Developmental expression of a cell-surface protein involved in calcium uptake and skeleton formation in sea urchin embryos

DEVELOPMENTAL

BIOLOGY

122,320-331

(1987)

Developmental Expression of a Cell-Surface Protein Involved in Calcium Uptake and Skeleton Formation in Sea Urchin Embryos MARYC.FARACH,'MARIAVALDIZAN,HELEN Department

of Biochemistry

and Molecular

R. PARK,GLENN L. DECKER,AND~ILLIAM

Biology, University of Texas, M. D. Anderson Hospital 6723 Bertner Avenue, Houston, Texas 77030

Received August 26, 1986; accepted in revised form

J. LENNARZ'

and Tumor Institute

at Hwustq

March 9, 1987

The developmental expression of a cell-surface protein involved in Ca2+ accumulation and skeleton formation in sea urchin embryos has been studied. In Strcmgylocentrotus purpuratus, this protein is present in the egg and in all cell types of the early embryo. After gastrulation, its synthesis and expression are restricted to the skeleton-forming primary mesenchyme cells. In Lytechinus pictus, the protein cannot be detected in eggs or in embryos until the mesenchyme blastula stage. Hybrid embryos demonstrate a pattern of expression indistinguishable from that of the species contributing the maternal genome, which suggests that early expression of the protein in S. purpuratus embryos is due to utilization of maternal transcripts from the egg. Later expression of this protein in primary mesenchyme cells is the result of cell-type-specific synthesis, likely encoded by embryonic transcripts. This cell-type-specific expression in primary mesenchyme cells correlates temporally with Ca2+ accumulation during skeleton formation in the embryo. o 1987 Academic Press, Inc.

Galau et al, 1977; Brandhorst, 1985), might therefore represent a developmental period of great interest with respect to the study of the distribution of lineage-specific markers. Our previous results demonstrated that the 1223 antigen is localized to the primary mesenchyme cells at the gastrula stage of development (Carson et al., 1985). At least two other markers for these cells at this stage have also been reported. Wessel and McClay (1985) described a mesenchyme-specific antigen, Meso I, that is first detected at about the time that primary mesenthyme cells delaminate from the wall of the blastula. In addition, Benson et ah, (1986) found that the glycoproteins comprising the spicule’s organic matrix are synthesized by primary mesenchyme cells at the time CaCOa is deposited into the embryonic skeleton. In this study, we have shown that expression of the 1223 antigen in cultured primary mesenchyme cells correlates temporally with the accumulation of Ca2+ into the growing spicule. We examined the pattern of synthesis and the cell-surface expression of this antigen in developing embryos, and we investigated distribution of this antigen with respect to cell type and relative abundance from egg through pluteus stages. The patterns of expression in Strongylocentrotus purpuratus and Lytechinus pictus embryos, as well as in hybrids of these two species, were also studied. The results of these studies provide the first example of a protein exhibiting a switch from maternal to embryonic expression accompanied by a change from cell-type-nonspecific to cell-type-specific expression.

INTRODUCTION

The calcium carbonate skeleton (spicule) of sea urchin embryos is produced by primary mesenchyme cells at the late gastrula stage of development. Utilizing a monoclonal antibody (MAb 1223) as a probe, we have identified a 130-kDa cell-surface protein that is involved in this biomineralization process. Addition of this monoclonal antibody to primary mesenchyme cells in culture was found to immediately inhibit spicule growth and Ca2+ accumulation (Carson et al., 1985). The results of 45Ca2+uptake experiments, performed under conditions in which Ca” conversion to insoluble CaC03 was nearly eliminated, suggested that the 1223 antigen is involved in Ca2+ uptake rather than the deposition process (Grant et al., 1985). Given these findings, we were interested in determining whether developmental expression of the 1223 antigen coincided with the onset of spicule formation during the late gastrula stage of development. Sea urchin eggs are known to contain stored maternal mRNAs (Davidson, 1976), which are responsible for much of the protein biosynthesis that occurs during the early developmental stages after fertilization (see Brandhorst, 1985). The time of gastrulation, when most of the mRNA being translated shifts from maternal to embryonic in origin (Brandhorst and Humphreys, 1972;

i Present address: Department of Physiology and Molecular physics, Baylor College of Medicine, Houston, TX 77030. ‘To whom correspondence should be addressed.

0012-1606/87$3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

Bio-

320

FARACH MATERIALS

AND

ET AL.

Expression

METHODS

Materials. Sea urchins (S. purpuratus and L. pictus), purchased from Pacific Biomarine Supply (Venice, CA), were maintained and embryos were prepared and cultured at 14°C as described previously (Heifetz and Lennarz, 1979). Developmental stages were described morphologically according to the criteria of Galileo and Morrill (1985). Materials used included Biotrans nylon membranes (0.2 pm), [1251]protein A, and Nalz51 (17 Ci/ mg) from ICN Biomedicals, Inc. (Irvine, CA); [3H]leucine (120-190 Ci/mmole) and 45CaC12(lo-40 mCi/mg) from Amersham Corp. (Arlington Heights, IL); tissue culture media, sera, and supplies from GIBCO (Grand Island, NY); Nitex filters from Tetko, Inc. (Elmsford, NY); fluorescein isothiocyanate-conjugated goat anti-mouse IgG, cyanogen bromide-activated Sepharose 4B and protein A-Sepharose 4B from Sigma Chemical Co. (St. Louis, MO); normal mouse IgG from Cappel Laboratories (West Chester, PA); and nitrocellulose from Schleicher and Schuell, Inc. (Keene, NH). All other chemicals used were reagent grade or better. Culture of primary mesenchyme cells. Cultures of primary mesenchyme cells (S. purpuratus) were prepared and incubated at 14°C as described (Carson et al., 1985). Attached cells were detached from tissue culture plates by gentle scraping after incubation for 15 min in Ca’+,Mg’+-free artificial seawater and then quantified using a Coulter counter. The total number of cells attached per dish was found to be 2-3 X lo5 and the plating efficiency of the primary mesenchyme cells was estimated as 10% .The attachment of primary mesenchyme cells was found to be complete within 12 hr after plating; the number of cells found to be attached to the dishes remained constant after this time. Production of hybrid embryos. Eggs (3 ml) from L. pictus were washed twice with filtered seawater by allowing them to settle and then were treated with trypsin (250 pg/ml final concentration) with gentle swirling for 10 min at 16°C. At this time, an excess of soybean trypsin inhibitor was added and the eggs were washed three times with a 25-fold excess volume of filtered seawater and diluted to approximately 100 ml. The eggs were inseminated with fresh S. purpuratus sperm (200 ~1). After 15 min, sperm were removed by allowing the eggs to settle in an excess of filtered seawater. After 5 hr at 14”C, approximately 45% of the zygotes had developed to the 4- to 8-cell stage. Unfertilized eggs were removed at the hatched blastula stage by passage through 80-pm Nitex filters, which selectively retained the embryos. Eggs from S. purpuratus females were harvested and allowed to incubate at 14°C for 16 hr before they were fertilized with fresh sperm from L. pi&us males. The ratio of sperm to egg used for insemination was as described above for the reverse cross. The low fertilization

of Cell-Surface

Protein

321

efficiency (12%)obtained in this cross required that fertilized eggs and early cleavage stage embryos be manually separated from unfertilized eggs under a dissection microscope. At hatched blastula and other later swimming stages, embryos were selected by several passages through Nitex filters as described above. Complete removal of unfertilized eggs from all preparations was determined by visual inspection using a dissection microscope. Metabolic labeling. Embryos were collected at various developmental stages and incubated with [3H]leucine for 2 hr with constant aeration as described previously (Carson et al., 1985). Labeling of cultures of primary mesenchyme cells was initiated 66 hr after plating, a time when both spicule growth and 45Ca2+ uptake are near maximal levels (Mintz et ah, 1981; Carson et al., 1985). [3H]Leucine was added to cultures to a final concentration of 10 &i/ml and the incubation continued for 2 hr as previously described (Carson et al., 1985). Immunojluorescence microscopy over development. Hybridoma 1223 was maintained and purified 1223 IgG was isolated from conditioned media as described (Carson et al., 1985). Chromatographically purified normal mouse IgG was used as a control. Unfertilized and fertilized eggs were washed twice and resuspended in Ca2+,M2+-free seawater containing 1.0 mM EDTA. Embryos at various stages of development were suspended in Ca2+,M$+-free seawater and then pipetted through a large-bore plastic pipet until dissociation into single cells was complete. To minimize cellular damage, dissociation was carefully monitored in a Nikon Diaphot phase-contrast microscope. All samples for indirect immunofluorescence studies were fixed for 30 min in 1.0% paraformaldehyde and treated with first and second antibody as previously described (Carson et al., 1985). Samples were examined and photographed with a Nikon Diaphot phase-contrast microscope equipped for epifluorescence. To permit visualization of MAb 1223 binding sites on primary mesenchyme cells at different stages of development within the blastocoel, basal laminar bags were prepared using a method similar to that of Harkey and Whiteley (1980) as described by Carson et al. (1985). The procedure was monitored in the phase microscope to minimize release of primary mesenchyme cells from the blastocoel. To avoid further dissociation, bag preparations were routinely attached to polylysine on glass slides by the method of Mazia et al. (1975). Measurement of cell-surface binding of MAb 12.23. Embryos were cultured as described above except that fertilization envelopes were eliminated by addition of 2 mM 3-amino-1,2,4-triazole as described by Foerder and Shapiro (1977). Equal aliquots (0.2 ml) were removed from a 1% embryo suspension from early cleavage

322

DEVELOPMENTALBIOLOGY

through prism stages and the embryos were dissociated in Ca2+,M2+-free artificial seawater. Under these conditions, less than 5% of the 1223 antigen is released to the supernatant. The resulting cells were gently pelleted and resuspended in the original volume of artificial seawater containing 1 mg/ml bovine serum albumin (BSA) and 10 mMNa1. One microgram of either MAb 1223 or preimmune mouse IgG was added and the volume was adjusted to 1 ml by addition of artificial seawater containing 1 mg/ml BSA and 10 mM NaI. The cells were incubated with antibody by gentle shaking at 4°C for 30 min and then washed extensively with the same solution as above. After the final wash, cells were again resuspended in this solution, and 1 X lo5 cpm of [‘251]protein A was added. After incubation for 30 min at 4°C the cells were washed twice by centrifugation, and the amount of bound radioactivity in the cell pellet was determined. The integrity of the cells was assessed by trypan blue exclusion. Synthesis of 1223 antigen over devetopment. Embryos labeled with [3H]leucine were homogenized with a Polytron tissue homogenizer (Brinkman Instruments Co., Westbury, NY) for 2 X 1 min at setting ‘7 in 2% cholate, 10 mMTris, pH 8.0, and a mixture of protease inhibitors (Carson et al., 1985). Solubilization was continued overnight with gentle stirring at 4°C. Samples were then applied batchwise to beads of Sepharose 4B to which preimmune mouse IgG had been coupled. The amount of labeled protein binding to these control beads was found to be minimal. Proteins not bound to the preimmune beads were incubated with beads containing covalently coupled MAb 1223 as described previously (Carson et al., 1985). The beads were then washed six times by centrifugation using buffer consisting of 0.54 M NaCl, 10 mM Tris-HCl, pH 8.0, and 0.1% Triton X100. Bound 1223 antigen was then eluted from the beads with 6 M guanidine HCl and 1% Triton X-100 and dialyzed exhaustively against 0.5% (w/v) cholate in 10 mM Tris-HCl, pH 8.0. The contribution of [3H]leucine-labeled 1223 antigen to the total pool of [3H]leucine-labeled proteins at each developmental stage was then assessed by separately precipitating the unbound proteins and the bound and eluted 1223 antigen twice with trichloroacetic acid as described (Farach et al., 1986). Greater than 95% of the radioactivity that was bound and subsequently eluted from the 1223 immunoaffinity resin was precipitable with trichloroacetic acid. The percentage of the total [3H]leucine-labeled material bound and eluted from the immunobeads was used to determine the percentage of de novo synthesis of the 1223 antigen over the course of development of the embryos. The same procedure was used to assess the amount of 1223 antigen synthesized de nouo in [3H]leucine-labeled spicule-forming cultures of primary mesenchyme cells.

VOLUME122,1987

Immunoblotting with MAb 1223. Samples of total solubilized embryonic protein (30 yg/dot) prepared as described above were dried onto 0.2-hrn nylon Biotrans membranes which were then soaked for 2 hr in a freshly prepared solution of 2 mM sodium acetate, 5 mM MOPS (3-[N-morpholinolpropanesulfonic acid), pH 7.5, containing 20% (v/v) ethanol. The membranes were then incubated overnight with shaking at 50°C in 10% (w/v) BSA in phosphate-buffered saline (PBS, blocking buffer). The blocking buffer was replaced with 50 ml of PBS and 2% (w/v) BSA containing 0.5 mg of MAb 1223, and the membranes were incubated for a minimum of 6 hr at 22°C. The membranes were then washed thoroughly with PBS containing 0.1% (v/v) Triton X-100, and lo6 epm of ‘““I-labeled goat anti-mouse IgG was added. Iodination of IgG was performed using the chloramine T method (Review 18, Amersham, Inc.). After 1 hr, the radioactive solution was removed, and the membranes were washed thoroughly with PBS and 0.1% (v/v) Triton X-100 and then processed for autoradiography. The individual radioactive spots were located on the Biotrans membrane, cut out, and counted in a gamma counter. Western blot analysis was performed using the following modifications of the published procedure (Beisiegel et al., 1982): Total solubilized proteins (50 Kg/ lane) from embryos at various developmental stages or from primary mesenchyme cells in spicule cultures were separated by SDS-PAGE on 10% gels as described previously (Carson et al., 1985) and transferred to nitrocellulose membranes overnight at 4°C using a Hoefer transfer apparatus (San Francisco, CA). Transfer efficiency was monitored by staining the polyacrylamide gel with Coomassie brilliant blue R after transfer. Membranes were incubated at 22°C for 30 min with a blocking solution of 2% (w/v) hemoglobin in 10 mMTrisHCl, pH 7.4, containing 0.9% (w/v) NaCl, 0.5% (v/v) NP40, and 0.1% (w/v) SDS. MAb 1223 (10 pg/ml) was then added in fresh blocking solution and incubation continued for 1 hr. The membranes were washed exhaustively with PBS and then with 10 mM Tris-HCl, pH 7.4, containing 0.9% (w/v) NaCl in the presence and absence of 0.1% SDS and 0.5% (v/v) NP-40. lz51-labeled goat antimouse IgG (lo6 cpm) was then added in blocking solution and incubation continued for 2 hr at room temperature. The membranes were washed as described above and were washed finally with a solution consisting of 10 mM Tris, pH 7.4, 0.5 M NaCl, and 0.5% (v/v) NP-40. The washed filter was dried and analyzed by autoradiography. Quantitation of radioactive bands was performed by direct scanning of the autoradiograph using a Quick Scan, Jr. densitometer (Helena Laboratories, Beaumont, TX). Other procedures. Protein was determined by the procedure of Lowry et al. (1951) using BSA as a standard.

323 SDS-PAGE was performed (Carson et al., 1985).

as previously

described

RESULTS

Synthesis and expression of the 12%’ antigen in S. purpratus embryos. The monoclonal antibody (MAb 1223)

was initially selected for further study because it exhibited higher binding to embryos of S. purpuratus at the gastrula stage than at the hatched blastula stage. Subsequently, we found that the 1223 antigen is present in both eggs and early cleavage stage embryos at levels higher than those at the hatched blastula stage. As shown in Fig. lA, when total solubilized proteins from embryos at various developmental stages were analyzed by the immunodot blot method for concentration of the 1223 antigen, a biphasic pattern of expression was seen. It is clear that the 1223 antigen is present at all developmental stages, and its concentration does not vary more than fivefold over the course of development. The relative concentration of the 1223 antigen in unfertilized eggs obtained from different females was somewhat variable; in some cases the amount in eggs was similar to that found at the gastrula stage, whereas in others it was closer to that found at the 4- to g-cell stage (as in Fig. 1A). In contrast to these measurements of total 1223 antigen in the egg and early embryos, the amount

1.0

A.

Total antigen

.a F a

.6

5

.2

if

.40 lll-idh

E

C

HBMBMG

Developmental

LG

Pr

PI

Stage

FI(;. 1. Binding of MAb 1223 to embryonic extracts and the cell surface over development. (A) Embryos at various developmental stages were soluhilized and analyzed for the presence of the 1223 antigen by immunodot hlots as described under Materials and Methods. Error bars represent the maximum deviation in the values shown obtained in four separate analyses of embryos with different parents. (B) Presence of 1223 antigen on the surface of cells from dissociated embryos was determined as described under Materials and Methods. Error bars represent duplicates. Surface assays were not performed at late gastrula or pluteus stages. E, unfertilized egg; C, cleavage stage (4-8 cell); HB, hatched blastula; MB, mesenchyme blastula; MG, midgastrula: LG, late gastrula; Pr, prism; Pl. pluteus. In both panels, preimmune controls gave constant background values that did not exceed 20% of the lowest value obtained with MAb 1223 (hatched blastula).

”

C

HB

Development

G

Pr

PI

Stage

FIN:. 2. Biosynthesis of 1223 antigen over the course of development. Embryos were incubated for 2 hr with [3H]leucine and the contribution of the 1223 antigen to the total pool of newly synthesized protein, determined as described under Materials and Methods. C, cleavage stage; HB, hatched blastula; G, mid-late gastrula; Pr, prism; PI, pluteus. In this particular experiment, the values for 1223 antigen biosynthesis for the various developmental stages were 0.4,0.26,0.52,0.8, and 0.67% of total de wwo synthesized protein, respectively. Error bars represent the range ohtained in duplicate measurements.

of antigen present on the surface of cells revealed an approximately threefold increase between fertilization and early cleavage (Fig. 1B). As shown in Fig. lB, subsequent to the early cleavage stages the amount of cellsurface antigen decreased and then increased again during posthatching development, that is, during differentiation of primary mesenchyme cells. Taken together, the biphasic patterns obtained for the developmental distribution of the total amount of 1223 antigen in embryo extracts (Fig. 1A) and on the surfaces of cells of dissociated embryos (Fig. 1B) suggested that, in addition to being present in eggs, the 1223 antigen might be synthesized at two distinct periods during development. To test this idea directly, embryos were labeled with [“Hlleucine for 2-hr intervals throughout development from early cleavage through pluteus stages. As shown in Fig. 2, de ?LOVO synthesis of the 1223 antigen was also found to be biphasic, with a minimum at the time of hatching followed by an increase to a maximum at the prism stage. In duplicate experiments, the maximum rate of synthesis was sometimes observed slightly earlier during the late gastrula stage. The contribution of the 1223 antigen to the total pool of de novo synthesized proteins varied from 0.2 to 0.8% in the experiment shown in Fig. 2, indicating that the 1223 antigen is a major protein being synthesized de nova in developing sea urchin embryos. Consistent with this, SDS-PAGE analysis of all [“Hlleucine-labeled proteins present in embryos showed the presence of labeled 130-kDa protein at all developmental stages (data not shown). It should be noted, however, that the mass of the 130-kDa protein in total embryo extracts is very low. Indeed, this antigen could not be detected in Coomasie-blue-stained gels of total embryonic proteins at any developmental stage. We have previously found that at the gastrula stage

324

DEVELOPMENTALBIOLOGY

the 1223 antigen is localized to the primary mesenchyme cells; little or none could be detected on the surface of other cell types (Carson et al., 1985). In view of the results shown in Figs. 1 and 2, we examined the pattern of 1223 antigen distribution by indirect immunofluorescence of fixed cells from dissociated embryos over the course of development. As shown in Figs. 3A and 3B, MAb 1223 binding in unfertilized eggs revealed diffuse immunofluorescence on the cell surface. No dramatic changes in the intensity of immunofluorescence could be detected at early cleavage stages (Figs. 3C and 3D), although quantitation of surface binding of MAb 1223 revealed a threefold increase in the amount of antigen on the surface (Fig. 1B). By the 4-cell stage the fluorescence was fairly uniform on the surfaces of all cells (Figs. 3C and 3D), although in some preparations a small percentage (12% )of the cells lacked any detectable fluorescence. After the unequal cleavages at the 16- to 32-cell stages, the 1223 antigen was detected on micromeres, mesomeres, and macromeres (Figs. 3E and 3F), and it remained widely distributed on the majority of cells through the hatched blastula stage (Figs. 3G and 3H). Subsequent to the hatched blastula stage, the distribution of the 1223 antigen on S. purpuratus embryos was found to change dramatically. In contrast to the diffuse surface fluorescence seen with early stage embryos, by the prism stage (Figs. 31 and 35) only a small number of cells in the field exhibited very strong fluorescence. The relative intensity of fluorescence observed was consistent with that predicted by the surface binding assays in Figs. 1B; that is, during cleavage stages, binding sites for MAb 1223 are distributed over the entire cell surface of the relatively large embryonic cells, whereas at the prism stage a similar number of binding sites are restricted to the surfaces of a few small cells. As shown in Figs. 3G and 3H, the presence of the 1223 antigen on the surfaces of all cells at early stages resulted in cell agglutination when MAb 1223 was added to dissociated cells at the hatched blastula stage. This effect was not observed when Fab fragments prepared from MAb 1223 IgG were used. Earlier it was found that MAb 1223 inhibited attachment of primary mesenchyme cells to culture dishes (Carson et ah, 1985). Our current findings suggest that MAb 1223 crosslinks epitopes present on opposing blastomere surfaces at the time of plating. Such crosslinking would be expected to entrap the primary mesenchyme cells in the large aggregates that form, and consequently they would not have the opportunity to adhere to the substratum. This is consistent with our observations that (1) MAb 1223 was unable to dislodge previously attached cells from culture dishes, and (2) Fab fragments prepared from MAb 1223 did not inhibit attachment of primary mesenchyme cells to culture dishes (data not shown).

VOLUME122,1987

Because of the dramatic change in the number of cells exhibiting fluorescence with MAb 1223 between the hatched blastula and gastrula stages, we examined the fluorescence pattern in embryos that were partially dissociated in a manner that preserves the basal lamina and the cells within it but that strips away most of the ectodermal cells (Harkey and Whiteley, 1980). As seen clearly in Figs. 4A-4H, strong fluorescence is exhibited by the primary mesenchyme cells, whereas the level of fluorescence on the ectodermal and endodermal cells is much lower. At the mesenchyme blastula stage (Figs. 4A and 4B) the partially dissociated embryos exhibit clusters of fluorescent primary mesenchyme cells that correspond in location and number to those found within the intact embryo (Giudice, 1973). Occasionally cells seen outside the clusters also show strong fluorescence. Immunostaining also reveals the characteristic ring-like structure that is formed by primary mesenchyme cells at the gastrula stage (Figs. 4C and 4D). By late gastrula stage (Fig. 4D), cellular processes emerge from the “ring” of cells to form a roughly triangular mass that mimics the shape of the embryo at the prism stage (Figs. 4E and 4F). Although, occasionally, dislocation of skeletal segments occurs (Fig. 4G), the major structural features of the cellular template that forms the pluteus stage skeleton can be clearly visualized within the blastocoel (Fig. 4H). In Table 1, the percentages of the total cells that exhibit strong fluorescence with MAb 1223 are shown from the midgastrula through pluteus stages. It is clear that beginning at the midgastrula stage, when spicule growth has started, MAb 1223 is a useful surface marker for primary mesenchyme cells. The value for the percentage of cells containing 1223 antigen (7.3%)agrees well with the value one can calculate for the percentage of spiculeforming primary mesenchyme cells in the midgastrula stage embryo (7.3%) (Davidson, 1986). At the earlier mesenchyme blastula stage, even though primary mesenchyme cells were present, the immunofluorescence of a small number of other cells precluded the use of MAb 1223 as a lineage-specific marker. As shown in Table 1, at stages after midgastrula, the percentage declines, as expected if the total number of cells increases but the number of primary mesenchyme cells remains unchanged. The localization of the 1223 antigen on the surface of primary mesenchyme cells during the later stages of embryonic development led us to ask whether its de nmo synthesis at these stages was also restricted to this cell type. If a quantitatively minor cell type of the embryo synthesizes all of a particular protein, then it follows that the percentage of the total protein represented by that protein should be much greater in that cell type than in the entire embryo. One can calculate that if 4.5%

FARACH

FIN. 3. Distribution of 1223 antigen on the surface of embryonic cells of S. prpurutus by indirect immunofluorescence. Fluorescence and phase micrographs are shown at each developmental stage. Embryos were dissociated prior to fixation and immunofluorescent staining was performed as described under Materials and Methods. (A, B) Unfertilized egg; (C, D) J-cell stage; (E. F) 16-cell stage; (G, H) hatched blastula stage; (I, J) prism stage; (K, L) prism stage treated with preimmune mouse I&. Magnification, X325.

326

DEVELOPMENTAL BIOLOGY

VOLUME 122, 1987

FIG: 4. Immunofluorescence localization of MAb 1223 hinding sites in partially dissociated S. INITJN~TYI~Wembryos attached to pal! FlLloi7 ?scence micrographs are shown at each developmental stage. (A, B) Mesenchyme hlastula; (C, D) middle and late gastrula respec tively; (E, F) prism stage; (G, H) pluteus stage. For all panels except (E, G), magnification x340. For (E, G) magnification X125

Isine. ages,

TABLE 1 THE PERVENTAGE OF CE:LLS EXFRICWNG THE: 1223 ANTIGEN DECKE.GES OVER DEVELOPMENT

Stage Midgastrula Late gastrula Prism Pluteus

Percentage of cells exhibiting strong immunofluorescence” 7.3 -i- 0.9 4.5 f 1.8 4.1 f 1.3 <4.0*

0 Embryos were dissociated and the percentage of cells demonstrating strong fluorescence was determined. At least 100 cells were counted in each experiment. Strongly fluorescent cells were defined by visual intensity; a typical field with three strongly fluorescent cells is shown in Fig. 3.1. h Values were ditficult to determine accurately because embryos could not he completely dissociated.

(Table 1) of the cells (i.e., the primary mesenchyme cells) of the late gastrula stage embryo synthesized all of the newly synthesized 1223 antigen (0.52% of total embryonic protein made by gastrula stage embryos, Fig. 2), then approximately 11% of the protein synthesized de nnuo by primary mesenchyme cells in culture should be immunoprecipitated with MAb 1223. In order to directly determine this percentage, cultures of primary mesenthyme cells were labeled with [“Hlleucine for 2 hr on the second day after plating as described under Materials and Methods, and the relative contribution of the 1223 antigen to de )~OUCI synthesized protein was determined. In several experiments, the amount of 1223 antigen synthesized by primary mesenchyme cells on the second day in culture was found to be 8 to 9% of the total [“Hlleucine-labeled protein. This agreement with the predicted value of 11% suggests that nearly all of the 1223 antigen synthesized by late gastrula stage embryos is made in the primary mesenchyme cells. E.rpressiorl of’ the MB antigen in embryos other seu urchin species. At the midgastrula

of several

stage of development, embryos of not only S. purpratus but also of two other sea urchin species, L. picks and Arbacia p~~ct~lutcr, express the 1223 antigen. In all three species 1223 antigen was found by indirect immunofluorescence to be restricted to 7-9X of the total cells, presumably the skeleton-forming primary mesenchyme cells. Interestingly, when embryos of L. pictus at the hatched blastula stage were examined by immunofluorescence, no staining above preimmune background was observed (data not shown). This result differed strikingly from that described earlier for S ~uqmrutus (see Figs. 3G and 3H), in which all cells of the hatched blastula stage embryo exhibited immunofluorescence with MAb 1223. To further investigate the different patterns of embryonic expression of the 1223 antigen in these two sea ur-

chin species, the amount of the 130-kDa protein present in extracts from the embryos was quantitated. Because the 1223 antigen has occasionally been observed in crossreactive 250-kDa and 50-kDa forms (Carson et ul., 1985), both of which would be detected upon immunodot analysis as in Fig. lA, we used Western blot analysis to confirm that only the 130-kDa protein was being measured. As shown in Figs. 5A and 6A, the I30-kDa protein is present in eggs and is expressed in a biphasic manner over the course of development of S. purpurtxtus embryos, as predicted from the results of the immunodot blot analysis shown in Fig. 1. In contrast, 1223 antigen could not be detected in eggs or embryos of L. picfus prior to the mesenchyme blastula stage (Figs. 5B and 6B). The 1223 antigen was not detected in sperm of either L. pi&s or S. purpxrwtus, nor in eggs or sperm of the starfish Putiria nli?liatn (data not shown). When hybrids of S purpumtus and L. pick embryos were produced and analyzed similarly by Western blots (Figs. 6C and 6D), the pattern of developmental expression was indistinguishable from that of the species contributing the maternal genome. This result indicates that the presence of the 130-kDa 1223 antigen in embryos prior to the mesenchyme blastula stage requires only that the egg be from S. purpuratus and is independent of the paternal species. E.xpwssim qf fh,e 12~3 untigen iw prima rg mesew yrtrcj cells cultured in Afro. Primary mesenchyme cells undergo a dramatic differentiation process, resulting in an

A

E C HB MG Pr PI b E CjiB&K%Pr 1 *

!‘l

20097-

6a43-

26-

Frc:. 5. Western blots of 1223 antigen in S. ~rurjxcr~tus and I,. /Gcftrs embryos. At the indicated developmental stages, embryos were solubilized and equal amounts of total embryonic protein were separated on SDS-PAGE gels, transferred to nitrocellulose, and immunoblotted with MAb 1223 (see Materials and Methods). As shown, the major antigenic form recognized by MAb 1223 is the 130-kDa protein. (A) S’. y~r~ncrnt/r.s; (B) L. ~)ict~s. Developmental stages are as indicated in the legend to Fig 1. Lanes 1 and 4 in (A) contain obvious doublets in the 130.kDa region; only the band migrating at the same position in all lanes (arrow) is included in the bar graph shown in Fig. 6.

328

DEVELOPMENTAL BIOLOGY 1.0

A.

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.4 L-L 1.0 .8 .6 .4 .2 E C HEMBMG iG Pr PI Developmental Stage

FIG. 6. Developmental expression of 130-kDa protein in embryos of various species. Expression of the 130-kDa protein was quantitated from densitometric recordings of autoradiographs of Western blots as described under Materials and Methods. (A) S. prpuratus embryos (developmental stages are those in Fig. 5A; assays were not performed at mesenchyme blastula or late gastrula stages; see Fig. 1A for complete developmental profile); (B) L. pictus embryos; (C) hybrid embryos (S. wrmwatus 8 X L. pictus P); (D) hybrid embryos (L. pictus 6 X S. WPpwatus P). Each panel represents data obtained using a single embryo culture derived from one male and one female parent.

assembly of spicules in vitro that parallels their development in viva (Carson et ah, 1985). In view of this, we investigated the expression of the 1223 antigen in these cells from the start of in vitro cultivation until large linear spicules were formed on the third day in culture. Western blots of total cellular extracts of cultured primary mesenchyme cells 66 hr after plating showed a strong signal for the 130 kDa protein (Fig. ‘7, lane 3). In contrast, a relatively low level of several immunoreactive proteins in this molecular weight range was detectable on Western blots of total cellular extracts at the time of plating (lane 1); no substantial increase in immunoreactive proteins in this molecular weight range was observed upon examination of attached primary mesenchyme cells 18 hr after plating (lane 2). The results indicate that the 1223 antigen is found in high levels in

VOLUME 122, 198’7

cultured primary mesenchyme cells only after postattachment differentiation, ImmunoAuorescence patterns obtained with MAb 1223 during the postattachment stages of differentiation of the primary mesenchyme cells in vitro supported the results of the Western blots, In Fig. 8A, it can be seen that by approximately 36 hr after attachment of primary mesenchyme cells (see Materials and Methods) the 1223 antigen was detected on the cell surfaces and filopodial networks. During the next 24 hr of rapid spicule growth (Fig. 8B), similar to the results obtained with partially dissociated embryos (Fig. 4), immunostaining revealed the presence of 1223 antigen throughout the cell surfaces, including the membranous sheath surrounding the spicule (Mintz et al, 1981). Expression of the 1223 antigen in vitro correlates temporally with an increase in Ca” accumulation. Previous studies using a mixture of all the embryonic cells from dissociated embryos maintained in culture (Mintz et al., 1981) revealed that 45Cazi2+ uptake increased at about the time of spicule formation. To examine Ca2+ uptake in isolated primary mesenchyme cells differentiating in culture in the absence of other cell types, uptake of 45Ca2+ was measured on 4 sequential days after plating. As shown in Fig. 9, 45Ca2t uptake increased progressively during Days 2 and 3, before falling off on Day 4. This

12 .

3

FIG. 7. Western blot of 1223 antigen made by primary mesenchyme cells in culture. Primary mesenchyme cells were removed from dishes and the relative level of 1223 antigen was determined on Day 1 and Day 3 in culture by Western blots. Each lane represents 50 fig of total protein (see Materials and Methods) probed with MAb 1223. Lane 1, cells at the time of plating, 2 hr after hatching from the fertilization envelope, Lane 2, 18 hr after plating; Lane 3,66 hr after plating.

FARACH ET AL.

Exprwsim

of

Cell-Surfuce Protci?c

329

FIN2. 8. Cell surface expression of 1223 antigen by cultured spicule-forming cells. (A) Attached 4%hr primary mesenchyme cell cultures .ing clusters and cell networks interconnected by cell-surface processes. Magnification, X700. (B) 72-hr primary mesenchyme cell cultures +-o produced elongated spicules. The intensity of fluorescence on these cells demonstrates that the 1223 antigen persists on the cell surface during the time elongated spicules are formed. Magnification, X350.

period of increasing 4’Ca2t uptake corresponded to the time that the 1223 antigen increased in abundance in Western blots (Fig. 7) and appeared on the cell surfaces of isolated primary mesenchyme cells (Fig. 8). Moreover, after the first day in culture, the processes of Ca2+ uptake and deposition into CaC03 seemed to be tightly coupled in primary mesenchyme cells producing spicules, because most of the ionic 45Ca2+taken up during a 2-hr labeling period was found to be present in an alkaline hypochlorite insoluble form (Fig. 9). The drastic reduction in 4hCa2+uptake observed on the fourth day may have been caused by cell death because at this time a significant number of the cells began to detach from the culture dishes. DISCUSSION

Primary mesenchyme cells of the sea urchin embryo participate in the assembly of the embryonic skeleton. Many, if not all, of these cells seem to be committed to this function very early in development because when

isolated micromeres, the predecessors of primary mesenchyme cells, are cultured in the absence of other cell types, they undergo differentiation and exhibit many of the behavioral characteristics of primary mesenchyme cells in viva (Okazaki, 1975; Harkey and Whiteley, 1980, 1983; McCarthy and Spiegel, 1983). In earlier studies, we identified a 130-kDa protein in S. purpuratus embryos that appears to be involved in the accumulation of Ca”’ that occurs during skeleton formation (Carson et al., 1985; Grant et al., 1985). In the current study, we have shown that this 130-kDa protein is specifically expressed by primary mesenchyme cells at the midgastrula stage of development. In addition, by using immunoblot techniques we have found that the expression of the 130kDa cell-surface protein does not correlate temporally with substratum attachment of primary mesenchyme cells in zdro; rather it correlates with the increase in Ca2+ uptake that occurs in these cells when spicule assembly is initiated. Based on these findings, coupled with our previous observations that MAb 1223 inhibits spicule growth and %a’+ uptake by primary mesenchyme cells

330

DEVELOPMENTAL

36.0 t

BIOLOGY

r-7

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s ‘Z g 16.0 P i 12.0 N 0” a.0 z 4.0 18

42

66

Hours

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90

FIG. 9. Ca2+ uptake correlates temporally with the expression of the 1223 antigen in r&o. Uptake of %a” into primary mesenchyme cells in culture was measured as described previously (Carson et al., 1985) (dotted bars). The amount of cell-associated %a’+ that had been incorporated into magnesium calcite was assessed by insolubility in alkaline hypochlorite (Mintz et al., 1981) (hatched bars). In each case uptake of added ‘%a’+ (25 &i) was measured for 2 hr beginning at the indicated time.

(Carson et al., 1985), it seems likely that the expression of the 1223 antigen is related to the acquisition of the ability of primary mesenchyme cells to accumulate Ca2+ during spicule formation. This putative function of the 130-kDa protein is consistent with the data demonstrating it to be a major protein (8% of the total) synthesized de nova by spicule-forming primary mesenchyme cells. That 50-60 primary mesenchyme cells in an embryo of 500-1000 cells invest almost one-tenth of their proteinsynthesizing machinery in the manufacture of one protein is not so surprising when one considers that the only apparent function of these cells is to construct a rather elaborate magnesium calcite skeleton. Consistent with this, the immunofluorescence patterns reveal that the 1223 antigen is widely distributed on the cell surfaces and associated filopodia of spicule-forming cells both in vivo and in vitro. The function of the 130-kDa protein found in the egg and synthesized in cleavage stage embryos during early development of S. purpuratus is unknown. However, it is interesting to note that calcium appears to be required for early postfertilization events in sea urchin embryos, including fusion of cortical granules with the plasma membrane (Vacquier, 1975; Decker and Lennarz, 1979; Baker et al., 1980). Experiments are currently underway in our laboratory to determine whether microinjection of MAb 1223 into unfertilized eggs has any consequences on the early developmental changes that occur upon fertilization in S. purpuratus.

VOLIJME 122, 1987

The 130-kDa 1223 antigen is not detected in preblastula stage embryos of L. pictus or in hybrid embryos where L. pictus provides the maternal genome. Thus, it is possible that the 1223 antigen serves no essential function in early embryonic development. Alternatively, it could be that in L. pictus the epitope recognized by MAb 1223 is not present until the mesenchyme blastula stage, even though the apoprotein might be made throughout development. Such an “epitope switch” between the blastula and gastrula stages is plausible because it is at this time in development that sea urchin embryos begin to rely upon the embryonic genome rather than stored maternal transcripts (Brandhorst, 1985). In fact, Wessel and McClay (1985) have described a monoclonal antibody that recognizes an epitope present on primary mesenchyme cells that first appears at the time of delamination of these cells, but is believed to be a post-translational modification of a protein synthesized much earlier in development. We have found that this antibody (lG8) precipitates a 130-kDa protein in our S. purpuratus embryo extracts, but that it has no effect on Ca2+ accumulation by cultured primary mesenchyme cells (M. C. Farach, G. Wessel, and W. J. Lennarz, unpublished results). The biphasic pattern of developmental synthesis and expression of the 130-kDa protein in S. purpuratus embryos, as well as the switch from its expression in the majority of embryonic cells to expression in a quantitatively minor cell lineage after the mesenchyme blastula stage, suggests that early expression of the 1223 antigen is the result of translation of maternal mRNAs stored in the egg. Indeed, this idea is supported by findings with hybrid embryos, where we found that the developmental pattern of expression of the protein was that of the species contributing the maternal genome. Given the restricted expression of paternal genes in interspecies hybrids of sea urchin embryos (Tufaro and Brandhorst, 1982; Showman and Whiteley, 1980), it seems clear that the 130-kDa protein seen in early embryos from S. purpuratus eggs is of maternal origin. It is likely that the general decrease in the intensity of cell surface fluorescence on the nonmesenchymal cells that is observed during and subsequent to gastrulation is associated with a dilution and depletion of the 1223 antigen and the maternal store of mRNA encoding it. Later production of the 130-kDa cell-surface protein in primary mesenchyme cells in S. purpuratus, and presumably in the other two sea urchin species examined, seems to be the result of cell-type-specific expression of embryonic mRNA for the 1223 protein. Although further studies will be necessary to establish the existence of two classes of 1223 mRNA, we can conclude that from gastrula through pluteus stages MAb 1223 is a useful marker (see Angerer and Davidson, 1985)

for spicule-forming primary mesenchyme cells. This contention is further supported by the observation that the number of primary mesenchyme cells detected using MAb 1223 binding is consistent with that estimated in previous studies (see Davidson, 1976, 1986). Given the unique distribution of the 1223 antigen, we plan to elucidate its structure and function, including the nature and processing of the epitope recognized by MAb 1223. We hope that purification and functional reconstitution of this protein in vitro will define its molecular role in Ca” accumulation and skeleton formation by primary mesenchyme cells. We arc grateful to Ms. Diana Welch for her help in preparing the manuscript. We also thank Mr. Dana Earles for culturing embryos, Mr. Matt Keast for his assistance in preparing the hybrids, and Dr Steve Nadler for determining conditions for antigen dissociation. We are especially grateful to Dr. Daniel Carson for performing the iodinated surface binding assays and for many helpful discussions. This work was supported in part by National Institutes of Health Grant HD-21483 to William J. Lcnnarz. Mary C. Farach was supported by a postdoctoral fellowship from the National Institutes of Health (HD 06509). William J. Lennarz, who is a Robert A. Welch Professor of (:hemistry, gratefully acknowledges the Robert A. Welch Foundation REFERENCES Amersham Review No. 18 “Radioiodination Techniques,” Pp. 45-48. Amcrsham/Searle, Arlington Heights, IL. APGERF,R, R. C., and DA~IOSON, E. H. (19851. Molecular indices of cell lineage specification in sea urchin embryos. Sciwcr 226, 1153-1160. B.UXK, P. F., KNI(:HT, D. E., and WIIITAIXK, M. J. (1980). The relation between ionized calcium and cortical granule esocytosis in eggs of the sea urchin Echi~(rs wc~/w(t~rs. P,oc. K. Sot. Lonrlo)( U 207, 149 161. BEISIE(:EL, U., SCHNEIDER, It’. J., BHO~X, M. S., and Gol,rwtwN, J. L. (19821. Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholcsterolemia. J. Hiol. (‘l/rrt/. 257, 13150-13156. BENSON, S C., BENSON, N. C., and \VII.T, F. (1986). The organic matrix of the skeletal spiculc of sea urchin embryos. J. Cf,ll Birjl. 102, 187% 1886. BRANIIIIOKST, B. P. (1985). The informational content of the echinoderm egg. I)r “Developmental Biology: A Comprehensive Synthesis” (L. Brow-der, Ed.1 Vol. 1, pp. 525-576. Plenum, New York. B~UNI~ORST, B. P., and HIXPIIKI;YS, T. (19721. Stabilities of nuclear and messenger RNA molecules in sea urchin embryos. J (‘cl/ Rio/. 53,474-488. CARSON, D. D., FARACH, M. C., EARIzS, D. S., DECKER, G. L., and LENN.-\Rz, W. J. (1985). A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin. Cell 41, 639648. D.q\-II)SON, E. H. ( 19761. “Gene Activity in Early Development,” 2nd cd. Pp. U-55, If% Academic Press, New York. D4\ ILISON, E. H. (1986). “Gene Activity in Early Development,” 3rd rd Pp. 213-246. Academic Press, New York.

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