DEVELOPMENTAL

BIOLOGY

Primary

143, 389-397

(1991)

Mesenchyme

Cell Migration Dermatan Sulfate

Requires a Chondroitin Proteoglycan

Sulfate/

Primary mesenchyme cell migration in the sea urchin embryo is inhihited by sulfate deprivation and exposure to exogenous $-D-xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary mesenchgmc cells of a very large, cell surface chondroitin sulfate/drrmatan sulfate proteoglpcan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective cells iv t~ifvo. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary mescnchgme cell migration since treatment of actively migrating cells with chondroitinh: 1991 Academic Press. Inc. ase ABC reversibly inhibited their migration i?/ c-itro.

that a dermatan sulfate proteoglycan (DSPG) isolated from bovine articular cartilage inhibited fibroblast attachment and spreading on fibronectin, while a chondroitin sulfate/keratan sulfate PG from the same source had no effect. CSPG metabolism has been correlated with active migration in two systems. After aortic endothelial cells have been grown to confluency, wounding of the layer stimulates cell migration. Kinsella and Wight (1986) reported that the profile of newly synthesized PGs changes from approximately 805% HSPG in postconfluent cultures to as much as 60% chondroitin sulfate/ dermatan sulfate proteoglycan (CS/DSPG) in wounded cultures, thus demonstrating an increase in CS/DSPG synthesis concomitant with cell migration. Funderburg and Markwald (1986), studying cardiac mesenchyme migration in collagen gels, found that HSPG remained pericellular, whereas a mesenchyme-specific CSPG was shed as a trail behind migrating cells. These results provide evidence that PGs are involved in mesenchyme cell migration. Overall, results from various cell lines have led to the suggestion that HSPG may be involved in cell attachment while CS/DSPG may promote cell detachment (Ruoslahti, 1988). The developing sea urchin embryo provides a morphologically simple model system for the study of mesenthyme cell migration. Early in development, a subpopulation of cells in the vegetal plate ingresses into the blastocoel as they undergo an epithelial-mesenchymal transition. These are the primary mesenchyme cells (PMCs), and following a lag period, they migrate along the basal lamina that lines the wall of the blastocoel. PMC migration irr ciao can be blocked by a number of environmental perturbations, including sulfate deprivation (Herbst, 1904; Karp and Solursh, 1974) and expo-

INTRODUCTION Morphogenesis in metazoans is punctuated by repeated episodes of mesenchymal cell migration through embryonic extracellular matrices. The regulation of these migrations within the embryo has proven difficult to elucidate, in large part due to the complexity of the migratory process, but also to the difficulty of obtaining adequate amounts of material for biochemical analysis. It is clear, however, from both in V~VOand in vitro studies that onset, maintenance, and termination of movement are dependent on the interactions between the plasma membrane of the migrating cell and the complex extracellular materials found throughout the embryo. Growing evidence from a number of vertebrate and invertebrate systems suggests that cell surface-associated proteoglycans (PGs) are present at sit.es of cellsubstratum attachment and may mediate or modulate cell-matrix interactions during migration. Observations by Culp and his colleagues on murine fibroblasts indicated that there is turnover of the proteoglycan constituents of dynamic cell-substratum attachment sites (Rollins and Culp, 1979; Lark and Culp, 1983). Other studies have shown that the addition of exogenous cartilage chondroitin sulfate proteoglycan (CSPG) not only inhibits cell attachment to fibronectin and collagen (Knox and Wells, 1979; Rich et (xl., 1981; Tsao and Eisenstein, 1981; Schmidt ct trl., 1987), but also migration by neural crest on fibronectin (Perris and Johansson, 1987; 1990). Additionally, the different glycosaminoglycan (GAG) constituents of PGs may have specific effects on cell behavior. Lewandowska et nl. (1987) have shown ’ Present address: versity of (‘alifornia,

department Berkeley,

of Molecular CA4 94720.

and Cell Biology.

IJni3x9

OOl”-1606/Yl Copyrigqht All rights

$3.00

CC’1991 by Academic Press. Inc. of reproduction in any form reserved.

390

DEVELOPMENTALBIOLOGY V0~~~~143,1991

sure to exogenous fi-D-xyloside (Akasaka et al., 1980; Solursh et ab, 1986), an inhibitor of normal proteoglycan biosynthesis. Although both of these treatments have morphological and biochemical effects on the extracellular matrix (ECM) of the blastocoel (Solursh and Katow, 1982; Solursh et al, 1986), they also cause defects at the cell surface that render PMCs nonmotile on blastocoelic ECM in vitro (Lane and Solursh, 1988). We previously reported that an extract prepared by treating normal PMCs with 1 M urea could restore migratory ability to sulfate-deprived or xyloside-treated PM&. The extract had activity across species lines but not across treatments [i.e., an extract prepared from sulfate-deprived PMC did not restore migratory ability to xyloside-treated cells (Lane and Solursh, 1988)]. Based on the results of the in vitro migration assays, we proposed that sulfate deprivation and exposure to exogenous xyloside affected a cell surface-associated PG that was synthesized by the PMC. In the present paper we report that the urea extract prepared from PMC contains a chondroitin sulfate/dermatan sulfate PG (CS/ DSPG) and present evidence that this component of the extract is required for primary mesenchyme cell migration. METHODS AND MATERIALS Embryo purpuratus

culture. Gametes from Strongylocentrotus or Lytechinus pictus adults were released by

intracoeloemic injection of 0.5 MKCl. Eggs were washed twice with artificial seawater (ASW), fertilized with a dilute sperm suspension, and cultured at 15°C (S. purpuratus) or 17°C (L. pictus) until the mesenchyme blastula stage. To generate migration-defective embryos, eggs were fertilized and cultured in sulfate-free seawater (SFSW) or in seawater containing 2.5-4 mMp-nitrophenyl-fl-D-xylopyranoside (XSW) as required. Radiolabeling. Embryos to be labeled with 35S0, were cultured in either reduced sulfate seawater (RSSW, 1 ASW:SSFSW, v/v) or RSSW containing 2.5-4 mM xyloside until the hatched blastula stage. The embryos were transferred to fresh SFSW or SFSW plus xyloside and cultured for 2 hr. Two labeling protocols were utilized. In the first, intact embryos were cultured in SFSW containing 300-350 PCi Na,SO,/ml (sp act 325-425 mCi/ mmol, New England Nuclear) for 6-7 hr. Under these conditions, PMC migrated from the vegetal plate in the embryos cultured in SFSW plus 35S0,. These embryos were then dissociated and the 35S0,-labeled blastocoelic ECM was collected. The cells were separated and extracted with urea (see below). Alternatively, mesenthyme blastulae were dissociated, separated into epithelial and mesenchymal populations as described

previously (Lane and Solursh, 1988), and then labeled for 6-7 hr with 300-350 PCi 35S0,. To label with 3H-labeled amino acids, embryos were cultured in ASW until hatched blastula stage, transferred to fresh ASW containing 5 &i/ml 3H-labeled amino acids (New England Nuclear), and cultured until migrating mesenchyme blastula stage. Embryo dissociation, cell isolation, and urea extraction. Nonlabeled embryos were washed twice and la-

beled embryos were washed 5~ with sterile ASW and dissociated as previously described (Lane and Solursh, 1988). The mixed embryonic cells were separated into epithelial and primary mesenchymal populations by exploiting the selective attachment of PMC to Integrid tissue culture dishes (Falcon No. 3025) in the presence of 2% horse serum (Venkatasubramanian and Solursh, 1984). After 90 min, the unattached cells were poured off and retained as epithelial cells. The two cell populations were washed 5~ with ASW and subsequently extracted for 1 hr with 1 M urea (ultra pure, ICN Biochemicals) in 0.05 M Tris-buffered CFSW, pH 8.1. The urea extract was centrifuged (12OOg, 7 min) to remove the detached cells, transferred to dialysis tubing, concentrated 50x using solid pellets of polyethylene glycol, and dialyzed against 100 vol of dilute ASW (1 ASW:9 H,O, v/v) to remove the urea. The concentrated extract was precipitated with 7 vol of 95% EtOH containing 1% NaOAc and stored at -20°C. The extracted cells were solubilized in 3% SDS, 20 mMTris containing protease inhibitors (10 mM EDTA, 10 mM NEM, and 1 mM PMSF), pH 7.9, precipitated with EtOH plus NaOAc, and stored at -20°C. Electrophoresis and jluorography. SDS-PAGE analysis of the urea extracts followed the methods of Laemmli (1970). The polyacrylamide concentration was 6.3% for the separation and 3.0% for the stacking gel. Approximately 1.2 x lo3 cpm of the EtOH precipitates was loaded in each lane. Gels were electrophoresed and stained with silver (Bio-Rad) before being embedded in salicylate for fluorography. Agarose-acrylamide composite gel electrophoresis was performed as described by Carney et al. (1986). Each lane was loaded with 1.5 x lo3 cpm. After fixation, the gels were embedded in PPO in EtOH (Carney et al., 1986), dried, and exposed to Kodak X-Omat-AR film with intensifying screens (Cronex Lightning Plus, DuPont) at -80°C. Enzymatic digestion. The EtOH precipitates of 35S0,labeled samples were centrifuged (lO,OOOg, 10 min, 4°C) and then resuspended in 0.1 M Tris, 30 mM NaOAc, 10 mM EDTA, 10 mM NEM, 1 mM PMSF, pH 7.3, for 1 hr. Chondroitinase ABC or AC was then added to make 0.1 U/150 ~1, and the samples were digested at 37°C for 3 hr. The reaction was stopped by boiling for 2 min, and the

LANE AND SOLUR~H

Mesenchyme

samples were concentrated in a speedvac concentrator and analyzed by SDS-PAGE and fluorography. Ion-exchange chrom,atography. Freshly isolated urea extract was concentrated 10X and dialyzed exhaustively against 8 Murea, 0.05 MTris buffer, protease inhibitors, pH 7.5 (column buffer), at room temperature. A 50-~1 aliquot of the resulting solution was analyzed for radioactivity using Aquasol II (New England Nuclear) and a Beckman LS 3801 scintillation counter. Fifty thousand counts per minute was loaded onto a 2-ml DEAESephacel (Pharmacia) column equilibrated with the column buffer and eluted using a linear 0 to 1.0 M NaCl gradient. Fractions of 1 ml were collected and analyzed for radioactivity, and the salt concentration was determined by measuring the conductivity of each fraction. Partial purification of the urea extract was carried out by loading 200,000 cpm on a 7-ml DEAE-Sephacel column and eluting bound materials stepwise with 0.25, 0.55, and 1.5 M NaCl. After the addition of carrier GAG (chondroitin sulfates A and C, 50 pg each) to the samples, the fractions were concentrated with polyethylene glycol, dialyzed against column buffer without urea, concentrated using a speedvac concentrator, and stored at -20°C. In vitro migration assay. Urea extracts prepared from normal PMC were fractionated stepwise on DEAE-Sephacel as described above. Fractions were concentrated and dialyzed against SFSW or XSW, depending on the cell type to be tested. PMC isolated from sulfate-deprived or xyloside-treated embryos were exposed to fractions and tested for the restoration of migratory ability as described previously (Lane and Solursh, 1988), except that cell behavior was scored as “net movement less than or equal to one cell diameter” or “net movement greater than one cell diameter.” The effect of chondroitinase ABC on cell migration was determined by treating cells migrating on fibronectin with the enzyme. The activity of chondroitinase ABC in ASW plus 0.05 M NaOAc, pH 8.1, 17°C was determined using chondroitin sulfate C (Sigma) as the standard and the method of Yamagata et al. (1968). Tissue culture dishes (Falcon No. 3001) were treated with human plasma fibronectin (BRL, 12 pg/ml CFSW) for 1 hr at 3’7°C. Excess solution was removed and the dish washed twice with CFSW. Isolated PMC were resuspended in CFSW, seeded onto the dish, and allowed to attach. After 45 min, the unattached cells were removed by gentle washing. ASW plus NaOAc was then added, and the attached cells were monitored for migration by microcinematography as described above. After l-l.5 hr of migration, the medium was changed to ASW containing 0.1 U chondroitinase ABC (ICN Biochemicals) per 100 microliters ASW, 0.05 M NaOAc, pH 8.1, 17°C. The cells were again monitored for migration.

Cell

391

Migrntion

11697-

66-

1

45-

FIG. 1. %O,-labeled blastocoelic ECM and PMC urea extract, from normal and xyloside-treated embryos S. purpuratus, analyzed by SDS-PAGE and fluorography. The blastocoelic ECM contains a sulfated, high-molecular-weight species (asterisk, ecm) which is affected by 2.5 mM xyloside (x-ecm). A sulfated, high-molecular-weight species is also present in the PMC urea extract (asterisk, pmc-ue), and this molecule is sensitive to xyloside (x-pmc-ue). Sensitivity to xyloside suggests that the sulfated species are serine-xylose linked proteoglgcans.

RESULTS

Electrophoresis and Juorography. The content of the crude extract prepared from the surface of normal mesenchyme cells was examined by electrophoresis and fluorography. The 35S0,-radiolabeled, urea-extracted matePMC were compared rials from normal 5’. purpuratus with an extract prepared from xyloside-treated PMC and with normal and xyloside-treated blastocoelic ECM by SDS-PAGE. As shown in the fluorogram in Fig. 1. the ECM of the blastocoel contains a very large molecular weight species (marked with an asterisk in the ecm lane) that is affected by 2.5 mM xyloside (x-ecm). The materials extracted from the surface of PMC include an even larger sulfated species (pmc-ue), and the synthesis of this molecule is also inhibited by exogenous xyloside (x-pmc-ue). The difference in the relative mobilities of these sulfated, xyloside-sensitive molecular species

392

DEVELOPMENTAL

BIOLOGY

leads us to believe that there may be distinct blastocoelit matrix and mesenchymal surface PGs in the embryo at mesenchyme blastula stage. In the in vitro migration assay, materials extracted from the surface of PMCs supported the migration of sulfate-deprived and xyloside-treated mesenchyme cells, while an epithelial urea extract did not (Lane and Solursh, 1988). This raised the possibility that a mesenchymal surface PG might be structurally different from an epithelial surface PG. To determine whether there were distinct epithelial and mesenchymal PGs, and whether these were cell surface-associated, 35S0,-labeled S. purpuratus PMC and epithelial urea extracts and SDS-solubilized, extracted cells were compared by SDS-PAGE and fluorography. A silver-stained gel is shown in Fig. 2A and the corresponding fluorogram in Fig. 2B. Silver staining reveals that there is little stainable protein in the urea extracts prepared from PMC (pmc-ue) and epithelial cells (ep-ue), and various protein assays (Lowrey, Pierce’s BCA, Bio-Rad) confirmed this finding. The extracted, SDS-solubilized epithelial and PMC cells are shown in lanes labeled ep-sds and pmc-sds, respectively. The fluorogram in Fig. 2B is more informative. The large, sulfated mesenchymal molecule in the stacking gel (pmc-ue), which does not stain with silver, appears to be quantitatively removed from the cells by urea extraction and is not seen in the extracted, solubilized PMC (pmc-sds). The epithelial cells also produce a large, sulfated species that is removed by 1 M urea (ep-ue), and this molecule is missing from extracted, solubilized epithelial cells (ep-sds). The synthesis of the epithelial molecule is sensitive to exogenous xyloside (not shown), and its relative mobility indicates that it is smaller than the mesenchymal species but comparable to the matrix species seen in Fig. 1. Matrix, epithelial, and PMC urea extracts isolated from embryos labeled with 3H- or 14C-labeled amino acids were analyzed by SDS-PAGE and fluorography (not shown). After long exposures, corresponding bands in the extracts and ECM were seen, demonstrating that peptide is present in the high-molecular-weight species. This result, combined with the high level of sulfate incorporation and the sensitivity to P-D-xyloside, suggests that the high-molecular-weight species are probably serinexylose-linked proteoglycan in nature. The effect of 4 mM /3-D-xyloside on the synthesis of the mesenchymal molecule was examined using agarose-acrylamide composite gel electrophoresis and fluorography. The urea extract isolated from normal 5’. purpuratus PMC includes a large, sulfated molecule (Fig. 3, lane a) that is not present in the extract prepared from xyloside-treated PMC (lane b). The presence of a single, discrete band indicates that the PG synthesized by the PMC is fairly homogeneous. Most of the sulfate in the

VOLUME

143,199l

205

116

45

A

B

FIG. 2. %O,-labeled epithelial and PMC urea extracts and SDSsoluhilized cells analyzed by SDS-PAGE (A) and fluorography (B) as described under Methods and Materials. In the silver-stained gel in panel A, the S. ~YW~WU~ZLS epithelial (ep-ue) and PMC (pmc-ue) urea extracts contain little protein. Following urea extraction, the cells were soluhilized in SDS, and many hands are apparent in the soluhilized epithelial (ep-sds) and PMCs (pmc-sds). The fluorogram from the gel in panel A is shown in panel B. A comparison of ep-ue with ep-sds and pmc-ue with pmc-sds reveals that 1 M urea quantitatively removes the high-molecular-weight, sulfated species seen in the stacking gel. Also, the proteoglycan removed from the PMC surface (pmc-ue) migrates more slowly than the epithelial species (ep-ue), suggesting that the two molecules are distinct.

xyloside urea extract migrates faster than the intact PG in lane a and the PG standard, RCAlDl, and probably represents free GAG chains. This result confirms the finding of Solursh et al. (1986) that the effect of exogenous xylosides on sea urchin embryos is to inhibit the synthesis of PGs by stimulating the synthesis of free GAGS. Chondroitinase digestions. The 35S04-labeled S. purpuratus PMC urea extract was subjected to chondroitinase AC and ABC digestion, and the sensitivity was monitored by SDS-PAGE and fluorography. Figure 4 shows that the xyloside-sensitive, high-molecular-weight proteoglycan in the PMC urea extract (pmc-ue) is partly sensitive to chondroitinase AC (ch.ABC) and highly

LANEANDSOLURSH

a 4

b

.

RCPG, slags * FIG. 3. 35S0,-laheled PMC urea extract from normal and xglosidetreated S. pcryrrotus cells, separated hy agarose-acrylamide composite gel clrctrophorrsis. Sulfated materials in the extract prepared from normal cells (lane a) migrate more slowly than the sulfated material isolated from PMC exposed to 4 rnbf xylosidc (lane b) and the proteoglycan standard, RCPG (rat chondrosareoma proteoglycan). These results suggest that the PMC urea extract contains a very large proteoglycan, whose synthesis is disrupted by exogenous xyloside. The sulfated materials in lane b probably represent free GAGS polymerized on the xyloside, which is known to serve as an acceptor in wrt,ehrate systems. The position of free chondroitin sulfate GAG standards (Sigma) is indicated on the left.

sensitive to chondroitinase ABC (ch.AC) digestion, indicating that the proteoglycan contains both chondroitin and dermatan sulfates. Iowexclttr~rge chromatography. To determine further the nature of the materials removed from the surface of cells were double laPMC by 1 M urea, S. purpurntus beled with 35S0, and 3H-laheled mixed amino acids, and the urea extract was fractionated on DEAE-Sephacel using a linear NaCl gradient. The elution profiles are shown in Fig. 5. 35S0, retained by the column elutes as two major peaks at 0.15 and 0.46 M NaCl. The incorporated amino acids eluted as one major peak at 0.15 M NaCl and several smaller peaks. Based on the DEAESephacel elution profile for incorporated 35S0,, nonlabeled PMC urea extract was fractionated on DEAESephacel by stepwise elution with 0.25, 0.55, and 1.5 M NaCl. These concentrations were chosen to separate the peaks of incorporated sulfate. Following concentration and dialysis into SFSW or XSW, these fractions were tested for the ability to restore migratory activity to defective cells in the i?r ?dtro migration assay as described previously (Lane and Solursh, 1988). The results of the recovery assays are given in Table 1. Only the material eluted between 0.25 and 0.55 M NaCl, which corresponds to one of the peaks of incorporated sulfate, promoted migration (36% migrated, as compared with 0%. in the low and high-salt fractions). The presence of

biological activity in this fraction supports the hypothesis that a proteoglycan in the urea extract plays a role in cell migration, since proteoglycans are typically eluted from DEAE-Sephacel by salt concentrations of 0.4 M or greater. 35S0,-labeled fracctions eluted stepwise from DEAESephacel and corresponding to the fractions tested in the i?z vitro migration assay were analyzed by SDSPAGE and fluorography. Figure 6 shows the results of electrophoresis, with the silver-stained gel in panel A and the corresponding fluorogram in panel B. The crude PMC urea extract (pmc-ue) contains a number of bands in the separating gel. Most, if not all, of these bands are present in the low salt cut (0.25 M NaCl) of the extract. The 0.55 M NaCl cut, which contained all of the biological activity, contains little stainable protein. The chondroitinase ABC digest of the 0.55 M NaCl cut stains darkly and fairly uniformly, which may be due to the crude enzyme preparation. The 1.5 M NaCl cut, which contains little stainable material, is shown in the last lane. The fluorographic results are depicted in panel 8.

11697-

45-

FIG. 4. Fluorogram of %W,-labeled PMC urea extract digested by chondroitinases. The high-molecular-Lveight proteoglycan (lane a) is digested hy chondroitinase ABC (lane b). Some of the high-molecularweight material is removed hg treatment with chondroitinase AC: (lane c). These results demonstrate that the PMC proteoglycan contains chondroitin and dormatan sulfates.

394

DEVELOPMENTAL

BIOLOGY

VOLUME

143,199l

ml FIG. 5. DEAE-Sephacel elution profile of %O, (-) and 3H-labeled amino acid (---) double-labeled PMC urea extract. The urea extract and column were prepared as described under Methods and Materials and eluted using a linear NaCl gradient ( * . e). Labeled amino acids eluted as a major peak at 0.15 M, whereas sulfated materials eluted as two major peaks at 0.15 and 0.46 M NaCl. Only the latter peak contained biological activity in the migration assay.

205

116 97

The sharp bands seen in the separating gel in the lane containing the crude PMC urea extract (pmc-ue) are present in the 0.25 M NaCl cut. The high-molecularweight species seenpreviously in the stacking gel is present predominantly in the 0.55 MNaCl cut. This cut also contains two broad smears in the separating gel, which were not seen in the crude extract, and may represent breakdown products of the high-molecular-weight species. As seen in the lane containing the chondroitinase ABC-digested 0.55 M cut, all three %O,-labeled bands present in the active fraction are digested by chondroitinase ABC. Some high-molecular-weight, sulfated material is present in the 1.5 MNaCl cut, although significantly less than in the 0.55 M NaCl cut. These results indicate that the active fraction isolated on DEAESephacel contains sulfated species of three distinctly

TABLE IN

1

PMC MIGRATION RECOVERY:EFFECTSOF FRACTIONATED PMC-UREA EXTRACTSEPARATEDONDEAE-SEPHACEL

VITRO

DEAE-Sephacel 0.25 0.55 1.50

M NaCl M NaCl M NaCl

fraction

7% Migrating” o.o* 36.0

o.ot

Nb 27 67 37

a Migration is defined as movement greater than one cell diameter. *Number of cells observed. * Using x2 and a two-way test of contingency, the difference between treatment with 0.25 and 0.55 M NaCl fractions is significant at P = 0.005. The number of trials was two. t Using x2 and a two-way test of contingency, the difference between treatment with the 1.00 and 0.55 M NaCl fractions is significant at P = 0.005. The number of trials was two.

66

45

* A

6

FIG. 6. SDSPAGE and fluorographic analysis of %O,-labeled pmc-ue fractionated on DEAE-Sephacel. The crude extract and the three fractions obtained by stepwise elution of the column with 0.25, 0.55, and 1.5 M NaCl are shown after electrophoresis by silver staining (A) and fluorography (B). The 0.25 MNaCl cut consists of a number of discrete bands, and some of these are sulfated. The 0.55 M NaCl cut, which displayed biological activity in the migration assay, contains material that stains with silver near the tops of the stacking and resolving gels. Three bands of sulfated material are present. There is high-molecular-weight material in the stacking gel, high-molecularweight material at the top of the resolving gel, and material between 90 and 200 kDa. All of the sulfated material is susceptible to chondroitinase ABC digestion. The two lower bands are not obvious in the crude extract and may represent breakdown products of the high-molecular-weight species. The two high-molecular-weight bands are present in the 1.5 M NaCl cut, but at greatly reduced levels.

different sizes, all of which are fairly large and all of which are sensitive to chondroitinase ABC. PMC. Finally, Chondroitinase treatment of migrating the effect of chondroitinase ABC was tested on L. pictus cells migrating in vitro. To avoid digestion of the substratum, cells were seeded onto fibronectin rather than blastocoelic ECM. Cells actively migrating on fibronectin in ASW plus NaOAc were treated with chondroitin-

TABLE2 EFFECTS

OF CHONDROITINASE

ABC

Treatment

ON IN

VITRO PMC

MIGRATION Nb

%a Migrating”

Controls + Chond’ase

ABC

(2 hr)

40.8 2x*

125 110

+ Chond’ase

ABC

(4 hr)

1.6

61

41.2t

51

Remove chond’ase ABC, recover (2 hr)

a Migration is defined as movement greater than one *Number of cells observed. * Using x2 and a two-way test of contingency, the tween enzyme-treated and control cells is significant at number of trials was five. t Using x2 and a two-way test of contingency, the tween enzyme-treated and recovered cells is significant The number of trials was two.

cell diameter. difference beP = 0.005. The difference beat P = 0.005.

ase ABC and the effects were monitored continuously by microcinematography. Cells continued to migrate for approximately 1.5 hr after the addition of the enzyme, when most migration had ceased (41% of control cells migrated, as opposed to 3% of treated cells; seeTable 2). After the cells had remained stationary for 1 hr, the enzyme was removed and the cells were returned to ASW. Within 1 hr, cells were again translocating on the fibronectin substrata (41% migrating). Control cells, which were treated identically except that no chondroitinase ABC was added, continued to migrate throughout the observation period (up to 6 hr). These results indicate that chondroitin and/or dermatan sulfate are required for PMC migration. DISCXJSSION

Previous results from testing xyloside-treated, sulfate-deprived, and normal PMC in a migration assay suggested a number of characteristics that could be used to identify the component of the crude urea extract that restored migratory ability to defective cells. The molecule should be reduced or absent from urea-extracted, solubilized cells; it should contain sulfate; and its synthesis should be altered by P-D-xylosides. Also, it may be produced only by the PMC and not by the epithelial cells, since a urea extract prepared from epithelial cells did not promote migration in vitro. Finally, if the active component corresponds to the 30-nm granules that Solursh and Katow (1982) reported were associated with the appendages of PMC in situ, the sulfated, xylosidesensitive component should have a very high molecular weight.

One component of the crude PMC urea extract met these criteria. Based on its incorporation of sulfate, and its sensitivities to xyloside and chondroitinases, the high-molecular-weight species has been identified as a CS/DSPG. Support for the identification of the mesenchymal CS/DSPG as the active component in the urea extract comes from two variations of the in vitro migration assay. When the urea extract was fractionated on DEAE-Sephacel, only the materials eluted between 0.25 and 0.55 M NaCl restored migratory capacity (Table 1). Since proteins and glycoproteins are normally eluted by 0.15 M NaCl, while proteoglycans are eluted by much higher salt concentrations, this supported the hypothesis (based on the xyloside sensitivity of the active component) that a proteoglycan in the urea extract was involved in cell migration. In addition, the reversible inhibition of PMC migration by chondroitinase ABC (Table 2) indicated that the proteoglycan should contain chondroitin sulfates, and the presence of these GAG chains is essential for the molecule’s biological activity. The effects of xyloside exposure and chondroitinase ABC digestion on in vitro migration lead us to believe that only an intact CS/DSPG proteoglycan functions in mediating or modulating mesenchyme cell migration. In vertebrate cells, xyloside treatment generally results in the synthesis and secretion of both the undersubstituted core protein and the free GAG chains. This also appears to be the case in the sea urchin, as xyloside treatment resulted in smaller sulfated species when the urea extract was analyzed on agarose-acrylamide gels (Fig. 3). The secretion of the undersubstituted core protein and free GAGS to the extracellular compartment in the embryo however does not support migration. Likewise, chondroitinase ABC treatment of PMC Irh vitro causes migratory cells to become reversibly stationary. This again indicates that the presence of the core protein is not sufficient for migration, and taken together, these results demonstrate that the GAG chains must be attached to the core protein for the proper functioning of the PG in migration. We attempted to determine the nature of the saccharide digestion products of the proteoglycan from L. pietus and S. purpurtrfus. Following pronase pretreatment and chondroitinase ABC or AC digestion as described by Solursh and Katow (1982, after Yamagata et ul., 1968), 35S0,-labeled digestion products were separated by descending paper chromatography (not shown). Labeled materials that comigrated with chondroitin 6-sulfate and chondroitin 4-sulfate were found in I,. picfus and also in S. ~ur~~)ur~fus,a result which was expected based on chondroitinase sensitivity. However, large amounts of label (-70%) remained at the origin, even after extended periods of digestion. Analysis of the structure of glycosaminoglycans from several marine invertebrates

396

DEVELOPMENTAL

BIOLOGY

has revealed that GAG composition may be more complex than that in vertebrates, with both highly branched structures and unusual sugar compositions being reported (Vieira and Mourao, 1988; Mourao and Perlin, 1987). The susceptibility of the synthesis of the PMC proteoglycan to exogenous xyloside suggests that the GAG linkage region is similar to the linkage region found in vertebrate proteoglycans. We suspect that the PMC proteoglycan may contain GAGS of unusual sugar composition or structure that resist complete degradation by commercially available chondroitinases. The exact nature of the GAGS remains to be elucidated. There are several mechanisms by which PGs associate with the plasma membrane (Hook et al., 1986; Ishihara et ab, 1987), including direct intercalation of the core protein in the phospholipid bilayer, peripheral association via either the core protein or a GAG chain with an integral membrane receptor, and covalent linkage through phosphatidylinositol with membrane lipid. The CS/DSPG present in the active fraction is removed from the PMC surface by treatment with 1 Murea in the presence or absence of several protease inhibitors (10 mM PMSF, 10 mM NEM, and 1 mM EDTA, not shown) and maintains its activity when added to migration-incompetent cells. This observation leads us to believe that the mesenchymal CS/DSPG can be neither an integral membrane nor a phosphatidylinositol-linked PG. It should now be possible to determine whether the PMC proteoglycan binds to the cell surface via the core protein or the GAGS, and ultimately what role the CS/ DSPG plays at the cell-matrix interface. It has been suggested that CS/DSPGs may promote cell migration in some systems by facilitating cell detachment from extracellular matrix (Kinsella and Wight, 1986; Ruoslahti, 1988), while in other systems CSPGs from a heterologous source have been reported to inhibit cell migration (Perris and Johansson, 1987, 1990). It is clear in the sea urchin that mesenchyme cell migration is dependent on an endogenous proteoglycan, and to our knowledge this is the only system in which the role of an endogenous CSPG in migration has been tested. We are currently investigating the morphological effects of the presence or absence of the cell surfaceassociated CS/DSPG on isolated PMC, as well as the effect of chondroitinase ABC. The primary mesenchyme cells should provide an ideal model system in which to study the role of cell surface proteoglycans in cell migration, including cell detachment. We thank Drs. Tamayuki Shinomura and Ahnders Franzen for their advice on proteoglycan biochemistry, Dr. Joseph Frankel for advice on statistical analysis, Dr. Jim J.-C. Lin for the use of equip merit, and the two reviewers for their criticisms and suggestions. This

VOLUME

143, 1991

research was supported by NIH Grants by the Graduate College of the University

HD

18577 and GM 07228 and of Iowa.

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