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PCC4azal teratocarcinoma stem cell differentiation in culture
DEVELOPMENTAL
BIOLOGY
75, 93-111 (1980)
PCC4azal Teratocarcinoma
Stem Cell Differentiation
II. Morphological CECILIA The Rockefeller
Characterization
W. Lo’ AND NORTON B. GILULA
University,
Received April
in Culture
1230 York Avenue, Neu! York, New York 10021
23, 1979; accepted in revised form August 2, 1979
The ultrastructural morphology of the PCC4azal embryonal carcinoma cells and their differentiated counterparts, endoderm-like cells and giant cells, was characterized and compared with that of the cells of embryoid bodies. The ultrastructure of the PCC4azal embryonal carcinoma cells is similar to that of the embryonal carcinoma cells of the embryoid body. These cells are small, with a large nucleus and relatively few cytoplasmic organelles. Gap junctions and modified adherens junctions are formed at some areas of intercellular contact between the embryonal carcinoma cells. The differentiated PCC4azal endoderm-like cells have a more developed cytoplasm, containing an extensive endoplasmic reticulum with large Golgi regions. Most striking is the de nouo appearance of epithelial-like junctional complexes which join the apical borders between the endoderm-like cells, thus polarizing the cell monolayer. The zonula occludens junctions of the junctional complex are extensive, consisting of six or more strands of tight junctional ridges. Terminal webs are present in the apical regions that are inserted into the zonula adherens region of the junctional complex. Gap junctions continue to join neighboring cells, and some gap junctions are intercalated within tight junctional ridges. The ultrastructure of the differentiated endodermal cells of the embryoid bodies is very similar to that of the PCC4azal endoderm-like cells. The embryoid body endodermal cells form similar junctional complexes which also contain continuous belts of tight junctions that are intercalated with gap junctions. As the PCCIazal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of lo-nm filaments, microtubules, and 7-nm microfilaments. The junctional complexes that were present during the endodermal stage are partially disassembled as the giant cells migrate apart. Thus, the differentiation process in this system is characterized by significant and distinctive morphological changes. INTRODUCTION
pare the cytodifferentiation ture to the differentiation
The differentiation of PCC4azal embryonal carcinoma cells, as described previously, is accompanied by distinct changes in enzyme properties and by the disappearance of a cell surface antigen (Lo and Gilula, 1980a). The morphological changes which the embryonal carcinoma cells undergo are striking, starting from a small epithelial cell to a giant fibroblast-like cell. It is of interest to study the ultrastructural morphology of these cells in order to (1) obtain an ultrastructural basis for the cytodifferentiation of the teratocarcinoma cells and (2) com-
obtained in culof the tumor in
vivo. In this study, the ultrastructure of the various cell types of the PCC4azal cells cultured in vitro and the ascites form of the original tumor line, the embryoid bodies, is characterized by freeze-fracture and thinsection electron microscopy. As differentiation to giant cells proceeds, specific elaboration of the cytoplasmic organelles and the cytoskeleton is observed and specialized cell surface contacts are developed. The PCC4azal embryonal carcinoma cells and the endoderm-like cells formed in vitro are compared to the embryo& carcinoma cells and the outer ring of differentiated endo-
’ Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
93 00%1606/80/030093-19$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction
in any form reserved.
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derm-like cells of the embryoid bodies formed in vivo. A preliminary report of this study was presented previously (Lo and Gilula, 1978). MATERIALS
AND
METHODS
Materials Eight percent aqueous glutaraldehyde (N2 sealed) and 4% aqueous osmium tetroxide (Polysciences Inc., Warrington, Pa.); analytical grade tannic acid and reagent grade glycerol (Mallinckrodt, St. Louis, MO.); uranyl acetate (Fisher Scientific Co.); Epon 812 (Ladd Research Industries, Inc., Burlington, Vt.); and gold alloy specimen carriers (Balzers High Vacuum Corp., Santa Ana, Calif.). All other materials used are as described previously (Lo and Gilula, 1980a). Microscopy (Thick Sections and Thin Sections) The PCC4azal cells and the embryoid bodies were maintained as described in the previous study (Lo and Gilula, 1980a). For microscopy, the PCC4azal cells were processed at room temperature in the tissue culture dishes and the embryoid bodies as a suspension using published procedures (Kalderon et al., (1977)). Briefly, the cells were fixed in 2.5%glutaraldehyde, postfixed in 1%osmium tetroxide, and stained en bloc with 0.125% tannic acid followed by uranyl acetate. The specimens were further processed by dehydration in a graded series of
ethanol and embedded in Epon 812. The embedded PCC4azal cell monolayers were peeled off the plastic culture dishes, and areas selected by light microscopy were punched out. The punched-out disks were then mounted on Beem capsules or reembedded in flat blocks. Thick sections were cut with glass knives, mounted on glass slides, and stained with toluidine blue. Photographs were taken on Kodak Plus-X film with a Zeiss photomicroscope II. Thin sections obtained with a diamond knife were poststained with uranyl acetate and lead citrate prior to examination in a Siemens 101 electron microscope at 80 kV. Freeze-Fracture Replication For freeze-fracture, the PCC4azal cells were grown on Balzers gold carriers for the double replica device. They were fixed as above and then treated with 25% glycerol in 0.1 M cacodylate buffer (pH 7.3) at 4°C overnight. Prior to freezing, the specimens were brought to room temperature, then sandwiched with an identical blank carrier and rapidly frozen in liquid nitrogen-cooled Freon 22. The frozen sandwiched carriers were mounted in the Balzers machine (Freeze Etch Unit BA 360 M), then forced open at -115°C and immediately shadowed with platinum and carbon. The replicas were cleaned with sodium hypochlorite and subsequently examined in a Siemens 101 electron microscope.
FIG. 1. Embryoid body ultrastructure. (a) Thin-section electron micrographs of an embryoid body. The outer layer of endoderm-like cells (EN) has a highly differentiated cytoplasm which contains many large lipidlike droplets. Junctional complexes join the outer layer of endoderm-like cells at their apical cell surfaces (arrow). Numerous microvilli project from the outer cell surface, and an amorphous material resembling a basal lamina (BL) is sometimes found beneath the endodermal cell layer. The embryo& carcinoma cells (EC) which are located on the inside have few organelles, and these cells are closely associated with each other as well as with the endoderm-like cells. x 7200. Inset: Thick section of an embryoid body. The endoderm-like cells (EN) form a closed vesicle, and the embryonal carcinoma cells (EC) are located within it. The bar in the inset represents 36.9 pm. (b) An enlargement of an area similar to that indicated by the arrow in a. A junctional complex (JC) between the apical surfaces of two endoderm-like cells. Elements of the junctional complex including a tight junction (TJ), a gap junction (GJ), and desmosomes (D) are present. Fibrous densities in the cytoplasm next to the junctional complex are elements of the terminal web. x 84,000. (c) Freeze-fracture replica of an embryoid body junctional complex region exposing the network of tight junctional ridges (black and white arrow). Gap junctional particles (black arrow) are intercalated within some regions of the tight junctions. x 6o.ooo.
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The embryoid bodies were fixed and glycerinated as above but in a suspension. They were then transferred onto paper disks as concentrated droplets and rapidly frozen in liquid nitrogen-cooled Freon 22. The frozen samples were mounted in the Balzers device and cut with a precooled knife at -115°C. The replicas were processed and examined as above. RESULTS
Embryoid
Bodies
The mouse teratocarcinoma can be adapted to growth in the peritoneum as floating aggregates called embryoid bodies (Stevens, 1960; Pierce et al., 1959). They are organized structures consisting of an outer layer of differentiated cells referred to as the endoderm and an inner mass of cells containing undifferentiated embryonal carcinoma cells (see inset, Fig. la). The embryonal carcinoma cells have a large nuclear to cytoplasmic ratio and contain few organelles, including a few spherical mitochondria with few cristae, some endoplasmic reticulum, primitive Golgi, occasional lipid droplets, and ribosomes (Fig. la). They are linked to one another by gap junctions and modified adherens junctions (data not shown). The endodermal cells in the outer layer are usually aligned end to end, forming a single cell layer often resembling a squamous epithelium. In some regions a pseudostratified arrangement of the endodermal cells is also present. The endodermal cells have numerous microvillar projections on their outer membrane which faces the external medium. The cytoplasm of endodermal cells contains well-developed Golgi regions, elongated mitochondria with moderate numbers of cristae, rough
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and smooth endoplasmic reticulum, ribosomes, glycogen granules, lipid-like droplets, and pinocytic vesicles (Fig. la). The cisternae of the endoplasmic reticulum often contain amorphous deposits. The endodermal cells are linked to one another at the outer cell borders by zonulae occludens or tight junctions, zonula adherens junctions, desmosomes, and gap junctions that are arranged in a junctional complex (Figs. la, b, and c). Similar to the junctional complex of a typical epithelium, there is a “tripartite” arrangement at the apical or “luminal” surface, starting with the zonula occludens or tight junction at the most apical region, followed by the zonula adherens junction, and then desmosomes (Figs. 4b, 5, 6b, and 7) (Farquhar and Palade, 1963; Friend and Gilula, 1972). Gap junctions are present at varying locations within this complex. Filaments of the terminal web of the junctional complex are associated with and terminate at the membranes of the junctional complex (Fig. lb). Freezefracture replicas of the embryoid bodies reveal that the tight junctions consist of a complex array of many tiers of interconnecting 8.5-nm ridges on the P fracture face (inner membrane half) and grooves on the E fracture face (outer membrane half). They form continuous belts of ridges and grooves around the entire apical border of the embryoid body. These tight junctional elements are often intercalated with aggregates of 8.5-nm gap junctional particles (on the P face) and pits (on the E face). An amorphous basal lamina frequently separates the outer endodermal layer from the inner mass of embryo& carcinoma cells. This lamina is not continuous beneath the entire endoderm layer, and in many regions
FIG. 2. Step cell or embryonal carcinoma cell. (a) Horizontal section. The embryonal carcinoma stem cell has a large nucleus with one or two nucleoli exposed in this plane of section. The cytoplasm is small, with few organelles. A rudimentary Golgi apparatus is visible at the top left (G). x 6060. (b) Vertical section. (The bottom of the culture dish or the substrate is indicated by the letter S. All subsequent vertical sections are similarly labeled.) The cuboidal cells are arranged as a single cell layer with some overlapping processes. Note the areas of specialized cell contact (arrows). A gap junction (small arrow) and a modified adherens junction region (large arrow) are present. X 6900.
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FIG. 3. Specialized intercellular junctions between embryonal carcinoma stem cells. (a) Freeze-fracture image of a large gap junction. A plaque of particle aggregates separated by particle-free aisles is present on the P face (P), while complementary pits are present on the E face (E). Note the isolated small aggregates of E-face pits in the upper-left region. X 60,000. (b) Thin section of modified adherens junctions. At this site of close membrane apposition, dense fibrous material accumulates on the cytoplasmic surfaces of both membranes. The accumulation is asymmetrical; it is heavier on the membrane of the cell to the right. The intercellular space narrows to 6-8 nm, and some dense staining material is present between the membranes. X 69,000. (cl Thin section of a gap junction. The two closely apposed membranes are separated by an intercellular space or gap of 2-4 nm, generating a septalaminar image. In this image, the gap is filled with electron-dense tannic acid stain. x 96,000.
the inner embryonal carcinoma cells are in direct contact with the endodermal cells. Embryoid bodies have also been reported to contain other cell types (Martin et al., 1977); however, with the embryoid bodies used in this study, only the embryonal carcinoma cells and the endodermal cells were unequivocally identified. It is possible that a small number of other cell types is present but is difficult to identify due to either their smaIl numbers or their morphological similarity to the endodermal or embryonal carcinoma cells.
PCC4azal
Stem Cells
Stem cells are closely packed and their nuclear to cytoplasmic ratio is characteristically high. The cytoplasm is relatively undifferentiated, containing many free ribosomes, a number of small spherical mitochondria, and a rudimentary Golgi. Elements of the endoplasmic reticulum are almost nonexistent. From verticle sections, it can be seen that embryonal carcinoma cells are arranged as a closely packed single layer of cuboidal
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FIG. 4. Cells from stage 1 of differentiation, elongation. Whenever possible, the same labeling is used in both a and b. (a) Horizontal section. The nucleus and cell body are elongated. The Golgi apparatus (G) is enlarged, and some endoplasmic reticulum (ER) is present. Microfilament bundles (MF) extend along the length of the cell, while others are associated with the cortical regions. The microfilaments in the cortical regions are inserted into a series of adherens junctions (arrows) to form a terminal web-like structure (TW). x 9120. (b) Vertical section. The change in cell shape is apparent in this plane (cf. Fig. 2b); the cell is flattened (squamous) and the nucleus is oblong. x 9240.
cells with some overlapping processes (Fig. 2b). Gap junctions and modified adherens junctions are formed in some areas of cellto-cell interaction (Figs. 3b and c). In
freeze-fracture replicas, the gap junctional particle aggregates consist of 8.5nm particles in the P fracture face and complementary pits in the E fracture face. The gap
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junctions are low in frequency and variable in size, containing from a few particles to over 900 particles per plaque (Kreutziger, 1968; McNutt and Weinstein, 1970; Chalcroft and Bullivant, 1970; Friend and Gilula, 1972). These particle aggregates and their complementary pits are loosely arranged in small islands which are separated by particle-free aisles (Fig. 3a). The modified adherens junctions formed are simple, consisting of close membrane appositions where the two apposed membranes are aligned in parallel and separated by an intercellular space of 6-8 nm (Fig. 3b). Fibrous densities are intimately associated with the cytoplasmic surfaces of both membranes, and sometimes the density is greater on one of the two membranes. PCC4azal
Differentiated
Cells
The ultrastructural characterization of the PCC4azal transformation to endodermlike cells and then to giant cells is presented in the same three-stage scheme as reported in the previous study (Lo and Gilula, 1980a). Stage 1: Cell elongation. With the onset of differentiation to endoderm-like cells, the cells flatten and are arranged as a monolayer of squamous epithelium (compare Fig. 2b with Fig. 4b). This results in the lengthening and apparent enlargement of the cell as seen in horizontal sections (Fig.
4a). Concomitant with this overall change in cell shape is a similar flattening and elongation of the nucleus of the cell (Fig. 4b). Within the cytoplasm Golgi elements are prominent. A system of smooth and rough endoplasmic reticulum is present (Fig. 4a) and a light flocculent material is distributed within its cisternae. At regions of the cell periphery, large pockets of intercellular space interrupt the attachment of the cells to the substrate such that only the central (somatic) portion of the cell is in contact with the culture dish. The peripheral regions of the cytoplasm decrease in thickness and the intercellular contacts are established at some distance from the substrate (Fig. 4b). Although the area of cell-to-cell contact is greatly reduced (compare Fig. 2a with Fig. 4b), each cell maintains intimate contact with its neighbors. At the regions of cell-tocell contact, junctional complexes join the adjacent cells. The junctional complex is formed de novo, and it is not yet completely developed at this stage. The ordering of the junctions within the complex is similar to that of a typical epithelium, with tight junctions at the luminal surface, followed by zonula adherens junctions, then desmosomes. Gap junctions are intercalated throughout the complex as before. The equivalent “luminal” side in this monolayer culture system is the apical surface of the
FIG. 5. Specialized intercellular junctions between differentiating cells of stage 1. (a-c) Thin-section morphology. (a) Gap junctions. At a region comparable to that depicted by the arrows in Fig. 4a, a series of gap junctions (GJ), intercalated by zonula adherens junctions (ZA), joins the cells. x 52,650. (b) High magnification of a gap junction similar to those in a. Note the septalaminar appearance and the dense material on the cytoplasmic surfaces of the junctional membranes. x 180,000. (c) Demosomes (macula adherens junctions). Several desmosomes (D) join two adjacent cells. Cytoplasmic densities along both membranes of the desmosomes are intimately associated with IO-nm tonofilaments (TF), and these are connected to each other via the tonofilaments. At these contacts, the 30-nm intercellular space contains a crystalline extracellular matrix that is bisected by a central dense lamina. x 36,000. (d, e) Freeze-fracture morphology. (d) Tight junction (ridge) formation. Tight junctional ridges are assembled by the linear aggregation of intramembrane particles that subsequently fuse. The black and white arrows (l-4) depict stages of transition, from aligned single particles in area 1 to completed ridges in area 4. Arrow 2 indicates an area where single particles and short segments of completed ridges are closely associated. In area 3, short completed ridges are aligned with one another. The black arrow points to a particle aggregate associated with a ridge; this aggregate may be related to a gap junction. x 60,000. (e) A plaque of gap junctional particles is closely associated with some tight junctional ridges (arrow) and complementary grooves. x 60,000.
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cells, where the basal surface is defined as being adjacent to the bottom of the culture dish. Each element of the junctional complex of these differentiating cells is described in detail below. Tight junctional elements are detected for the first time in freeze-fracture replicas of these cells at this stage. The de novo assembly of the 8.5~nm intramembrane tight junctional ridges (Friend and Gilula, 1972; Claude and Goodenough, 1973; Staehelin, 1973) involves much of the lateral membrane surfaces. Various structural arrangements are present that can be translated into a possible sequence of assembly events (Fig. 5d). These include the following: (1) Individual 8.5-nm particles align in linear arrays; (2) the linear particle aggregates fuse to generate short ridge segments; and (3) short ridge segments fuse to form more extended ridges that are randomly oriented. Similar schemes of tight junctional ridge formation have previously been described in other systems (Elias and Friend, 1976; Montesano et al., 1975). During this assembly process, gap junctional particle aggregates are closely associated with the tight junctional ridges (Fig. 5d), and they are frequently quite large. They have a characteristic septalaminar arrangement in thin sections (Fig. 5b) and their particle packing in freeze-fracture replicas is similar to the gap junctional particle arrangement of stem cells (Fig. 3a). Gap junctions of similar size and morphology also are present in regions some distance from the complex. Microfilaments and microfilament bundles are distributed throughout the cytoplasm, and frequently they are aligned in parallel and in close association with the cell borders (Fig. 4a). They insert into regions of the zonula adherens junctions to form a rudimentary terminal web similar to that in various epithelia (Figs. 4a and 5a) (Farquhar and Palade, 1963,1965). Many desmosomes are frequently found in series below the zonula adherens junc-
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tions (Fig. 5~). Tonofilament bundles interconnect the characteristic cytoplasmic dense plaques, resulting in the formation of linked chains of desmosomes. Stage 2: Cell enlargement. The differentiating endoderm-like cells are larger in size (compare Fig. 4a with Fig. 6a). The changes in the cytoplasm that were initiated with the onset of differentiation are more apparent at this stage (Fig. 6a). These include further enlargement of the Golgi regions and the endoplasmic reticulum and the presence of phagolysosomal vesicles. At the apical regions of the cells, junctional complexes consisting of tight junctions, terminal web attachment elements (zonula adherens), and desmosomes have further developed, and they are now elaborate and mature structures. Their de novo assembly which was initiated at stage 1 is now complete. The tight junctions, which previously consisted of short lengths of ridges and grooves which were randomly oriented, are now composed of extensive lengths of ridges and grooves arranged in parallel tiers of 6 to 10 interconnected strands (Fig. 7). They form continuous belts which surround the entire apical borders of each cell. In addition to this belt-like arrangement of the tight junction within the junctional complex, tight junctions in plaque-like domains exist in regions below the complex (Fig. 7). These consist of localized areas of concentric arrays of interconnected tight junctional strands. This arrangement of tight junctions has previously been described in some epithelia and is referred to as a “compartmentalized” junction (Staehelin et al., 1973; Friend and Gilula, 1972; Orci et al., 1973). Gap junctions are intercalated within both types of tight junctions, and they are found at a lower frequency than during the time of tight junctional ridge formation. The terminal webs associated with the zonula adherens junctions are well developed, consisting of a thick assembly of microfilaments. Below the belts of occluding
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FIG. 6. Stage 2 of differentiation, cell enlargement. (a) Horizontal section. The shape of this large cell is polygonal. The endoplasmic reticulum (ER) is dilated, and a flocculent matrix is dispersed within its cisternae. There are large numbers of mitochondria, and the cells are joined by extensive junctional complexes (arrow). x 4800. (b) Desmosome (D) between the differentiating cells. Cytoplasmic plaques with their associated tonofilaments (TF) are increased in density. Note that the desmosome to the left bends at a sharp angle. x 36,000.
zonula adherens junctions, desmosomes continue to join adjacent cells. The fibrous cytoplasmic plaques of the desmosomes have greatly increased in density, and their cytoplasmic membranes are no longer always aligned in straight lines. The opposing membranes, while remaining parallel, often undulate with bends and curves. These des-
mosomes resemble the “modified desmosomes” of the frog skin (Fig. 6b) (Farquhar and Palade, 1965). Stage 3: Terminal differentiation of giant cells. The differentiating cells have greatly enlarged and are further flattened, especially in peripheral regions of the cell away from the nucleus (Figs. 8b and c).
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FIG. 7. Freeze-fracture morphology of specialized junctions in later differentiating cells (stages 2-3). Mature tight junctions coexisting in two arrangements: a belt-like zonula occludens, and a plaque-like macula occludens. In the upper region, a complex array of several parallel tiers of ridges on the P face (P) and their complementary grooves on the E face (E) characterizes the belt-like arrangement. Short strands at an angle to the axis of the belt connect the tiers. At the lower left, several sequestered domains of grooves in the E face represent the plaque-like or macula configuration of tight junctional elements. In this arrangement, one continuous strand usually defines the boundary of the tight junction domain. x 60,000. Inset: Intermediate stage of development of the zonula occludens junction. Short segments of completed ridges exist in semiparallel arrays. Some of the strands branch at an angle to connect parallel ridges. The arrows indicate closely associated gap junctional particle aggregates, some of which are sequestered within these ridges. x 75,000.
Some of these regions are so thin that they are but a fraction of the original thickness of stem cells (compare Figs. 2b and 8~). Most of the cellular organelles are located in the perinuclear region of the cytoplasm (Fig. 8a). The endoplasmic reticulum is extremely distended and completely filled with a dense amorphous matrix. Golgi re-
gions are large and abundant. The mitochondria are very long and greatly increased in number. The organelles seem to be geographically organized in a nonrandom fashion, where some regions of the perinuclear cytoplasm are predominantly occupied by one type of organelle, i.e., an entire region of distended endoplasmic re-
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ticulum, or mitochondria, etc. The phagolysosomes are quite large and filled with various dense elements. Lipid-like droplets are present, consisting of membrane-bound vesicles filled with an amorphous mass. At the periphery of the cell, a few mitochondria are present and a system of anastomosing endoplasmic reticulum extends throughout the complex array of cytoplasmic fibers (Fig. 9c). Many of the regions of close cell-to-cell contact are disassembled as a result of increased cell motility (Lo and Gilula, 1980a). The cells closely interact with one another via a series of cytoplasmic interdigitations (Fig. 9a, inset). The finger-like interdigitation results from the insertion of a thin pseudopod-like structure into an invagination of the adjacent cell (Fig. 9a). Zonula adherens junctions continue to join some regions of contact, and they are the insertion site for the extensive terminal web elements which are still present (Fig. 9a). Due to the association of many filaments with the membrane and the undulation of
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the opposing membranes themselves, it is difficult to resolve the thin-section characteristics of the junctions that are present in the regions of cell-to-cell contact. Some punctate contacts can be tentatively identified as tight junctions (Figs. 8d and e), although some of these may, in fact, be gap junctions. A more precise identification awaits additional freeze-fracture information. The most striking features of the differentiated giant cell are the large number of cytoplasmic fibers and their complex arrangements. Stress fibers seen in the light microscope (Fig. 9a, inset) are readily identified in thin sections as thick cables of 7nm microfilaments which extend in straight lines for long distances in the cytoplasm (Fig. 9d) (Buckley and Porter, 1967; Goldman et al., 1975). The bundles of microfilaments have dense bodies or bands along their length. These cables sometimes intersect, and at the regions of juncture, microtubules sometimes extend through and between adjacent bundles (Fig. 9d). Microtu-
FIG. 8. Giant cells. (a) Horizontal section. This cell is extremely large; only a small portion of the perinuclear region of the cytoplasm is shown here. The endoplasmic reticulum (ER) is dilated and completely filled with a dense matrix. The Golgi components (G) are well developed. Many mitochondria, lipid-like vesicles (L), and lysosomal (LY) elements are also present in this region. x 6400. (b-e) Vertical section. (b) A region of a giant cell containing a small portion of the nucleus and perinuclear cytoplasm (cf. Figs. 2b and 4b). The nucleus is pleiomorphic in shape. This section plane provides a different view of the arrangement of cytoplasmic organelles (cf. a). The endoplasmic reticulum (ER) is in a parallel array, with no visible dilation. The Golgi (G) is arranged as stacks of flattened vesicles, and a large phagolysosome (PL) extends from the apical to the basal region of the cell. x 7600. (c) A small region of the peripheral cytoplasm of the giant cell some distance from the nucleus. The cell is exceedingly thin in this region (cf. Figs. 2b and 4b). Few cytoplasmic organelles are present. Filament bundles are the most prominent cytoplasmic components. x 12,000. (d) Region of contact between the peripheral regions of two cells (arrows). These are not two cell processes or microvilli. The areas of contact are filled with microfiiaments that are related to the terminal web-like (TW) structure. x 24,000. (e) High magnification of the region of contact in d (asterisk). The two arrows outline a small focal region of membrane contact between these giant cells. X 132,000. FIG. 9. Characteristic features of a differentiated giant cell. (a) A series of zonula adherens (ZA) junctions joins two giant cells. Parallel arrays of 7-nm microfiiaments insert into cytoplasmic densities along the membranes of the adherens structures. The microfilaments of one cell are strikingly aligned with those of its neighbor. In addition, a cell process of the upper cell is invaginated into the cytoplasm of the adjacent cell, perhaps stabilizing their association. x 36,000. Inset: This light micrograph shows a region where three giant cells are in intimate contact. Note the interdigitation of the cytoplasmic borders and the many stress fibers in the cytoplasm. x 200. (b) Bundles of lo-nm filaments are distributed throughout the cytoplasm. x 48,000. (c) A complex array of microtubules (large arrow) and lo-nm filaments (small arrow) intermingles in the peripheral cytoplasm. Note the anastomosing endoplasmic reticulum (ER). x 43,500. (d) Stress fibers are comprised of tightly packed microfilament bundles. At the left, the bundles merge and microtubules are closely associated with this area. Note the periodic dark banding distributed along the fiber bundles (arrow). x 11.400.
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e FIG. 8. a-e.
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bules usually exist as single fibers that are widely distributed in the cytoplasm (Fig. 9c). Many lo-nm filaments are also present, and they are arranged in loose bundles which twist and turn. The interplay of these three types of filaments forms a complex cytoskeletal network which extends throughout the enormous expanse of cytoplasm, providing these cells with a distinct fibroblast-like morphology. DISCUSSION
In this study the morphological characterization of the PCC4azal cells and the embryoid bodies provides a basis for understanding the cellular changes that are associated with the differentiation process in the PCC4azal system and relating the PCC4azal differentiated cells to the cellular phenotypes in the embryoid bodies. The morphological transformation of the PCC4azal stem cells is characterized by several major changes, including an increase in cell size, a change in cell shape, the development of a junctional complex that includes the de novo formation of a zonula occludens, an alteration in cellular metabolism and biosynthetic activity as indicated by cytoplasmic organelle development, and the genesis of an extensive cytoskeletal framework. Comparison of the embryoid bodies with the PCC4azal cells has shown that the stem cells of the embryoid bodies are identical in morphology to the PCC4azal embryonal carcinoma cells that are maintained in culture, and the differentiated endodermal cells of the embryoid body are very similar, if not identical, to the PCC4azal endoderm-like cells that are generated in culture. Since in previous studies it has been demonstrated that the endodermal cells and other differentiated cell types of teratocarcinomes are derived from embryo& carcinoma cells, the differentiation of PCC4azal embryonal carcinoma cells to similar endoderm-like cells described in this study probably parallels the in vivo differentiation of embryonal carcinoma cells to endoderm in the embryoid
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bodies (Kleinsmith and Pierce, 1964; Martin and Evans, 1975). In previous studies it has been suggested that embryonal carcinoma cells originate from embryonic ectodermal cells (Damjanov and Solter, 1974) or primordial germ cells (Pierce and Beal, 1964; Pierce et al., 1967), since all three cell types are morphologically very similar and have similar capabilities of differentiation into many cell types. However, in a recent study, evidence has been presented to suggest that embryonal carcinoma cells are probably derived from somatic cells and not germ cells (Mintz et al., 1978). In this study, the morphology of the embryonal carcinoma cells of the embryoid body was observed to be similar to that of the PCC4azal embryonal carcinoma cells, and this is consistent with the fact that the PCC4azal cells were originally derived from embryoid bodies (Jakob et al., 1973). The true in vivo equivalence to this stem cell phenotype awaits further characterization. The term “endodermal” has been used by many investigators to describe the differentiated outer layer of cells of the embryoid body. The basis for this distinction is primarily morphological; the endoderm of the embryoid body is thought to be related to the outer layer of extraembryonic endoderm formed around the inner cell mass of the mouse embryo (Martin, 1975). In this study we have utilized the same general morphological criteria for adopting the term “endoderm-like” for the stage 1 and stage 2 differentiated PCC4azal cells. Furthermore, it is now possible to include several additional specific morphological criteria for applying this endodermal distinction. These include the extensive development of specific cytoplasmic organelles and the de novo formation of junctional complexes, in particular the zonula occludens element. A definitive clarification of these cells as being equivalent to the extraembryonic endoderm of the mouse embryo will require additional characterization.
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The de novo formation of junctional complexes is the clearest indication by morphology of a new cell type arising from the differentiation of PCC4azal embryonal carcinoma cells in vitro or embryonal carcinoma cells of the embryoid body in vivo. The arrangement of highly specialized junctions in a complex is frequently found in well-differentiated epithelia, where the continuous belts of occluding junctions form a permeability seal (Farquhar and Palade, 1963; Friend and Gilula, 1972; Claude and Goodenough, 1973). In fact, an effective permeability seal must exist in the embryoid bodies since many of them are swollen with fluid, somewhat resembling a blastocyst with a pumped-up blastocoelic cavity. Similarly, large intercellular spaces formed by the detachment of the peripheral cytoplasm of the PCC4azal endodermal cells from the substratum may result from the accumulation of fluid following the formation of a permeability barrier. The cells formed from the endoderm-like cells are labeled “giant” simply to emphasize their large size and extremely low nuclear to cytoplasmic ratio. The giant cell is distinctively fibroblastic in morphology, and its cytoplasmic characteristics appear to be the result of further elaboration of the cytoplasmic differentiation that was initiated at stage 1. In particular, the Golgi elements and the endoplasmic reticulum, which are still somewhat rudimentary at the endoderm-like stages, are well developed at the giant cell stage. The development of these organelles probably reflects a major change in the biosynthetic activity that accompanies this differentiation. The complex array of microfilaments, microtubules, and lo-nm filaments in the giant cells also appears for the first time in the endoderm-like cells. These cytoskeletal elements may be essential for the maintenance of cell shape and motility, as well as for the coordination of the various metabolic activities within the cytoplasm. Cell-to-cell interactions between giant cells are limited, and thin-section profiles suggest that some
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tight junctions may still exist at this stage. Finally, these cells appear to be a terminally differentiated cell type that can be maintained indefinitely in culture without detectable proliferation or changes in their morphology. At many regions of cell-to-cell contact, gap junctions are present between stem cells and between endoderm-like cells. Gap junctions contain intercellular channels which mediate the direct cytoplasmic transfer of ions and low molecular weight metabolites between cells (Payton et al., 1969; Gilula et al., 1972; Bennett, 1973; Gilula, 1977). It has been suggested that gap junctional channels may provide an intercellular pathway for the generation of morphogenetic gradients during embryogenesis (Wolpert, 1978). In a companion study the stem cells and endoderm-like cells were found to be linked via such functional communication pathways (Lo and Gilula, 1980b). Although it was difficult to resolve the junctional contacts between giant cells, observations in the companion study also indicated that gap junctions probably exist between giant cells, since they are able to transfer tritiated nucleotides in metabolic coupling (Lo and Gilula, 1980b). The original conversion of the stem cells to endoderm-like cells and of endodermlike cells to giant cells may provide a good model system for studying (1) the de nova assembly of tight junctions and their subsequent disassembly and (2) the de novo assembly of cytoskeletal structure which results in the conversion of a cubodial epithelial cell (almost devoid of cytoskeletal elements) to a large fibroblastic cell (containing a massive cytoskeletal network complete with stress fibers). In addition, the identification of the biosynthetic product(s) of the giant cells in the future may help to clarify the relevance of this differentiation process to normal mouse embryogenesis in vivo. We would like to thank Ms. Asneth Kloesman and Ms. Kathy Wall for help in the preparation of figures
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and Mrs. Madeleine Naylor for secretarial assistance. This research was supported by grants from the United States Public Health Service (HL 16507 and GM 24753) and The Rockefeller Foundation (RF 70095). N. B. Gilula is the recipient of a Research Career Development Award (HL 00110). REFERENCES BENNETT, M. V. L. (1973). Function of electrotonic junctions in embryonic and adult tissues. Fed. Proc. 32,65-75.
BUCKLEY, I. K., and PORTER,K. R. (1967). Cytoplasmic fib& in living cultured cells. Protoplasma 64, 349-380. CHALCROFT,J. P., and BULLIVANT, S. (1970). An interpretation of liver cell membrane and junction structure based on observations of freeze-fracture replicas of both sides of the fracture. J. Cell Biol. 47,49-60.
CLAUDE, P. L., and GOODENOUCH,D. A. (1973). Fracture faces of zonulae occludentes from tight and leaky epithelia. J. Cell Biol. 58, 390-400. DAMJANOV, I., and SOLTER,L. (1974). Ultrastructure of murine teratocarcinomas. Zrz“Teratomas and Differentiation,” pp. 209-220. Academic Press, New York. ELIAS, P. M., and FRIEND, D. S. (1976). Vitamin A induced mucous metaplasia. J. Cell Biol. 68, 173188. FARQUHAR,M. G., and PALADE, G. E. (1965). Cell junctions in amphibian skin. J. Cell Biol. 26, 263291. FARQUHAR,M. G., and PALADE, G. E. (1963). Junctional complexes in various epithelia. J. Cell Biol. 17,375-412. FRIEND, D. S., and GILULA, N. B. (1972). Variations in tight and gap junctions in mammalian tissues. J. Cell Biol. 53, 758-776. GILULA, N. B. (1977). Gap junctions and cell communication. In “International Cell Biology (19761977)” (B. R. Brinkley and K. R. Porter, eds.), pp. 61-69. The Rockefeller University Press, New York. GILULA, N. B., REEVES, R. O., and STEINBACH, A. (1972). Metabolic coupling, ionic coupling and cell contacts. Nature (London) 235,262-265. GOLDMAN, R. D., LAZARIDES, E., POLLACK, R., and WEBER, K. (1975). The distribution of actin in nonmuscle cells. Exp. Cell Res. 90.333-350. JAKOB, H., BOON, L., GAILLARD, J., NICOLAS, J. F., and JACOB,F. (1973). Teratocarcinome de la sourisisolement, culture et proprietb de cellules a potential&es multiples. Ann. Microbial. (Inst. Pasteur) 124B, 269-282.
KALDERON, N., EPSTEIN, M. L., and GILULA, N. B. (1977). Cell-to-cell communication and myogenesie. J. Cell Biol. 75, 788-806. KLEINSMITH, L. J., and PIERCE, G. B., JR. (1964).
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Multipotentiality of single embryonal carcinoma cells. Cancer Res. 24, 1544-1552. KREUTZIGER,G. 0. (1968). Freeze-etching of intercellular junctions of mouse liver. 26th Proceedings of the Electron Microscopy Society of America, p. 234. LO, C. W., and GILULA, N. B. (1978). Differentiation and junctional communication properties of teratocarcinoma cells and the early mouse embryo. J. Cell Biol. 79,18a. LO, C. W., and GILULA, N. B. (1980a). PCCIazal teratocarcinoma stem cell differentiation in culture. I. Biochemical studies. Develop. Biol. 75, 78-92. LO, C. W., and GILULA, N. B. (1980b). PCC4azal teratocarcinoma stem cell differentiation in culture. III. Cell-to-cell communication properties. Develop. Biol. 75, 112-120. MARTIN, G. R. (1975). Teratocarcinomas as a model system for the study of embryogenesis and neoplasia. Cell 5, 229-243. MARTIN, G. R., and EVANS, M. J. (1975). Differentiation of clonal lines of teratocarcinoma cells Formation of embryoid bodies in vitro. Proc. Nat. Acad. Sci. USA 72,1441-1445. MARTIN, G. R., WILEY, L. M., and DAMJANOV, I. (1977). The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Develop. Biol. 61,230-244. MCNUTT, N. S., and WEINSTEIN, R. S. (1970). The ultrastructure of the nexus. A correlated thin section and freeze-cleave study. J. Cell Biol. 47, 666-688. MINTZ, B., CRONMILLER,C., and CUSTER,P. (1978). Somatic cell origin of teratocarcinomas. Proc. Nat. Acad. Sci. USA 75,2834-2838. MONTESANO,R., FRIEND, D. S., PERRELET, A., and ORCI, L. (1975). In vivo assembly of tight junctions in fetal rat liver. J. Cell Biol. 67, 310-319. ORCI,L., AMHERDT,M., HENGUIN, J. C., LAMBERT,A. C., UNGER, R. H., and RENOLD, A. E. (1973). Pronase effect in pancreatic beta cell secretion and morphology. Science 180, 647-649. PAYTON, B., BENNETT, M. V. L., and PAPPAS,G. D. (1969). Permeability and structure of junctional membranes of an electrontonic synapse. Science 166, 1641-1643. PIERCE,G. B., and BEAL, T. F. (1964). The ultrastructure of primordial germinal cells of the fetal testes of embryonal carcinoma cells of mice. Cancer Res. 24, 1553-1567. PIERCE,G., DIXON, F., and VERNEY, E. (1959). Testicular teratomas. II. Teratocarcinoma as an ascitic tumor. Cancer Res. 12, 584-589. PIERCE, G. B., STEVENS,L. C., and NAKANE, P. K. (1967). Ultrastructural analysis of the early development of teratocarcinomas. J. Nat. Cancer Inst. 39, 755-773.
STAEHELIN, L. A. (1973). Further observations on the tine structure of freeze-cleaved tight junctions. J.
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Cell Sci. 13, 763-786. STEVENS, L. C. (1960). Embryonic potency of embryoid bodies derived from a transplantable testicular teratoma of the mouse. Develop. Biol. 2, 285297.
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WOLPERT, L. (1978). Gap junctions: Channels for communication in development. In “Intercellular Junctions and Synapses” (J. Feldman, N. B. Gilula, and J. D. Pitts, eds.), pp. 83-94. Chapman and Hall, London.
BIOLOGY
75, 93-111 (1980)
PCC4azal Teratocarcinoma
Stem Cell Differentiation
II. Morphological CECILIA The Rockefeller
Characterization
W. Lo’ AND NORTON B. GILULA
University,
Received April
in Culture
1230 York Avenue, Neu! York, New York 10021
23, 1979; accepted in revised form August 2, 1979
The ultrastructural morphology of the PCC4azal embryonal carcinoma cells and their differentiated counterparts, endoderm-like cells and giant cells, was characterized and compared with that of the cells of embryoid bodies. The ultrastructure of the PCC4azal embryonal carcinoma cells is similar to that of the embryonal carcinoma cells of the embryoid body. These cells are small, with a large nucleus and relatively few cytoplasmic organelles. Gap junctions and modified adherens junctions are formed at some areas of intercellular contact between the embryonal carcinoma cells. The differentiated PCC4azal endoderm-like cells have a more developed cytoplasm, containing an extensive endoplasmic reticulum with large Golgi regions. Most striking is the de nouo appearance of epithelial-like junctional complexes which join the apical borders between the endoderm-like cells, thus polarizing the cell monolayer. The zonula occludens junctions of the junctional complex are extensive, consisting of six or more strands of tight junctional ridges. Terminal webs are present in the apical regions that are inserted into the zonula adherens region of the junctional complex. Gap junctions continue to join neighboring cells, and some gap junctions are intercalated within tight junctional ridges. The ultrastructure of the differentiated endodermal cells of the embryoid bodies is very similar to that of the PCC4azal endoderm-like cells. The embryoid body endodermal cells form similar junctional complexes which also contain continuous belts of tight junctions that are intercalated with gap junctions. As the PCCIazal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of lo-nm filaments, microtubules, and 7-nm microfilaments. The junctional complexes that were present during the endodermal stage are partially disassembled as the giant cells migrate apart. Thus, the differentiation process in this system is characterized by significant and distinctive morphological changes. INTRODUCTION
pare the cytodifferentiation ture to the differentiation
The differentiation of PCC4azal embryonal carcinoma cells, as described previously, is accompanied by distinct changes in enzyme properties and by the disappearance of a cell surface antigen (Lo and Gilula, 1980a). The morphological changes which the embryonal carcinoma cells undergo are striking, starting from a small epithelial cell to a giant fibroblast-like cell. It is of interest to study the ultrastructural morphology of these cells in order to (1) obtain an ultrastructural basis for the cytodifferentiation of the teratocarcinoma cells and (2) com-
obtained in culof the tumor in
vivo. In this study, the ultrastructure of the various cell types of the PCC4azal cells cultured in vitro and the ascites form of the original tumor line, the embryoid bodies, is characterized by freeze-fracture and thinsection electron microscopy. As differentiation to giant cells proceeds, specific elaboration of the cytoplasmic organelles and the cytoskeleton is observed and specialized cell surface contacts are developed. The PCC4azal embryonal carcinoma cells and the endoderm-like cells formed in vitro are compared to the embryo& carcinoma cells and the outer ring of differentiated endo-
’ Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
93 00%1606/80/030093-19$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction
in any form reserved.
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derm-like cells of the embryoid bodies formed in vivo. A preliminary report of this study was presented previously (Lo and Gilula, 1978). MATERIALS
AND
METHODS
Materials Eight percent aqueous glutaraldehyde (N2 sealed) and 4% aqueous osmium tetroxide (Polysciences Inc., Warrington, Pa.); analytical grade tannic acid and reagent grade glycerol (Mallinckrodt, St. Louis, MO.); uranyl acetate (Fisher Scientific Co.); Epon 812 (Ladd Research Industries, Inc., Burlington, Vt.); and gold alloy specimen carriers (Balzers High Vacuum Corp., Santa Ana, Calif.). All other materials used are as described previously (Lo and Gilula, 1980a). Microscopy (Thick Sections and Thin Sections) The PCC4azal cells and the embryoid bodies were maintained as described in the previous study (Lo and Gilula, 1980a). For microscopy, the PCC4azal cells were processed at room temperature in the tissue culture dishes and the embryoid bodies as a suspension using published procedures (Kalderon et al., (1977)). Briefly, the cells were fixed in 2.5%glutaraldehyde, postfixed in 1%osmium tetroxide, and stained en bloc with 0.125% tannic acid followed by uranyl acetate. The specimens were further processed by dehydration in a graded series of
ethanol and embedded in Epon 812. The embedded PCC4azal cell monolayers were peeled off the plastic culture dishes, and areas selected by light microscopy were punched out. The punched-out disks were then mounted on Beem capsules or reembedded in flat blocks. Thick sections were cut with glass knives, mounted on glass slides, and stained with toluidine blue. Photographs were taken on Kodak Plus-X film with a Zeiss photomicroscope II. Thin sections obtained with a diamond knife were poststained with uranyl acetate and lead citrate prior to examination in a Siemens 101 electron microscope at 80 kV. Freeze-Fracture Replication For freeze-fracture, the PCC4azal cells were grown on Balzers gold carriers for the double replica device. They were fixed as above and then treated with 25% glycerol in 0.1 M cacodylate buffer (pH 7.3) at 4°C overnight. Prior to freezing, the specimens were brought to room temperature, then sandwiched with an identical blank carrier and rapidly frozen in liquid nitrogen-cooled Freon 22. The frozen sandwiched carriers were mounted in the Balzers machine (Freeze Etch Unit BA 360 M), then forced open at -115°C and immediately shadowed with platinum and carbon. The replicas were cleaned with sodium hypochlorite and subsequently examined in a Siemens 101 electron microscope.
FIG. 1. Embryoid body ultrastructure. (a) Thin-section electron micrographs of an embryoid body. The outer layer of endoderm-like cells (EN) has a highly differentiated cytoplasm which contains many large lipidlike droplets. Junctional complexes join the outer layer of endoderm-like cells at their apical cell surfaces (arrow). Numerous microvilli project from the outer cell surface, and an amorphous material resembling a basal lamina (BL) is sometimes found beneath the endodermal cell layer. The embryo& carcinoma cells (EC) which are located on the inside have few organelles, and these cells are closely associated with each other as well as with the endoderm-like cells. x 7200. Inset: Thick section of an embryoid body. The endoderm-like cells (EN) form a closed vesicle, and the embryonal carcinoma cells (EC) are located within it. The bar in the inset represents 36.9 pm. (b) An enlargement of an area similar to that indicated by the arrow in a. A junctional complex (JC) between the apical surfaces of two endoderm-like cells. Elements of the junctional complex including a tight junction (TJ), a gap junction (GJ), and desmosomes (D) are present. Fibrous densities in the cytoplasm next to the junctional complex are elements of the terminal web. x 84,000. (c) Freeze-fracture replica of an embryoid body junctional complex region exposing the network of tight junctional ridges (black and white arrow). Gap junctional particles (black arrow) are intercalated within some regions of the tight junctions. x 6o.ooo.
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The embryoid bodies were fixed and glycerinated as above but in a suspension. They were then transferred onto paper disks as concentrated droplets and rapidly frozen in liquid nitrogen-cooled Freon 22. The frozen samples were mounted in the Balzers device and cut with a precooled knife at -115°C. The replicas were processed and examined as above. RESULTS
Embryoid
Bodies
The mouse teratocarcinoma can be adapted to growth in the peritoneum as floating aggregates called embryoid bodies (Stevens, 1960; Pierce et al., 1959). They are organized structures consisting of an outer layer of differentiated cells referred to as the endoderm and an inner mass of cells containing undifferentiated embryonal carcinoma cells (see inset, Fig. la). The embryonal carcinoma cells have a large nuclear to cytoplasmic ratio and contain few organelles, including a few spherical mitochondria with few cristae, some endoplasmic reticulum, primitive Golgi, occasional lipid droplets, and ribosomes (Fig. la). They are linked to one another by gap junctions and modified adherens junctions (data not shown). The endodermal cells in the outer layer are usually aligned end to end, forming a single cell layer often resembling a squamous epithelium. In some regions a pseudostratified arrangement of the endodermal cells is also present. The endodermal cells have numerous microvillar projections on their outer membrane which faces the external medium. The cytoplasm of endodermal cells contains well-developed Golgi regions, elongated mitochondria with moderate numbers of cristae, rough
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and smooth endoplasmic reticulum, ribosomes, glycogen granules, lipid-like droplets, and pinocytic vesicles (Fig. la). The cisternae of the endoplasmic reticulum often contain amorphous deposits. The endodermal cells are linked to one another at the outer cell borders by zonulae occludens or tight junctions, zonula adherens junctions, desmosomes, and gap junctions that are arranged in a junctional complex (Figs. la, b, and c). Similar to the junctional complex of a typical epithelium, there is a “tripartite” arrangement at the apical or “luminal” surface, starting with the zonula occludens or tight junction at the most apical region, followed by the zonula adherens junction, and then desmosomes (Figs. 4b, 5, 6b, and 7) (Farquhar and Palade, 1963; Friend and Gilula, 1972). Gap junctions are present at varying locations within this complex. Filaments of the terminal web of the junctional complex are associated with and terminate at the membranes of the junctional complex (Fig. lb). Freezefracture replicas of the embryoid bodies reveal that the tight junctions consist of a complex array of many tiers of interconnecting 8.5-nm ridges on the P fracture face (inner membrane half) and grooves on the E fracture face (outer membrane half). They form continuous belts of ridges and grooves around the entire apical border of the embryoid body. These tight junctional elements are often intercalated with aggregates of 8.5-nm gap junctional particles (on the P face) and pits (on the E face). An amorphous basal lamina frequently separates the outer endodermal layer from the inner mass of embryo& carcinoma cells. This lamina is not continuous beneath the entire endoderm layer, and in many regions
FIG. 2. Step cell or embryonal carcinoma cell. (a) Horizontal section. The embryonal carcinoma stem cell has a large nucleus with one or two nucleoli exposed in this plane of section. The cytoplasm is small, with few organelles. A rudimentary Golgi apparatus is visible at the top left (G). x 6060. (b) Vertical section. (The bottom of the culture dish or the substrate is indicated by the letter S. All subsequent vertical sections are similarly labeled.) The cuboidal cells are arranged as a single cell layer with some overlapping processes. Note the areas of specialized cell contact (arrows). A gap junction (small arrow) and a modified adherens junction region (large arrow) are present. X 6900.
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FIG. 3. Specialized intercellular junctions between embryonal carcinoma stem cells. (a) Freeze-fracture image of a large gap junction. A plaque of particle aggregates separated by particle-free aisles is present on the P face (P), while complementary pits are present on the E face (E). Note the isolated small aggregates of E-face pits in the upper-left region. X 60,000. (b) Thin section of modified adherens junctions. At this site of close membrane apposition, dense fibrous material accumulates on the cytoplasmic surfaces of both membranes. The accumulation is asymmetrical; it is heavier on the membrane of the cell to the right. The intercellular space narrows to 6-8 nm, and some dense staining material is present between the membranes. X 69,000. (cl Thin section of a gap junction. The two closely apposed membranes are separated by an intercellular space or gap of 2-4 nm, generating a septalaminar image. In this image, the gap is filled with electron-dense tannic acid stain. x 96,000.
the inner embryonal carcinoma cells are in direct contact with the endodermal cells. Embryoid bodies have also been reported to contain other cell types (Martin et al., 1977); however, with the embryoid bodies used in this study, only the embryonal carcinoma cells and the endodermal cells were unequivocally identified. It is possible that a small number of other cell types is present but is difficult to identify due to either their smaIl numbers or their morphological similarity to the endodermal or embryonal carcinoma cells.
PCC4azal
Stem Cells
Stem cells are closely packed and their nuclear to cytoplasmic ratio is characteristically high. The cytoplasm is relatively undifferentiated, containing many free ribosomes, a number of small spherical mitochondria, and a rudimentary Golgi. Elements of the endoplasmic reticulum are almost nonexistent. From verticle sections, it can be seen that embryonal carcinoma cells are arranged as a closely packed single layer of cuboidal
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FIG. 4. Cells from stage 1 of differentiation, elongation. Whenever possible, the same labeling is used in both a and b. (a) Horizontal section. The nucleus and cell body are elongated. The Golgi apparatus (G) is enlarged, and some endoplasmic reticulum (ER) is present. Microfilament bundles (MF) extend along the length of the cell, while others are associated with the cortical regions. The microfilaments in the cortical regions are inserted into a series of adherens junctions (arrows) to form a terminal web-like structure (TW). x 9120. (b) Vertical section. The change in cell shape is apparent in this plane (cf. Fig. 2b); the cell is flattened (squamous) and the nucleus is oblong. x 9240.
cells with some overlapping processes (Fig. 2b). Gap junctions and modified adherens junctions are formed in some areas of cellto-cell interaction (Figs. 3b and c). In
freeze-fracture replicas, the gap junctional particle aggregates consist of 8.5nm particles in the P fracture face and complementary pits in the E fracture face. The gap
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junctions are low in frequency and variable in size, containing from a few particles to over 900 particles per plaque (Kreutziger, 1968; McNutt and Weinstein, 1970; Chalcroft and Bullivant, 1970; Friend and Gilula, 1972). These particle aggregates and their complementary pits are loosely arranged in small islands which are separated by particle-free aisles (Fig. 3a). The modified adherens junctions formed are simple, consisting of close membrane appositions where the two apposed membranes are aligned in parallel and separated by an intercellular space of 6-8 nm (Fig. 3b). Fibrous densities are intimately associated with the cytoplasmic surfaces of both membranes, and sometimes the density is greater on one of the two membranes. PCC4azal
Differentiated
Cells
The ultrastructural characterization of the PCC4azal transformation to endodermlike cells and then to giant cells is presented in the same three-stage scheme as reported in the previous study (Lo and Gilula, 1980a). Stage 1: Cell elongation. With the onset of differentiation to endoderm-like cells, the cells flatten and are arranged as a monolayer of squamous epithelium (compare Fig. 2b with Fig. 4b). This results in the lengthening and apparent enlargement of the cell as seen in horizontal sections (Fig.
4a). Concomitant with this overall change in cell shape is a similar flattening and elongation of the nucleus of the cell (Fig. 4b). Within the cytoplasm Golgi elements are prominent. A system of smooth and rough endoplasmic reticulum is present (Fig. 4a) and a light flocculent material is distributed within its cisternae. At regions of the cell periphery, large pockets of intercellular space interrupt the attachment of the cells to the substrate such that only the central (somatic) portion of the cell is in contact with the culture dish. The peripheral regions of the cytoplasm decrease in thickness and the intercellular contacts are established at some distance from the substrate (Fig. 4b). Although the area of cell-to-cell contact is greatly reduced (compare Fig. 2a with Fig. 4b), each cell maintains intimate contact with its neighbors. At the regions of cell-tocell contact, junctional complexes join the adjacent cells. The junctional complex is formed de novo, and it is not yet completely developed at this stage. The ordering of the junctions within the complex is similar to that of a typical epithelium, with tight junctions at the luminal surface, followed by zonula adherens junctions, then desmosomes. Gap junctions are intercalated throughout the complex as before. The equivalent “luminal” side in this monolayer culture system is the apical surface of the
FIG. 5. Specialized intercellular junctions between differentiating cells of stage 1. (a-c) Thin-section morphology. (a) Gap junctions. At a region comparable to that depicted by the arrows in Fig. 4a, a series of gap junctions (GJ), intercalated by zonula adherens junctions (ZA), joins the cells. x 52,650. (b) High magnification of a gap junction similar to those in a. Note the septalaminar appearance and the dense material on the cytoplasmic surfaces of the junctional membranes. x 180,000. (c) Demosomes (macula adherens junctions). Several desmosomes (D) join two adjacent cells. Cytoplasmic densities along both membranes of the desmosomes are intimately associated with IO-nm tonofilaments (TF), and these are connected to each other via the tonofilaments. At these contacts, the 30-nm intercellular space contains a crystalline extracellular matrix that is bisected by a central dense lamina. x 36,000. (d, e) Freeze-fracture morphology. (d) Tight junction (ridge) formation. Tight junctional ridges are assembled by the linear aggregation of intramembrane particles that subsequently fuse. The black and white arrows (l-4) depict stages of transition, from aligned single particles in area 1 to completed ridges in area 4. Arrow 2 indicates an area where single particles and short segments of completed ridges are closely associated. In area 3, short completed ridges are aligned with one another. The black arrow points to a particle aggregate associated with a ridge; this aggregate may be related to a gap junction. x 60,000. (e) A plaque of gap junctional particles is closely associated with some tight junctional ridges (arrow) and complementary grooves. x 60,000.
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FIG. 5. a-e.
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cells, where the basal surface is defined as being adjacent to the bottom of the culture dish. Each element of the junctional complex of these differentiating cells is described in detail below. Tight junctional elements are detected for the first time in freeze-fracture replicas of these cells at this stage. The de novo assembly of the 8.5~nm intramembrane tight junctional ridges (Friend and Gilula, 1972; Claude and Goodenough, 1973; Staehelin, 1973) involves much of the lateral membrane surfaces. Various structural arrangements are present that can be translated into a possible sequence of assembly events (Fig. 5d). These include the following: (1) Individual 8.5-nm particles align in linear arrays; (2) the linear particle aggregates fuse to generate short ridge segments; and (3) short ridge segments fuse to form more extended ridges that are randomly oriented. Similar schemes of tight junctional ridge formation have previously been described in other systems (Elias and Friend, 1976; Montesano et al., 1975). During this assembly process, gap junctional particle aggregates are closely associated with the tight junctional ridges (Fig. 5d), and they are frequently quite large. They have a characteristic septalaminar arrangement in thin sections (Fig. 5b) and their particle packing in freeze-fracture replicas is similar to the gap junctional particle arrangement of stem cells (Fig. 3a). Gap junctions of similar size and morphology also are present in regions some distance from the complex. Microfilaments and microfilament bundles are distributed throughout the cytoplasm, and frequently they are aligned in parallel and in close association with the cell borders (Fig. 4a). They insert into regions of the zonula adherens junctions to form a rudimentary terminal web similar to that in various epithelia (Figs. 4a and 5a) (Farquhar and Palade, 1963,1965). Many desmosomes are frequently found in series below the zonula adherens junc-
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tions (Fig. 5~). Tonofilament bundles interconnect the characteristic cytoplasmic dense plaques, resulting in the formation of linked chains of desmosomes. Stage 2: Cell enlargement. The differentiating endoderm-like cells are larger in size (compare Fig. 4a with Fig. 6a). The changes in the cytoplasm that were initiated with the onset of differentiation are more apparent at this stage (Fig. 6a). These include further enlargement of the Golgi regions and the endoplasmic reticulum and the presence of phagolysosomal vesicles. At the apical regions of the cells, junctional complexes consisting of tight junctions, terminal web attachment elements (zonula adherens), and desmosomes have further developed, and they are now elaborate and mature structures. Their de novo assembly which was initiated at stage 1 is now complete. The tight junctions, which previously consisted of short lengths of ridges and grooves which were randomly oriented, are now composed of extensive lengths of ridges and grooves arranged in parallel tiers of 6 to 10 interconnected strands (Fig. 7). They form continuous belts which surround the entire apical borders of each cell. In addition to this belt-like arrangement of the tight junction within the junctional complex, tight junctions in plaque-like domains exist in regions below the complex (Fig. 7). These consist of localized areas of concentric arrays of interconnected tight junctional strands. This arrangement of tight junctions has previously been described in some epithelia and is referred to as a “compartmentalized” junction (Staehelin et al., 1973; Friend and Gilula, 1972; Orci et al., 1973). Gap junctions are intercalated within both types of tight junctions, and they are found at a lower frequency than during the time of tight junctional ridge formation. The terminal webs associated with the zonula adherens junctions are well developed, consisting of a thick assembly of microfilaments. Below the belts of occluding
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FIG. 6. Stage 2 of differentiation, cell enlargement. (a) Horizontal section. The shape of this large cell is polygonal. The endoplasmic reticulum (ER) is dilated, and a flocculent matrix is dispersed within its cisternae. There are large numbers of mitochondria, and the cells are joined by extensive junctional complexes (arrow). x 4800. (b) Desmosome (D) between the differentiating cells. Cytoplasmic plaques with their associated tonofilaments (TF) are increased in density. Note that the desmosome to the left bends at a sharp angle. x 36,000.
zonula adherens junctions, desmosomes continue to join adjacent cells. The fibrous cytoplasmic plaques of the desmosomes have greatly increased in density, and their cytoplasmic membranes are no longer always aligned in straight lines. The opposing membranes, while remaining parallel, often undulate with bends and curves. These des-
mosomes resemble the “modified desmosomes” of the frog skin (Fig. 6b) (Farquhar and Palade, 1965). Stage 3: Terminal differentiation of giant cells. The differentiating cells have greatly enlarged and are further flattened, especially in peripheral regions of the cell away from the nucleus (Figs. 8b and c).
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FIG. 7. Freeze-fracture morphology of specialized junctions in later differentiating cells (stages 2-3). Mature tight junctions coexisting in two arrangements: a belt-like zonula occludens, and a plaque-like macula occludens. In the upper region, a complex array of several parallel tiers of ridges on the P face (P) and their complementary grooves on the E face (E) characterizes the belt-like arrangement. Short strands at an angle to the axis of the belt connect the tiers. At the lower left, several sequestered domains of grooves in the E face represent the plaque-like or macula configuration of tight junctional elements. In this arrangement, one continuous strand usually defines the boundary of the tight junction domain. x 60,000. Inset: Intermediate stage of development of the zonula occludens junction. Short segments of completed ridges exist in semiparallel arrays. Some of the strands branch at an angle to connect parallel ridges. The arrows indicate closely associated gap junctional particle aggregates, some of which are sequestered within these ridges. x 75,000.
Some of these regions are so thin that they are but a fraction of the original thickness of stem cells (compare Figs. 2b and 8~). Most of the cellular organelles are located in the perinuclear region of the cytoplasm (Fig. 8a). The endoplasmic reticulum is extremely distended and completely filled with a dense amorphous matrix. Golgi re-
gions are large and abundant. The mitochondria are very long and greatly increased in number. The organelles seem to be geographically organized in a nonrandom fashion, where some regions of the perinuclear cytoplasm are predominantly occupied by one type of organelle, i.e., an entire region of distended endoplasmic re-
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ticulum, or mitochondria, etc. The phagolysosomes are quite large and filled with various dense elements. Lipid-like droplets are present, consisting of membrane-bound vesicles filled with an amorphous mass. At the periphery of the cell, a few mitochondria are present and a system of anastomosing endoplasmic reticulum extends throughout the complex array of cytoplasmic fibers (Fig. 9c). Many of the regions of close cell-to-cell contact are disassembled as a result of increased cell motility (Lo and Gilula, 1980a). The cells closely interact with one another via a series of cytoplasmic interdigitations (Fig. 9a, inset). The finger-like interdigitation results from the insertion of a thin pseudopod-like structure into an invagination of the adjacent cell (Fig. 9a). Zonula adherens junctions continue to join some regions of contact, and they are the insertion site for the extensive terminal web elements which are still present (Fig. 9a). Due to the association of many filaments with the membrane and the undulation of
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the opposing membranes themselves, it is difficult to resolve the thin-section characteristics of the junctions that are present in the regions of cell-to-cell contact. Some punctate contacts can be tentatively identified as tight junctions (Figs. 8d and e), although some of these may, in fact, be gap junctions. A more precise identification awaits additional freeze-fracture information. The most striking features of the differentiated giant cell are the large number of cytoplasmic fibers and their complex arrangements. Stress fibers seen in the light microscope (Fig. 9a, inset) are readily identified in thin sections as thick cables of 7nm microfilaments which extend in straight lines for long distances in the cytoplasm (Fig. 9d) (Buckley and Porter, 1967; Goldman et al., 1975). The bundles of microfilaments have dense bodies or bands along their length. These cables sometimes intersect, and at the regions of juncture, microtubules sometimes extend through and between adjacent bundles (Fig. 9d). Microtu-
FIG. 8. Giant cells. (a) Horizontal section. This cell is extremely large; only a small portion of the perinuclear region of the cytoplasm is shown here. The endoplasmic reticulum (ER) is dilated and completely filled with a dense matrix. The Golgi components (G) are well developed. Many mitochondria, lipid-like vesicles (L), and lysosomal (LY) elements are also present in this region. x 6400. (b-e) Vertical section. (b) A region of a giant cell containing a small portion of the nucleus and perinuclear cytoplasm (cf. Figs. 2b and 4b). The nucleus is pleiomorphic in shape. This section plane provides a different view of the arrangement of cytoplasmic organelles (cf. a). The endoplasmic reticulum (ER) is in a parallel array, with no visible dilation. The Golgi (G) is arranged as stacks of flattened vesicles, and a large phagolysosome (PL) extends from the apical to the basal region of the cell. x 7600. (c) A small region of the peripheral cytoplasm of the giant cell some distance from the nucleus. The cell is exceedingly thin in this region (cf. Figs. 2b and 4b). Few cytoplasmic organelles are present. Filament bundles are the most prominent cytoplasmic components. x 12,000. (d) Region of contact between the peripheral regions of two cells (arrows). These are not two cell processes or microvilli. The areas of contact are filled with microfiiaments that are related to the terminal web-like (TW) structure. x 24,000. (e) High magnification of the region of contact in d (asterisk). The two arrows outline a small focal region of membrane contact between these giant cells. X 132,000. FIG. 9. Characteristic features of a differentiated giant cell. (a) A series of zonula adherens (ZA) junctions joins two giant cells. Parallel arrays of 7-nm microfiiaments insert into cytoplasmic densities along the membranes of the adherens structures. The microfilaments of one cell are strikingly aligned with those of its neighbor. In addition, a cell process of the upper cell is invaginated into the cytoplasm of the adjacent cell, perhaps stabilizing their association. x 36,000. Inset: This light micrograph shows a region where three giant cells are in intimate contact. Note the interdigitation of the cytoplasmic borders and the many stress fibers in the cytoplasm. x 200. (b) Bundles of lo-nm filaments are distributed throughout the cytoplasm. x 48,000. (c) A complex array of microtubules (large arrow) and lo-nm filaments (small arrow) intermingles in the peripheral cytoplasm. Note the anastomosing endoplasmic reticulum (ER). x 43,500. (d) Stress fibers are comprised of tightly packed microfilament bundles. At the left, the bundles merge and microtubules are closely associated with this area. Note the periodic dark banding distributed along the fiber bundles (arrow). x 11.400.
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e FIG. 8. a-e.
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bules usually exist as single fibers that are widely distributed in the cytoplasm (Fig. 9c). Many lo-nm filaments are also present, and they are arranged in loose bundles which twist and turn. The interplay of these three types of filaments forms a complex cytoskeletal network which extends throughout the enormous expanse of cytoplasm, providing these cells with a distinct fibroblast-like morphology. DISCUSSION
In this study the morphological characterization of the PCC4azal cells and the embryoid bodies provides a basis for understanding the cellular changes that are associated with the differentiation process in the PCC4azal system and relating the PCC4azal differentiated cells to the cellular phenotypes in the embryoid bodies. The morphological transformation of the PCC4azal stem cells is characterized by several major changes, including an increase in cell size, a change in cell shape, the development of a junctional complex that includes the de novo formation of a zonula occludens, an alteration in cellular metabolism and biosynthetic activity as indicated by cytoplasmic organelle development, and the genesis of an extensive cytoskeletal framework. Comparison of the embryoid bodies with the PCC4azal cells has shown that the stem cells of the embryoid bodies are identical in morphology to the PCC4azal embryonal carcinoma cells that are maintained in culture, and the differentiated endodermal cells of the embryoid body are very similar, if not identical, to the PCC4azal endoderm-like cells that are generated in culture. Since in previous studies it has been demonstrated that the endodermal cells and other differentiated cell types of teratocarcinomes are derived from embryo& carcinoma cells, the differentiation of PCC4azal embryonal carcinoma cells to similar endoderm-like cells described in this study probably parallels the in vivo differentiation of embryonal carcinoma cells to endoderm in the embryoid
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bodies (Kleinsmith and Pierce, 1964; Martin and Evans, 1975). In previous studies it has been suggested that embryonal carcinoma cells originate from embryonic ectodermal cells (Damjanov and Solter, 1974) or primordial germ cells (Pierce and Beal, 1964; Pierce et al., 1967), since all three cell types are morphologically very similar and have similar capabilities of differentiation into many cell types. However, in a recent study, evidence has been presented to suggest that embryonal carcinoma cells are probably derived from somatic cells and not germ cells (Mintz et al., 1978). In this study, the morphology of the embryonal carcinoma cells of the embryoid body was observed to be similar to that of the PCC4azal embryonal carcinoma cells, and this is consistent with the fact that the PCC4azal cells were originally derived from embryoid bodies (Jakob et al., 1973). The true in vivo equivalence to this stem cell phenotype awaits further characterization. The term “endodermal” has been used by many investigators to describe the differentiated outer layer of cells of the embryoid body. The basis for this distinction is primarily morphological; the endoderm of the embryoid body is thought to be related to the outer layer of extraembryonic endoderm formed around the inner cell mass of the mouse embryo (Martin, 1975). In this study we have utilized the same general morphological criteria for adopting the term “endoderm-like” for the stage 1 and stage 2 differentiated PCC4azal cells. Furthermore, it is now possible to include several additional specific morphological criteria for applying this endodermal distinction. These include the extensive development of specific cytoplasmic organelles and the de novo formation of junctional complexes, in particular the zonula occludens element. A definitive clarification of these cells as being equivalent to the extraembryonic endoderm of the mouse embryo will require additional characterization.
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The de novo formation of junctional complexes is the clearest indication by morphology of a new cell type arising from the differentiation of PCC4azal embryonal carcinoma cells in vitro or embryonal carcinoma cells of the embryoid body in vivo. The arrangement of highly specialized junctions in a complex is frequently found in well-differentiated epithelia, where the continuous belts of occluding junctions form a permeability seal (Farquhar and Palade, 1963; Friend and Gilula, 1972; Claude and Goodenough, 1973). In fact, an effective permeability seal must exist in the embryoid bodies since many of them are swollen with fluid, somewhat resembling a blastocyst with a pumped-up blastocoelic cavity. Similarly, large intercellular spaces formed by the detachment of the peripheral cytoplasm of the PCC4azal endodermal cells from the substratum may result from the accumulation of fluid following the formation of a permeability barrier. The cells formed from the endoderm-like cells are labeled “giant” simply to emphasize their large size and extremely low nuclear to cytoplasmic ratio. The giant cell is distinctively fibroblastic in morphology, and its cytoplasmic characteristics appear to be the result of further elaboration of the cytoplasmic differentiation that was initiated at stage 1. In particular, the Golgi elements and the endoplasmic reticulum, which are still somewhat rudimentary at the endoderm-like stages, are well developed at the giant cell stage. The development of these organelles probably reflects a major change in the biosynthetic activity that accompanies this differentiation. The complex array of microfilaments, microtubules, and lo-nm filaments in the giant cells also appears for the first time in the endoderm-like cells. These cytoskeletal elements may be essential for the maintenance of cell shape and motility, as well as for the coordination of the various metabolic activities within the cytoplasm. Cell-to-cell interactions between giant cells are limited, and thin-section profiles suggest that some
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tight junctions may still exist at this stage. Finally, these cells appear to be a terminally differentiated cell type that can be maintained indefinitely in culture without detectable proliferation or changes in their morphology. At many regions of cell-to-cell contact, gap junctions are present between stem cells and between endoderm-like cells. Gap junctions contain intercellular channels which mediate the direct cytoplasmic transfer of ions and low molecular weight metabolites between cells (Payton et al., 1969; Gilula et al., 1972; Bennett, 1973; Gilula, 1977). It has been suggested that gap junctional channels may provide an intercellular pathway for the generation of morphogenetic gradients during embryogenesis (Wolpert, 1978). In a companion study the stem cells and endoderm-like cells were found to be linked via such functional communication pathways (Lo and Gilula, 1980b). Although it was difficult to resolve the junctional contacts between giant cells, observations in the companion study also indicated that gap junctions probably exist between giant cells, since they are able to transfer tritiated nucleotides in metabolic coupling (Lo and Gilula, 1980b). The original conversion of the stem cells to endoderm-like cells and of endodermlike cells to giant cells may provide a good model system for studying (1) the de nova assembly of tight junctions and their subsequent disassembly and (2) the de novo assembly of cytoskeletal structure which results in the conversion of a cubodial epithelial cell (almost devoid of cytoskeletal elements) to a large fibroblastic cell (containing a massive cytoskeletal network complete with stress fibers). In addition, the identification of the biosynthetic product(s) of the giant cells in the future may help to clarify the relevance of this differentiation process to normal mouse embryogenesis in vivo. We would like to thank Ms. Asneth Kloesman and Ms. Kathy Wall for help in the preparation of figures
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and Mrs. Madeleine Naylor for secretarial assistance. This research was supported by grants from the United States Public Health Service (HL 16507 and GM 24753) and The Rockefeller Foundation (RF 70095). N. B. Gilula is the recipient of a Research Career Development Award (HL 00110). REFERENCES BENNETT, M. V. L. (1973). Function of electrotonic junctions in embryonic and adult tissues. Fed. Proc. 32,65-75.
BUCKLEY, I. K., and PORTER,K. R. (1967). Cytoplasmic fib& in living cultured cells. Protoplasma 64, 349-380. CHALCROFT,J. P., and BULLIVANT, S. (1970). An interpretation of liver cell membrane and junction structure based on observations of freeze-fracture replicas of both sides of the fracture. J. Cell Biol. 47,49-60.
CLAUDE, P. L., and GOODENOUCH,D. A. (1973). Fracture faces of zonulae occludentes from tight and leaky epithelia. J. Cell Biol. 58, 390-400. DAMJANOV, I., and SOLTER,L. (1974). Ultrastructure of murine teratocarcinomas. Zrz“Teratomas and Differentiation,” pp. 209-220. Academic Press, New York. ELIAS, P. M., and FRIEND, D. S. (1976). Vitamin A induced mucous metaplasia. J. Cell Biol. 68, 173188. FARQUHAR,M. G., and PALADE, G. E. (1965). Cell junctions in amphibian skin. J. Cell Biol. 26, 263291. FARQUHAR,M. G., and PALADE, G. E. (1963). Junctional complexes in various epithelia. J. Cell Biol. 17,375-412. FRIEND, D. S., and GILULA, N. B. (1972). Variations in tight and gap junctions in mammalian tissues. J. Cell Biol. 53, 758-776. GILULA, N. B. (1977). Gap junctions and cell communication. In “International Cell Biology (19761977)” (B. R. Brinkley and K. R. Porter, eds.), pp. 61-69. The Rockefeller University Press, New York. GILULA, N. B., REEVES, R. O., and STEINBACH, A. (1972). Metabolic coupling, ionic coupling and cell contacts. Nature (London) 235,262-265. GOLDMAN, R. D., LAZARIDES, E., POLLACK, R., and WEBER, K. (1975). The distribution of actin in nonmuscle cells. Exp. Cell Res. 90.333-350. JAKOB, H., BOON, L., GAILLARD, J., NICOLAS, J. F., and JACOB,F. (1973). Teratocarcinome de la sourisisolement, culture et proprietb de cellules a potential&es multiples. Ann. Microbial. (Inst. Pasteur) 124B, 269-282.
KALDERON, N., EPSTEIN, M. L., and GILULA, N. B. (1977). Cell-to-cell communication and myogenesie. J. Cell Biol. 75, 788-806. KLEINSMITH, L. J., and PIERCE, G. B., JR. (1964).
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Multipotentiality of single embryonal carcinoma cells. Cancer Res. 24, 1544-1552. KREUTZIGER,G. 0. (1968). Freeze-etching of intercellular junctions of mouse liver. 26th Proceedings of the Electron Microscopy Society of America, p. 234. LO, C. W., and GILULA, N. B. (1978). Differentiation and junctional communication properties of teratocarcinoma cells and the early mouse embryo. J. Cell Biol. 79,18a. LO, C. W., and GILULA, N. B. (1980a). PCCIazal teratocarcinoma stem cell differentiation in culture. I. Biochemical studies. Develop. Biol. 75, 78-92. LO, C. W., and GILULA, N. B. (1980b). PCC4azal teratocarcinoma stem cell differentiation in culture. III. Cell-to-cell communication properties. Develop. Biol. 75, 112-120. MARTIN, G. R. (1975). Teratocarcinomas as a model system for the study of embryogenesis and neoplasia. Cell 5, 229-243. MARTIN, G. R., and EVANS, M. J. (1975). Differentiation of clonal lines of teratocarcinoma cells Formation of embryoid bodies in vitro. Proc. Nat. Acad. Sci. USA 72,1441-1445. MARTIN, G. R., WILEY, L. M., and DAMJANOV, I. (1977). The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Develop. Biol. 61,230-244. MCNUTT, N. S., and WEINSTEIN, R. S. (1970). The ultrastructure of the nexus. A correlated thin section and freeze-cleave study. J. Cell Biol. 47, 666-688. MINTZ, B., CRONMILLER,C., and CUSTER,P. (1978). Somatic cell origin of teratocarcinomas. Proc. Nat. Acad. Sci. USA 75,2834-2838. MONTESANO,R., FRIEND, D. S., PERRELET, A., and ORCI, L. (1975). In vivo assembly of tight junctions in fetal rat liver. J. Cell Biol. 67, 310-319. ORCI,L., AMHERDT,M., HENGUIN, J. C., LAMBERT,A. C., UNGER, R. H., and RENOLD, A. E. (1973). Pronase effect in pancreatic beta cell secretion and morphology. Science 180, 647-649. PAYTON, B., BENNETT, M. V. L., and PAPPAS,G. D. (1969). Permeability and structure of junctional membranes of an electrontonic synapse. Science 166, 1641-1643. PIERCE,G. B., and BEAL, T. F. (1964). The ultrastructure of primordial germinal cells of the fetal testes of embryonal carcinoma cells of mice. Cancer Res. 24, 1553-1567. PIERCE,G., DIXON, F., and VERNEY, E. (1959). Testicular teratomas. II. Teratocarcinoma as an ascitic tumor. Cancer Res. 12, 584-589. PIERCE, G. B., STEVENS,L. C., and NAKANE, P. K. (1967). Ultrastructural analysis of the early development of teratocarcinomas. J. Nat. Cancer Inst. 39, 755-773.
STAEHELIN, L. A. (1973). Further observations on the tine structure of freeze-cleaved tight junctions. J.
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Cell Sci. 13, 763-786. STEVENS, L. C. (1960). Embryonic potency of embryoid bodies derived from a transplantable testicular teratoma of the mouse. Develop. Biol. 2, 285297.
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WOLPERT, L. (1978). Gap junctions: Channels for communication in development. In “Intercellular Junctions and Synapses” (J. Feldman, N. B. Gilula, and J. D. Pitts, eds.), pp. 83-94. Chapman and Hall, London.