva. Thus because the timing of behavioral events,
the antigen expression, and the morphological
changes
were similar, we felt we could use the cultured cells in
the cell-binding
assay with confidence of cell stage.
Measurement
of Cell-Cell and Cell-Substrate
A$inity
To quantify the adhesive interactions
exhibited by
the primary mesenchyme cells, binding assays were
performed that measured affinities between cells or
between cells and substrate materials (McClay et ak,
1981). The properties of the cell-binding
assay are
shown in Fig. 2. Labeled midgastrula
probe cells were
tested against gastrula monolayers using a series of
centrifugal
forces to separate probes from attached
monolayers. At 4°C the percentage binding versus
relative centrifugal
force profile shows a decline in
cell-to-cell binding over the range of centrifugal forces
applied. At low centrifugal
forces, 100% of the test
cells remain bound to the monolayers. A removal force
I
I
25
50
100
RCF
200
500
1000
FIG. 2. Percentage binding versus relative centrifugal
force (RCF).
Binding of dissociated gastrula cells to gastrula monolayers at two
different temperatures.
Percentage binding over a range of relative
centrifugal
removal forces was determined by the cell binding assay
(see Materials
and Methods). The 24°C binding profile represents
metabolically
stabilized cell-cell contacts (see text). Data reported
in this paper were obtained from assays performed at 4°C. The 4’C
RCF value giving 50% binding (2OOg) can be used to estimate a
median dislodgement force (McClay et aL, 1981).
VOLUME 107, 1985
of 2009 reduces the number of test cells bound to about
50%. By 10009 all but 20% of the test cells have been
removed. In contrast, when the probe cells and monolayers were incubated at 24°C for 15 min, the probe
cells were much harder to remove. One-hundred percent
of the probe cells remained bound to the monolayers
even at removal forces of 200g. Binding was only
reduced to 65% at the maximum
removal force of
10009. Thus even a short incubation at 24°C appears
to stabilize the adhesive interactions
between probe
and monolayer cells.
Cell-To-Cell Afinity
Changes
To examine the relationship
of developing mesenthyme cells with other cells of the embryo, confluent
monolayers of dissociated early gastrula cells were
used as substrates in a series of assays. Radiolabeled
mesenchyme probe cells were added to monolayercovered microtiter
wells at 4°C and centrifuged
into
contact with the substrate at log. The plates were
inverted and centrifuged in this inverted position at a
relative centrifugal
force of 50g. Table 1 shows that
prior to mesenchyme cell ingression, newly isolated or
cultured micromeres had an affinity for gastrula cells.
This affinity was reduced in migratory-stage
mesenthyme cells. The decrease in cell-to-cell
affinity was
exhibited by postingressive
mesenchyme cells of both
Arbacia and Str~ylocentrotus.
By contrast, when other
cells of the embryo were tested against the same
gastrula monolayers, a high degree of binding was
measured. All through gastrulation
this affinity was
retained both by isolated ectoderm and by endodermal
cells. Thus only descendents of micromeres lost an
affinity for other cells and this loss occurred at the
mesenchyme blastula stage.
From profiles of binding versus centrifugal
force
applied, Table 3 shows that 16-cell-stage micromeres
resisted a removal force of about 7 X 10m5dyn per cell.
Migratory-stage
mesenchyme cells lost the ability to
bind and the force dropped to less than 10m7dyn per
cell (the median number of cells bound at lg was less
than 50%). The calculated binding affinity for gastrula
cells (ectoderm and endoderm) remained unchanged
over this same developmental interval.
The Interaction between Cells and the Basal Lamina
The basal lamina appears during cleavage as the
blastocoel forms (Wessel et al., 1984). Each blastomere
is in contact with the basal lamina which thickens and
gains structural integrity by the mesenchyme blastula
stage (Gibbons et aZ., 1969). Early mesodermal cells
are in association with the outer face of the basal
lamina. Upon ingression into the blastocoel, primary
mesenchyme cells establish close contact with the
FINK AND MCCLAY
Sea Urchin
TABLE 1
AFFINITY TO GASTRULA MONOLAYERS”
Percentage
Probe cell type
16-Cell-state micromeres
Migratory
mesenchymed
Uncultured mesenchymed
Dissociated blastulae
Dissociated gastrulae
Arbucia
82
22
27
66
86
i 11 (5)C
f 9 (4)
f 11 (2)
(1)
+ 7 (3)
bindingb
S. purpuratus
88 + 9 (3)
46 31 3 (2)
ND”
ND
ND
a Monolayers
made from single-cell suspensions generated upon
dissociation of early gastrulae (predominantly
ectoderm and endoderm
cells, with few mesodermal cells).
bBinding measured in microtiter
well assay (see Materials
and
Methods). A removal force of 50g was applied; assays were performed
at 4°C. Arbacia cells were tested against Arbacia monolayers;
S.
pzqwuratus cells against S. purpuratus monolayers.
‘Data presented are means + SD of binding assays performed on
different days. Numbers in parentheses are the number of experiments. Values for each experiment are the mean binding for at least
six replicates. Within an experiment, errors were 10% or less.
d Cells were isolated as 16-cell-stage micromeres and raised in
vitro, or isolated from mesenchyme blastulae (uncultured).
e Not determined.
inner face of the basal lamina and the extracellular
matrix which serve as a substrate for their subsequent
migrations (Okazaki, 1965). To determine whether
mesenchyme cells undergo affinity changes for the
basal lamina, preparations of basal lamina, isolated as
bags from late mesenchyme blastula stage embryos
and then fragmented, were bound as substrates to
microtiter wells of assay plates. Sixteen-cell-stage micromeres of both Arbacia and Strongylocentrotus exhibited some degree of binding to the basal laminar
fragments (Table 2). Arbacia micromeres that had
been cultured to the early blastula stage showed no
significant increase in binding affinity for basal laminae.
At the migratory stage, the strength of the measurable
binding affinity increased for the mesenchyme cells
that normally use the basal lamina as a migratory
substrate. Dissociated blastula cells and gastrula cells
(ectoderm and endoderm) both showed an affinity for
basal lamina that did not change with developmental
time. The binding strength of early micromeres was
calculated to be on the order of lo-? dyn per cell, and
for wandering mesenchyme cells it was about 10e5dyn
per cell (Table 3), representing a large increase in
binding affinity of wandering mesenchyme cells for the
basal lamina. The increase appears to occur very rapidly
in development; the duration of primary mesenchyme
cell ingression in vivo is 1 hr, and cells cultured to just
prior to ingression do not demonstrate this increased
affinity. Cells cultured to just after the ingression, or
primary mesenchyme cells isolated after ingression in
Cell Reco,qnitim
71
Changes
vivo both demonstrate the increased affinity for basal
lamina.
Characterization of sea urchin basal lamina has
identified several components that are cross reactive
with those of vertebrate basal laminae (Spiegel et al,
1980; Wessel et al, 1934). To determine whether the
measured increase in binding affinity for complete
basal lamina was due to a subset of these components,
a series of binding assays was performed. The binding
affinity of l&cell-stage micromeres and migratory
mesenchyme cells were similar for laminin, collagen,
chondroitin sulfate, and hyaluronic acid (Table 4).
When micromeres and wandering mesenchyme were
tested against fibronectin, however, the older cells had
a higher affinity for this component. This increased
affinity for fibronectin was exhibited by micromeres
cultured through the wandering mesenchyme cell stage
as well as mesenchyme cells newly isolated from postmesenchyme blastula stage embryos (Table 4). The
horse serum used for culture had no apparent effect
on the cultured cells because the affinity change correlated with time of mesenchyme blastula stage rather
than inclusion of, or length of time in the presence of,
serum.
Cell-Hgalin Binding Strength
In a previous study we examined the relationship of
mesenchyme cells to hyalin (McClay and Fink, 1982).
Sixteen-cell-stage micromeres and presumptive primary
mesenchyme cells showed a high degree of binding to
TABLE 2
AFFINITY TO BASAL LAMINA~
Percentage
Arbacia
Probe cell type
16-Cell-stage micromeres
Morula stage mesenchyme
Migratory
mesenchymed
Dissociated blastulae
Dissociated gastrulae
cellsd
19
23
55
23
20
+ 12 (4)c
(1)
+ 6 (3)
+ 4 (2)
-t 8 (2)
bindingb
S. purpuratus
20 + 9
ND”
58 + 10
47
52
(2)
(2)
(1)
(1)
a Substrates were complete, isolated basal laminae.
bBinding measured in microtiter
well assay (see Materials
and
Methods). A removal force of 50g was applied; assays were performed
at 4°C. A&a&a cells were tested against Adacia
basal lamina; S.
pqmratus
cells against S. purpratus
basal lamina.
‘Data presented are means f SD of binding assays performed on
different days. Numbers in parentheses are the number of experiments. Values for each experiment are the mean binding for at least
four, and usually six, replicates. Within an experiment, errors were
10% or less.
‘Cells were isolated as 16-cell-stage micromeres and raised in
culture to the morula stage (prior to ingression) or to the migratory
stage (after ingression was completed).
e Not determined.
72
DEVELOPMENTALBIOLOGY
TABLE 3
ESTIMATESOF BINDING
VOLUME107, 1985
STRENGTH”
Force* required to dislodge cells from:
Cell type
Hyalin
Monolayers
Basal lamina
16-Cell-stage micromeres
Migratory-stage mesenchyme cells
Gastrula ectoderm and endoderm
5.8 x 10-5
1.2 x 1o-7 (15%)
5.0 x 1o-6
6.8 x 1o-5
1.2 X 1O-7(46%)
5.0 x w5
4.8 x 1o-7 (20%)
1.5 x 1o-5
5.0 x 10-7
“As measured by a series of adhesion assays performed under different centrifugal removal forces. See Materials and Methods, and
Fig. 2.
*Dislodgement force, in dynes, is calculated for that centrifugal force that removes 50% of the test cells from the substrate.
‘Number in parentheses is the maximum binding achieved (when less than 50%), and the binding strength estimate, in dynes, is for that
value.
hyalin that was lost at the time of mesenchyme ingression. Table 3 shows that 16-cell-stage micromeres were
dislodged from hyalin at about 10e5 dyn per cell;
wandering mesenchyme cells were released at about
lo-’ dyn per cell.
DISCUSSION
The results of the binding studies show that primary
mesenchyme cells, at the mesenchyme blastula stage,
TABLE 4
AFFINITY
TO ISOLATED
Substrate”
Fibronectin
Laminin
Collagen
Hyaluronic acid
Chondroitin sulfate
BASAL
LAMINA
COMPONENTS
Probe cells*
Micromeres’
Cultured mesenchyme
Micromeres
Uncultured mesenchyme
Micromeres
Cultured mesenchyme
Micromeres
Cultured mesenchyme
Micromeres
Cultured mesenchyme
Micromeres
Cultured mesenchyme
% Binding”
20 t
72 *
18 +
55 2
26 k
31 +
28 It
31 +
41 +
34
46 f
55
9 (4)d
17 (3)
7 (2)
13 (3)
10 (3)
21 (2)
14 (3)
15 (2)
9 (2)
(1)
8 (2)
(1)
‘Sources and concentrations of substrates are described under
Materials and Methods.
bThree different probe cells were used: (1) 16-cell stage micromeres
(Micromeres), (2) mesenchyme cells isolated from mesenchyme blastula stage embryos (Uncultured mesenchyme), and (3) mesenchyme
cells that were isolated as micromeres and raised in culture (Cultured
mesenchyme).
“Binding measured in microtiter well assay (see Materials and
Methods section). A removal force of 5Og was applied; assays were
performed at 4°C.
‘Data presented are means k SD of binding assays performed on
different days. Numbers in parentheses are the number of experiments. Values for each experiment are the mean binding for six
replicates. Within an experiment, errors were 10% or less.
ePresence or absence of exogenous serum did not affect the results.
lose an affinity for hyalin and for other cells. At the
same time they gain a measurable affinity for basal
lamina that may be specific for fibronectin (Fig. 3).
Ectodermal and endodermal cells, under the same
conditions, showed no such developmentally regulated
changes in affinity (i.e., dissociated blastulae as early
nonmesenchymal test cells, and combined ectoderm
and endoderm from the gastrula stage, displayed no
changes in their affinities for hyalin, embryonic monolayers, or basal lamina). Thus the changes in initial
binding behavior appear to reflect specific affinity
changes of primary mesenchyme cells at the time of
ingression.
To measure the timing of the affinity changes two
cell populations were used. Micromeres were isolated
from the 16-cell stage and primary mesenchyme cells
were isolated from the mesenchyme blastula stage. To
examine the precision of the affinity changes we used
cultured micromeres. Several lines of evidence suggested that the cultured mesenchyme cells are comparable to those in whole embryos, and that the
cultured cells undergo the affinity changes just as
ingression occurs in vivo. First, the behavior and
FIG. 3. Changes in adhesive interactions with developmental time.
As presumptive primary mesenchyme cells (P) begin to ingress to
the interior of the embryo, they lose affinities for hyalin (H) and for
neighboring blastomeres, and gain an affinity for the basal lamina
(BL). Nonmesenchymal blastomeres (E) exhibit no such developmentally regulated changes in affinity.
FINK AND MCCLAY
Sea Urchin
timing of cultured mesenchyme cells paralleled normal
development. Second, monoclonal antibodies specific
for postingressive primary mesenchyme cells recognized
cultured cells at the precise stage this antigen was
expressed in vivo. Third, cells grown in vitro to the
primary mesenchyme cell stage had the same altered
affinity properties as the corresponding cells obtained
from whole embryos (Table 4; McClay and Fink, 1982).
Fourth, micromeres, in the presence or absence of
serum, or cells raised in culture (2 to 6 hr) in the
presence of serum for any time up to the ingression
event behaved in the same way as uncultured micromeres. This argues that the presence of serum in the
culture medium has no artifactual effect on the affinity
changes. Thus it would appear that cells grown in
culture accurately reflect the developmental changes
of their counterparts in vivo. We therefore conclude
that the affinity changes of primary mesenchyme cells
occur coincident with mesenchyme cell ingression.
The cell-binding assays in this study last about 10
min each and were carried out at 4°C in an effort to
restrict observations to initial binding events only.
Under these conditions other adhesion-associated
events (such as cell junction formation) probably do
not occur. Quantitation of the dislodgement forces
showed that cells had an affinity for one another in
the range of 10-5-10-’ dyn per cell or less. When cells
were allowed to warm to 24°C for even a brief time, a
dramatic increase in binding affinity was measured
(Fig. 2). This high level of binding probably represents
any of a number of stabilization events such as the
formation of cell junctions, cell shape change to increase
surface area of contact between cells, or secretion and
adhesion to extracellular matrix materials. Any of
these events might also participate in primary mesenchyme morphogenesis but in the present context
even the earliest events in the adhesive process reflect
potentially important morphogenetic properties.
In the binding studies cell orientation cannot be
controlled when the cells settle into contact with the
substrate. Thus, if blastomeres have only a portion of
their surface area that can bind to the substrate, such
blastomeres as a population would have a reduced
affinity for the substrate relative to blastomeres that
had binding components covering the entire surface.
It is possible, therefore, that some of the affinity
changes observed could be due to some kind of a mosaic
restriction of surface binding sites. The alternative to
this possibility is a change in the identity or quantity
of molecular components on the surface of the primary
mesenchyme cells at ingression. In either event, the
mesenchyme cells increase in their affinity for one
substrate while decreasing in their affinity for two
Cell Recognition
Changes
73
other substrates. Thus, at least two changes must occur
and these changes must be at the surface of the
primary mesenchyme cells since the experimental substrates used (hyalin, cell monolayers, and basal lamina)
were the same throughout.
The developmentally regulated changes in cell-cell
and cell-matrix binding reported here support the
Gustafson and Wolpert (1967) model of morphogenesis.
The sudden change in cell affinities observed here may
not be unique to sea urchin primary mesenchyme cells.
Other organisms, for example, display similar epithelial-mesenchymal morphogenetic shifts. In vertebrate
embryos neural crest cells break away from neighboring
cells in the neural folds as they attain the migratory
morphology of mesenchyme cells (Tosney, 1978). Also,
at gastrulation in the chick, mesenchyme cells break
away from the epiblast of the primitive streak and
undergo an epithelial-mesenchymal
change (Solursh
and Revel, 1978). For these cell types, and for others,
changes in cell affinities may occur at specific times to
represent a general mechanism that precedes or accompanies morphogenetic movements.
Mechanistically, movement of a cell on a substratum
can be modeled as the transient formation and breakage
of localized bonds. The initial affinities measured by
the binding assay may represent such bonds. If this
were the case then the observed specific increase in
binding to fibronectin may have importance for the
mechanism by which primary mesenchyme cells move
on the wall of the blastocoel. Several studies have
correlated fibronectin location with coincidental morphogenetic movement. In amphibian embryos, for example, fibronectin first appears on the roof of the
blastocoel along the pathway of mesoderm cell migration (Boucaut and Darribere, 1983; Lee et cd., 1984). In
the chick fibronectin has been detected along the path
of mesoderm migration in the primitive streak (Critchley et ah, 1979). Later in development fibronectin has
been observed in the pathway of neural crest migration
(Rovasio et al., 1983). In the sea urchin fibronectin has
been localized, by immunofluorescence, to the surface
of the blastocoel and to primary mesenchyme cells
(Katow et aZ., 1982; Wessel et aL, 1984). The demonstration of a developmentally regulated increase in
affinity for fibronectin thus lends further support to
the hypothesis that fibronectin serves as a substrate
for morphogenetic movements.
We thank G. Cannon for technical
assistance in monoclonal
antibody production. Laminin was the kind gift of H. Kleinman. We
thank M. Truschel, K. Lakoski, G. Wessel, and D. Papaj for critical
comments on the manuscript.
This work was supported by NIH
Grant HD14483 (D.R.M.) and a COCOS Foundation
Predoctoral
Training Grant in Morphology (R.D.F.).
74
DEVELOPMENTALBIOLOGY
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