Cell surface changes occurring during sea urchin embryonic development monitored by quantitative agglutination with plant lectins

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. AN rights of reproduction in any form reserved

Experimental Cell Research 84 (1974) 191-198

CELL SURFACE CHANGES OCCURRING EMBRYONIC

DEVELOPMENT

MONITORED

AGGLUTINATION SUSAN W. KRACH,l

A. GREEN,’

DURING

SEA URCHIN

BY QUANTITATIVE

WITH PLANT LECTINS G. L. NICOLSON2 and S. B. OPPENHEIMER’*

‘Department of Biology, California State University, Northridge, Northridge, Calif. 91324, and Vancer Council Laboratory, The Salk Institute for Biological Studies, San Diego, Calif. 92112, USA

SUMMARY Plant lectins have been widely used to investigate the nature and functional significance of carbohydrate-containing cell surface receptor sites on normal, embryonic and tumor cells. In the present study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicated that carbohydrate-containing lectin-binding sites change during sea urchin embryonic development. Sea urchin embryo cells are 26 +_5 % more agglutinable with Con A at early developmental stages (day 1) than at later ones with a marked decline in agglutinability between day 2 and 3. Agglutinability with Ricinus communis agglutinin (RCA) may also be greater in young embryos. Normal embryonic sea urchin cells are not agglutinated with wheat germ agglutinin (WGA) unless the cells are trypsinized. The results suggest that changes in the amount, mobility or distribution of Con A-binding (containing rx-n-mannose-like or cr-n-glucose-like residues) and RCA-binding (containing p-n-galactose or p-N-acetyl-ngalactosamine-like residues) receptor sites occur with differentiation in sea urchin embryos, while WGA-binding receptor sites (containing N-acetyl-D-glucosamine-like residues) are displayed in a state which precludes agglutination at all developmental stages studied. The results are discussed with respect to the cell types present during sea urchin embryo development and provide quantitative evidence for the contention that specific changes in carbohydrate-containing cell surface sites occur with differentiation and maturation.

Cell surface carbohydrates appear to play important roles in cellular adhesion (l-51 and other cell contact phenomena [Sll]. In recent years, the plant lectins have been used to investigate amount [9, 12-141,distribution [15, 16, 171 and mobility [18, 191 of sugarcontaining cell surface receptor sites on a wide variety of normal, embryonic and tumor cell types [6, 7, 9, 11-271. Over 800 plant species contain hemagglutinins, although only a small number of these lectins have been isolateLand purified [27]. Recently, KleinSchuster & Moscona [24] examined the agglutinability of embryonic and fetal chick * Reprint requests should be sent to Dr Oppenheimer. 13-741811

retina cells by concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA). They showed that ConA readily agglutinated retina cells from early embryos, but was less effective on cells from the later (fetal) stages.They also found that WGA did not agglutinate retina cells at any developmental stage (unless the cells were previously trypsinized), while RCA agglutinated cells of all developmental ages to the same degree. ConA is specific for receptor sites containing a-D-mannoselike or Cc-D-glucose-likeresidues [28], while WGA and RCA exhibit IV-acetyl-D-glucosamine and /?-D-galactose- (and ,&iV-acetylD-galactosamine)-like specificities, respecExptl CeN Res 84 (1974)

192 Krach et al. tively [29, 301. Therefore, Kleinschuster & Moscona’s studies provided evidence of changes in the display of specific carbohydrate-containing receptor sites on the surface of embryonic cells that correlated with differentiation and maturation. In the present study we present evidence for changes in carbohydrate-containing lectin-binding sites during sea urchin embryonic development using a quantitative assay for measuring agglutination [3 l] with Con A, WGA and RCA. Our results indicate that sea urchin embryonic cells are more agglutinable with Con A and possibly by RCA at early developmental stages compared with later ones. Agglutination with WGA was negligible for all developmental stages examined. This study provides quantitative evidence for the contention that specific changes in carbohydrate-containing cell surface sites occur during differentiation and maturation.

MATERIALS

AND METHODS

Reagents and media Concanavalin A (Con A), dithiothreitol (DTT, Cleland’s Reagent) and N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES buffer) were A grade and obtained from CalBiochem, San Diego, Calif. Deoxyribonuclease I (DNAse), 1 x crystallized and lyophilized was obtained from Sigma Chemical Co., St Louis, MO. Other materials were obtained from the indicated sources: pancreatic trypsin, 1 : 250, Difco Labs., Detroit, Mich.; Isoton, Coulter Electronics, Hialeah, Fla; Hanks balanced salts solution, GIBCo., Grand Island, N.Y. Calcium-magnesium-free sea water (CMF-SW) was prepared as follows: 27.0 g NaCl, 1.0 g Na,SO,, 0.8 g KCI, 0.18 g NaHCO, and 0.01 g phenol red were dissolved in 1 1 of distilled water.

Preparation of cell suspensions Gametes of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus were obtained as previously described [31]. Eggs were washed 3 x with millipore-filtered sea water (MF-SW), filtered through silk gauze and fertilized with freshly diluted sperm susp&sions. MF-SW containing excess sperm- was decanted, and the fertilized embryos were transferred to Petri plates with fresh MF-SW and maintained at 17°C. Exptl Cell Res 84 (1974)

9-h urchins: In order to obtain healthy, viable single cells from 9-h embryos. each 10 ml of sentlv packed eggs resuspended in 50 ml of MF-SW was combined with 50 ml of 0.02 M DTT in MF-SW (adjusted to pH 9.1). The suspension was slowly stirred for 4 min and added to 2 1 of 0.02 M Trisbuffered sea water (pH 8.0). The eggs were allowed to settle and washed again with 1 1 of Tris-buffered sea water. Each 10 ml of DTT-treated eggs was suspended in 75-100 ml of Tris-buffered (0.02 M, pH 8.0) MF-SW; 0.054.10 ml of undiluted semen was added and the suspension was slowly stirred. After 45 set fertilization was complete and the suspension was immediately added to 2 1 of MF-SW to urevent clumping of the newly fertilized zygotes. This procedure effectively prevented formation of fertilization membranes [32] and enabled procurement of healthy 9-h embryonic cells by the procedure which follows. Dissociation of 9-h embryos was accomnlished according to a modification of the method of Guidice [33]. Embryos were washed 3 x with CMF-SW, incubated with 1.0-1.5 ml 0.01 M EGTA in 0.02 M Tris CMF-SW (nH 8.0). and gentlv oioetted (IO x ) with a Pasteur pipette. ‘Ten mlCMF-SW was added to the suspension; the cells were centrifuged and resuspended in CMF-SW. Agglutination experiments (described below) were performed with these 9-h embryo cells. In all cases the rate and extent of agglutination with ConA, WGA and RCA was lower than that observed for l- and 2-day embryo cells. Since it was necessary to use DTT and EGTA to effectively obtain 9-h cells, and since these compounds could result in cell surface alterations, the results with 9-h cells cannot be properly compared with those from older embryos described below. One- to 7-day urchins: One- to 7-day-old sea urchin embryos were dissociated by washing (3 x ) in CMFSW and allowed to incubate in CMF-SW at 17°C until the embryos exhibited characteristic stickiness. The incubation time in CMF-SW required for proper dissociation varied directlv with embrvonic aee. The embryos were gently sedimented, resuspended-in l.O1.5 ml CMF-SW and eentlv oioetted (about 10 x ) to produce a suspension of ‘healthy’ (see viability criteria below) single cells.

Lectin preparations RCA was isolated from R. communis beans and prepared as previously described [30]. In brief, the beans were blended and extracted in phosphate buffer; agglutinins were precipitated with ammonium sulfate, dialysed and applied to an Agarose A-O.5 m (Biorad) column. After washing with nhosohate buffer. the agglutinins were eluted by either -D-galactose ‘or a linear gradient of lactose in phosphate buffer. When the linear lactose gradient was used, one peak was obtained on the affinity column. This peakontainti 2 agglutinins with approximate mol. wts of 120 000 and 60 000 with t!?-D-galactose-like and p-n-galactoselike plus b-N-acetyl-D-galactosamine-like specificities, respectively [30]. One ml aliquots of purified R. communis agglutinins were frozen and extensively dialysed in NaCl-sodium phosphate buffer imme-

Cell surface changes during development

193

diately prior to use. After separating the agglutinins on a 45 x 0.9 cm Biogel P-150 column, preliminary experiments suggest that the 60 000 mol. wt peak is most active in agglutinating sea urchin embryo cells. WGA was prepared according to the method of Burger & Goldberg [29]. In brief, wheat germ lipase was ground and homogenized in distilled water, immersed in a 63°C water bath for 15 min, centrifuged and filtered. The supematant was precipitated with ammonium sulfate at O”C, centrifuged and the pellet containing agglutinin activity was redissolved in distilled water. The lectin was extensively dialysed and centrifuged to remove any orecioitate which formed. The-dialysate was applied to- a Sephadex G-75 column and eluted with water. The peak of N-acetyl-o-glucosamine specific agglutinating activity representing purified 26 000 mol. wt WGA was frozen in 1 ml aliquots until just prior to use.

Agglutination

assay

Agglutination was measured using a auantitative and exiremely reliable agglutination assay [31]. Using a Model B Coulter Counter (Coulter Electronics. Hialeah, Fla) or a Model 112 LT Celloscope (Particle Data, Inc., Elmhurst, Ill.) agglutination was measured by the disappearance of single cells in a rotating suspension as described below. The settings which counted single cells and excluded most clumps and debris were as follows: 9 h urchin cells, l/amplification = 16 and l/aperture current = 1 with a window of 20-70 (Coulter Counter); I-7-day urchin cells, l/amplification = 4 and i/aperture current = 0.354 with a window of 20-90 (Coulter Counter); current = l/2 and gain = 48 with a window of 50-900 (Celloscope). Sarcoma 180 cells were counted with current = 1 and gain = 8 i/2 with a window of 100-500 (Celloscope). Cells were suspended in MF-SW (pH 7.8), CMFSW (pH 7.8) or Hepes buffered (pH 7.4) Hanks balanced salts solution (Sarcoma 180 cells) with or without varying concentrations of Con A,’ RCA or WGA. DNAase (10 ualml) was sometimes added to the suspensions, but had no effect upon agglutination. Aliquots (0.2 ml) were placed in 1 dram screw cap vials on a gyratory shaker with a 49 inch diameter of rotation and rotated at 68 rum and 17°C. At various time points, the vials were piaced on ice, diluted with 10 ml MF-SW (urchin cells) or Isoton (Sarcoma 180 cells) and counted with an electronic particle counter at the settings described above.

Fig. 1. Abscissa: time (min); ordinate: % agglutination. Effect of lectin concentrations on the kinetics of agglutination. Embryos were dissociated in CMF-SW and cells were rotated with the following lectins: (A) Con A, I: 1 mg/ml; 2: 20 pg/ml; 3: 5 pg/ml; 4: CMFSW without additions; (B) RCA, I: 50 pg/ml; 2: 20 pg/ml; 3: 5 ,ug/ml; 4: CMF-SW without additions; CC) WGA. 1: 50 iuaiml: 2: 20 us/ml: 3: 5 ualml: 4: &F-SW ‘without additions. Agglutination
of the assay procedure. The maximum variation for mean values obtained for a given time point in a repeated experiment was 10%. Mean values for points in repeated experiRESULTS ments differed by an average of 5 %. RepliReliability of data cate determinations using 2 vials/point indiAll experknents in this study were repeated cated that maximum variation in single cell at least 3 times. The range of values from re- number in a given experiment was less than peated experiments for each point is given on 4%. The average variation of replicate vials the graphs. Results (figs l-3) were highly was 2 %. This error includes errors and variareproducible under the standard conditions tions introduced by differences in the geoExptl Cell Res 84 (1974)

194

Krach et al.

12

r3 40‘ 30 20 -

B

Fig. 2. Abscissa: time (min); ordinate: % agglutination. Kinetics of agglutination of cells dissociated from embryos of different ages. (A) l-day embryo gastrula cells; (f?) 2-day embryo prism cells; (C) 3-day embryo pluteus cells; (0) 4-day embryo cells; (E) 7-day embryo cells: 1: Con A, 20 pug/ml; 2: RCA, 20 pug/ml; 3: WGA, 20 pg/ml. Embryos were dissociated and rotated as described and the % agglutination was determined as the % of single cells which have agglutinated under the standard conditions described in text. Each point gives the range of values obtained in 3 repeated experiments. In each experiment duplicate vials were used for each time point. Values have been corrected for background aggregation occurring in CMF-SW.

metry of vials, pipetting and in the electronic particle counters. The most satisfactory method of dissociating I-7-day sea urchins involved incubations in CMF-SW followed by gentle pipetting. Agglutination experiments were also performed in this medium since the natural aggregation (in the absence of added lectins) was reduced, yet still varied with cell batches in CMF-SW. This enabled more accurate Exptl Cell Res 84 (1974)

measurement of lectin-mediated agglutination. The ConA, RCA and WGA samples used did not require exogenously added divalent cations for maximal agglutinating activity. Agglutination results (% agglutination) with ConA, RCA and WGA were similar, regardless of whether the experiments were performed in CMF-SW or normal sea water. Hapten inhibition experiments with c(methyl-D-glucoside, N-acetyl-D-glucosamine, N-acetyI-D-galactosamine and D-galactose indicated that the purified lectins used in these experiments agglutinated cells with appropriate sugar specificities (as previously described [7, 20, 21, 27, 301). In all experiments, a decrease in the numbers of single cells over time was the result of clumping of viable cells and not cell lysis. Cell viability and agglutination was checked microscopically using the nigrosin dye exclusion test and observation of cellular motility and electronically with particle counter debris windows as previously reported [l, 2, 31, 34, 351. Stage specific agglutination with lectim

The rate and extent of agglutination of CMFSW dissociated sea urchin embryo cells at different stages of development varied with the concentration of lectin used. Fig. 1 shows typical concentration curves obtained for sea urchin embryo cells. After correcting for background aggregation in CMF-SW, by 60 min 37 % of the cells agglutinated when the ConA concentration was 1 mg/ml, while the percent agglutination for 20 pg/ml and 5 pug/ml was 32 and 21, respectively. Results with RCA were qualitatively similar, though total agglutination was always -*wer than that observed with ConA. By 60 min (after correcting for background aggregation in CMF-SW) 19, 9, and 7% of the cells were agglutinated by 50, 20, and 5 ,ug RCA/ml,

respectively (fig. 1). As mentioned previously, little agglutination occurred with WGA at concentrations of 5-50 pg/ml under the standard conditions of the assay procedure (fig. 1). Fig. 1 also gives the results for natural aggregation in CMF-SW in the absence of added lectin. The other graphs given here show percent agglutination in the presence of lectin corrected for background aggregation in CMF-SW. When agglutination of different developmental stages of sea urchin embryo cells with Con A (20 fig/ml) was examined kinetically, 47 Y0 of the cells from 1 day embryos agglutinated by 60 min, while the percent agglutination during this time period for 2-, 3-, 4-, and 7-day cells was 37, 18, 22, and 21 %, respectively. That is, ConA-mediated agglutination decreased by 10 % from day 1 to day 2 and by an additional 17 % from day 2 to day 3. Agglutination was relatively constant from day 3 through day 7, remaining at about 20 % (fig. 2). RCA agglutinated cells dissociated from young embryos (l-4 days) (about 15 % agglutination by 60 min with 20 ,ug RCA/ml) to a greater extent than cells from 7 day embryos. By 7 days, agglutination with RCA was virtually negligible. In all cases,agglutination with RCA was significantly less than that observed with ConA at similar concentrations and under the standard conditions of the assay procedure (fig. 2). WGA was the least effective lectin used in this study. At all developmental stages studied, WGA-mediated agglutination (20 ,ug WGA/ml) was negligible (less than 2%). The results for all lectins are summarized in fig. 3 using the data obtained after 60 min incubation with 20 ,ug of each lectin/ml. In all cases examined WGA exhibited little, if any, agglutinating effect on CMF-SW dissociated sea urchin embryo cells. Fourand 5-day old sea urchin embryo cells were

Cell surface changes during development

195

I 30:\ 1.-’I

I

20

I

2

3

4

5

6

7

Fig. 3. Abscissa: embryo age (days); ordinate: % agglutination. Summary of age-dependent agglutination with lectins. (1) ConA, 20 ,ug/ml; (2) RCA, 20 pg/ml; (3) WGA, 20 pg/ml. Embryos were dissociated and rotated as described and the % agglutination was determined after 60 min under the standard conditions described in text. Each point gives the range of values obtained in 3 repeated experiments. In each experiment, duplicate vials were used for each time point. Values have been corrected for background aggregation occurring in CMF-SW.

agglutinated (20 % in 60 min) by 50 ,ug WGA/ml if these cells were first treated with 0.07% trypsin in CMF-SW for 20 min. At 20 ,ug WGA/ml about 5 % agglutinated, while at 10 pugWGA/ml little, if any, agglutination occurred. Untrypsinized cells did not agglutinate (less than 2 %) with any WGA concentration used (figs l-3). The WGA sample used was active since by 60 min 50 % of a suspension of Sarcoma 180 cells were agglutinated by 50 pg WGA/ml. It has long been known that this lectin effectively agglutinates neoplastic cells [6, 13, 20, 27, 291. Exptl Cell Res 84 (1974)

196

Bach

et al.

Qualitatively similar agglutination results were obtained at a wide range of lectin and cell concentrations. That is, agglutination of sea urchin embryo cells by ConA and RCA was greater at 24 h than at 7 days, regardless of the concentrations of cells or lectins used under the standard conditions of the assay procedure. This suggests that simple changes in cell membrane surface area could not explain the observed results.

DISCUSSION Previous results have shown that embryonic cells are agglutinable with lectins specific for carbohydrate-containing cell surface receptor sites [23-25, 311. Cell surface carbohydrates appear to play important roles in surface phenomena such as cell adhesion [l-5] and possibly density-dependent inhibition of growth [5, 71. Malignant cells which often show reduced cellular adhesion [36] and reduced density-dependent inhibition of growth exhibit greatly increased agglutinability with lectins [27, 371. Malignant cells may represent a regression to an embryonic state in terms of their cell surface properties. It has been suggested that the agglutinability of both neoplastic cells and embryonic cells with lectins may be related to the reappearance of embryonal antigens on the surfaces of cancer cells [24, 381. Kleinschuster & Moscona [24] have recently found that Con A readily agglutinated chick retina cells from early embryonic stages, but was less effective on cells from later stages. They also found that WGA did not agglutinate retina cells at any developmental stage (unless the cells were briefly trypsinized), while RCA agglutinated cells of all developmental ages to the same degree. Their results suggested that during differentiation and maturation specific cell surface changes occurred with respect to the amount, Exptl Cell Res 84 (1974)

mobility or distribution of Con A receptor sites. Similar changes in RCA or WGA receptor sites did not occur. It remains to be seen if such specific cell surface changes are correlated with differentiation and maturation in other developing embryos under different experimental conditions. Such information is important to understanding the problems associated with differentiation and with malignancy. The present study provides quantitative evidence for the contention that specific changes in carbohydrate-containing cell surface sites occur during differentiation and maturation. We previously demonstrated that sea urchin embryo cells are agglutinated with Con A 1311. Here we show that with differentiation these cells become markedly less agglutinable with this lectin. These results suggest several possibilities: (a) As new membrane is synthesized during development less Con A receptors are incorporated into surface membrane, (b) ConA receptor sites may become masked during later developmental stages, making them less accessible for interaction with this lectin [20, 291, (c) during differentiation or maturation ConA receptor sites move in the plane of the cell surface from a clustered to a more random distribution [ 15-171. This rearrangement of receptor sites would effectively reduce lectinmediated agglutination without affecting the binding of lectin to the cells [ 15, 171. (d) Finally, the mobility of ConA receptors is reduced during differentiation preventing lectin-induced rearrangements in the distribution of ConA receptors [18, 191. Such changes in the nature, distribution or mobility of Con A receptor sites may play important roles in determining the, social hehavior of these embryonic cells. Indeed, like malignant cells, certain populations of early sea urchin embryo cells (l-2 days old) are actively migratory (primary and secondary

Cell surface changes during development

mesenchyme cells). Whether the observed changes in agglutinability with Con A can be attributed to cell surface changes in all the cells of the embryo or only in a specific cell population remains to be determined. A direct correlation may exist between active migratory ability during gastrulation or malignancy and the display of Con A receptor sites. Though agglutination with RCA was less than that observed with ConA, a change in cell surface RCA receptor sites also appears to occur by the 7th day of development; WGA-binding sites appear to be presented in a manner that precludes agglutination at any of the stagesexamined under the standard conditions of the assay procedure. Since trypsinization increased agglutinability with WGA, it appears that WGA receptor sites are present, but in a distribution which precludes lectin-mediated agglutination [ 1% 191.It should be emphasized that the experiments in this study were performed with CMF-SW dissociated cells and agglutination was performed in CMF-SW under the standard conditions of the assay procedure. The effects of different dissociation procedures, different agglutination media and different conditions on the stage-dependent agglutination of sea urchin embryo cells remains to be examined. It should be noted that cell agglutination is a complex phenomenon that is determined by a variety of interrelated cell surface parameters, any one of which could change cell agglutinability [39]. These parameters inelude the biochemical nature of the agglutinating molecules and the cell surface agglutination sites, the number of molecules directly involved in agglutination which is related to .$the totaL number of available (or accessible) binding sites and the number of sites occupied, the mobility and distribution of the binding sites and the rate of endocytosis of surface agglutination receptor sites. The

197

structure of the cell surface also affects the agglutinability in any given cell system. Cell surface structures such as microvilli may enhance cell agglutination by literally trapping cells more effectively or by presenting specialized surfaces to other cells that are different from the remaining membrane surface. Cell rigidity or deformability [40] may affect agglutinability by determining the amount of effective surface area that can be presented to adjacent cells through a rebuction in the local membrane radius of curvature. Cell charge repulsive forces are important in reducing spontaneous cell aggregation [5]; therefore, a change in surface charge density or cell zeta potential would affect cell agglutination. Finally, cytoplasmic peripheral membrane components (such as microtubule or microfilament components attached to the membrane inner surface) may determine, in part, cell rigidity and the mobility and topographic distribution of agglutination sites [41]. We thank Dr V. D. Vacquier for helpful assistance with 9-h embryos. This work was supported by USPHS research grant CA-12920 from NC1 (to S. B. 0.) and NSF grant (GE-34178) from the Human Cell Biology Program and grants from the New York Cancer Research Institute and Armand Hammer Fund (to G. L. N.).

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