Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."     

Cellular junctions of myelinated nerves (Review)

Myelinated nerves are specifically designed to allow the efficient and rapid propagation of action potentials. Myelinating glial cells contain several types of cellular junctions that are found between the myelin lamellas themselves in specialized regions of non-compact myelin and between the myelin membrane and the underlying axon. These include most of the junctional specializations found in epithelial cells, including tight, gap and adherens junctions. However, whereas in epithelial cells these junctions are formed between different cells, in myelinating glia these so called autotypic junctions are found between membrane lamellae of the same cell. In addition, myelinating glial cells form a heterotypic septate-like junction with the axon around the nodes of Ranvier and, in the peripheral nerve system, contact the basal lamina, which surrounds myelinating Schwann cells. This short review discusses the structure, molecular composition and function of the junctions present in myelinating cells, concentrating on the axo-glial junction.     

Carbon tetrachloride induced proliferation of tight junctions in the rat liver as revealed by freeze-fracturing

Application of carbon tetrachloride produced a progressive proliferation of tight junctions in the rat liver. This system proved to be rapid and highly reproducable and affords the opportunity for tracing the fate of tight junctions in freeze-fracture replicas, facilitating investigations on their formation and function. Beginning on day one carbon tetrachloride treatments resulted in the progressive loosening and fragmentation of the junctional meshwork. After three to four days the membrane outside the zonulae occludentes was extensively filled with proliferated discrete junctional elements often forming complex configurations. From the fifth day on the zonulae occludentes were restricted again predominantly around the bile canaliculus margins. But the junctional meshwork of the zonulae occludentes remained loosened in comparison to those in the control rats. It could be shown that tight junction proliferation on the lateral surface of the plasmalemma occurred both through de novo formation from discrete centers of growth by addition of intramembranous particles and through reorganization of preexistent junctional strands of the fragmented zonulae occludentes bodies. Whereas the large gap junctions close associated with the zonulae occludentes remained more or less unaffected during the experiments, small gap junctions increased in number after five days and were located at the margin or in the tight junction domain. It is assumed that the degeneration of the tight junctions served as a pool for intramembranous particles which form the gap junctions. The results of these observations are discussed in relation to those obtained in other systems.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Relationship of sertoli-sertoli tight junctions to ectoplasmic specialization in conventional and en face views

Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system

Summary Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6–7 nm particles soley near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions) contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia.The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neural lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.     

Reciprocal influence of connexins and apical junction proteins on their expressions and functions

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

A new angle on blood–CNS interfaces: A role for connexins?

Neuronal signaling in the CNS depends on the microenvironment around synapses and axons. To prevent fluctuations in blood composition affecting the interstitial fluid and CSF, two barriers, the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB), are interposed between the blood and the brain/CSF compartment. Brain capillary endothelial cells (ECs) constitute the BBB whereas choroid plexus epithelial (CPE) cells form the BCSFB. The anatomical basis of these barriers is located at the level of an intercellular junctional complex that impedes paracellular diffusion. Tight and adherens junctions are known as the principal constituents of this junctional complex. Transmembrane connexins (Cxs) are the prime building blocks of plasma membrane hemichannels that combine to form intercellular gap junctions (GJ). Although Cxs co-exist within the junctional complex, their influence on tight/adherens junctions and their role in barrier function of BBB ECs and CPE has been mostly ignored. Here, we review current knowledge on the role of Cxs in the BBB, BCSFB and other interfaces that subside within the CNS. We conclude that Cxs are a rather unexplored but promising target for influencing CNS barrier function.     

Interaction of filipin with junctional membrane at different stages of the junction's life history

The relationship of filipin-sterol complexes to tight and gap junctions during their formation, maturation, internalization, and degradation was studied in separate cell lines. Filipin-sterol complexes tended to be excluded from mature junctions in tight junction forming COLO 316 cells and gap junction forming SW-13 cells. Once internalized, unlabeled junctional membrane appeared to fuse with heavily labeled vesicles, presumably lysosomes. Although the absence of filipin-sterol complexes from junctional membrane does not necessarily reflect the absolute sterol content of this membrane, the fact that filipin-sterol complexes are largely excluded from these areas indicates that this membrane is different from surrounding membrane. The absence of filipin-sterol complexes also permits the visualization of 'mixing' of this specialized unlabeled membrane domain with other filipin labeled membrane systems.     

The structure and function of myelin: from inert membrane to perfusion pump

The goal of this overview is to propose a novel structure/function model of central nervous system myelin. Although myelin is known to be a compact multilamellar structure that wraps around axons, the biologic role this structure plays in the nervous system remains an enigma. One means of ascertaining myelin's biologic role is by analyzing its structure. The recent discovery of tight junctions in myelin may be the key that unlocks the mysterious black box of myelin structure/function. Tight junctions in other cell types are invariably adjacent to adherens junctions, with both of these junctional plaques playing critical roles in paracellular barrier function, i.e., adhesion of cell membranes, signal transduction, and fluid movement between cells via aqueous pores and channels. The application of current knowledge about junctional plaques to myelin is an original concept. This knowledge, taken together with evidence from studies of normal and pathologic myelin, supports the possibility that a primary function of junctional plaques in myelin is to perfuse the periaxonal space.     

A possible correlation between ultrastructure and function in the thin descending and ascending limbs of the loop of Henle of rabbit kidney

Summary An ultrastructural study of the thin loops of Henle has been made in the renal papilla of the rabbit. Animals in different states of water balance were used but no morphological difference was observed in the loops obtained from animals in different experimental groupings. The cytoplasm of the squamous cells lining the limbs was characterised by a paucity of organelles. Descending and ascending limbs were distinguishable. A distinct morphological difference was seen in the junctional regions of cell processes of the descending and ascending thin limbs of the loop. The ascending limb processes were joined by continuous tight junctions whereas the descending limb junctional regions invariably showed a space of at least 70 Å between adjacent processes. It is suggested that there may be a correlation between the structure of these junctional regions and the different permeability characteristics of the two limbs. The thin ascending limb must, on physiological evidence, be relatively impermeable with reference to the thin descending limb.The author wishes to thank Professor F. R. Johnson for his advice and assistance, and Mr. R. F. Birchenough, Mr. P. L. Hyam and Mr. J. Manston for valuable technical assistance.     

Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost-multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.     

VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Occluding-like junctions at mesaxons of central myelin in Anolis carolinensis are not 'tight'. A freeze-fracture-protein tracer analysis.

The junctional complexes of the myelin sheath of central nervous system axons in the American chameleon, Anolis carolinensis, exhibit an intramembrane ridge and groove construction in freeze-fracture replicas that has usually been interpreted in other organisms as evidence for an occluding or tight intercellular junction. Close examination of PF fracture face ridges, however, shows them to be made up of discontinuous rows of particles of variable length separated by frequent gaps of non-uniform width. Introduction of horseradish peroxidase into the intercellular milieu of the lizard central nervous system is followed by appearance of this protein in interlamellar spaces of the myelin sheath and in the intercellular spaces containing focal membrane fusions that correspond precisely in position and center-to-center spacing to the ridges and grooves in platinum replicas of the same tissue. Since the junctional ridges on PF fracture faces in these mesaxonal junctional complexes are conspicuously discontinuous and since the areas within the myelin sheath where these junctional complexes are located inner and outer mesaxons) are readily permeated by exogenous protein tracer, it is concluded that the junctional complexes of central myelin mesaxons, heretofore incorrectly interpreted as functionally tight, are actually very leaky and probably contribute only to the structural stability of the myelin sheath architecture.     

A quantitative study of intramembrane changes during cell junctional breakdown in the dystrophic rat retinal pigment epithelium

Previous electron microscope freeze-fracture and tracer studies have revealed that intercellular junctions in the retinal pigment epithelium (RPE) of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy [5] break down between three and six postnatal weeks [6, 7]. In this study quantitative computer techniques were used to analyze the freeze-fracture changes in the dystrophic RPE. The following parameters were measured: length of tight junctional strands/micron2; number of tight junctional strand anastomoses/micron2; number of gap junctional aggregates/micron2; area of gap junctional aggregates/micron2; and density of background intramembrane particles/micron2. At three postnatal weeks, the dystrophic junctional complex membrane is similar to normal, but at 10 weeks and later there are dramatic decreases in tight junctional strand length/micron2 and number of anastomoses/micron2, as well as in the number/micron2 and area of gap junctions/micron2, while the density of background particles/micron2 is dramatically increased. Correlational analysis revealed that changes in gap and tight junctions were significantly related to each other and to the increase in background particle density. The diameter of background particles within the normal and post-breakdown dystrophic junctions was measured in order to see whether the dispersal of gap and tight junctional particles (8-10 nm) into the surrounding membrane contributes to the increased particle density. These measures showed that background particles in all size ranges were more numerous in the dystrophic RPE, but that the largest increase was in the smallest diameter particles (6-7 nm). Thus, while gap and tight junctional sized particles contribute to the increase, particles from other sources may also be involved. Particle density of apical and basal membranes in the normal and in the 10 week and older dystrophic RPE was analyzed to study the effects of tight junctional breakdown on the distribution of intramembrane particles. These measures showed that particle density was greater basally than apically in the normal RPE and that particle density in both membranes decreased slightly in the dystrophic RPE, but that their ratio remained unchanged. It has been shown previously that even a single intact tight junctional strand is sufficient to maintain differences in particle density between apical and basal surfaces [14, 15] and in the majority of abnormal dystrophic junctional complexes at least one tight junctional strand remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural organization of the tight junctions

Tight junctions are the most apical organelle of the apical junctional complex and are primarily involved in the regulation of paracellular permeability and membrane polarity. Extensive research in the past two decades has identified not only the individual molecules of the tight junctions but also their mutual interactions, which are the focus of the present review article. While a complete map of the interactions among the tight junction molecules is probably far from being complete, the available evidence already allows outlining the general molecular architecture of the tight junctions. Here, with the aim of gaining deeper mechanistic understanding of tight junction assembly, regulation and function, we have subdivided the known molecular interactions into four major clusters that are centered on cell surface, polarity, cytoskeletal and signaling molecules.     

PCC4azal teratocarcinoma stem cell differentiation in culture: II. Morphological characterization

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 novo 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 PCC4azal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of 10-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.