DEVELOPMENTAL

BIOLOGY

70, 89-104 (1979)

Analyses of Cell Surface and Secreted Proteins of Primary Cultures Mouse Extraembryonic Membranes ANTON M. JETTEN, M.E.R. Roche Institute

of Molecular

JETTEN,AND MICHAEL Biology,

I. SHERMAN*

Nutley, NeLc Jersey 07110

Received August 23, 1978; accepted in revised form January

8, 1979

Procedures have been developed for primary culture of 13th day mouse parietal and visceral endoderm, yolk sac mesoderm, and amnion cells. We have analyzed cell surface and secreted proteins of these cultures by labeling the cells with radioactive iodine, glucosamine, or amino acids, and/or by immunofluorescence. Cell surface and secreted proteins of visceral endoderm, yolk sac mesoderm, and amnion cells resemble each other closely, whereas parietal endoderm cells are strikingly different. Unlike the other cell types, parietal endoderm cells synthesize and secrete substantial quantities of a protein tentatively identified as procollagen. These cells also secrete a number of other glycoproteins not observed in the media from the other cultures. It is proposed that the procollagen and one or more of the other unique, secreted glycoproteins are normally constituents of Reichert’s membrane. Compared to the other cultures, parietal endoderm cells appear to be deficient in production of LETS protein. However, parietal endoderm-Reichert’s membrane complexes analyzed by immunofluorescence directly after dissection from the uterus show an abundant association with LETS protein. It is not clear whether this LETS protein is actually synthesized by the parietal endoderm cells themselves. If so, it is possible that this protein is rapidly degraded after its secretion in parietal endoderm primary cultures. The studies reported here represent a fist step in the characterization of cell surface properties of embryonic and extraembryonic cell types. The information already accumulated should be useful in investigations aimed at identification of cells derived from blastocysts and teratocarcinomas in vitro. INTRODUCTION

We have recently analyzed the extraembryonic tissues of 11th and 13th day mouse conceptuses for the presence of a variety of enzymes (Sherman and Atienza-Samols, 1979). In that study, we found that distinctions could be drawn between amnion, yolk sac mesoderm, visceral endoderm, and parietal endoderm on the basis of enzyme content. However, there are limitations to the use of these enzyme systems as markers for monitoring development of extraembryonic tissues. For example, some of the enzymes under study are not qualitative markers, i.e., they are present in all extraembryonic tissues but show substantial quantitative differences among the tissues. Such markers might be of limited use in

efforts to trace the origin, location, and development of the various cell types at early stages when it might not be possible to obtain pure preparations, but only a mixture of cell types differing in specific enzyme activities. Studies on cell surface proteins (CSP)’ have suggested that each cell type has its own characteristic “signature” (e.g., Glick et al., 1973; Hynes and Wyke, 1975; Hynes et al., 1976; Jetten, A., Atienza-Samols, S., and Sherman, M., in preparation) and that the CSP composition is susceptible to change when cells undergo alterations in state, e.g., during transformation (Brady and Fishman, 1975; Nicolson, 1975; Hynes, 1976) or differentiation (Hynes et al., 1976; Zetter and Martin, 1978; Jetten et al., 1979).

’ To whom dressed.

’ Abbreviations used: CSP, cell surface protein(s); PBS, phosphate-buffered saline, solution A of Dulbecco; CIG, cold-insoluble globulin.

requests

for reprints

should

be ad-

89 0012-1606/79/050089-16$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

DEVELOPMENTAL BIOLOGY VOLUME70,1979

90

Therefore, in the hope of finding specific CSP for each of the extraembryonic cell types under study, we have endeavored to characterize the proteins on the surface of visceral and parietal endoderm, yolk sac mesoderm, and amnion cells, both by electrophoresis of labeled CSP and by immunofluorescence with the use of selected antisera. We have also begun to investigate the nature of proteins secreted by these cells. These analyses require successful culture of the different extraembryonic cell types. This communication describes procedures for preparing primary cultures of extraembryonic cells and compares the CSP and the secreted proteins of the different cell types. MATERIALS

AND

METHODS

Preparation of Primary Cultures We used 13th gestation day amnion, yolk sac, and parietal endoderm cells to generate primary cultures. Yolk sacs were separated into mesoderm and endoderm layers as described below. Amnion and yolk sac mesoderm cells were washed in phosphatebuffered saline (PBS; solution A of Dulbecco) and incubated with 0.05% trypsin0.02% EDTA (Gibco) for 10 min at 37°C with shaking to disaggregate them into single cells and small clumps. After being pelleted at low speed, the cells were suspended in NCTC-109 medium (Microbiological Associates) supplemented with 10% heat-inactivated (56°C 20 min) fetal calf serum (Microbiological Associates), 100 U/ml each of penicillin and kanamycin, and 100 pg/ml streptomycin (complete NCTC-109 medium). DNase (bovine testicular, Sigma Chemical Co.) was added to a final concentration of approximately 50 U/ml. After 5 min of incubation at 37”C, the cells were pelleted, resuspended in complete NCTC109 medium, and seeded in 35- or 60-mm tissue culture dishes (Falcon Plastics, Oxnard, Calif.). To obtain pure sheets of visceral endoderm and yolk sac mesoderm cells, we sub-

mitted yolk sacs to a procedure modified from that used by West et al. (1977). For best results with primary cultures of visceral endoderm cells, yolk sacs were washed with PBS and incubated for 1.5 hr at 4°C in a 0.5% trypsin/2.5% pancreatin mixture in Hank’s basic salt solution. Before separation of the mesoderm and visceral endoderm sheets with watchmakers’ forceps, the trypsin-pancreatin-treated yolk sacs were incubated for l-2 hr at 37’C in complete NCTC-109 medium. The separated visceral endoderm layers were washed with PBS and cells were disaggregated by incubation with trypsin-EDTA for 2 min. DNase treatment and sedimentation of the cells were carried out as described above. The cells were densely seeded in tissue culture dishes. Parietal endoderm sheets, which had been dissected away from the trophoblast layer with watchmakers’ forceps, were washed with PBS and treated with trypsinEDTA and then DNase as described for visceral endoderm cells. The cells were seeded at a moderate density in tissue culture dishes which had been precoated for 24 hr with conditioned medium from stationary phase PYS-2 cells (Lehman et al., 1974) as described by Atienza-Samols and Sherman (1978). By these procedures, the cells from five 13th day conceptuses were generally adequate to give moderately dense cultures in a single 35-mm tissue culture dish.

E&erase Ebctrophoretic

Analysis

After 24 hr in culture, visceral endoderm and yolk sac mesoderm cells were washed with PBS, collected with a rubber policeman, and stored at -70°C. The cells were thawed, suspended in a small volume of water, and homogenized. After protein analysis (Lowry et al., 1951), the homogenates were diluted to 1 mg/ml and frozen once again at -7O’C. Samples (100 ~1) were electrophoresed along with 13th day visceral endoderm tissue homogenates for comparative purposes and tested for non-

JETTEN, JETTEN, AND SHERMAN

Mouse Extraembryonic

specific esterase activity with cw-naphthyl acetate and a-naphthyl butyrate substrates as described by Sherman (1972). The stained gel profiles were scanned with a densitometer (Ortec, Inc., Oak Ridge, Term.). Labeling

of Primary

Cultures

After 24 hr of culture, dead and unattached cells were removed and fresh medium was added. At that time, glycoproteins were labeled with glucosamine, CSP were labeled by radioiodination, or total proteins were labeled with proline and/or leucine. In the first procedure, cells were incubated for 24 hr in the presence of D-[l14C]glucosamine (1 pCi/ml; 45 mCi/mmole, New England Nuclear, Boston) and then washed twice with PBS. Cells were harvested with a rubber policeman into PBS containing 2 rnM phenylmethylsulfonylfluoride (Calbiochem, La Jolla, Calif.), centrifuged, and solubilized in electrophoresis sample buffer (0.08 M Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 2 mM phenylmethylsulfonylfluoride, and 0.1 M dithiothreitol). Lactoperoxidase-catalyzed iodination of CSP was performed essentially as described by Hynes (1973). Briefly, cells grown in 35 or 60-mm culture dishes were washed three times with PBS and labeled in 0.5 or 1.2 ml, respectively, of PBS containing 400 @/ml sodium [ 12”I]iodide (carrier-free, New England Nuclear), 20 pg/ml lactoperoxidase (Calbiochem), 5 mM glucose, and 0.1 U/ml glucose oxidase (Worthington, Freehold, N.J.). Incubation was carried out for 10 min at room temperature. The reaction was stopped by the addition of phosphatebuffered sodium iodide (PBS with sodium iodide substituted for sodium chloride at the same molarity). Following two washes with the same buffer, cells were collected into phosphate-buffered sodium iodide containing 2 mM phenylmethylsulfonylfluoride. The cells were pelleted and‘boiled for 5 min in electrophoresis sample buffer.

Cell Surface Proteins

91

To label total cell and secreted proteins, cells were grown in complete NCTC-109 medium supplemented with 1 &i/ml L[ “C]proline (ca. 225 mCi/mmole), 1 @/ml L-[‘4C]leucine (ca. 270 mCi/mmole), or a combination of both (the specific proline and leucine radioactivities, taking into account the unlabled amino acid content in the medium, were ca. 17.5 and ca. 6.3 mCi/ mmole, respectively). After 24 hr of incubation, media containing labeled secreted proteins were collected, dialyzed for 24 hr against water, lyophilized, and solubilized in electrophoresis sample buffer. The cells were collected, washed three times in PBS containing 2 m.M phenylmethylsulfonylfluoride, and then solubilized in electrophoresis sample buffer. Polyacrylamide

Gel Electrophoresis

The labeled cell extracts or secreted proteins were subjected to electrophoresis on 7.5% polyacrylamide vertical slab gels using the buffer system described by Laemmli (1970). In general, samples labeled with [‘4C]amino acids or glucosamine contained ca. 25,000 cpm, while radioiodinated samples contained ca. 80,000 cpm. Electrophoresis was terminated when the bromphenol blue marker had reached the bottom of the gel. Gels were processed and prepared for fluorography as described by Bonner and Laskey (1974). Kodak X-Omat R film was used with exposure times for 14C-labeled and ““I-labeled gels of 4 and 2 days, respectively. In some cases autoradiograms were scanned with an Ortec densitometer. Immunoprecipitation Immunoprecipitation studies were carried out with the use of Staphylococcus aureus (strain Cowan I) as described by Kessler (1975). In brief, cells from a 35-mm tissue culture dish, labeled with [‘“Clproline, were washed and solubilized in 0.3 ml of 0.5% NP40 in NET buffer (Kessler, 1975). After solubilization, cell extracts were centrifuged at 48,000 g and the pellet was dis-

92

DEVELOPMENTALBIOLOGY

carded. To the supernatant fraction was added 10 ~1 of a rabbit nonimmune serum or a rabbit anti-TSD4 procollagen antiserum [TSD-4 is a mouse teratocarcinomaderived cell line (Little et al., 1977)] obtained from Dr. Charles Little, Department of Ophthamology, University of Pittsburgh School of Medicine. [This antiserum also appears to cross-react with the large external transformation sensitive (LETS) protein (Dr. R. Church, personal communication), but since cultured parietal endoderm cells appear to possess at most small amounts of LETS protein (see Figs. 3 and 6), this cross-reactivity would not be expected to interfere with the analysis.] After incubation at 4°C for 15 min, 200 ~1 of a 10% solution of S. aureus prepared as a bacterial immunoadsorbent (Kessler, 1975) was added. After a further 15min incubation period at 4°C the suspension was centrifuged at low speed. The pellet was washed three times to remove unbound proteins. The adsorbed antigen-antibody complexes were then solubilized by boiling for 5 min in electrophoresis sample buffer. After centrifugation to remove the bacteria, the supernatant fractions were stored at -70°C prior to electrophoresis. Immunofluorescence The LETS protein was detected by the technique of Chen et al. (1976). Briefly, cells were grown as monolayers on 12-mm glass coverslips, fixed in 2% paraformaldehyde in PBS for 15 min, washed with PBS, and covered with 10 ~1 rabbit anti-human cold-insoluble globulin (CIG) antibody (diluted 1:40), generously provided by Dr. L. B. Chen (Sidney Farber Cancer Institute, Boston). After 15 min of incubation at 37°C coverslips were washed extensively with PBS and incubated further for 15 min with 10 ~1 of fluorescein-conjugated goat antirabbit IgG (1:20 dilution; Meloy, Springfield, Va.). Coverslips were then washed and mounted on microscope slides with Gelvatol 20/30 (Monsanto Co., St. Louis,

VOLUME 70,1979

MO.). Fluorescence was observed by use of a Leitz Orthoplan microscope equipped with Ploem illumination. An antiserum, raised in a rabbit against an acellular preparation consisting of the Reichert’s membrane-like material secreted by endoderm cells differentiating from the embryonal carcinoma cell line PSA-I, was obtained from Dr. R. Oshima (University of California, San Diego). This antiserum was used in the same way as described for the antihuman CIG antibody, except that the coverslips were treated with normal goat serum prior to exposure to the fluoresceinconjugated antibody. In several of these were experiments, control coverslips treated in an identical fashion except that preimmune rabbit serum was substituted for the antisera. Plasminogen

Activator

Plasminogen activator activity was monitored in primary cultures by use of the fibrin-agar overlay technique described by Beers et al. (1975). RESULTS

Production of Primary Cultures traembryonic Membranes

of Ex-

In our efforts to prepare primary cultures of extraembryonic cell types, we experienced no difficulties in generating cultures of yolk sac mesoderm and amnion; routine proteolytic disaggregation and incubation procedures sufficed. Under similar conditions, however, visceral and parietal endoderm cell cultures were inadequate for the kinds of analyses we wished to perform. We were successful in preparing healthy-looking visceral endoderm cultures only when we shortened the period of trypsin-pancreatin treatment of yolk sacs from 2-3 hr (West et al., 1977; Sherman and AtienzaSamols, 1979) to 1.5 hr; a subsequent l- to 2-hr incubation of the yolk sacs in culture medium prior to the separation by dissection of the mesodermal and endodermal moieties led to further improvements in

JETTEN, JET-TEN, AND SHERMAN

Mouse Extraembryonic

numbers of surviving cells and culture morphology. Occasionally, even when these steps were taken, some cultures were visibly contaminated with cells with a fibroblastic appearance, presumably from the yolk sac mesoderm layer; these cultures were discarded. Generally, however, the cells have an epithelioid morphology (Fig. 1D) and express G esterase activity (Fig. 2) characteristic of visceral endoderm cells (Sherman and Atienza-Samols, 1979). We experienced initially a different problem with parietal endoderm cells. Although adequate numbers of cells remained viable after disaggregation with trypsin-EDTA, the cells were poorly adherent to glass or plastic, and the vast majority remained rounded up and failed to spread (see, e.g., Fig. 9A). We overcame this by using culture dishes previously coated with conditioned

FIG. 1. Morphology parietal

endoderm;

Cell Surface Proteins

93

medium from the teratocarcinoma-derived parietal endoderm-like cell line, PYS-2 (Lehman et al., 1974). We found that coated dishes promote spreading of parietal endoderm cells, just as they do for endoderm cells developing from isolated inner cell masses of the blastocyst (Atienza-Samols and Sherman, 1978). In two experiments, we used the procedures described above for preparing primary cultures of the various cell types and we determined cell numbers. The percentages of cells seeded which had attached to the culture dish and were viable (by the trypan blue exclusion test) after 24 hr in vitro were as follows: amnion, 20-30%; yolk sac mesoderm, 40-60%; yolk sac endoderm, l&30%; parietal endoderm, 30-50%. The net number of viable cells in the cultures did not change by more than *25% follow-

of primary cultures of extraembryonic (D) visceral endoderm.

tissues. (A) Amnion;

(B) yolk sac mesoderm; (C)

DEVELOPMENTAL BIOLOGY

VOLUME 70,1979

GEL LENGTH (cm) FIG. 2. Nonspecific esterase activities in primary cultures of visceral endoderm and yolk sac mesoderm cells. Densitometric profiles of activity from (b) visceral endoderm and (c) yolk sac mesoderm cell culture homogenates are compared with that of (a) a homogenate of 13th day visceral endoderm cells. Enzyme bands are designated by letters according to Sherman (1972). The stained gel is shown in the inset. Note that the visceral endoderm cultured cells express G esterase activity, whereas yolk sac mesoderm cells, as expected (Sherman and AtienzaSamols, 1979), do not. Note also that F esterase, an enzyme of maternal origin (Sherman and Chew, 1972), is not detected in visceral endoderm cells after the 24-hr culture period.

JETTEN, JETTEN, AND SHERMAN

Mouse Extraembryonic

ing a second 24-hr incubation period. Of all the cultures, only yolk sac mesoderm consistently showed a capability for continued growth for several days. The morphologies of the primary cultures are illustrated in Fig. 1. Amnion (Fig. 1A) and yolk sac mesoderm cells (Fig. 1B) are both fibroblastic but are distinguishable in that the former cells are characteristically flatter and more irregular than the latter. The amnion cultures contain few cells with a distinct epithelioid appearance, which may indicate that the ectodermal layer of the amnion is poorly represented. Figures 1C and D provide examples of the morphologies of parietal and visceral endoderm cells, respectively. Both cell types are epithelioid. Surface Protein Analyses In Fig. 3 are presented fluorographs of electrophoretic protein profiles from extraembryonic tissues labeled by radioiodination (A) or with [‘4C]glucosamine (B). Since the proteins visualized by the latter procedure are largely glycoproteins and not necessarily restricted to the cell surface, whereas those detected by the former procedure are principally CSP (glycosylated and nonglycosylated) with exposed tyrosine and histidine residues (Phillips and Morrison, 1971), it is not surprising that the “‘1 and [‘4C]glucosamine profiles from the same cell type are different, both quantitatively and qualitatively. With few exceptions and regardless of the labeling method, the protein profiles for amnion, yolk sac mesoderm, and visceral endoderm cells are similar to each other but different from those of parietal endoderm. For example, all profiles but those of parietal endoderm possess a heavy band in the 230,000-dalton range (arrow in Fig. 3); this band is very faint or absent in the latter profiles. The iodination patterns for amnion, yolk sac mesoderm, and visceral endoderm cells all contain prominent bands with approximate molecular weights of

Cell Surface Proteins

95

125,000 (band 1) and 115,000 (band 2) daltons and two or more bands in the range of 85,000 daltons (band 3). The glucosamine profiles contain two notable bands in common in the 135,000- to 140,000-dalton range (bands 7 and 8), and others with molecular weights of approximately 120,000 (band 9), 95,000 (band lo), and 48,000 (band 11) daltons. The visceral endoderm profiles differ reproducibly from those of yolk sac mesoderm and amnion in that band 7 is more heavily labeled than band 8 in visceral endoderm preparations, whereas the labeling intensity is reversed in the other two cell types. Furthermore, the visceral endoderm radioiodination profile contains a group of bands in the 55,000-dalton region (region 6) in common with parietal endoderm cells but not apparent in the yolk sac mesoderm or amnion profiles. The parietal endoderm profiles include several bands which are not evident in those profiles from the other cell types. The three most prominent bands in the radioiodination pattern have approximate molecular weights of 170,000, 120,000, and 55,000 daltons (bands 4, 5, and 6, respectively). Of these, only the bands in region 6 are shared, as mentioned, with visceral endoderm cells. The five most heavily labeled bands in the [‘4C]glucosamine profile for parietal endoderm cells consist of one with a molecular weight greater than 250,000 daltons (band 12; a band of similar molecular weight is also present in parietal endoderm radioiodination profiles), three consecutive bands with approximate molecular weights of 210,000, 190,000, and 170,000 daltons (region 13), and a heavily labeled area in the 120,000-dalton range (region 14) which is resolved into a doublet on other gels. Total Protein Analyses derm Cells

of Parietal

Endo-

When we analyzed proteins synthesized by parietal endoderm cultures incubated with [‘4C]proline or [‘4C]leucine, we observe a very heavily labeled protein with an

96

DEVELOPMENTALBIOLOGY

VOLUME lo,1979

-

200

-

130

-95

-68 -43

FIG. 3. Autoradiograms of cell surface protein electrophoretic profies. Cells were labeled by (A) lactoperoxidase-catalyzed iodination or with (B) [i4C]glucosamine. Proteins were extracted and run on 7.5% polyacrylamide slab gels as described in Materials and Methods. The arrow indicates the position of the LETS glycoprotein, and numbers are placed beside bands that are referred to in the text. The profdes shown here are representative of several such analyses. In general, the observed patterns were very reproducibile; however, some minor tissue-specific differences are present in the illustrated profiles but are not referred to in the text because they were not evident on all the gels. The scale on the right indicates the position of migration of molecular weight standard proteins, which, in descending order, were myosin, ,&galactosidase, phosphorylase a, bovine serum albumin, and ovalbumin. Molecular weight estimates in this and subsequent gels are flO%.

approximate molecular weight of 170,000 daltons (Fig. 4). It is notable that this protein is proportionately much more intensely labeled by proline than by leucine, indicating a high proline or hydroxyproline content. When a similar extract from parietal endoderm cultures was challenged with an antiserum to procollagen and the bound antigenic material subsequently analyzed

by electrophoresis, it was apparent that the 170,000-dalton protein had been selectively extracted (Fig. 4). No proteins were precipitated when a control, preimmune serum was used (not shown).

Secreted Proteins Primary cultures of amnion, mesoderm, and visceral endoderm cells labeled with

JETTEN, JETTEN, AND SHERMAN

Mouse Extraembryonic

r

four tissues contain a weakly labeled band in the 230,000-dalton range. We studied proteins secreted by parietal endoderm cells more closely by comparing profiles obtained when primary cultures were labeled with [ 14C]glucosamine, [“Clproline, or [14C]leucine (Fig. 6). The major bands observed in Fig. 5 are also present in the amino acid-labeled profiles in this experiment (bands 1, 2, 3, and 5). Prominent bands with molecular weights of 65,000 (band 4) and 40,000 (band 6) daltons are also evident in this preparation. It is apparent that the 170,000- and 95,000-dalton species are more heavily labeled with proline than with leucine. The profile of glucosamine-labeled proteins from parietal endoderm cells is, in general, similar to that of the amino acid labeling profiles, indicating that most, or all, of the secreted proteins are glycoproteins. It is notable, however,

‘RO

-EU I M

I

I

I

I

G

P

B

0

97

Cell Surface Proteins

I

I

I

I

J

2

4

6

8

IO

GEL LENGTH (cm) FIG. 4. Electrophoretic analyses of total parietal endoderm proteins labeled with [%]proline or [‘“Clleucine. Labeling and electrophoresis were performed as described in Materials and Methods. The autoradiagrams were scanned with a densitometer. The dashed line in the upper profile represents the radioactivity proftie of gel electrophoresis of antigens adsorbed out by the bacterial immunoadsorption procedure using an anti-procollagen antiserum (see Materials and Methods). (M) Myosin (200,000 daltons), (G) /?-galactosidase (130,000 daltons), (P) phosphorylase (95,000 daltons), (B) bovine serum albumin (68,000 daltons), and (0) ovalbumin (43,000 daltons) were used as molecular weight markers.

[14C]leucine and [‘4C]proline secrete similar populations of proteins: The major species have molecular weights of approximately 150,000, 140,000, and 50,000 daltons (Fig. 5). The most heavily labeled proteins secreted by parietal endoderm cells have molecular weights of about 170,000, 120,000, 95,000, and 50,000 daltons. Of these, only the latter protein appears to be in common with those secreted by the other tissues. Profiles of all

I 0

I 2

I 4 GEL LENGTH

I 8

1 6

I IO

(cm)

FIG. 5. Electrophoretic analyses of secreted proteins labeled with [‘%]proline and [‘%]leucine. The secreted proteins of primary cultures of (a) parietal endoderm, (b) amnion, (c) yolk sac mesoderm, and (d) visceral endoderm were labeled and subjected to electrophoresis as described in Materials and Methods. The autoradiograms were scanned with a densitometer. M, G, P, B, and 0 represent molecular weight markers as described in the legend to Fig. 4.

98

DEVELOPMENTAL BIOLOGY VOLUME70,1979

oresce intensely (Fig. 7 and Table l), whereas fluorescence is not observed with preimmune serum (not shown). The cell surface distribution of the LETS protein is different for the fibroblastic and epithelioid cell types: In the former case, the pattern is one of thick fibers running across the cell surface (Figs. 7A and B); visceral endoderm cells, on the other hand, show a finer meshwork of LETS protein (Fig. 7C). Parietal endoderm cells, unlike the other extraembryonic cell types, possess only occasional and faint fluorescence on their exposed surfaces (Fig. 7D). However, when we analyzed intact parietal endoderm layers dissected from the uterus, i.e., still attached to their secreted Reichert’s membrane, for LETS protein, strong fluorescence was observed at the contact areas of the cells (Figs. 8A and B). The possible significance of this observation will be discussed below. We have also tested primary cultures of amnion, yolk sac mesoderm, visceral endoderm, and parietal endoderm for reactivity with a presumptive anti-Reichert’s membrane antiserum. Only parietal endoderm FIG. 6. Electrophoretic analyses of proteins secells react strongly with this antiserum at creted by parietal endoderm cells. Secreted proteins a dilution of 1:60 (Table 1). In many cases were labeled by (A) [r4C]glucosamine, (B) [‘%]proline, (but only in parietal endoderm cultures) and (C) [‘%]leucine. Numbers on the left-hand side identify bands discussed in the text. The scale on the there is evidence by immunofluorescence of right represents apparent molecular weights x 1O-3 large networks of reactive material, even on based on molecular weights of standard proteins reareas of the coverslip devoid of cells (Fig. ferred to in the legend to Fig. 3. 9).

- 200

-

130

-95

-68

-43

A

B

C

that the proteins in the 170,000 (band l)and 95,000 (band 3)-dalton regions are relatively weakly labeled with glucosamine.

Immunofluorescence Studies Following upon the observation that amnion, visceral endoderm, and yolk sac mesoderm cells possess a prominently labeled CSP band with the molecular weight of the LETS protein, 230,000 daltons, we tested for the presence of LETS protein on cells in primary culture by immunofluorescence, using anti-human CIG as a probe. These studies indicate that amnion, yolk sac mesoderm, and visceral endoderm cells flu-

Plasminogen Activator Analyses In previous studies (Sherman et al., 1976) we reported that most parietal endoderm activator, cells secrete plasminogen whereas cultured cells from the amnion and the total yolk sac (i.e., without prior separation of the layers) do not. In light of our observations that visceral endoderm cells fare poorly in culture, particularly in competition with yolk sac mesoderm, we felt it prudent to reassess the activities of the cells cultured from the separated layers of the yolk sac. Table 1 illustrates that our earlier observations are still valid: In primary cul-

JETTEN, JETTEN, AND SHERMAN

Mouse Extraembryonic

99

Cell Surface Proteins

FIG. 7. Indirect immunofluorescence of cells from primary cultures of extraembryonic membranes reacted with anti-human CIG antiserum. (A) Amnion; (B) yolk sac mesoderm; (C) visceral endoderm; (D) parietal endoderm. Note that endoderm cella do not flatten on the coverslip in the absence of conditioned medium from PYS-2 cells. Conditioned medium was not used to promote spreading of parietal endoderm cells in this experiment because the active factors do not adhere to glass (Atienza-Samols and Sherman, 1978) and also because we wished to avoid false positives due to the possible presence of LETS protein in the conditioned medium. TABLE 1 COMPARISONOFCHARACTERISTICSOFPRIMARYCULTURESOFI~~~DAYEMBRYONICMEMBRANES Cell type

Amnion Yolk sac mesoderm Visceral endoderm Parietal endoderm

Presence of LETS protein determined by Iodination”

Immunofluorescence’

++ ++ + +

++ ++ ++ +

Reaction with anti-basement membrane antiserumh + ++

Percentage of cells producing plasminogen activator’

0 0 0.9 39.8

w2m umw (2/235) (81/204)

LI++ = Intense band in the autoradiograph in the 230,000-dalton range; + = moderate band; + = very faint band. * Antisera were used as described in Materials and Methods. ++ = Strong fluorescence; + = moderate to weak fluorescence; - = no fluorescence. ’ Twenty-four hours after their initiation, sparsely seeded primary cultures were assayed for plasminogen activator secretion by the fibrin-agar overlay procedure. The cells were monitored for the production of halos of fibrinolysis 7 hr after overlay. The ratio of plasminogen activator-positive cells to total number counted is given in parentheses.

100

DEVELOPMENTAL BIOLOGY VOLUME70,1979 that with culture beyond 48 hr, the morphology of visceral endoderm cells tends to become progressively more fibroblastic, whereas that of parietal endoderm cells remains epithelioid (unpublished observations). This change in visceral endoderm culture morphology is most likely not due to overgrowth by contaminating yolk sac mesoderm since the transformation takes place in the absence of an increase in cell numbers. It will be interesting to determine whether characteristic properties of visceral endoderm cells, such as production of esterase G (see Fig. 2), persist after morphological changes are observed. The most striking result of our experiments is that CSP and secreted proteins of parietal endoderm cells differ dramatically

FIG. 8. Indirect immunofluorescence of intact parietal endoderm layers reacted with anti-human CIG antiserum. Sheets of parietal endoderm cells adhering to Reichert’s membrane were dissected from 13th day conceptuses and were carried with forceps through washing solutions, fixative, and antisera as described for our routine immunofluorescence analyses. The sheets were then flattened on a slide with forceps and inspected by (A) phase-contrast microscopy and by (B) fluorescence microscopy to test for immunofluorescence. In control experiments in which preimmune serum was used, no fluorescence was observed (not shown). ture, substantial numbers of parietal endoderm cells secrete plasminogen activator, whereas very few or no cells from the other extraembryonic cultures are positive. DISCUSSION

We have described in this report techniques for generating and comparing primary cultures of four extraembryonic cell types. Although these cultures show relatively little net increase in cell numbers with time, the cells are healthy in appearance over the first 48 hr, the period throughout which the studies described here were performed. It is, perhaps, notable

FIG. 9. Indirect immunofluorescence of parietal endoderm cells stained with anti-basement membrane antiserum. Parietal endoderm cultures were prepared as described in Materials and Methods except that culture dishes were not precoated with conditioned medium. Note that the network of reactive material in the center of photograph B is not obviously cell associated (A) Phase-contrast optics; (B) fluorescence optics.

JETTEN, JETTEN, AND SHERMAN

Mouse Extraembryonic Cell Surface Proteins

from those of visceral endoderm, yolk sac mesoderm, and amnion cells in several respects. These differences extend to overall profiles of glucosamine-labeled proteins and radioiodinated CSP, secreted proteins, plasminogen activator production, and surface expression of basement membrane and LETS protein antigens. Visceral and parietal endoderm cells show only minor similarities in surface properties: Both react to some extent with a basement membrane antiserum, and in the radioiodination profiles, there is a region around 60,000 daltons containing a pattern of common bands that are not apparent in yolk sac mesoderm and amnion profiles. Overall, however, visceral endoderm CSP and secreted proteins resemble those of yolk sac mesoderm and amnion very closely, much more so than those of parietal endoderm. This is, perhaps, surprising in view of (a) the common origin from a primitive endoderm progenitor of visceral and parietal endoderm cells (see Rossant and Papaioannou, 1977) and (b) the morphological resemblance of visceral endoderm cells to parietal endoderm cells during the period of study and their dissimilarity to the other, fibroblastic cell types. It is possible that the CSP of parietal and visceral endoderm cells differ because the surface of the former cell type is obscured by a secreted matrix, the components of w,hich dominate the radioiodination and glucosamine profiles. In vivo, rodent parietal endoderm cells secrete a thick basement membrane called Reichert’s membrane (Pierce et al., 1962; Pierce, 1966). This material consists of collagen and/or procollagen (Minor et al., 1976a) as well as one or more glycoprotein(s) (Johnson and Starcher, 1972; Minor et al., 1976b). The LETS protein might be either a component of Reichert’s membrane or closely associated with it (Fig. 8; Wartiovaara et al., 1979). Reichert’s membrane from rat conceptuses continues to be synthesized in vitro

101

by intact sheets of parietal endoderm plus Reichert’s membrane, either isolated or in contact with trophoblast cells (Clark et al., 1975a,b; Minor et al., 1976a-c). While we must stress that we do not have definitive evidence that primary cultures of parietal endoderm cells synthesize and secrete the components of Reichert’s membrane [such evidence would require identification of newly synthesized type IV collagen or procollagen and glycoprotein(s) cross-reacting with the appropriate antiserum], we believe it possible, on the basis of our studies, that this does occur. The following observations support the view that cultured parietal endoderm cells synthesize and secrete a 170,000-dalton protein which is the pro (11 chain of procollagen: (a) We observe a 170,000-dalton protein in total cell extracts (Fig. 4), as a secreted protein (Figs. 5 and 6), and on the surface (Fig. 3) of parietal endoderm cells; (b) the molecular weight is very close to that (160,000 daltons) of pro (Y chains observed by Minor et al. (1976a) to be a component of rat Reichert’s membrane; (c) the 170,000-dalton protein in total cell extracts has a very high proline/ leucine ratio, and in fact, it is by far the species incorporating the most proline in the entire extract; (d) the secreted 170,000dalton protein also has a high proline/leutine ratio and appears to label weakly with glucosamine; and (e) it is selectively immunoadsorbed with an anti-procollagen antiserum. It is of further significance that a prominent, 95,000-dalton protein with a high proline/leucine ratio is also present in the secreted protein profile; prccollagens synthesized by chick embryo cranial bones (Byers et al., 1975; Fessler et al., 1975) and a mouse teratoma cell line, TSD-4 (Little et al., 1977), possess a pepsin-resistant triple helical region containing three a-chain subunits, each with a molecular weight of 90,000 to 100,000 daltons. If we are in fact observing procollagen (170,000 daltons) and collagen (95,000 daltdns) in our cultures, our results would sup-

102

DEVELOPMENTALBIOLOGY

port the view of Minor et al. (1976a) that most, if not all, of the collagen secreted by parietal endoderm cells is in the procollagen form. However, breakdown of the putative pro a chains to a 95,000-dalton protein was not observed by Minor et al. under conditions in which the procollagen was becoming part of the newly forming basement membrane material. Furthermore, when these investigators treated their pro a chains with pepsin, they observed a product with a higher molecular weight, between 125,000 and 140,000 daltons. These discrepancies with our observations might result from an increased susceptibility to proteolysis of procollagen secreted into the culture medium rather than fixed into a membrane (see below); the difference in presumptive a-chain size might be a species difference or due to incomplete proteolysis of the nonhelical regions of the rat pro a chains under the conditions used by Minor et al. (1976a) in their studies. Our data do not provide information concerning the nature of the glycoprotein constituent(s) of Reichert’s membrane, and their identity remains obscure. There are a number of glycoproteins secreted by parietal endoderm cells in notable amounts (see Fig. 6); the glycoprotein with a molecular weight of 120,000 to 125,000 daltons might be a particularly promising candidate for a Reichert’s membrane component since it appears to be secreted in substantial amounts by parietal endoderm cells (band 2 in Fig. 6) but not by the other cell types (Fig. 5). Also, both the cell-associated glucosamine and CSP profiles for parietal endoderm cells contain a very heavily labeled band or group of bands in the 120,000- to 125,000-dalton region. In order for basement membranes to be formed, the appropriate constituents need be not only secreted but also assembled into the appropriate complexes. Although parietal endoderm cells react with an antibasement membrane antiserum, we cannot be certain that this represents synthesis

VOLUME To,1979

and assembly of new membrane. Minor et al. (1976b) have demonstrated that in parietal endoderm organ cultures, new membrane can only be deposited against preexisting membrane, and we might have removed much of this material in preparing our cultures. On the other hand, positive staining of acellular sheets on the surface of the coverslips (Fig. 9) might suggest that the cultures contain a substantial amount of residual basement membrane synthesized in utero. Zetter and Martin (1978) demonstrated that cells of the inner cell mass of the mouse blastocyst secrete LETS protein just prior to their differentiation into primitive endoderm. With the appearance of the outer ring of primitive endoderm cells, reactivity with anti-CIG antiserum disappears. On the other hand, these authors reported that PYS cells, a teratocarcinoma-derived cell line closely resembling parietal endoderm (Lehman et al., 1974; Jetten, A., AtienzaSamols, S., and Sherman, M. I., in preparation), do secrete substantial amounts of LETS protein. Also, Wartiovaara et al. (1979) have observed the association of LETS protein with the parietal endodermReichert’s membrane layer of the conceptus. We have detected ample amounts of LETS protein production by our visceral endoderm cultures (Figs. 3 and 7), but evidence for LETS protein production by our parietal endoderm cultures is scanty: There is little suggestion of surface LETS protein either by radioiodination (Fig. 3A) or by immunofluorescence (Fig. 7); the glucosamine profile in Fig. 3 suggests that little LETS protein is inside the cell, and the secreted protein profiles (Figs. 5 and 6) do not indicate the presence of substantial amounts of a 230,000-dalton protein. On the other hand, we concur that there is good evidence of parietal endoderm-Reichert’s membrane-associated LETS protein in utero (Fig. B), although we do not know anything about the source of the LETS protein under those conditions. It is, of

JETTEN, JETTEN, AND SHERMAN

Mou !se Extraembryonic

course, possible that large amounts of LETS protein are synthesized and immediately secreted by cultured parietal endoderm cells but then quickly degraded by plasminogen activator (Table 1) and/or other proteases in the culture medium. Finally, we have for the first time provided a detailed description of the CSP and secreted proteins of several extraembryonic cell types. However, we have merely begun to properly characterize and identify these proteins. We are curious but know little, for instance, about the heavily labeled 50,000dalton glycoprotein secreted by all the cell cultures (Fig. 5) and what its relationship is to a glycoprotein with a similar molecular weight that is prominent in all glucosamine profiles except that of parietal endoderm (Fig. 3B). We might, perhaps, be able to pinpoint more subtle differences in CSP among the various cell types by two-dimensional electrophoretic analyses. Nevertheless, the information that we do have available in this report has allowed us to better characterize a number of blastocyst- and teratocarcinoma-derived cell lines. These results will be presented elsewhere (Jetten et al., 1979; Jetten, A., Atienza-Samols, S., and Sherman, M. I., in preparation). We wish to thank Drs. Robert Oshima and Lan Bo Chen for generous gifts of antisera and Drs. Martin Sellens and David Webb for helpful comments on the manuscript. REFERENCES ATIENZA-SAMOLS, S. B., and SHERMAN, M. I. (1978). Outgrowth promoting factor for the inner cell mass of the mouse blastocyst. Develop. Biol. 66,220-231. BEERS, W. H., STRICKLAND, S., and REICH, E. (1975). Ovarian plasminogen activator: Relationship to ovulation and hormonal regulation. Cell 6, 387-394. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids. Eur. J. Biochem. 46, 83-88. BRADY, R. A., and FISHMAN, P. H. (1975). Membranes of transformed mammalian cells. Zn “Biochemistry of Cell Walls and Membranes” (C. F. Fox, ed.), pp. 61-96. University Park Press, Baltimore, BYERS, P. H., CLICK, E. M., HARPER, E., and BORNSTEIN, P. (1975). Interchain disulfide bonds in procollagen are located in a large nontriple-helical

Cell Surface Proteins

103

COOH-terminal domain. Proc. Nat. Acad. Sci. USA 72,3009-3013. CHEN, L. B., GALLIMORE, P. H., and MCDOUGALL, J. K. (1976). Correlation between tumor induction and the large external transformation sensitive protein on the cell surface. Proc. Nat. Acad. Sci. USA 73, 3570-3574. CLARK, C. C., TOMICHEK, E. A., KOSZALKA, T. R., MINOR, R. R., and KEFALIDES, N. A. (1975a). The embryonic rat parietal yolk sac. J. Biol. Chem. 250, 5259-5267. CLARK, C. C., MINOR, R. R., KOSZALKA, T. R., BRENT, R. L., and KEFALIDES, N. A. (1975b). The embryonic rat parietal yolk sac. Changes in the morphology and composition of its basement membrane during development. Develop. Biol. 46, 243-261. FESSLER, L. I., MORRIS, N. P., and FESSLER, J. H. (1975). Procollagen: Biological scission of amino and carboxy extension peptides. Proc. Nat. Acad. Sci. USA 72,4905-4909. GLICK, M. C., KIMHI, Y., and LITTAUER, U. Z. (1973). Glycopeptides from surface membranes of neuroblastoma cells. Proc. Nat. Acad. Sci. USA 70,16821687. HYNES, R. 0. (1976). Cell surface proteins and malignant transformation. Biochim. Biophys. Acta 458, 73-107. HYNES, R. O., and WYKE, J. A. (1975). Alterations in surface proteins in chicken cells transformed by temperature-sensitive mutants of Rous sarcoma virus. Virology 64.492-504. HYNES, R. O., MARTIN, G. S., SHEARER, M., CRITCHLEY, D. R., and EPSTEIN, C. J. (1976). Viral transformation of rat myoblasts: Effects on fusion and surface properties. Develop. Biol. 48, 35-46. JETTEN, A. M., JETTEN, M. E. R., and SHERMAN, M. I. (1979). Stimulation of differentiation of several embryonal carcinoma cell lines by retinoic acid (submitted for publication). JOHNSON, L. D., and STARCHER, B. C. (1972). Epithelial basement membranes: The isolation and identification of a soluble component. Biochim. Biophys. Acta 290, 158-167. KESSLER, S. W. (1975). Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: Parameters of the interaction of antibody-antigen complexes with protein A. J. Zmmunot. 115, 1617-1624. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. LEHMAN, J. M., SPEERS, W. C., SWARTZENDRUBER, D. E., and PIERCE, G. B. (1974). Neoplastic differentiation: Characteristics of cell lines derived from a murine teratocarcinoma. J. Cell. Physiol. 84, 1328. LITTLE, C. D., CHURCH, R. L., MILLER, R. A., and RUDDLE, F. H. (1977). Procollagen and collagen

104

DEVELOPMENTALBIOLOGY

produced by a teratocarcinoma-derived cell line, TSDI: Evidence for a new molecular form of collagen. Cell 10,287-295. LOWRY,0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MINOR, R. R., CLARK, C., STRAUSE,E., KOSZALKA,T., BRENT, R., and KEFALIDES, N. (1976a). Basement membrane procollagen is not converted to collagen in organ cultures of parietal yolk sac endoderm. J. Biol. Chem. 251,1789-1794. MINOR, R. R., How, P. S., KOSZALKA,T. R., BRENT, R. L., and KEFALIDES,N. A. (1976b). Organ cultures of the embryonic rat parietal yolk sac. I. Morphologic and autoradiographic studies of the deposition of the collagen and noncollagen glycoprotein components of the basement membrane. Develop. Biol. 48,344-364. MINOR, R. R., STRAUSE, E. L., KOSZALKA, T. R., BRENT, R. L., and KEFALIDES, N. A. (1976c). Organ cultures of the embryonic parietal yolk sac. II. Synthesis, accumulation, and turnover of collagen and noncollagen basement membrane proteins. Develop. Biol. 48, 365-376. NICOLSON,J. L. (1976). Trans-membrane control of the receptors on normal and tumor cells. Biochim. Biophys. Acta 458, l-72. PHILLIPS, D. R., and MORRISON,M. (1971). Exposed protein on the intact human erythrocyte. Biochemistry 10, 1766-1771. PIERCE, G. B. (1966). The development of basement membranes of the mouse embryo. Develop. Biol. 13,

VOLUME 70, 1979 231-249.

PIERCE, G. B., MIDGLEY, A. R., SRI RAM, J., and

FELDMAN,J. D. (1962). Parietal yolk sac carcinoma: Clue to the histogenesis of Reichert’s membrane of the mouse embryo. Amer. J. Pathol. 41, 549-566. ROSSANT, J., and PAPAIOANNOU, V. E. (1977). The biology of embryogenesis. In “Concepts in Mammalian Embryology” (M. I. Sherman, ed.), pp. l-36. MIT Press, Cambridge, Mass. SHERMAN, M. I. (1972). Biochemistry of differentiation of mouse trophoblast: Esterase. Exp. Cell Res. 75, 449-459. SHERMAN,M. I., and ATIENZA-SAMOLS,S. B. (1979). Enzyme analyses of mouse extraembryonic tissues. J. Embryol. Exp. Morph., in press. SHERMAN,M. I., and CHEW,N. J. (1972). Detection of maternal esterase in mouse embryonic tissues. Proc. Nat. Acad. Sci. USA 69,2551-2555. SHERMAN, M. I., STRICKLAND, S., and REICH, E. (1976). Differentiation of early mouse embryonic and teratocarcinoma cells in vitro: Plasminogen activator production. Cancer Res. 36,4208-4216. WARTIOVAARA, J., LEIVO, I., VIRTANEN, I., VAHERI, A., and GRAHAM, C. F. (1978). Cell surface and extracellular matrix glycoprotein fibronectin: Expression in embryogenesis and in teratocarcinoma differentiation. Ann. N. Y. Acad. Sci. 312, 132-141. WEST, J. D., FRELS, W. I., CHAPMAN, V: M., and PAPAIOANNOU (1977). Preferential expression of the maternally derived X chromosome in the mouse yolk sac. Cell 12,873~882.