An Ovomucin-Like Protein on the Surface of Migrating Primordial Germ Cells of the Chick and Rat

Development 122, 915-923 (1996) 915 Printed in Great Britain © The Company of Biologists Limited 1996 DEV3249

An ovomucin-like protein on the surface of migrating primordial germ cells of the chick and rat

W. Halfter1,*, B. Schurer1, H.-M. Hasselhorn2, B. Christ2, E. Gimpel2 and H. H. Epperlein3 1University of Pittsburgh, Department of Neurobiology, 842 Scaife Hall, Pittsburgh, PA 15261, USA 2University of Freiburg, Department of Anatomy, Freiburg, Germany 3University of Dresden, Department of Anatomy, Dresden, Germany *Author for correspondence

SUMMARY

A mucin was discovered on the surface of migratory pri- is localized on the cell surface. The anti-OLP antibodies mordial germ cells (PGCs) from chick and rat embryos by also stained PGCs in the gonads of the rat embryo, showing means of two monoclonal antibodies. The protein was that the expression of this antigen on PGCs is phylogenet- found to be identical or closely related to ovomucin, a ically conserved. Ovomucin isolated from vitelline 600×103 relative molecular mass glycoprotein, and a major membrane prevented adhesion of ﬁbroblasts but not PGCs constituent of the vitelline membrane of the avian yolk. when used as a substratum in vitro. The anti-adhesive Based on its resemblance to ovomucin it is referred to as quality of the mucin resides in the sialic acid residues of the ovomucin-like protein (OLP). The OLP was expressed on carbohydrate side chains. We propose that OLP has a PGCs from E3 to E7 female, and from E3 to E12 male chick similar anti-adhesive quality as the ovomucin from vitelline embryos as the PGCs migrate and colonize the gonadal membrane, and that this anti-adhesive property is ridges. After the PGCs have settled in the gonads, they no important to prevent precocious adhesion of migrating longer express OLP. In tissue cultures of dissociated cells PGCs to blood vessel walls and to connective tissue in the from E6 gonads, OLP was present only on cells that were mesentery as they migrate toward the gonadal ridges. positive for PAS staining, the standard histological method to identify PGCs in the chick embryo. Since unﬁxed PGCs Key words: primordial germ cells, gonad development, cell were recognized by the antibodies, at least part of the OLP migration, mucins, chick, rat

INTRODUCTION Up to E6, the two gonads are indistinguishable, but from E7 onwards, the right gonad is abandoned by germ cells in the Primordial germ cells (PGCs) are an undifferentiated popula- female embryo and is visibly smaller than the left gonad tion of cells in the embryo that serve as precursors of germ (Ukeshima and Fujimoto, 1991; Ukeshima, 1994). cells. PGCs are generated outside the embryo and enter the The mechanism underlying the migration and the targeting embryonic gonadal anlage first by following routes along of PGCs to the genital ridges is still unknown. Several studies blood vessels and then by migrating through the mesenchyme showed that PGCs have the ability for ameboid movement of the hindgut. At their target, the genital ridges, PGCs undergo (Kuwana and Fujimoto, 1983), and can react chemotactically further differentiation and develop, according to the genetic to genital ridge tissue in vitro (Kuwana et al., 1986; Godin et background of the gonadal mesenchyme, into spermatogonia al., 1990; Godin and Wylie, 1991). Further, the PGCs are able or oogonia (for review, see Wylie and Heasman, 1993; to migrate using fibronectin as a substratum (Alvarez-Buylla Kuwana, 1993). and Merchant-Larios, 1986; ffrench-Constant et al., 1991), and In the chick embryo, the PGCs fist appear in the hypoblast fibronectin is found in the migratory route of PGCs (Fujimoto of the germinal crescent, constituting a narrow band near the et al., 1985). A hallmark of PGCs, however, is their reluctance head fold. From stage 10 to 16, the PGCs enter the vasculature to spread in tissue culture and their tendency to get washed and circulate throughout the entire embryo. By stage 17, they away even by mild rinsing (Buehr and McLaren, 1993) indi- exit the omphalomesenteric arteries into the splanchnic cating a weak adhesivity to the underlying matrix. mesoderm and migrate through the mesenchyme of the dorsal Here, we describe a cell surface mucin, ovomucin-like mesentery to the genital ridges, which are recognizable as two protein (OLP), on migrating chick and rat PGCs that may small bulges medial to the mesonephros. By stage 22, the cells provide an explanation for the weak adhesion of these cells to start to settle into the genital ridges and differentiate to oogonia the underlying matrix. Mucins are a subclass of glycoproteins and spermatogonia at later stages of development (Swift, 1914; with multiple O-linked carbohydrate side chains on a protein Meyer, 1964; Fujimoto et al., 1976; Ando and Fujimoto, 1983). core with tandem repeat peptide units (Strous and Dekker, 916 W. Halfter and others

1992). Mucins provide lubrication to mucous-type epithelia MEM (N-methylmaleimide; Sigma) to limit proteolysis. The enzyme- and modulate the cell and substratum adhesion of the mucin- treated samples were then extracted with DNase/4 M urea and boiled expressing cells (Hilkens et al., 1992). We propose that the in sample buffer as described above. Samples of ovomucin from OLP on the surface of PGCs facilitates the extensive migration vitelline membrane were also treated with 0.6 Units endoglycosidase of the cells on their way to the genital ridges by preventing F/N-glycosidase F (Sigma) for 14 hours at 37¡C after reductive alky- adhesion of the PGCs to blood vessel walls and to the mes- lation and in the presence of 0.1% Triton X-100. Samples of bovine submaxillary gland mucin (Sigma) were also tested for anti-OLP enchyme. immunoreactivity in western blots. Ovomucin isolation MATERIALS AND METHODS Vitelline membranes from freshly laid eggs were collected as described (Back et al., 1984). The ovomucin was enriched by extract- Monoclonal antibody (mAb) production ing the vitelline membranes from 15 eggs in 50 ml 8 M urea overnight The mAbs against OLP were isolated in two fusions against skin according to the method described by Kido and Doi (1988). The extracellular matrix. The matrix was isolated from the back skin of extract was centrifuged for 20 minutes at 15,000 rpm and the gelati- E6 chick embryos. Skin segments were dissected, treated with 2% nous pellet was collected. For further purification, the crude ovomucin Triton X-100 in distilled water for 30 minutes, and freed of the epi- was dissolved by stirring in 50 ml 8 M urea with 10 mM mercap- thelial cell layer by a stream of Ca2+- and Mg2+-free Hank’s solution toethanol for 4 hours (Robinson and Monsey, 1971). The reduced (CMF) over the skin surface. The matrix was further treated with 50 mucin was alkylated with 15 mM jodoacetamide for 1 hour in the dark µg/ml DNase (Sigma, St. Louis, MO) for 1 hour, and rinsed several and clarified by centrifugation at 15,000 rpm for 20 minutes. Four ml times. For immunization, the skin matrix of 20 E6 chick embryos was of Q-Sepharose was added to the mucin solution. After 1 hour of suspended in 200 µl CMF, mixed with the same amount of complete shaking, the beads were filtered and extensively washed in 4 M urea Freunds adjuvant and injected intraperitoneally into Balb/C mice. plus 0.5 M NaCl. The mucin was eluted from the beads by 20 ml 1.5 Boosts were given after 4 and 2 week intervals in incomplete M NaCl in 4 M urea. The eluate was dialyzed against water and adjuvant. Four days before fusion, a final boost without adjuvant was lyophilized. Fractions were tested by gel electrophoresis. Ovomucin given. The spleen cells were fused with X63Ag8.653 myeloma cells containing fractions were collected, lyophilized and taken up in CMF. (Kearny et al., 1979) as described by Fazekas et al. (1980). Hybridoma The concentration of the mucin was estimated by the dye-binding cells were raised in RPMI medium supplemented with 10% FCS and method of Minamide and Bamburg (1990). The isolated protein was 10% Origen cloning factor (Igen, Rockville, MD). Wells with clones separated by SDS-PAGE, and the protein bands were detected by were screened by immunostaining of sections from E6 chick trunk staining the gels with an Alcian blue/silver staining developed for the with hybridoma cell culture supernatants (see below). Positive clones detection of proteoglycans and mucins (Moller et al., 1993). were subcloned twice with the limited dilution method. Two Ovomucin from egg white was isolated by ultracentrifugation as hybridoma clones from two fusions were used in this study: clones described by Sleigh et al. (1973). 7B7 and 1B3. The antibodies were both IgMs. A monoclonal antibody to the Stage Specific Embryonic Antigen, SSEA-1 (clone MC-480, Histology Solter and Knowles, 1978) was obtained as hybridoma supernatant Trunks from E3 to E16 chick and E15 rat embryos were fixed in 4% from the Developmental Studies Hybridoma Bank maintained by the paraformaldehyde in 0.1 M potassium phosphate buffer pH 7.4 for 1 Department of Pharmacology and Molecular Science, John Hopkins hour. The bodies were washed in CMF and cryoprotected with 30% University School of Medicine, Baltimore, MD, and the Department sucrose for 4 hours. The specimens were sectioned after embedding of Biology, University of Iowa, Iowa City, IA, under contract NO.- and mounting in OCT compound (Miles, Elkhart, IN) with a Reichert HD-2-3144 from the NICHD. An anti-laminin antibody (clone 3H11) Jung (Nussloch, FRG) cryostat at 25 µm. Sections were mounted on served as reference for the distribution of basal laminae in the chick gelatin/chrome alum-subbed slides and incubated with supernatants embryo (Halfter, 1993). from the hybridoma cell cultures for 1 hour. After 3 rinses, the sections were incubated with a labeled secondary antibody for another Gel electrophoresis and western blotting hour. As secondary antibody, a Cy-3-labeled, affinity-purified µ- Gonads and mesonephros from E6 chick embryos, and vitelline chain-specific goat anti-mouse antibody was used for the 7B7, 1B3 membrane from E0 embryos were isolated, incubated with 50 µg/ml and the SSEA-1 mAbs. For the 3H11 anti-laminin mAb, a Cy-3- DNase I (Sigma) and extracted with 8 M urea for 30 minutes. The labeled goat anti-mouse (H+L) antibody was used (Jackson Immuno extract was centrifuged at 10,000 rpm for 2 minutes and the super- Research, West Grove, PA). After two final rinses, the specimens natant collected. The samples were boiled in sample buffer for 2 were examined with an epifluorescence microscope (Zeiss, minutes (Laemmli, 1970). The proteins were separated by a 3.6-14% Thornwood, NY). PGCs in rat embryo sections were detected by his- SDS-PAGE and stained with colloidal Coomassie brilliant blue tochemical staining for alkaline phosphatase (Chiquoine, 1956; (Sigma) or Alcian blue as described (Moller, 1993). For western blots, Merchant-Larios et al., 1985). In a series of experiments, 200 µl- gels were electrophoretically transferred to nitrocellulose filters samples of each of the 2 anti-OLP and the anti-SSEA-1 hybridoma (Towbin et al., 1979). After blocking with 5% skimmed milk in supernatants were mixed with 5µg of the Lewis-x trisaccharide TBS/Tween (Tris-buffered saline with 0.5% Tween 20), the blots fucosyl-lactosamine (Sigma) and then applied to the sections. The per- were incubated with the hybridoma supernatants for 1 hour. The blots sistence or the abolishment of staining indicated whether the anti- were rinsed three times in TBS/Tween, incubated with 1:5000 diluted bodies recognized the Lewis-x trisaccharide epitope or not. To check (TBS/Tween) alkaline phosphatase-conjugated µ-chain-specific goat whether the mAbs also recognize glycolipids, sections were rinsed anti-mouse (Jackson Laboratories) for 1 hour and finally developed with methanol and methanol/chloroform prior to the antibody incu- with NBT/BCIP as described previously (Halfter, 1993). Some tissue bation. samples were pretreated with chondroitinase ABC, keratanase, hyaluronidase (Boehringer Mannheim, Indianapolis, IN), heparitinase Tissue culture and cell adhesion assays (Seikagaku, Rockville, MA) and 0.2 Units neuraminidase (Sigma) for Gonads from E6 chick embryos were dissected and incubated with 1 to 3 hours at 37¡C under buffer and pH conditions recommended O.O5% trypsin, 0.53 mM EDTA (Gibco/BRL) for 15 minutes at 37oC. by the manufacturers. Enzyme digestions were performed in the After washing in 2% BSA/Dulbecco’s modified Eagle’s medium presence of 1 mM PMSF (phenylmethyl sulfonyl fluoride; Sigma) and (DMEM) and the addition of DNase I to a final concentration of 20 Ovomucin on migrating primordial germ cells 917

µg/ml, the gonads were dissociated in culture medium (DMEM) with lane 3) suggested that it could be identical or related to 10% new born calf serum) by trituration. The cells were plated onto ovomucin, a prominent high molecular mass, sialylated con- dishes coated with polylysine. After 24 hours in vitro, the cultures stituent of the vitelline membrane (Kido and Doi, 1988). To were fixed in 4% paraformaldehyde in CMF for 15 minutes. The verify the identity or close relationship of the antigen to cultures were rinsed with CMF and stained with 7B7 or 1B3 mAbs ovomucin, ovomucin was isolated from vitelline membrane. as described above. Periodic acid-Schiff (PAS) staining of primordial Purified ovomucin isolated from vitelline membrane showed germ cells was performed according to standard procedures (Meyer, 1960). up as a diffuse smear with a relative molecular mass of × 3 Cell adhesion assays on purified ovomucin, BSA and fibronectin- approximately 700 10 . The abundance of sialic acid in coated dishes were performed using chick embryo dermal fibroblasts ovomucin was reflected by its very weak staining with and cells from E6 chick gonads. The cells were dissociated as Coomassie brilliant blue (Fig. 1, lane 4) and its strong described above. The culture dishes were coated with nitrocellulose staining with the cationic dye Alcian blue (Fig. 1, lane 5). (Lagenaur and Lemmon, 1987) followed by 20 µg/ml ovomucin, 20 Further, because of the presence of sialic acid in mucins, µg/ml BSA (Sigma) or 20 µg/ml fibronectin (Gibco/BRL). Other ovomucin is highly negatively charged and binds strongly to nitrocellulose-treated dishes were coated with mixtures of fibronectin Q-Sepharose, an anion exchange matrix. The binding to Q- µ µ µ (2.5 g/ml) with ovomucin (5 g/ml) or BSA (20 g/ml). Excess Sepharose was used to purify ovomucin from vitelline binding sites were blocked with 2% BSA/DMEM for 15 minutes. In membrane. The purified ovomucin was then probed for reac- some cases, the substrata were treated with 0.1 U/ml neuraminidase for 1 hour in CMF at 37oC. Finally, the dishes were rinsed 3 times tivity with the two mAbs. Western blots showed that the with 2% BSA/DMEM and the cells were pipetted onto the substrata. purified ovomucin from vitelline membrane was highly After 2 hours at 37¡C, non-adherent cells were rinsed off. After two immunoreactive with the 7B7 and 1B3 mAbs (Fig. 1, lane 6). more rinses, the cultures were fixed with 2.5% glutaraldehyde. The The identical molecular mass of the antigen in all tissue cell nuclei were stained with bisbenzimide (Sigma), and the number samples, the identical diffuse banding pattern of the antigen of bound cells per unit surface area were estimated by counting the in western blots and the strong immunoreactivity to purified fluorescent nuclei. PGCs were counted after staining with PAS. The ovomucin suggest that the 7B7 and 1B3 mAbs recognize a cell counts were expressed as a percentage using the number of cells adhering to the control substratum fibronectin as 100%. The experiments were performed in triplicate and repeated in at least three inde- pendent assays. To enrich PGCs by panning (DeFelici and Pesce, 1995), dissociated cells from E6 gonads were incubated with 7B7 or 1B3 hybridoma supernatant for 1 hr. The cells were spun down and washed twice in culture medium. The cells were suspended in 1 ml of culture medium/10% new born calf serum and plated onto dishes coated with 20 µg/ml µ-chain-specific goat anti-mouse antibody. The dishes had been pre-treated with nitrocellulose (Lagenaur and Lemmon, 1989). Two hours after plating, non-adherent cells were washed off, and the dishes were rinsed two more times. PGCs were detected by PAS staining.

RESULTS

Two mAbs, clones 7B7 and 1B3, were isolated that recognized migrating PGCs in the chick and rat embryo. Western blot analysis and purification of the antigen Fig. 1. Western blots and gels stained with Coomassie and Alcian In western blots using gonadal tissue (Fig. 1, lane 1), blue showing the identity of the antigen recognized by the 7B7 and mesonephros (Fig. 1, lane 2) and cartilage (not shown) from E6, 1B3 mAbs as ovomucin or a protein closely related to ovomucin. In western blots using samples of E6 chick gonads (lane 1), or vitelline membrane (Fig. 1, lane 3) from E0 chick embryos, mesonephros (lane 2) and E0 vitelline membrane (lane 3), the 7B7 3 the mAbs labeled a single band with a relative molecular mass mAb recognized a single band of approximately 700×10 Mr. The of approximately 700×103. The diffuse banding indicated that abundance in vitelline membrane (lane 3) and the high molecular the antigen is highly glycosylated and could either be a proteo- mass of the antigen suggested that the antigen might be ovomucin. glycan or a mucin. Its identity as a proteoglycan was excluded An ovomucin-enriched fraction from vitelline membrane separated by the fact that the antigen did not shift in molecular mass after by PAGE is shown in lanes 4 and 5. The high molecular mass the samples had been treated with chondroitinase, heparitinase, ovomucin is barely detectable by Coomassie blue staining (lane 4), keratanase or hyaluronidase (not shown). The carbohydrate but easily identified after a combined staining with Coomassie and chains did not seem to be N-linked, since the molecular mass Alcian blue (lane 5). Ovomucin, when solubilized and purified by of the antigen did not shift when samples from vitelline ion exchange chromatography, was highly immunoreactive for the 7B7 antibody (lane 6). The immunoreactivity and the molecular membrane were treated with endoglycosidase F/N-glycosidase mass of ovomucin were unaffected by treatment of the samples with F (Fig. 1, lane 7). The immunoreactivity of the antigen was endoglycosidase F/N-glycosidase F (lane 7), but were reduced by weaker and the molecular mass was reduced, when the samples treatment with neuraminidase (lane 8). The molecular mass markers 3 were treated with neuraminidase (Fig. 1, lane 8). were non-reduced α2 macroglobulin (725×10 Mr) and myosin 3 The abundance of the antigen in vitelline membrane (Fig. 1, (200×10 Mr). 918 W. Halfter and others

A B A B

C D

Fig. 3. Adjacent sections of E5 gonads (G) stained with the 1B3 anti- Fig. 2. The anti-OLP mAb 7B7 recognizes PGCs in the developing OLP (A,C,E,F) and the anti-SSEA-1 mAb (B,D). Both mAbs stain chick embryo. Cross sections through the lumbar region of an E5 PGCs in the embryonic gonads (A,B). The PGC staining by the anti- chick embryo, stained with an antibody to laminin (A), shows the SSEA-1 antibody was blocked by incubating the antibody with localization of the gonadal ridges (arrow) next to the mesonephros. A Lewis-x trisaccharide (D), whereas the staining by the anti-OLP high power view of one of the gonadal ridges, stained for laminin, is antibody was not blocked by the saccharide (C). The OLP-staining of shown in B. The adjacent section has been stained with the 7B7 mAb PGCs was, however, blocked by incubating the anti-OLP mAb with (C). Note the presence of labeled cells in the gonadal ridges. The 5 µg of ovomucin, purified from vitelline membrane by ion exchange gonads of an E7 chick embryo stained with the 7B7 antibody is chromatography (F). Bar, 50 µm. shown in D. The number of labeled cells has increased. Bar (A) 100 µm; (B-D) 50 µm. single molecule, and that this molecule is ovomucin or a OLP is present on the surface of migrating PGCs mucin closely related to ovomucin. Taking into consideration Transverse sections through the trunk of the chick embryo that ovomucin may not represent a single protein, rather a showed OLP immunoreactivity in several locations: OLP was family of similar mucins, we will refer to the antigen on the found around the notochord (Fig. 5A), in developing cartilage, PGCs recognized by the 7B7 and 1B3 mAbs as ovomucin- in the mucosal lining of the gut, the mesonephros (Fig. 5A), in like protein (OLP). Ovomucin isolated from egg white the cornea, in the dermis and in infoldings of the epidermal (Sleigh et al., 1973) was only very weakly immunoreactive surface, such as the nasal pit (not shown). for the mAbs, even at concentration 10 times higher than A few large cells expressing OLP on their surface were ovomucin from vitelline membrane, indicating that the two detected in the E5 gonadal ridges (Fig. 2C). OLP-expressing components are not identical (not shown). To see whether the cells were found in much greater number in the gonads of the anti-OLP mAbs detect other mucins as well, we probed more advanced E7 embryos (Fig. 2D). The immunofluor- mucin from submaxillary glands in western blots. Results escence delineated the surface of the cells, suggesting a local- showed that the anti-OLP mAbs did not recognize the sub- ization on the cell surface. The large size and the distribution maxillary gland-derived mucin (not shown). of the OLP-expressing cells suggested that they are PGCs col- Ovomucin on migrating primordial germ cells 919 onizing the gonadal anlagen. The identity of the OLP-positive The distribution of OLP-positive cells during development cells as PGCs was confirmed by comparing the distribution of provided further evidence that they were migratory PGCs. The the labeled cells with PGCs labeled immunocytochemically by first OLP-positive cells were found between E3 and E4 in the the anti-SSEA-1 antibody (Fig. 3), by tissue culture experiments (Fig. 4) and by following the distribution of OLP-positive cells during development (Fig. 5). A B The size and the location of the OLP- positive cells (Fig. 3A) were analogous to the size and location of PGCs labeled with the anti-SSEA-1 antibody, an antibody that recognizes the Lewis-X trisaccharide fucosyl-lactosamine (Fig. 3B). The presence of the Lewis-X epitope has previously been used to detect migrating PGCs in the mouse and rat embryo (Solter and Knowles, 1978). The staining of the PGCs C D with the anti-SSEA-1 antibody could be blocked by incubation of the mAb with Lewis-X trisaccharide as shown in Fig. 3D, whereas the labeling of the PGCs with the anti-OLP antibodies could not be blocked by this trisaccharide (Fig. 3C), showing that the two types of antibodies react to different epitopes. The labeling of PGCs and all the other structures stained by the anti-OLP mAbs, however, was abolished when the 7B7 and 1B3 mAbs were pre- F incubated with purified ovomucin from E vitelline membrane, further supporting the idea that the antigen recognized by the anti- OLP mAbs is identical to ovomucin from vitelline membrane (Fig. 3F). Treatment of the sections with methanol or methanol/chloroform prior to antibody staining did not affect the anti-OLP labeling in cartilage, dermis, kidney or PGCs, indicating that the anti-OLP mAbs do not recognize glycolipids (not shown). H When single cell cultures from E6 chick G gonads were immunostained with the anti- OLP antibodies, only a fraction of the cells were immunoreactive (Fig. 4A,C,E). The immunoreactive cells were large, located on top of a monolayer of gonad-derived fibroblasts and did not adhere very well to the culture dishes or to the underlying fibroblasts. In many cases, the OLP- immunoreactive cells aggregated with each Fig. 4. Tissue culture confirmed that the cells recognized by the anti-OLP mAbs are PGCs. other (Fig. 4C,E). Staining of the Cells from E6 chick gonads were dissociated and cultured for 24 hours. The cultures were immunoreactive cells with PAS (Fig. 4F), stained with the 7B7 mAb. Only a minority of the cells are immunoreactive (A,C,E). The which is specific for PGCs of the chick labeled cells were large and differed from the majority of fibroblast-like cells that cover the embryo (Meyer, 1960), showed that the dish. The labeled cells did not spread onto the substratum and in most of the cases, were PAS-positive cells were identical to the located on top of the fibroblast monolayer. The OLP-positive cells formed often aggregates cells labeled with the two mAbs (Fig. (C,E). The cells were also labeled by incubating unfixed cultures with the antibodies (A), showing that the antigen is located on the cell surface. Double labeling of the cultures with 4E,F). Since unfixed cells were stained the 7B7 mAb and PAS staining showed that the cells positive for 7B7 (E) were also with the anti-OLP mAbs (Fig. 4A), at least positive for PAS (F), confirming the identity of the 7B7-positive cells as PGCs. PAS- a portion of the antigen is localized on the positive PGCs were enriched by plating dissociated cells from E6 gonadal ridge onto surface of these cells. This surface local- purified ovomucin substratum (G), or by incubating dissociated gonadal cells with an OLP ization was used to immunopurify PGCs by mAb and binding the antibody-labeled cells to a culture dish coated with a µ-chain-specific panning (Fig. 4H). anti-mouse antibody (H). Bar, (A-F) 25 µm; (G,H) 100 µm. 920 W. Halfter and others mesentery, dorsal to the gut. By E4/E4.5, the first OLP- conclude that the cells identified by the anti-OLP mAbs are immunoreactive cells were found at the edges of the gonadal migratory PGCs. ridges (Fig. 5A,B,C), while most of the OLP-expressing cells To investigate whether OLP is also present on PGCs of rat were still in the mesenteries (Fig. 5A). The distribution of embryos, sections through the gonadal region of E15 rat OLP-positive cells at this stage showed a trail of immunore- embryos were stained with 7B7 and 1B3 antibodies (Fig. active cells from the mesenteries leading to the gonadal ridges 6C,D,E). The PGCs in the rat embryo were visualized by (Fig. 5B,C). During further development, the number of alkaline phosphatase histochemistry (Fig. 6A,B). As shown in labeled cells in the gonads increased and reached a maximum Fig. 6C,D,E, the mAbs stained the PGCs in the gonads and in between E6 and E7 (Fig. 5D,E). The number of OLP-positive the dorsal mesentery on the way to the gonads. The anti-OLP cells declined, and they were finally no longer detectable in staining in the rat also delineated the surface of the PGCs (Fig. gonads of male embryos by E12 (Fig. 5F), and in the female 6D,E). embryo by E7 (Fig. 5G). The distribution of OLP-immunoreactive cells matched precisely the distribution of migratory Ovomucin as a substratum for cell adhesion PGCs as reported in previous publications (Swift, 1914; To find out whether ovomucin or related mucins have adhesive Meyer, 1964; Fujimoto et al., 1976). Based on our studies, we or non-adhesive properties for mesenchymal and gonadal cells, ovomucin was isolated from vitelline membrane and used as a substratum to test its adhesive or anti-adhesive quality for A B dermal fibroblasts and gonadal cells in vitro (Figs 7, 8, 9). Culture dishes were coated with ovomucin that had been purified by ion exchange-chromatography. Dermal fibroblasts and dissociated cells from the chick E6 gonads were plated A B

C D E C

D E F G

Fig. 5. The expression of OLP on the surface of PGCs is developmentally regulated and restricted to PGCs that are en route to Fig. 6. OLP was also detectable on the surface of PGCs of the rat the gonadal ridges or in the process of colonizing the gonads. The embryo. Cross sections through the lumbar region of an E15 rat ﬁrst OLP-positive PGCs were detected in the dorsal mesentery embryo were stained with alkaline phosphatase to label the PGCs between E3 and E4 (A; arrows). A stream of labeled cells leading and the gonads (A,B). An adjacent section was stained with the 7B7 into the gonadal ridges were found at E5 (B, arrow; C). The antibody (C). The same cells that were stained with alkaline maximum number of labeled cells in the gonads was found between phosphatase were immunoreactive for the 7B7 antibody. A high E6 (D) and E8 (E). OLP-positive cells decreased in number by E12 power micrograph shows that the PGC labeling delineated the in the male (F) and by E7 in the female (G) chick embryo. N, membrane of the cells (D,E). Labeled cells were also found in the notochord; M, mesonephros. Bar, (A) 100 µm; (B-G) 50 µm. mesentery (E). (A) 200 µm; (B,C) 100 µm; (D,E) 50 µm. Ovomucin on migrating primordial germ cells 921

Fig. 9. Graphic representation of cell adhesion assays demonstrating adhesion of dermal fibroblasts to fibronectin (FN) and mixtures of Fig. 7. Graphic representation of the adhesion of fibroblasts and fibronectin with BSA (FN+BSA) and ovomucin (FN+OV). Cell gonadal cells to fibronectin, BSA and ovomucin. Dermal fibroblasts adhesion to fibronectin was inhibited when it was mixed with 20 (left) adhere strongly to a fibronectin substratum (FN), but not to µg/ml BSA, by about 40%. When fibronectin was mixed with only 5 bovine serum albumin (BSA) or ovomucin-coated dishes (OV). µg/ml ovomucin, cell adhesion was blocked by over 80%. The anti- Dissociated cells from E6 chick gonads (right) adhere well to adhesive activity of ovomucin was reversed by treatment of the fibronectin (FN). About 30% of these adherent cells are PGCs as fibronectin/ovomucin substratum with neuraminidase determined by PAS staining (not shown). Only a few of the gonadal (FN+OV+NEU). Treatment of fibronectin alone with neuraminidase cells adhere to BSA (BSA). A larger number of gonadal cells adhere (FN+NEU) led to only a minor decline in cell adhesion. The to ovomucin (OV). 63% of the cells adhering to ovomucin were experiments show that ovomucin inhibits cell adhesion to fibronectin PGCs (not shown). by means of its neuraminic acid residues in the carbohydrate chains. onto these dishes. The adhesion of cells to ovomucin was To detect whether ovomucin can counteract the adhesive compared with the cell adhesion to fibronectin as an adhesive quality of fibronectin, mixtures of fibronectin and ovomucin and BSA as a non-adhesive control substratum. As shown in were used as substrata for dermal fibroblasts (Fig.8). Results Fig. 7, a large number of dermal fibroblasts and gonadal cells showed that by increasing the ratio of ovomucin to fibronectin, adhered to fibronectin, whereas few dermal fibroblasts and a smaller number of fibroblasts were able to adhere to the sub- gonadal cells were able to adhere to ovomucin and BSA. PAS stratum. For example, less than 5% of the cells bound to a sub- staining of the gonadal cultures showed that 30±8% of the cells stratum of equal quantities of fibronectin and ovomucin, adhering to fibronectin were PGCs. Gonadal cells bound to the compared to 100% when fibronectin was present alone (Figs ovomucin substratum (Fig. 7) in greater number than the 8, 9). This reduction of cell attachment was not due to the dermal fibroblasts. PAS staining showed that 63±10% of the dilution of fibronectin, since mixing fibronectin with BSA at cells that adhered to ovomucin were PGCs (Fig. 4G). Thus, two times higher concentration than ovomucin reduced cell PGCs can adhere to ovomucin, whereas fibroblasts cannot. adhesion by less than 40% compared to fibronectin alone (Fig. 9). The anti-adhesive activity of ovomucin in mixtures with fibronectin was abolished by treatment with neuraminidase, showing that the sialic acid residues are most likely responsible for the anti-adhesive activity of ovomucin (Fig. 9). Treatment of fibronectin with neuraminidase had no effect on the adhesion of cells to this substratum (Fig. 9).

DISCUSSION

OLP in the chick embryo Fig. 8. Graphic representation of cell adhesion assays demonstrating This study demonstrates the presence of a mucin on the cell that the adhesion of dermal fibroblasts to fibronectin is inhibited by surface of PGCs as they migrate to and colonize the gonadal ovomucin. The base of culture dishes were coated with fibronectin ridges of the chick embryo. Mucins represent a special class of alone and mixtures of fibronectin (FN) and ovomucin (OV). The glycoproteins, whose carbohydrate portion constitutes between concentration of ovomucin in the mixtures was held constant at 5 50 and 80% of their molecular mass. The abundance of sialic µg/ml, whereas the concentration of fibronectin varied at 20, 10, 5 µ acid residues and the occasional sulfation of the carbohydrate and 2.5 g/ml. Cell adhesion to the experimental substrata was chains provide the mucins with a net negative charge, which expressed as a percentage relative to 100% cell adhesion to fibronectin at a concentration of 2.5 µg/ml. The data show that by is the reason for their staining with Alcian blue, their high decreasing the ratio of fibronectin to ovomucin (FN/OV), cell buoyant density during CsCl gradient centrifugation and their adhesion dropped. For example, the combination of ovomucin with strong binding to anion exchange matrices. The heterogeneity fibronectin in a ration of 1:1 (5 FN/5 OV) led to a drop in cell in the carbohydrate side chains results in the diffuse banding adhesivity of over 90% compared with cell adhesion to fibronectin of mucin in SDS-PAGE. Reductive alkylation in high molar alone. urea is required to solubilize mucins, since mucin monomers 922 W. Halfter and others are frequently connected to each other by intermolecular PGCs in the chick and rat embryo, by staining of the disulfide bridges. ovomucin-positive cells from chick gonads with PAS in vitro, One proposed function of mucins is to provide lubrication and by the co-localization of ovomucin-positive cells with and protection to mucous-type epithelia. The abundance of alkaline phosphatase-positive PGCs in the rat gonads. The mucins on the surface of tumor cells was supposed to be presence of OLP on the surface of PGCs was demonstrated by responsible for the escape of some tumor cells from the attack staining live cells with the antibodies, and by using the anti- by the body’s immune system and may even be related to the OLP mAbs to enrich PAS-positive PGCs by panning. The cell metastatic spreading capability of some of the tumors (for surface expression of the OLP by PGCs was transient and dis- review see Hilkens et al., 1992; Strous and Dekker, 1992). appeared in the female chick embryo by day 7 and by E12 in The mucin on the surface of the PGCs was identified as the male embryo. The temporal expression of OLP on PGCs ovomucin or a protein closely related to ovomucin. In western was similar to that of the Lewis-x antigen, also referred to as blots, the antigen appeared as a high relative molecular mass Stage-Specific Antigen (SSEA; Solter and Knowles, 1978). smear of 700×103. The antigen was present in gonads and in However, whereas the anti-SSEA-1 antibody staining was the mesonephros and, most abundantly, in the vitelline blocked by incubating the mAb with Lewis-x trisaccharide, the membrane, a membrane that covers the avian egg yolk. In all anti-OLP staining was not blocked, showing that the anti-OLP tissue that showed anti-OLP staining, such as gonads, mAbs recognize a different epitope. Another antigen was mesonephros, cartilage and vitelline membrane, only a single recently identified on PGCs of the chick embryo (Maeda et al., band with identical molecular mass was detected in western 1994). In western blots, the antigen shows up a sharp band with blots. Likewise, the banding pattern in western blots as a a relative molecular mass of 109×103, which is different from diffuse smear was identical for all tissues. The western blot OLP. data indicate that the anti-OLP mAbs recognize an epitope of a single molecule, rather than an epitope that is present on Ovomucin has anti-adhesive activity for fibroblasts several proteins. Furthermore, lipid extraction from tissue but not for PGCs sections with methanol or methanol/chloroform did not affect The limited amount of embryonic gonadal tissue precluded an the anti-OLP labeling, indicating that the antigen is not present isolation of OLP from gonads for cell adhesion or tissue culture in glycolipids. experiments. To find out how mesenchymal and gonadal cells The composition of the vitelline membrane has previously react to ovomucin-type glycoproteins, ovomucin was isolated been studied in detail (Back et al., 1984; Kido and Doi, 1988). from vitelline membrane and assayed for adhesive or anti- 3 Its largest constituent is ovomucin, a 700×10 Mr sialylated adhesive activity in vitro. Adhesion assays using dermal mucin. Ovomucin is highly negatively charged as shown by its fibroblasts and dissociated cells from E6 chick gonads showed staining with Alcian blue and its strong affinity to Q- that ovomucin does not support fibroblast adhesion. Moreover, Sepharose, an anion exchange matrix. Solubilized ovomucin experiments using mixtures of fibronectin with ovomucin as a was readily purified from vitelline membrane by ion exchange substratum showed that ovomucin counteracted the adhesive chromatography, and the purified ovomucin turned out to be activity of fibronectin. This anti-adhesive activity was not a highly immunoreactive for the 7B7 and 1B3 mAbs. Further- non-specific effect, since dilution of fibronectin with BSA even more, the immunohistochemical staining of cartilage, kidney at higher concentrations lowered the fibronectin-dependent and gonads in tissue sections was blocked by pre-incubating fibroblasts adhesion to a much lesser degree. Furthermore, we the anti-OLP mAbs with purified ovomucin from vitelline could show that the anti-adhesive activity of ovomucin is membrane. Our studies strongly suggest that the 700×10 Μr dependent on the presence of sialic acid on its carbohydrate antigen in embryonic kidney, cartilage and on the surface of side chains. While the present experiments do not provide migrating PGCs that is recognized by the 7B7 and 1B3 mAbs direct evidence that ovomucin on PGCs inhibit their adhesion is identical to ovomucin from vitelline membrane. Since to the embryonic mesenchyme, our cell adhesion assays ovomucin may exist as a family of related proteins, it is strongly suggest that cells expressing ovomucin or a similar unknown whether the ovomucin from PGCs and from vitelline type of molecule on their surface adhere less strongly to the membrane are identical in all molecular details. We, therefore, surrounding fibroblast/mesenchymal tissue. The anti-adhesive referred to the surface antigen on PGCs recognized by the 7B7 quality of ovomucin is evident even in the presence of large and 1B3 mAbs as ovomucin-like protein (OLP). Ovomucin is quantities of fibronectin, the most abundant cell adhesion also known as a constituent of the egg white (Robinson and protein in the embryonic mesenchyme. Monsey, 1971; Sleigh et al., 1973). The faint immunoreactiv- In all vertebrates studied so far, PGCs are generated during ity of purified ovomucin from egg white with the 7B7 and the early embryonic development in the extra-embryonic areas. To 1B3 antibodies, however, indicated that ovomucin from colonize the gonads, the PGCs travel through the embryo via vitelline membrane and egg white are not identical and blood vessels and via the dorsal mesentery to reach the gonadal supported the idea that the ovomucins represent a family of ridges. It is still unknown how PGCs colonize this location of similar but yet distinctive glycoproteins. the embryo. It is also unclear how the PGCs migrate through mesenchymal areas of the embryo that have abundant OLP on migrating PGCs fibronectin without being stopped. Based on the present results, Immunocytochemistry showed that PGCs in the early stages of we propose that the anti-adhesive ovomucin on the cell surface gonad colonization and on their route to the gonads express provides a means to avoid precocious attachment of the PGCs OLP on their surface. The identity of the OLP-immunoreac- to blood vessels and to fibronectin-rich mesenchymal tissues. tive cells as PGCs was confirmed by showing that the location Consistent with the function of ovomucin, to prevent adhesion of the OLP-positive cells matched the location of migrating of the PGCs on their way to the gonads, is its temporal Ovomucin on migrating primordial germ cells 923 expression at only those stages when the PGCs are migratory. membrane-associated mucins and their adhesion-modulating property. When the PGCs have reached their target, the mucin is no Trends Biochem. Sci. 17, 359-363. longer expressed. The poor adhesion of mucin-expressing Kearney J. F., Radbruch A., Liesegang B. and Rajewsky K. (1979). A mouse myeloma cell line that has lost immunoglobulin expression but tumor cells and mucin-transfected cells is consistent with the permits the construction of antibody-secreting hybridoma cell lines. J. hypothesis of the anti-adhesive function of an ovomucin-like Immunol. 123, 1548-1558. antigen on PGCs (for review see Hilkens et al., 1992). While Kido S. and Doi Y. (1988). Separation and properties of the inner and outer ovomucin inhibits fibroblasts adhesion, the present study also layers of the vitelline membrane of hen’s eggs. Poultry Sci. 67, 476-486. Kuwana T. and Fujimoto T. (1983). Active locomotion of human primordial showed that ovomucin allows the attachment of PGCs. Since germ cells in vitro. Anat. Rec. 205, 21-26. PGCs tend to aggregate in vitro and in vivo, our data suggest Kuwana T., Maeda-Suga H. and Fujimoto T. (1986). Attraction of that cell surface ovomucin could be responsible for the mutual primordial germ cells by gonadal anlage in vitro. Anat. Rec. 215, 403-406. aggregation of PGCs. Whether ovomucin has a substrate or Kuwana T. (1993). Migration of avian primordial germ cells toward the receptor function in the gonadal ridge targeting remains to be gonadal anlage. Dev. Growth Differ. 35, 237-243. Laemmli U. K. (197). Cleavage of structural proteins during the assembly of seen. the head of bacteriophage T4. Nature 227, 680-685. Lagenaur K. and Lemmon V. (1987). An L1-like molecule, 8D9 antigen, is a We would like to thank Dr R. Tucker for critical reading of the potent substrate for neurite extension. Proc. Natl. Acad. Sci. USA 84, 7753- manuscript. The work was supported by the grant NBS-9021474 from 7757. NSF to W.H. and B. S., and by the grant Ep 8/4-2 from DFG to H. Maeda S., Ohsako S., Kurohmaru M., Hayashi Y. and Nishida T. (1994). H. E. and H.-M. H. from DFG. 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