Transepithelial calcium transport in the chick chorioallantoic membrane I. Isolation and characterization of chorionic ectoderm cells

Calcium is a major nutritional requirement for the developing chicken embryo. As extensive mineralization of the embryonic skeleton begins, after day 10 of incubation, large amounts of calcium are mobilized from the calcite stored in the eggshell. Nearly 80% of the approximately 140-180 mg of calcium in the day 20-21 hatchling chicken is derived from the eggshell (Johnston and Comar, 1955; Romanoff, 1961) and is transported into the embryonic bloodstream by the chorioallantoic membrane (CAM) (Clark and Simkiss, 1980; Terepka et al., 1976; Tuan, 1987).

The CAM is an extra-embryonic, placenta-like tissue consisting of three distinct cellular layers: an ectodermal layer facing the eggshell, a sparsely populated mesodermal layer, and an endodermal layer lining the allantois (Leeson and Leeson, 1963; Narbaitz, 1972; Packard and Packard, 1984). The epithelial cells of the CAM ectoderm mobilize and transport eggshell calcium into the embryonic vasculature in a development-specific manner, starting at day 10 and peaking around day 17 of incubation (Garrison and Terepka, 1972a,b; Johnston and Comar, 1955; Terepka et al., 1969). Calcium accumulates in the chick embryo in ovo through a specific, temperature-sensitive CAM-mediated pathway at a very high rate (113.2 nmol/cm² per h; Crooks and Simkiss, 1975). Previous in vitro and in vivo studies have shown that CAM calcium transport is a unidirectional, active process that is highly specific for Ca²⁺ (Garrison and Terepka, 1972a,b; Terepka et al., 1976) and is regulated by both vitamin K (Tuan, 1979) and the availability of eggshell calcium for transport (Dunn et al., 1981; Tuan, 1983). The specific, cellular mechanism of transepithelial calcium transport in the CAM, however, is not understood.

The ectodermal region of the CAM undergoes distinct morphological changes as calcium transport activity increases (Coleman and Terepka, 1972c). By day 10 of development, the CAM surrounds the embryo and adheres to the acellular, inner shell membrane (ISM) found just inside the eggshell; the differentiated CAM ectoderm adheres tightly to the ISM such that the structural integrity and calcium transport activity of the CAM are lost if the ISM is mechanically removed (Garrison and Terepka, 1972a,b; Moriarty and Terepka, 1969; Terepka et al., 1969). As calcium transport activity increases, cells of the CAM ectoderm undergo specific morphological changes. The region of the CAM in contact with the ISM consists of two distinct cell types: (1) villus cavity (VC) cells, which are apparently secretory and perhaps involved in eggshell solubilization (Narbaitz, 1987; Narbaitz et al., 1981); and (2) capillary covering (CC) cells, which make up most of the CAM surface. The cells of the CAM are connected by apical tight junctions, and, coincident with the onset of calcium transport, the CC cells develop long, thin cytoplasmic extensions that form a barrier between the eggshell calcium reserve and the embryonic circulation (Coleman and Terepka, 1972a-c). As calcium transport increases, the cytoplasmic extensions become extensive and narrow so that the capillaries approach the eggshell calcium reserve more closely (Coleman and Terepka, 1972c). By day 15, prior to the peak of transport activity (Crooks and Simkiss, 1975; Terepka et al., 1969; Tuan, 1980), the CC cells contain few membranous organelles, and lack endoplasmic reticulum and Golgi apparatus in particular (Coleman and Terepka, 1972c). In conjunction with these distinct morphological changes, the CAM ectoderm expresses biochemical activities associated with calcium transport, including a vitamin K-dependent Ca²⁺-binding protein (transcalcin) (Tuan, 1987; Tuan et al., 1978a-c; Tuan and Scott, 1977), and an enhanced Ca²⁺-activated, Mg ²⁺-dependent ATPase activity (CaATPase) (Tuan and Knowles, 1984). Each of these bio-chemical activities has been functionally linked to CAM Ca²⁺ transport in vitro and in vivo (Tuan et al., 1986b).

The relatively simple cytoarchitecture of the CAM, and the availability of specific markers, make CC cells ideal candidates for the in vitro assessment of transepithelial calcium transport; however, as pointed out by previous researchers, there are several difficulties related to tissue inaccesibility and the presence of the adherent ISM (Garrison and Terepka, 1972a; Terepka et al., 1969). In order to delineate the cellular pathway involved in the transcellular mobilization of calcium across the CAM ectoderm, we sought to obtain cell preparations suitable for in vitro analyses and manipulations. We present here a straight-forward method for the preparation of the CAM ectoderm cells implicated in calcium transport. These cells are isolated specifically from the CAM ectoderm and retain surface transcalcin expression, elevated CaATPase activity, and the rapid single-step calcium influx pathway characteristic of CAM ectoderm in vivo. Our results indicate that isolated primary ectoderm cells may be useful for the in vitro characterization of the calcium transport function of the CAM.

Fertilized chicken embryos (Truslow Farms, Chesterfield, MD) were kept at 37.5°C in a humidified egg incubator for the number of days desired. Shell-less embryos were prepared on incubation day 3 and cultured as described by Tuan (1980) after Dunn and Boone (1976).

The CAMs of normal embryos incubated in ovo were prepared for cell isolation by first preparing tissue discs. The egg surface was disinfected with 70% ethanol, and the eggshell was broken above the air-space to expose the outer surface of the ISM. A section of the exposed ISM and underlying CAM were cut and removed as were the embryo, yolk, albumen and allantoic fluid. The bulk of the CAM remained attached to the eggshell. After being cut into halves, the eggshell was carefully separated from the shell membranes and CAM. The tissue was placed, ISM side up, on Parafilm (4 layers thick) and cut into 2 cm² circles, ‘cookie cutter style’, with a sharp cork-borer. The resulting tissue discs were placed in ice-cold Hanks’ Balanced Salt Solution with 10 mM HEPES (pH 7.2) (HBSSH) until the shell membranes were thoroughly wetted. The CAM discs were carefully separated from the ISM using fine forceps and transferred to a fresh container of cold HBSSH.

CAM tissue samples were processed for electron microscopy as described by Tuan et al. (1986a). Briefly, tissue was removed from embryos, rinsed in ice-cold HBSSH and then ice-cold 0.12 M sodium cacodylate (pH 7.4). Samples were fixed overnight in 0.25% glutaraldehyde (EM Science, Cherry Hill, NJ) in cacodylate buffer. Samples were rinsed, post-fixed in OsO₄ for 1 hour and embedded in Epon. Sections were stained with lead and uranyl acetate and viewed using a JEOL 100B transmission electron microscope.

Tissue specimens were mounted into O-rings with the ectodermal surface up and rinsed in HBSSH followed by ice-cold 0.12 M sodium cacodylate buffer (pH 7.4). Tissue was then fixed overnight with 0.25% glutaraldehyde (EM Science) in the same buffer, rinsed, post-fixed for 1 h with 1% OsO₄, rinsed, dehydrated through ethanol, and critical point dried into 100% CO₂. The samples were mounted onto aluminum stubs, sputter-coated with gold, and observed using a JEOL 35-C scanning electron microscope.

Embryos were dissected as described above for tissue disc preparation. After removal of the eggshell, the CAM tissue, with shell membranes attached, was rinsed in room temperature HBSSH until the ISM began to separate from the CAM. Small pieces of ISM were left in place to mark the chorionic side, and the tissue was drained and placed onto a 0.45 μm pore size, UV-sterilized, nitrocellulose (NC) sheet (Millipore, Bedford, MA) with the wetted shell membranes facing upward. After the allantoic cells had thoroughly adhered to the NC, the CAM was peeled away leaving the allantoic cells behind. The tissue was returned to HBSSH and the remaining pieces of ISM removed. The CAM fragment was then minced with scissors and digested with 0.1% trypsin (Sigma, St. Louis, MO), 10 mM EDTA (Sigma) in HBSSH at 37°C in a stirring flask. After 30 min, aliquots of digested material were assessed every 10 min using a hemocytometer. When numerous small clumps of cells (5-10 cells per clump) were evident, the digestion was stopped by the addition of soybean trypsin inhibitor (Sigma) and 0.05% deoxyribonuclease (Sigma) for 15 min at 37°C. Tissue remnants were removed, and the suspended cells were centrifuged at 300 g in an IEC (Boston, MA) centrifuge for 20 min to collect the ectoderm cells. The cell pellet was resuspended in 10% calf serum, 5 μM hemin, Ham’s F-12 medium (GIBCO-BRL; Gaithersburg, MD) to a total cell density of 1×10⁶ to 2 ×10⁶/ml. Fibroblasts were allowed to adhere to tissue culture dishes for 30 min at 37°C, then the non-adherent cells were plated, 1 ml of cells per 35 mm diameter well, on dishes coated with Matrigel (1/10 diluted; Collaborative Research, Bedford, MA) and cultured at 37°C with 5% CO₂.

Tissue sections for immunofluorescence were prepared from day 14 normal CAM. The tissue was prepared as described previously (Ono and Tuan, 1991). Briefly, pieces of CAM were fixed in a modified Carnoy’s fixative, embedded in paraffin, sectioned at 8 μm thickness, cleared and rehydrated. Tissue sections for hematoxylin and eosin staining were similarly prepared except that fixation was carried out in Bouin’s fixative (Humason, 1967).

CAM from day 14 normal embryos was removed and frozen-embedded at −70°C in Tissue-Tek O.C.T. Compound (Miles Inc., Diagnostics Div.; Elhart, IN). Sections were cut at 12 μm thickness and −20°C using a Tissue-Tek microtome (Miles Inc.; Naperville, IL), then fixed for 30 s in −20°C acetone, and air-dried on glass microscope slides. CaATPase histochemistry was carried out as previously described (Tuan and Knowles, 1984) after the method of Ando et al. (1981).

Antiserum to the CAM-specific calcium-binding protein, transcalcin (Tuan et al., 1978a; Tuan, 1987), was thawed from −80°C storage and diluted (1/50 for immunofluorescence and 1/200 for immunohistochemistry) with 10 mM Tris-HCl buffered 0.9% saline (pH 8.0, TBS) to which 0.05% Tween-20, 500 mM additional NaCl and 3% BSA (Fraction V, from Sigma) were added (TBSTSB). The serum was incubated at 37°C for 30 min with an acetone powder prepared from day 8 normal chick CAM to adsorb non-specific immunoglobulins. The acetone powder was subsequently removed by centrifugation and the supernatant recovered.

Tissue sections for anti-transcalcin immunofluorescence were blocked with TBSTSB for 30 min at 37°C. Blocking solution was removed and the sections were treated with the prepared, 1/50 diluted antiserum at 37°C for 30 min. The sections were rinsed extensively with TBSTSB to remove unbound and non-specifically bound antibodies. Secondary goat FITC-conjugated anti-rabbit IgG (SIGMA), diluted 1/1000 in TBSTSB, was added and the sections were incubated for 30 min at room temperature. Samples were rinsed extensively with TBSTSB and then twice with TBS. Sections were mounted in glycerol-vinyl-ethanol mounting solution (Zymed, South San Francisco, CA) and viewed using an Olympus BH-2 microscope with epi-illumination fluorescence.

Cultured CAM ectoderm cells were prepared as described above, rinsed with Dulbecco’s phosphate buffered saline (pH 7.2, PBS), fixed for 30 min at 4°C with 4% paraformaldehyde in PBS (pH 7.2), stained for transcalcin using the prepared, 1/200 diluted transcalcin antiserum, and processed immunohistochemically using a Zymed HISTOSTAIN-SP kit (with TBSTSB substituted for PBS in antibody and rinse solutions).

Tissue discs were prepared as described above then transferred to cold HBSSH with 1 mM CaCl₂ added (HBSSH+). At time zero, samples were transferred to HBSSH+ containing tracer amounts of ⁴⁵Ca²⁺ and [³H]inulin (ICN, Costa Mesa, CA) added. At various time points up to 30 min, the discs were removed and rinsed (5 s) in fresh, ice-cold HBSSH with 1 mM EGTA added to remove excess external Ca²⁺ and 0.1% D-glucose to remove excess inulin. The tissue was then placed in 1 ml of Amersham (Arlington Heights, IL) NCS tissue solubilizer for 10 h, followed by neutralization with acetic acid. Calcium and inulin accumulation were determined by liquid scintillation counting in ACS II scintillation cocktail (Amersham). Comparative values were determined by conversion of DPM to volumetric equivalents, and the data were standardized to the surface area of the ectodermal face of the tissue discs.

Incorporation of calcium by isolated CAM ectoderm cells was carried out using a modification of a procedure described previously for CAM microsomal calcium uptake (Tuan et al., 1986a). Briefly, cells were isolated and cultured as described above. Cells were released from tissue culture dishes into suspension using a non-enzymatic cell dissociation solution (Sigma), and collected by centrifugation at 300 g in an IEC centrifuge for 20 min. Cell pellets were resuspended into Hanks’ Balanced Salt Solution with HBSS at the temperature indicated in the text, and the cells were dispersed. Cell densities were determined using a hemocytometer. At time zero, calcium was added to the cell suspension (final concentration of 0.1 or 1.0 mM as indicated) with ⁴⁵Ca²⁺ as a tracer; at various time points, aliquots of the cell suspension were centrifuged through 1.04 g/cm³ sp. gr. mineral oil (Dow Corning) to pellet the cells. ⁴⁵Ca²⁺ accumulation was determined by liquid scintillation counting. In some experiments, the harvested cells were exposed to either 1 mM EGTA or 2 mM CaCl₂ to remove or compete external ⁴⁵Ca²⁺ label prior to collection. Calcium flux was determined either by direct calculation of the difference between calcium uptake at 30 s and 2 min or by indirect calculation from coefficients determined by mathematical curve fitting of the kinetic profile.

Curve fitting was carried out using Enzfitter software (Leather-barrow, 1987) and matched to the general equation: uptake = (A/λ₁)(1−e^−λ1t)+(B/λ₂)(1−e^−λ2t)+…, where A, B, λ₁, λ₂,…are constants and each additional term represents the mathematical values of various cellular compartments that may be involved in calcium uptake. Curve fit data were used to calculate the calcium flux rates and compartment sizes of the system after the method of Borle (1990) and using the equations derived by Uchikawa and Borle (1981). Briefly:

and

and

where ρ represents steady-state calcium flux rate in nmol/h for either 10⁶ cells or 1 cm² of ectodermal surface area; S represents compartment size in nmoles of calcium in either 10⁶ cells or 1 cm² of ectodermal surface area; C_e is the external medium calcium; C_i is the calcium in cellular compartment(s); and C_T is the total calcium at the end of the assay, in nmoles of calcium per 10⁶ cells or 1 cm² of ectodermal surface area. In the case of two compartment mammillary uptake, the intermediate value, X (equivalent to the per h rate constant of calcium flux from compartment one into the medium), was calculated prior to determining the other values.

To begin our study, we first confirmed the sparse distribution of cytosolic structures in CAM CC cells by TEM. Fig. 1A shows the ultrastructure of a section of day 14 CAM tissue including the apical region of a CC cell; consistent with previous descriptions (see Introduction), the cytosol of the CC cell appeared sparse with few membranous organelles. The general structure of the CAM/ISM interface, as seen by TEM, is shown diagrammatically in Fig. 1B.

To isolate calcium-transporting ectodermal cells from the chick CAM, it was necessary: first, to separate the cell layer from two adherent, acellular structures (i.e. the ISM and the basement membrane), and next to purify the CC cells from several contaminating cell types (i.e. allantoic cells, fibroblasts, blood cells, endothelial cells, etc.). We initially sought to release the CAM from the ISM by enzymatic digestion, but found that it was equally effective to simply soak the ISM with buffered saline until the two layers separated, and the coloration of the ISM turned from white to gray. The result of wetting the ISM prior to removal from the CAM is summarized in the SEM micrographs of Fig. 2. Fig. 2A represents the ectodermal CAM surface of a chicken embryo maintained in shell-less culture. The CAM of the shell-less embryo was never attached to the ISM and, therefore, served as a reference for the general morphology of the intact ectodermal layer. The shell-less CAM preparation showed a distinctive, cobblestone-like pattern of ectoderm cells. Fig. 2B shows a normal CAM removed from the ISM without wetting. The delicate CC cells were clearly destroyed and the extensive CAM capillary network exposed, a result consistent with the reported loss of calcium transport function upon mechanical removal of the ISM (see Introduction). Fig. 2C shows a CAM prepared as described above with the ISM thoroughly wetted before removal. The CC cells appeared intact, compared to the cells seen in Fig. 2B, and appeared similar to those of the SL embryo shown in Fig. 2A. Large CC cells were seen surrounding VC cells in a cobblestone-like arrangement with approximately 10⁶ CC cells per cm² of ectoderm surface.

We also asssessed functional differences in calcium uptake between normal CAM tissue discs, prepared with or without wetting of the ISM prior to its removal. As shown in Fig. 3, a fluid-phase marker, [³H]inulin, was used in combination with ⁴⁵Ca²⁺ to examine the extent to which tissue disc preparations accumulated calcium in a specific manner. The data showed that the rate of inulin accumulation by CAM tissue discs was the same with or without intact ectoderm. Calcium uptake by CAM tissue discs with disrupted ectoderm followed kinetics similar to inulin uptake, indicating that, in this case, calcium uptake was largely accounted for by a fluid-phase component alone. On the other hand, tissue discs with intact CAM ectoderm accumulated calcium in a manner that was consistent with a specific calcium-uptake mechanism like that seen in ovo (Crooks and Simkiss, 1975), where sustained and elevated levels of uptake above fluid-phase levels were evident. The morphological and physiological properties of these CAM tissue discs thus demonstrated the usefulness of such preparations for the further isolation of calcium transport cells or for direct, in vitro experimentation.

To prepare CAM ectoderm cells for experimental analysis, CAM tissue discs were further processed and digested. After careful removal of the hydrated ISM, the tissue was drained, and allantoic derivative endoderm cells were removed by placing the tissue, allantoic side down, onto NC. The endoderm adhered to the NC and the ectoderm could be peeled away. This peeling procedure cleaved the tissue down the mesodermal layer and left the ectodermal cell layer intact. Fig. 4A shows a histological section of a CAM sample immediately after dissection from the embryo with the attached ISM. Fig. 4B shows a similar piece of tissue after removal of the ISM and stripping of the allantoic derivative cells. The chorionic ectoderm cells were subsequently prepared for culturing by partial enzymatic digestion of the tissue fragments that remained after ISM separation and endoderm removal. To avoid contamination of the culture by large numbers of capillary endothelial cells or fibroblasts, samples were digested with trypsin/EDTA until small clumps of 5 to 10 cells became evident. The ectoderm cells were separated from the remaining tissue fragments, collected and fibroblasts were removed by preplating onto tissue culture plastic.

To characterize the CAM cell preparations, the degree to which isolated cells maintained markers and activities characteristic of the CAM ectoderm was assessed. As shown in Fig. 5, the cell surface calcium-binding protein, transcalcin, localized to the CAM ectodermal region. CaATPase cytohistochemistry, as shown in Fig. 6, demonstrated that the cells of the CAM ectoderm contain a high level of activity relative to the surrounding tissue. Transcalcin also localized to a subset of cultured CAM cells that had been isolated by extensive digestion of whole CAM (i.e. without prior removal of the allantoic-derivative, endoderm cells). As shown in Fig. 7A, the mixed population of cells released from the intact CAM contained an expectedly small subset of cells that were strongly positive for transcalcin. In Fig. 7B, a similarly stained culture of preferentially isolated CAM ectoderm cells is shown; a high proportion (> 80% in each preparation) of cells with surface transcalcin were seen by immunohistochemistry.

We also examined the distribution of CaATPase activity in cultured CAM cells. Fig. 8A shows histochemical staining of a mixed cell population isolated by extensive digestion from intact CAM. Various levels of CaATPase activity were seen; a small subset of the cells were intensely stained. Fig. 8B shows that, in the enriched preparation of CAM ectoderm cells, most of the cells exhibited intense CaAT-Pase activity staining relative to the few remaining fibroblasts. The observation of two distinct CAM ectoderm markers, cell surface transcalcin and elevated CaATPase activity, clearly indicated that cells prepared by the selective isolation procedure described in this report represent the population of CAM ectoderm cells presumed to be responsible for eggshell calcium transport.

To ascertain functional similarities between cells in vitro and the CAM in vivo, we analyzed and compared the kinetics and temperature dependence of calcium uptake activity in circular pieces of CAM tissue and in isolated CAM ectodermal cells. Using our observation that the ectodermal face of the CAM was made up of approximately 10⁶ cells per cm² of surface area, we compared the results of our in vitro characterizations to CAM calcium uptake seen in ovo. Fig. 9A depicts the kinetic profiles of calcium uptake by embryonic CAM tissue in ovo, CAM tissue discs, and isolated CAM ectodermal cells at 37°C. The uptake for all three preparations followed similar kinetics: rapid accumulation reaching a plateau in about 15 min. Decreasing the assay temperature to 23°C had an inhibitory effect on calcium uptake in each type of preparation, and, as shown in Fig. 9B, the initial rate of ⁴⁵Ca²⁺ accumulation was directly dependent on assay temperature.

Since information derived from the kinetic profiles of ⁴⁵Ca²⁺ uptake can be analyzed to assess the compartmentalization of Ca²⁺, we used computer curve fitting analysis to look at the routes of calcium uptake in each type of CAM preparation. As summarized in Table 1, a single mathematical term best accounted for each uptake curve, i.e. the shapes of the ⁴⁵Ca²⁺ accumulation curves in ovo, in tissue discs and in isolated cells were all consistent with a rapid influx of calcium into a single cellular compartment.

Calcium uptake by the CAM is an important, developmentally regulated function of the chick embryo. Research on the mechanism of CAM calcium transport has been limited due to the lack of suitable in vitro preparations. Previous studies of the physiological and mechanistic aspects of CAM calcium transport have relied on tissue preparations with the ectodermal cells obscured by the maternally derived ISM (e.g. see Crooks and Simkiss, 1975; Crooks et al., 1976; Garrison and Terepka 1972a,b; Terepka et al., 1969, 1976; Tuan, 1980; Tuan et al., 1986a,b; Tuan and Zrike, 1978), fixed and sectioned material (e.g. see Coleman and Terepka, 1972a-c), subcellular membrane preparations (Tuan et al., 1986a,b), or biochemical activities (e.g. see Tuan, 1984; Tuan et al., 1978b,c, 1986a,b;,Tuan and Knowles, 1984; Tuan and Scott, 1977; Tuan and Zrike, 1978). We present here a method to isolate and culture CAM ectoderm cells for the investigation of calcium homeostasis at the cellular level. We also demonstrate the in vitro maintenance of a calcium influx pathway that is characteristic of the rapid accumulation of calcium seen in ovo.

The preparation of CAM tissue discs with intact ectodermal surfaces was possible due to the nature of the interaction between the CAM and the ISM. During the course of development, as embryonic respiration increases, the ISM progressively dehydrates (Seymour and Piiper, 1988; Seymour and Visschedijk, 1988). Dehydration of the ISM roughly parallels the increase in its adhesion to the CAM, and, since simple hydration of the ISM prior to removal from the CAM was sufficient to allow the maintenance of the integrity of the CAM ectodermal surface, dehydration may account for CAM to ISM adhesion. Careful removal of the wetted ISM allowed direct access to the CAM ectoderm and made the tissue suitable for further in vitro manipulations.

As shown in Fig. 2, SEM analysis of the ectodermal surface of CAM preparations showed a coblestone-like pattern of cells in the shell-less chick embryos, similar to that previously reported by Dunn and Fitzharris (1979). When the ISM was hydrated prior to its removal from the CAMs of embryos developing in ovo, a cobblestone-like arrangement of cells was also observed. Although the cells of the shell-less CAM appeared smaller than those of the normal CAM, a difference that may reflect changes in morphology modulated by the presence of the ISM and eggshell (see Coleman and Terepka, 1972c; Tuan, 1983), the preparations were remarkably similar. Tissue discs may prove useful in the characterization of calcium uptake and transport by the CAM in a multicellular, in vitro system that should closely simulate in vivo conditions. It must be emphasized, however, that the underlying endothelial cells also transport calcium, and care must be taken to account for uptake into each cell layer.

Tissue discs of CAM have been shown to exhibit rapid, active and unidirectional calcium transport. The first component of this transport is uptake of calcium across the apical membrane of the CAM ectoderm. Crooks and Simkiss (1975) demonstrated that tracer ⁴⁵Ca²⁺ accumulation from the eggshell facing surface of the ISM was approximately equal to the accumulation of calcium in the embryo (i.e. net flux into the CAM from the eggshell equals flux out of the CAM into the embryonic compartments). In addition, Terepka and co-workers showed in vitro that the reverse flux of Ca²⁺ from the CAM back toward the eggshell was nearly negligible and that the CAM continued to accumulate calcium up substantial concentration gradients (see Terepka et al., 1976). Therefore, Ca²⁺ taken up by the rapid apical pathway of the CAM ectoderm seems destined for transport. Since isolated CAM ectoderm cells maintain a rapid Ca²⁺ uptake pathway similar to that seen in ovo, these cells may represent an exciting in vitro system for analysis of CAM calcium homeostasis.

It should be pointed out that we have attempted to analyze calcium transit through monolayer CAM ectoderm cells cultured on permeable Transwell membranes (Costar; Cambridge, MA), to simulate an epithelial cell layer (see Breitfield et al., 1989). Unfortunately, isolated CAM cells did not proliferate to form confluent monolayers in vitro, and we were unable to establish a suitable transport system. We were also unable to obtain viable, CAM ectoderm cells from embryos older than incubation day 15, although, during development in ovo, transport activity does not peak until around day 17. Both these observations are consistent with the morphological descriptions of ectoderm cells reported by Coleman and Terepka (1972c): the CC cells become terminally differentiated during development as calcium transport activity begins to increase in the CAM. Since mature CC cells lack membranous biosynthetic organelles (Coleman and Terepka, 1972c), the cells may simply be non-proliferative and recover poorly from enzymatic dissociation. Thus, it was not unexpected that the most successful cell isolations were those using embryos between days 12 and 15 of incubation, a period corresponding to the early phase of increasing calcium transport activity (Tuan et al., 1986a), but prior to the complete loss of cellular biosynthetic machinery (Coleman and Terepka, 1972c). In fact, in terms of the developmental profile of calcium uptake activity in isolated cells, we have observed the highest level of activity in day 13 to 15 CAM (data not shown). In this study, CAM cells were not harvested before day 10 of incubation, since CAM calcium transport does not normally start until later (Tuan, 1987). It should be noted that we have recently analyzed the calcium-uptake characteristics of the human choriocarcinoma cell line, JEG-3 (Tuan et al., 1991). The JEG-3 cells are derived from an epithelial choriocarcinoma and appear to exhibit calcium-transport-related activities similar to those of isolated CAM cells. JEG-3 cells may be useful in the direct analysis of transepithelial calcium transport by cells of extra-embryonic membranes.

Taken together, the presence of two known functional biochemical components of calcium transport in the isolated CAM cells, along with their ability to accumulate calcium rapidly in a manner similar to that seen in ovo and in tissue discs in vitro, strongly suggest that the isolated primary CAM ectoderm cells represent the calcium transport component of the CAM chorionic epithelium. The CAM ectoderm cells should, therefore, constitute a valid primary culture system for characterizing the cellular mechanism of CAM calcium transport and investigating calcium homeostasis in this transport epithelium. In the accompanying paper, we have characterized the calcium influx phase of the CAM transepithelial calcium transport pathway by analyzing the cellular distribution of calcium in isolated CAM ectoderm cells during the initial stages of calcium accumulation.

The authors thank David Kreitzer for EM analyses. This work was supported in part by grants from the NIH (HD15822 and HD21355), March of Dimes Birth Defects Foundation (1-1146), USDA (88-37200 and 90-37200-5265), and the Orthopaedic Research and Education Foundation.