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INTRODUCTION
Extracellular matrix (ECM) plays important roles in develop-ment
not only by providing structural support to cells but alsoby
influencing such cellular processes as cell proliferation,
celldifferentiation, cell adhesion and cell migration (see review
byHay, 1991). In order to study the influences of ECM on
cellmigration under in vivo conditions, we have utilized the
freshwater invertebrate,
Hydra vulgaris as a model system foranalysis. Hydra was chosen
because of its simple bodystructure and its high capacity for
regeneration. Structurally,hydra is composed of a gastric tube with
a head at one poleand a foot process at the other pole. Its entire
body wall isformed by an epithelial bilayer with an intervening
ECMtermed the mesoglea (Campbell and Bode, 1983). Hydramesoglea is
known to contain the major ECM components (i.e.fibronectin,
laminin, type IV collagen and heparan sulfate pro-teoglycan) found
in vertebrate and more complex invertebratespecies (Sarras et al.,
1991a). Recent functional studies havedemonstrated that these ECM
components play an importantrole in hydra head regeneration (Sarras
et al., 1991b) and inhydra cell aggregate development (Sarras et
al., 1993).
The regional cell differentiation pattern of hydra isdependent
on migration of interstitial cells (I-cells). Under in
vitro conditions, Day and Lenhoff (1981) have demonstratedthat
hydra cells attach to and spread on isolated mesoglea.More
recently, Agosti and Stidwill (1991) have demonstratedthat hydra
nematocytes attach to and migrate on substratacoated with isolated
mesoglea or coated with purified ECMcomponents such as type IV
collagen and laminin. Hydranematocytes have also been shown to bind
to fibronectin in aRGD-dependent manner (Ziegler and Stidwill,
1992). Previousin vivo studies by Campbell and Marcum (1980) also
indicatedthat nematocytes migrate between ectodermal
epitheliomuscu-lar cells via cell-cell contact guidance mechanisms.
While allprevious in vivo studies have indicated that cell
migration inhydra depends on cell-cell interactions (Campbell
andMarcum, 1980) and chemotaxic gradients (Teragawa andBode, 1991),
we propose that cell-ECM interactions are alsocritical to this
process.
In order to determine if hydra cell-ECM interactions dooccur in
situ, we have developed an in vivo bioassay thatcombines a number
of previously published procedures. Oneprocedure involves the use
of hydra grafting techniques thatallow quantification of the
migration of I-cells from a donorhydra (basal half of graft) to a
host hydra (apical half of graft)(Teragawa and Bode, 1990, 1991).
This technique was thencombined with two other procedures. In one
case, hydra grafts
425Development 120, 425-432 (1994)Printed in Great Britain © The
Company of Biologists Limited 1994
Interstitial cell (I-cell) migration in hydra is essential
forestablishment of the regional cell differentiation pattern inthe
organism. All previous in vivo studies have indicatedthat cell
migration in hydra is a result of cell-cell interac-tions and
chemotaxic gradients. Recently, in vitro celladhesion studies
indicated that isolated nematocytes couldbind to substrata coated
with isolated hydra mesoglea,fibronectin and type IV collagen.
Under these conditions,nematocytes could be observed to migrate on
some of theseextracellular matrix components. By modifying
previouslydescribed hydra grafting techniques, two procedures
weredeveloped to test specifically the role of extracellular
matrixcomponents during in vivo I-cell migration in hydra. In
oneapproach, the extracellular matrix structure of the apicalhalf
of the hydra graft was perturbed using
β-aminopropi-onitrile and β-xyloside. In the second approach,
grafts were
treated with fibronectin, RGDS synthetic peptide andantibody to
fibronectin after grafting was performed. Inboth cases, I-cell
migration from the basal half to the apicalhalf of the grafts was
quantitatively analyzed. Statisticalanalysis indicated that
β-aminopropionitrile, fibronectin,RGDS synthetic peptide and
antibody to fibronectin allwere inhibitory to I-cell migration as
compared to theirrespective controls.
β-xyloside treatment had no effect on interstitial
cellmigration. These results indicate the potential importanceof
cell-extracellular matrix interactions during in vivo I-cell
migration in hydra.
Key words: Hydra, mesoglea, extracellular matrix,
fibronectin,collagen, proteoglycans, cell migration
SUMMARY
Cell-extracellular matrix interactions under in vivo conditions
during
interstitial cell migration in
Hydra vulgaris
Xiaoming Zhang and Michael P. Sarras Jr*
Department of Anatomy and Cell Biology, University of Kansas
Medical Center, 3901 Rainbow Blvd, Kansas City, Kansas 66160-7400,
USA
*Author for correspondence
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426
were made using animals in which the structural integrity ofthe
mesoglea was perturbed using drugs that interfere with thesynthesis
and processing of matrix components. In the secondcase, procedures
were employed that allow one to introducemacromolecules between the
epithelium and mesogleautilizing a dimethylsulfoxide (DMSO)-loading
procedure(Fraser et al., 1987). Passage of macromolecules
betweenepithelial cells in hydra is normally prevented by
septatejunctions (Wood and Kuda, 1980), but low levels of DMSOhave
been shown to temporarily open these junctions (HansBode, personal
communication). We utilized this DMSOloading procedure to introduce
macromolecules into hydragrafts that could potentially interfere
with normal cell-ECMinteractions (e.g. ECM components, synthetic
peptide, or anti-bodies to ECM components). Using these approaches,
thecurrent study therefore focused on the effect of alterations
inECM structure and the role of fibronectin on in vivo
I-cellmigration in hydra.
MATERIALS AND METHODS
Culture of animalsHydra vulgaris (previously named Hydra
attenuata) were used in allexperiments. Animals were cultured in
hydra medium (HM) as pre-viously described by Sarras et al.
(1991a).
Depletion of I-cells in hydra using hydroxyurea and use ofdrugs
to alter mesoglea structureTo deplete hydra of I-cells, similar
size hydra polyps (about 30 pergroup) were incubated in HM
containing 0.01 M hydroxyurea (HU;Sigma Chemical Company, St Louis,
MO) for 5 days with repeatedsolution changes. Animals were then
transferred into fresh HM(without HU) for 2 days to allow recovery
of the polyps from HUtreatment prior to use. Under these
conditions, the I-cell population isreduced by approximately 99% as
monitored by macerate analysis andas reported by Bode et al. (1976)
and Teragawa and Bode (1990). I-cell-depleted hydra were used as
host grafts (apical half of graft) inthe cell migration assay.
To disrupt mesoglea structure, two protocols were followed. In
thefirst, polyps were treated with either 0.05 mM
β-aminopropionitrile(β-APN) (Sigma) or 0.01 mM p-nitrophenyl
β-D-xylopyranoside (β-xyloside; Sigma) for 15 days. 0.01 M HU was
added to the abovesolutions at day 11. A 2-day recovery period in
HM was employedafter 15 days of drug and HU treatment before
grafting wasperformed. In the second protocol, although the length
of treatmenttime was the same as previously described, the sequence
of drugtreatment was reversed (i.e. polyps were first treated with
HU and thenwith β-APN or β-xyloside) before grafting was performed.
Previousbiochemical and autoradiography studies have shown that
mesogleastructure is altered under these conditions of β-APN and
β-xylosidetreatment (Sarras et al., 1991b). Two different sequences
of HU andβ-APN or β-xyloside treatment were followed because of the
concernthat initial treatment with the mesoglea perturbing drugs
would com-pletely inhibit cell migration, thereby preventing the
depletion of I-cells during HU treatment. As will be discussed in
the results section,this was not the case.
Labeling of I-cells with 5-bromo-2′-deoxyuridineThe labeling
procedure was carried out according to Teragawa andBode (1990,
1991). Briefly, animals were injected through thehypostome into the
gastric cavity with an aqueous solution contain-ing 1.0 mM
5-bromo-2′-deoxyuridine (BrdU; Sigma) and 1-2% Indiaink (Pelikan
C11/1431a, Bio/medical Specialties). As an analog of
thymidine, BrdU is incorporated into DNA during S-phase.
BrdU-labeled cells can be distinguished from non-BrdU cells with
immuno-histochemical staining using antibody against BrdU. In
hydra, bothectodermal and endodermal cells can be labeled after 12
hoursfollowing BrdU injection (Teragawa and Bode, 1990). India ink
canbe phagocytosed by endodermal epithelial cells and, thus, was
usedin our grafting experiments to distinguish the graft junction
linebetween BrdU-labeled tissues and those non-BrdU labeled. In
orderto reduce experimental variations, efforts were taken to
select similarsize polyps without buds for each experiment. Buds
were removedbefore grafting from any animal that entered the
budding process afterbeing selected and during pregrafting
treatment. Considering the S-phase in hydra interstitial cells is
12 hours (Campbell and David,1974), BrdU injections were carried
out 12 hours before grafting andrepeated 1 hour before grafting
began. These BrdU-injected animalswere used as I-cell donors (basal
half of graft) in hydra grafts.
Grafting proceduresThe grafting procedure is illustrated in Fig.
1. For these experiments,the division between the apical and the
basal halves is determined bythe boundary between the gastric
region and the budding region asdescribed by Campbell and Bode
(1983). The grafting techniques thatwe employed were based on the
procedures described by Teragawaand Bode (1990, 1991) with the
following modifications: (1) the basalhalf of a BrdU/ink-labeled
hydra polyp was always grafted to theapical half of a
non-BrdU/ink-labeled polyp that had been either HUor HU and drug
treated; (2) for any animals that grew buds during thepregrafting
treatment, all buds were removed before animals weregrafted; and
(3) grafted tissues were held together on a fishing linewith two
end pieces of parafilm for 2 hours before any furthertreatment was
performed.
Treatment of hydra grafts with reagentsHydra grafts were removed
from the fishing lines 2 hours aftergrafting and transferred into
microtiter plates (Nunc, Denmark) with1 graft per well. The
microtiter plates were chilled to 4°C by placingthem on an ice
water bath. The hydra medium in the wells wereremoved by aspiration
with a tuberculin syringe and reagent solutionscontaining 5% DMSO
were added into each well with 10 µl/well.DMSO was used to
introduce reagents between the epithelium andthe mesoglea because
it is able to open septate junctions betweenhydra epithelial cells
(Fraser et al., 1987; Hans Bode, personal com-munication). The
following reagents were tested in grafting treat-ments: bovine
serum albumin (BSA; Sigma) at 0.05 mg/ml,fibronectin (Collaborative
research Inc. Bedford, MS) at 0.05 mg/ml,GRGDSP (Gly, Arg, Gly,
Asp, Ser, Pro) and GRGESP (Gly, Arg,Gly, Glu, Ser, Pro) synthetic
peptides (Telios, San Diego, CA) at 0.5mg/ml, and non-immune rabbit
serum or polyclonal antibody againsthuman plasma fibronectin (ICN
Biomedicals) at 1:10 dilution. DMSOloading of reagents was carried
out on ice for 30 minutes before thesolutions were removed and
fresh non-DMSO-containing reagentsolutions were added to the wells.
This latter step was needed (1) toprevent any leakage of the loaded
reagents from the inter-epithelialspace before septate junctions
closed and (2) to dilute the residualDMSO in hydra body. Hydra
grafts remained on ice for additional 15minutes after DMSO
treatment to allow septate junction closure andrecovery. The
non-DMSO reagent solutions were changed one moretime to further
dilute residual DMSO and hydra grafts were left in thefinal change
of non-DMSO reagent solutions at 18°C until 24 hourshad elapsed
from the initial time of reagent loading.
Immunocytochemistry and quantitative analysis of in vivoI-cell
migrationAt 24 hours after grafting and reagent treatment, hydra
grafts wereprocessed for immunohistochemistry staining following
the proce-dures described by Teragawa and Bode (1990). Specimens
wereexamined with a bright-field light microscope (Nikon) fitted
with a
X. Zhang and M. P. Sarras
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427Effect of ECM on I-cell migration in hydra
camera lucida attachment. The junction line between the apical
andbasal half of the grafts was recognized by the ink labeling of
the basalgraft. Migration was considered if a BrdU-positive cell
appearedabove the junction line in the apical graft. Because the
body wall ofthe hydra body column is composed of an epithelial
bilayer, twoepithelial bilayers are compressed together in
whole-mount prepara-tions. By focusing through the two epithelial
bilayers in hydra whole-mount preparations, all BrdU-positive
I-cells in the apical graft wereindividually identified and traced
using the camera lucida drawingattachment. Cells in these camera
lucida drawings were then countedfor quantitative analysis. In
order to avoid experimental error createdby any ambiguity in the
ink line, the apical half was divided into tenequal regions. Any
BrdU-positive cells that appeared in the regionnext to the ink line
in the apical half were not counted as migratedcells. A minimum of
three grafts was used per parameter in eachexperiment and all
experiments were repeated at least three times. Thetotal number of
BrdU-positive cells within each apical graft wascounted and these
numbers were normalized with each experiment aspercent of control.
Only the percent of control was used when datafrom different
experiments was combined for statistical analysis.Based on the
combined percent of control, either a student t-test or anANOVA
test was used to compare the control and experimentalgroups
depending on the number of groups analyzed. A P value ≤0.05was set
as the level of significant difference.
Ultrastructural analysis of DMSO-loaded hydraTo examine the
effect of DMSO loading on the attachment of epithe-lial cells to
the extracellular matrix, scanning (SEM) and transmission(TEM)
electron microscopy was performed. Hydra were DMSOloaded with BSA
or test molecules such as fibronectin and thenprocessed for SEM or
TEM at various time points (0-24 hours aftertreatment) as
previously described by Sarras et al. (1993). For SEManalysis,
hydra were freeze-fractured (Sarras et al., 1993) to allow
theepithelial bilayer to be observed in either a transverse or
coronalplane.
RESULTS
Perturbation of mesoglea collagen cross-linking byβ-APN
inhibited I-cell migrationThe hydra grafting method utilized in
this study is illustratedin Fig. 1. With this method, BrdU-positive
cells appearing inthe host hydra (apical half of the grafts) are
solely due tomigration of cells from the donor hydra (basal half of
the graft).Illustrations of these grafts and the appearance of
migratedcells in the apical half of the grafts is shown in Figs. 2
and 3.The total number of BrdU-positive cells within the apical
halfwas used for quantitative analysis of cell migration. Under
theBrdU-labeling conditions used, the present study focused on
I-cell migration. As shown in Fig. 4, I-cell migration was
sig-nificantly reduced in host (apical) grafts treated with
β-APN.Because β-APN treatment reduced but did not totally inhibit
I-cell migration, the order in which hydra were treated with HUand
β-APN did not significantly alter the final results. Incontrast, no
significant differences were observed betweencontrol and
β-xyloside-treated groups. These results indicatedthe relative
importance of collagen cross linking versus pro-teoglycan
processing during I-cell migration in hydra.
In addition to the total number of migrated cells, the
distanceof cell migration was also analyzed. Apical grafts were
dividedinto four zones longitudinally and BrdU-positive cells
withineach zone were counted and plotted. To determine if the
absolute distance of cell migration was affected, migrated
cellswithin each specific zone were compared between control
andexperimental groups. The results indicated that, although
thetotal number of migrated cells was reduced in the β-APN-treated
apical half, cells were still able to migrate to all fourregions
(most proximal to most distal). Therefore, β-APNtreatment did not
reduce the maximal distance cells could beseen to migrate, but it
did reduce the number of cells that couldeffectively migrate.
Fibronectin, antibody to fibronectin and RGDSsynthetic peptide
inhibited I-cell migration in hydragraftsBrief exposure to DMSO has
been shown to open the septatejunctions temporarily between hydra
epithelial cells (Fraser etal., 1987; Hans Bode, personal
communication). Thisprocedure was used in the present study to
introduce macro-molecules between epithelial cells so that their
effect on in vivoI-cell migration could be analyzed. As shown in
Fig. 5, I-cellmigration was inhibited in hydra grafts treated with
fibronectin,RGD peptide and antibody to fibronectin as compared to
their
A
B
A
B
BrdU treated
Graft
HU or drugtreated
DMSO loadreagents
Localize withantiBrdU andanalyze withcameralucida
Fig. 1. Illustration of the in vivo I-cell migration assay
whichinvolves hydra grafting techniques. Group A hydra were treated
with0.01 M hydroxyurea (HU) for 5 days to eliminate their
I-cellpopulation. A subgroup of hydra polyps were double treated
with0.05 mM β-APN or 0.01 mM β-xyloside for 15 days to perturb
theirmesoglea structure and then with HU for 5 days. A second
subgroupof hydra were treated with this order reversed (i.e. 5 days
HUtreatment followed by 15 days of β-APN or β-xyloside
treatment).Group B hydra were labeled with 1 mM BrdU containing 1%
Indiaink at 12 hours and 1 hour prior to grafting. The basal half
of BrdU-labeled polyps was always grafted with the apical half of
HU-treatedor double-treated polyps and the grafts were kept in
hydra mediumfor 24 hours. The determination of the cutting line
separating apicaland basal grafts is described in ‘Materials and
Methods’. In aseparate set of experiments, non-β-APN- or
non-β-xyloside-treatedhydra grafts were treated on ice for 30
minutes with reagents(fibronectin, synthetic peptides or
antibodies) containing 5% DMSOat 2 hours after grafting. After
washes in DMSO-free reagentsolutions, hydra grafts were incubated a
total of 24 hours in eachreagent solution. All grafts were
processed for immunocytochemicalstaining using BrdU antibody and
viewed with a light microscopecontaining a camera lucida
attachment.
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428
respective controls (BSA, RGE peptide andnon-immune serum).
Attachment of epithelial cells to theextracellular matrix was
not disruptedwith the drugs or reagents used inthese studiesTo
determine if the inhibition of I-cellmigration could be a secondary
effect resultingfrom a disruption of attachment of epithelialcells
to the mesoglea, ultrastructural studieswere performed. It was
previously shown thattreatment of hydra with β-APN or
β-xylosidealtered the structure of hydra extracellularmatrix
(mesoglea) but did not disrupt theattachment of epithelial cells to
the mesoglea.This analysis was expanded in the currentstudy to
evaluate the effect of the DMSO-loading procedure on the attachment
of epithe-lial cells to the mesoglea. As shown in Fig. 6,at the
concentrations used in these studies,DMSO loading of molecules such
asfibronectin (0.05 mg/ml) did not cause a dis-ruption of the
attachment of epithelial cells tothe extracellular matrix as
evaluated by SEMor TEM analysis.
DISCUSSION
Among all hydra cell types, interstitial cells arethe fastest
dividing subgroup of cells with a S-phase of about 12 hours
(Campbell and David,1974). This feature gave us the
opportunityspecifically to analyze I-cell migration withinhydra
grafts. Previous studies have shown thattreatment of hydra with HU
for 5 days depletesthe majority of their I-cell population (Bode
etal., 1976) and, therefore, when the apicalhalves of these hydra
are grafted to the basalhalves of normal hydra, an I-cell
populationgradient is created. This gradient results in ahigher
number of cells migrating from basalhalf to the apical half as
compared to hydragrafts formed between non-HU-treated
polyps(Teragawa and Bode, 1990). The combinationof this HU-induced
cell migration pattern andthe BrdU labeling of I-cells granted us
anunique model system to determine if perturba-tion of ECM
structure could affect I-cellmigration. It should be noted that
while othercell types do migrate in hydra (e.g. nemato-cytes), the
current study only focused on I-cellmigration because of the
techniques employed.
In regard to our pharmacological studies, β-APN has been shown
to interfere with collagencross linking by inhibiting lysyl oxidase
(Pageand Benditt, 1972; Wilmarth and Froines,1992). Previous
studies have established thatβ-APN does affect mesoglea structure
(Sarraset al., 1993) and collagen cross linking in hydra
X. Zhang and M. P. Sarras
Fig. 2. Whole-mount preparations of control (A,C,D) and
experimental (B) graftsstained with antibody to BrdU are shown. An
experimental graft DMSO-loaded withfibronectin (0.05 mg/ml) is
shown in B while the control shown represents a graftedDMSO-loaded
with BSA at the same concentration. At low magnification, the
juncturepoint between the apical (the host half for cell migration)
and basal (donor half whichwas injected with BrdU and ink-labelled)
halves of the graft are indicated by the arrows(shown in A and B).
BrdU-labelled nuclei of I-cells, which had migrated from the
basalto the apical half, are indicated by the arrowheads in A and
B. At higher magnification,BrdU-labelled I-cell nuclei in the
apical half could be distinguished from one another(C). These
I-cells reside in the epithelial bilayer of the tubular body column
of hydra.By focusing through the two epithelial bilayers, which are
compressed together inwhole-mount preparation, I-cells can be
identified and the total number that hadmigrated into the apical
half of the graft counted. As viewed at the periphery of thegraft
using phase-contrast microscopy (D), some I-cells (arrowheads) are
seen injuxtaposition to the extracellular matrix (arrows), which
appears as a clear line at thebase of the ectodermal cell layer in
these preparations. Bar for A and B, 125 µm. Barfor C and D, 25
µm.
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429Effect of ECM on I-cell migration in hydra
(Sarras et al., 1991b). In addition, previous in vitro studies
haveshown that, when used as a substratum, collagens
(especiallytype IV collagen) promote migration of various cell
types suchas bronchial epithelial cells (Rickard et al., 1993),
neural crestcells (Perris et al., 1991) and hydra nematocytes
(Agosti andStidwill, 1990). Under in vivo conditions, type I, III
and IVcollagen were shown to appear along the neural crest
migratorypathways during development of chick embryos (Perris et
al.,1991). This indicated a role for these ECM components
inmediating neural crest cell migration during embryonic
devel-opment. Therefore, in the current study, the reduction of
I-cell
migration in β-APN-treated grafts can be interpreted as
theresult of alterations in mesoglea structure related to the
per-turbation of collagen processing. β-xyloside has been shownto
interfere with the addition of glucosylaminoglycan (GAG)chains to
proteoglycan core proteins (Lelongt et al., 1988).When added to
culture medium, this reagent inhibited themigration of primary
mesenchyme cells in sea urchin embryos(Lane and Solursh, 1988).
While β-xyloside treatment doesinhibit hydra head regeneration
(Sarras et al., 1991b) and mor-phogenesis of hydra cell aggregates
(Sarras et al., 1993), itstreatment had no affect on I-cell
migration in hydra grafts. Wecan therefore eliminate alterations in
cell migratory patterns asthe basis for β-xyloside’s inhibitory
effect on general mor-phogenesis in hydra. Overall, our results
indicate that, in hydra,I-cell migration is more sensitive to
alterations in collagenstructure than alterations in proteoglycan
structure.
Under in vitro conditions, fibronectin has been shown topromote
cell adhesion and cell migration in neural crest cells(Perris et
al., 1989; Dufour et al., 1988), keratinocytes (Sarretet al., 1992)
and smooth muscle cells (Naito et al., 1992).Hydra nematocytes have
been shown to bind to a fibronectin-coated substratum and this
binding is known to be RGD
Fig. 3. Two sample camera lucida drawings of a control graft (A)
anda graft treated with β-APN (B). For quantitative analysis of
I-cellmigration, the apical half and the juncture point with the
basal half wasdrawn for each graft. The junction line between the
two graft halvescould be identified under the microscope by the ink
particle labeling ofthe basal half. This juncture point is
indicated by the arrowheads in thecamera lucida drawings shown in
this figure. The asterisk (*) indicatestentacles of the apical half
of the graft. Bar, 180 µm.
Treatment of apical halves
Analysis of total number of BrdU positive cellscounted within
the apical halves
Per
cent
of c
ontr
ol (
HU
gro
up)
Fig. 4. The effect of drugs that perturb ECM structure on in
vivo I-cell migration. Hydra polyps used for apical grafts were
treated witheither 0.05 mM β-APN or 0.01 mM β-xyloside for 15 days
and with0.01 M HU for 5 days (HU+β-APN or HU+β-XYL). For details,
see‘Materials and Methods’. Asterisks indicate groups
statisticallydifferent from controls with P≤0.05.
Per
cent
of c
ontr
ol g
roup
Per
cent
of c
ontr
ol g
roup
Analysis of total number of BrdU positive cellscounted within
the apical halves
Treatment of grafts (mg/ml)
Treatment of apical grafts (dilution)
Fig. 5. The effect of DMSO-loading with fibronectin (FN),
syntheticpeptides (RGD or RGE) and antibody to fibronectin
(Anti-FN) on I-cell migration. (A) FN and RGD peptide significantly
reduced I-cellmigration as compared to BSA or RGE controls. (B)
Anti-FN alsosignificantly inhibited I-cell migration as compared to
non-immuneserum (Non-Immune) controls. I-cell migration was
analyzed asdescribed in ‘Materials and Methods’ and in the figure
legend of Fig.3. Asterisks indicate groups significantly different
from controls(P≤0.05).
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430 X. Zhang and M. P. Sarras
Fig. 6. Ultrastructural analysis of the effect of the
DMSO-loading procedure on the attachment of epithelial cells to the
extracellular matrix inhydra. Control (A,C,E; BSA, 0.05 mg/ml) and
experimental (B,D,F; fibronectin, 0.05 mg/ml) specimens are shown.
As shown by SEM at low(A,B) and intermediate (C,D) magnification,
neither BSA or fibronectin at the concentrations used in this study
resulted in any apparentdisruption of the attachment of ectodermal
(Ec) or endodermal epithelial cells to the hydra extracellular
matrix (arrowheads indicate theattachment sites of epithelial cells
to the hydra mesoglea). The attachment of epithelial cells to the
extracellular matrix was confirmed by TEManalysis of BSA- (E) and
fibronectin- (F) treated specimens. The close association of the
epithelial plasma membrane to the mesoglea (M) isindicated by the
arrowheads in E and F. Bar for A and B, 10 µm. Bar for C and D, 2.5
µm. Bar for E and F, 233 nm.
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431Effect of ECM on I-cell migration in hydra
dependent (Ziegler and Stidwill, 1991). Although nematocytescan
bind to fibronectin, studies by Agosti and Stidwill (1990)have
shown that mature nematocytes migrate poorly on thisECM component.
The current study revealed that under in vivoconditions, I-cell
migration can be inhibited by macromole-cules that can compete with
cell-fibronectin interactions (e.g.intact fibronectin, RGD peptide
or antibody to fibronectin).
The inhibitory effect of fibronectin and antibody tofibronectin
on I-cell migration may seem contradictory. Anumber of
interpretations can be proposed for this result. Forexample, these
results could result from a competition betweenendogenous mesoglea
fibronectin and exogenously addedsoluble fibronectin. In this case,
it can be proposed that normalI-cell migration is dependent on the
interaction of cell surfacefibronectin receptors with fibronectin
molecules that areinsoluble and bound within the three-dimensional
structure ofthe hydra ECM. The presence of hydra cell surface
receptorsfor fibronectin is supported by in vitro cell adhesion
studies byZiegler and Stidwill (1992). Exogenously applied
solublefibronectin would compete for I-cell ECM receptors
andinterfere with normal contact guidance mechanism andtherefore
result in an inhibition of cell migration. In contrast,antibody to
fibronectin could mask endogenously boundfibronectin and thereby
interfere with the ability of I-cell ECMreceptors to bind to
fibronectin in the ECM. These proposalsare supported by the fact
that RGD peptide could also inhibitI-cell migration. The RGD amino
acid sequence is known tobe a cell binding domain for fibronectin
and can bind tointegrin receptors (see review by Akiyama et al.,
1990; Hynes,1992). When studied in an in vitro assay with isolated
hydranematocytes, this peptide was shown to inhibit
nematocytebinding to fibronectin (Ziegler and Stidwill, 1992).
Theseobservations can now be extended to the in vivo situation
inthe case of migrating I-cells. The inhibitory affect of
RGDpeptide on I-cell migration appeared to be specific since
theinactive peptide RGE had no effect on I-cell migration.
Although the total number of migrating cells is reduced
aftertreatment with β-APN, fibronectin, anti-fibronectin antibody
orRGDS synthetic peptides, the maximal distance of I-cellmigration,
however, was not affected. One explanation for thisresult is that
the migration of particular subpopulations of I-cells was
significantly inhibited by these reagents while othersubpopulations
of I-cells were only marginally affected or werenot affected at
all. While the immunocytochemical proceduresutilized in this study
present I-cells as a morphologicallyhomogeneous group, they are in
fact a heterogeneous cell pop-ulation composed of multipotent stem
cells and various celllineage precursor cells (Heimfeld and Bode,
1986a,b). Thesesubpopulations of cells could be selectively
sensitive to theperturbing reagents used due to the expression of
specific cellsurface receptors for ECM components.
Several mechanisms have been proposed to explain cellmigration
in hydra. These mechanisms include (1) the mechan-ical forces
resulting from tissue changes during contraction andexpansion of
polyps (Teragawa and Bode, 1990), (2) cell-cellinteractions
involved in contact guidance (Campbell andMarcum, 1980) and (3)
external cues such as chemotacticsignaling that may be related to
the head activator gradientalong the longitudinal axis of the
organism (Teragawa andBode, 1991). In addition to these proposed
mechanisms, ourdata indicate the potential role cell-ECM
interactions during in
vivo I-cell migration. The inhibition of I-cell
migrationobserved in this study could reflect a direct interaction
of I-cells with the ECM or could result from alterations in
thenormal attachment of epithelial cells with the ECM, which
thencauses a secondary inhibitory effect on I-cell migration. In
thelatter case, altered epithelial attachment to the ECM
wouldresult in a perturbation of cell-cell interactions and/or
chemo-tactic signaling systems that normally occur during
I-cellmigration. The ultrastructural analyses performed in this
studyhowever, indicate that, under the conditions used, no
disrup-tion in the attachment of epithelial cells with the
mesogleacould be observed. While this in itself does not
excludepotential secondary inhibitory effects, taken in concert,
all ofthe data presented in the current study is consistent with
adirect interaction of I-cells with ECM components. As a finalnote
regarding epithelial-ECM interactions in hydra, it shouldbe noted
that DMSO loading of higher concentrations offibronectin (e.g. 0.1
mg/ml) can cause a rapid dissociation ofhydra cells in the adult
organism (data not shown). Thissuggests that, while
epithelial-fibronectin interactions may bea component of
epithelial-ECM attachment in hydra, theseinteractions were not
affected by the concentration of reagentsused in the present study.
Although the exact mechanismsunderlying in vivo I-cell migration in
hydra are not yet clear,the current study and others do point to
the presence of specificECM cell surface receptors within the
different hydra celltypes. In this regard, Ziegler and Stidwill
(1992) have isolatedintegrin-like plasma membrane proteins from
nematocyteswith binding affinity for fibronectin. Further studies
will berequired to identify the full spectrum of ECM cell
surfacebinding proteins among the different hydra cell types and
todetermine their respective roles in the process of
patternformation in this organism.
The authors wish to thank Dr Hans R. Bode for his
continuoussupport and Drs Lynne Littlefield and Hiroshi Shimizi for
their helpand suggestions regarding hydra grafting and quantitative
analysis ofin vivo I-cell migration. The authors also wish to thank
the technicalsupport of Jacquelyn K. Huff in regard to the
ultrastructural studiespresented in this article. The studies
described in this article weresupported by funds provided by NIH
(RR06500) and the InternationalJuvenile Diabetes Foundation
Inc.
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(Accepted 6 November 1993)
X. Zhang and M. P. Sarras