Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Loss and reappearance of gap junctions in regenerating liver

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.     

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.     

ASSEMBLY OF GAP JUNCTIONS DURING AMPHIBIAN NEURULATION

Sequential thin-section, tracer (K-pyroantimonate, lanthanum, ruthenium red, and horseradish peroxidase), and freeze-fracture studies were conducted on embryos and larvae of Rana pipiens to determine the steps involved in gap junction assembly during neurulation. The zonulae occludentes, which join contiguous neuroepithelial cells, fragment into solitary domains as the neural groove deepens. These plaque-like contacts also become permeable to a variety of tracers at this juncture. Where the ridges of these domains intersect, numerous 85-Å participles apparently pile up against tight junctional remnants, creating arrays recognizable as gap junctions. With neural fold closure, the remaining tight junctional elements disappear and are replaced by macular gap junctions. Well below the junctional complex, gap junctions form independent of any visible, preexisting structure. Small, variegated clusters, containing 4–30 particles located in flat, particle-free regions, characterize this area. The number of particles within these arrays increases and they subsequently blend together into a polygonally packed aggregate resembling a gap junction. The assembly process in both apical and basal regions conforms with the concept of translational movement of particles within a fluid plasma membrane.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Junctions in the central nervous system of the cat

Interendothelial membrane contacts in different segments of brain blood vessels were investigated by the freeze-etching technique. The study demonstrated that the endothelial cells of the pre- and postcapillary segments were coupled by elaborate zonulae occludentes. These tight junction formations encompassed gap junctions of different sizes and distribution. The globules of the pre- and postcapillary tight junctions revealed a great fragility which led to an atypical distribution of the sealing elements. In the "typical" brain capillaries the endothelial cells were connected by continuous tight junctions. In contrast to the structural continuity of these formations the fenestrated segments of capillaries in the choroid plexus and area postrema demonstrated discontinuous fasciae occludentes. They were composed of rows of individual particles which should be regarded primarily as focal in nature.     

Reformation of gap and tight junctions in regenerating liver after cholestasis

Summary Morphometric analysis of the alterations in interhepatocyte junctions induced by bile duct ligation revealed that after 48 h, during which time the serum bilirubin increased 6 to 8 fold, the membrane area occupied by gap junctions on the apico-lateral and medio-lateral sides decreased from 3.6% in controls to 0.02% in the ligated group. The strands of the zonulae occludentes were reduced in number and showed increased discontinuities.Within 45 min of recanalization of the common bile duct, clusters of particles appeared within and adjacent to the tight junctional areas or in the lateral hepatocyte membrane. Subsequently, the particle aggregations localized in the apico-lateral membrane areas increased in number and size becoming finally indistinguishable from those of controls within 96 h after the onset of recanalization. The zonulae occludentes also rearranged and reestablished their original structure during this period. The serum bilirubin fell to normal within 24 h of recanalization. It is concluded that metabolic and ultrastructural restitution associated with the recanalization of the ligated bile duct have no strict temporal correlation to one another.These studies provide further evidence that alterations in gap and tight junctions induced by pathological processes, e.g. during bile duct ligation, are completely reversible when regeneration occurs.Summer student from Harvard Medical School, Boston (USA)     

Cell junctions in the epithelium of Bowman's capsule

Summary The intercellular connections between the epithelial cells of Bowman's capsule were investigated. It could be demonstrated that typical zonulae occludentes (tight junctions) are present in the species (rat, hamster, and Tupaia) studied. Freeze-fracturing shows a network of anastomizing strands; some species variations are described. In the rat two strands are common. In the golden hamster mostly two to four and occasionally five strands occur. In Tupaia regularly three tight junction strands are found and also gap junctions associated with the zonulae occludentes. In thin sections the goniometric analysis confirms the freeze-fracturing results and reveals attachment zones of macular shape, which are classified as intermediate junctions and desmosomes. The functional role of these cell junctions observed in the epithelium of Bowman's capsule is discussed.     

THE SPLITTING OF HEPATOCYTE GAP JUNCTIONS AND ZONULAE OCCLUDENTES WITH HYPERTONIC DISACCHARIDES

Mouse livers were perfused in situ through the portal vein with the disaccharides sucrose, lactose, maltose, and cellobiose in hypertonic concentrations (0.5 M). This treatment resulted in plasmolysis of the hepatocytes and splitting of the gap junctions and zonulae occludentes. The junctions split symmetrically, leaving a half-junction on each of the two separated cells. The process of junction splitting is followed using the freeze-fracture technique, since the junctional membranes are indistinguishable from the nonjunctional membranes in thin sections once the splitting occurs. The split junctions are also studied using the freeze-etch technique, allowing a view of the gap junction extracellular surface normally sequestered within the 2-nm "gap." The monosaccharides sorbitol and mannitol did not split the junctions during the times studied (2 min), but substitution of the chloride ion with propionate in the perfusion mixture did result in junction splitting. An envelope of morphologically distinct particles surrounding freeze-fractured gap junctions is also described.     

Gap junctions and zonulae occludentes of hepatocytes during biliary atresia in the lamprey

Gap junctions and zonulae occludentes of hepatocytes were examined in thin sections and freeze-fracture replicas from livers of larval and juvenile adult lampreys and during the phase of metamorphosis when bile ducts and bile canaliculi disappear (biliary atresia). Larvae possess zonulae occludentes at the canaliculi which are composed of one to five (mean = 2.81) junctional strands that provide a bile-blood barrier. Morphometry demonstrates that during biliary atresia the decreases in number of junctional strands and apico-basal depth of the zonulae occludentes are accompanied by an increase in the frequency of gaps or interruptions in the strands and in a breakdown of the bile-blood barrier. The zonulae occludentes completely disappear during metamorphosis and are not found in the adult liver. Gap junctions of the larval liver occupy 1% of the surface of the plasma membrane and have a mean area of 0.167 micron 2 but, following an initial decline in these parameters during early biliary atresia, they rise sharply in later stages of metamorphosis and in adults are 3.2% and 0.502 micron 2, respectively. The events of alteration in junctional morphology during lamprey biliary atresia is in many ways comparable to the changes in gap junctions and zonulae occludentes during experimental and pathological intra- and extrahepatic cholestasis in mammals.     

The tight junctions of rabbit choroid plexus and ciliary body epithelia. A comparative study using the double replica freeze-fracture technique

The spatial arrangement of tight junctions in choroid plexus and ciliary body rabbit epithelia has been determined by studying freeze-fracture complementary replicas. In the choroid plexus epithelium, the interruptions of the junctional P-face fibrils were measured to be 14% of their total length. In the ciliary body epithelium, where the fibrils were found to be more fragmented than in the choroid plexus, the P-face fibril interruptions accounted for 12 % of the total length of the zonulae occludentes sealing the non-pigmented cells and 30% in the focal linear tight junctions connecting the non-pigmented and pigmented cells at their apices. In both epithelia, the interruptions of the ridges are precisely complemented by particles or short bars of similar length found in the E-face furrows. Consequently, it is possible to conclude that the junctional fibrils are continuous in these two epithelia. For the zonulae occludentes, this continuity appears to be inconsistent with the ‘leaky’ properties of these epithelia shown by some physiological investigations.     

Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles

Summary Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland.In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit.Aggregates of particles related to desmosomes are found in the deeper meshes of the zonulae occludentes or close to them.     

Cell junctions in the early chick embryo--a freeze etch study

Cell junctions in the early chick embryo have been examined in freeze-etch specimen. Well developed zonulae occludentes are found in the epiblast as early as stage 4. Large gap junctions are also found in the epiblast at this stage. In those cells which have left the surface to form mesenchymal structures (Hensen's node, juxtanodal mesenchyme, primitive streak mesenchyme), one finds not only gap, but also tight, junctions. These junctions do not form continuous belts, but appear as fragments, often reduced to single strands, of typical tight junctions. They probably correspond to the focal tight junctions described earlier in sectioned material. The origin and possible significance of these contacts is discussed, and it is suggested that they represent remnants of junctions between neighboring cells in the epiblast. These junctional remnants slowly disappear by “dilution,” either through cell division and/or cell movement. The appearance of newly formed gap junctions is also described.     

Different functions of tight junctions in the ascidian branchial basket

The stigmatal cells in the branchial basket of ascidians from a number of genera have been examined as to the nature and distribution of their intercellular junctions. The branchial wall consists of ciliated and parietal cells; the ciliated cells are arranged in seven rows and are associated by junctions with other cells in the same row as well as with those in adjacent rows. They are also associated by junctions with peripheral parietal cells. Junctions between adjacent ciliated cells in all cases exhibit tight junctions or zonulae occludentes. However, these cell borders also possess fasciae or zonulae adhaerentes if they are in the same row and the ciliary rootlets insert-into these junctions. If the cells are in adjacent rows they exhibit adhaerentes junctions only in species belonging to the orders Phlebobranchiata and Aplousobranchiata. In contrast, if the cells in adjacent rows belong to the order Stolidobranchiata. they never exhibit any adhaerentes junctions and the ciliary rootlets of the basal bodies from the cilia insert instead into the tight junctions and the non-junctional membrane below them. At the homologous junctional borders between adjacent parietal cells and also at heterologous junctional borders between parietal and ciliated cells, tight junctions alone occur, with no co-existing adhaerentes junctions along their lateral borders. Again, fibrils from ciliary rootlets insert into zonulae occludentes. This shows that tight junctions are capable both of forming permeability barriers, in that they can be seen to prevent the entry of exogenous tracers such as lanthanum, and of acting as adhesive devices.     

Junctional diversity in two regions of the epidermis of Oikopleura dioica (Tunicata,Larvacea)

Summary A simple continuous epithelium surrounds the body of the pelagic larvacean. It consists of two zones of cells: oikoplast cells and flattened cells. The oikoplast cells are columnar and produce a thick extracellular house that ensheathes the body of the organism. These cells are joined laterally by wide tight junctions (zonulae occludentes). The tail of the animal is surrounded by exceedingly thin cells which are joined by narrow tight junctions under which lie intermediate junctions (zonulae adhaerentes) and gap junctions. A web of fibrous material inserts into the intermediate junctions. The transitional cells between the two epithelial zones have one lateral border with a wide tight junction, and the other lateral border with a narrow tight junction and a wide intermediate junction. In freeze-fracture replicas, the wide tight junction has a number of anastomosing ridges, in comparison with the narrow tight junction, which usually consists of only a single row of intramembranous particles. In replicas, the thin epithelial cells show unusual parallel arrays of particles in clusters on their apical plasma membranes. This simple epithelium, therefore, exhibits striking differences between the two cellular zones, in the structural characteristics of both the lateral borders and the apical membrane.     

The formation and distribution of intercellular junctions in the rhesus monkey optic cup: The early development of the cilio-iridic and sensory retinas

The eyes of prenatal monkeys from 30 to 102 ± 2 days old were examined by light microscopy, conventional electron microscopy, and the freeze-fracturing technique. At 30 days, invagination of the optic vesicle has begun, and the inner and outer walls of the forming optic cup are closely apposed anteriorly; invagination is complete at 45 days. By 58 days, the rudiment of the ciliary body and iris has appeared; at 71 days, primitive ciliary processes are present and retinal photoreceptors begin to differentiate. The distribution of intercellular junctions varies both in different regions of the optic cup and at different stages of development. At 30 days, adjacent ventricular and adjacent pigmented cells are joined throughout the optic cup by zonulae adhaerentes and gap junctions. The anterior region of the cup, however, contains two additional junctional specializations: (1) fasciae occludentes between ventricular cells and (2) intermediate and gap junctions between the apposing luminal surfaces of ventricular and pigmented cells. By 36 days the fasciae occludentes between ventricular cells in the anterior optic cup become zonular, signaling the morphological development of the blood-aqueous barrier. In the posterior optic cup, zonulae occludentes appear between adjacent pigmented cells at 36 days; furthermore, with the continuing obliteration of the optic ventricle, luminal junctions spread toward the optic stalk but do not reach the optic disc until 45 days, when invagination is complete. Between 58 and 102 days there are no further changes in the distribution of the junctions anteriorly between the primitive cilio-iridial epithelial cells, whereas in the posterior optic cup the luminal gap and intermediate junctions between pigmented cells and differentiating photoreceptors decrease in number and finally disappear. Two main conclusions can be drawn from this study. (1) In the optic cup, intermediate junctions are consistently present in regions of the plasma membrane which later contain junctional complexes. The temporal and spatial pattern of junctional development suggests that intermediate junctions are necessary for the establishment of tight and gap junctions. (2) Twenty days before the ciliary body-iris anlage becomes visible in the light microscope, the distribution of junctions in the anterior part of the optic cup is identical to that in the adult cilio-iridic retina. The time-honored view that the cilio-iridic retina appears late in development is, therefore, no longer tenable. In the monkey, the optic cup is divided into a cilio-iridic and a sensory region soon after the onset of invagination.     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

A new occludens-like junction linking endothelial cells of small capillaries (probably venules) of rat jejunum.

Freeze-cleave replicas of small capillaries of rat jejunum have revealed the presence of a new type of junction linking endothelial cells. This new junction reveals tight junctions (zonulae occludentes) in that the adjacent plasma membranes are held closely together along lines of attachment organized in the form of a loose, but frequently discontinuous network. In contrast to tight junctions, the A-face ridges possess a very low profile, and only at low shadowing angles can a repeating, particulate substructure occasionally be resolved. The shallow B-face furrows lack any particulate components. Images of cross-fractured focal points of attachment suggest that the external leaflets of adjacent membranes are closely apposed but not actually fused, as is the case with zonulae occludentes. It appears that this new type of endothelial junction is characteristic of small venules. Thus we propose that it be termed small venule endothelial junction.     

FRACTURE FACES OF OSMOTICALLY DISRUPTED ZONULAE OCCLUDENTES

Exposing the mucosal epithelium of the toad urinary bladder to 240 mM urea in Ringer's solution is known to cause a dramatic increase in the permeability of the zonulae occludentes and the appearance of distended, bubble-like compartments within these junctions. Examination of such osmotically disrupted junctions with the freeze-fracture technique reveals that these bubbles result from a distention of the compartments existing within the meshwork of interconnecting fibrils characteristic of the zonulae occludentes in this epithelium. Frequent discontinuities in the meshwork of fibrils are also found after osmotic disruption of the junction. These observations indicate the essential role of these fibrils in maintaining the characteristic properties of the zonula occludens as a site of cell-to-cell attachment and as a permeability seal.     

HEXAGONAL ARRAY OF SUBUNITS IN TIGHT JUNCTIONS SEPARATED FROM ISOLATED RAT LIVER PLASMA MEMBRANES

Solubilization of isolated rat liver plasma membranes in 1% deoxycholate and centrifugation yielded a fraction (pellet) that consisted mainly of tight junctions (zonulae occludentes). An hexagonal array of subunits similar to that previously found in a number of the unfractionated plasma membranes was demonstrated in all the membrane sheets of these preparations by negative staining. It is concluded that the hexagonal subunit pattern is present in the tight junctions, and that this structural differentiation may be related to the intercellular diffusion afforded by the junctional membrane.