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.     

Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates

The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role.     

Cell junctions in amphioxus (Cephalochordata): a thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent 'poorly developed' septate junctions.     

Gap junctions suggest epithelial conduction within the comb plates of the ctenophore Pleurobrachia bachei

Intercellular gap junctions occur between the ciliated cells that make up the comb plates of the ctenophore Pleurobrachia. Similar junctions are found within the ciliated grooves which run from the apical organ to the first plate of each comb row, as well as throughout the endoderm of the meridional canals. Gap junctions were not found in the ectodermal tissue between the comb rows. The distribution of junctions suggests that excitation conduction within the ciliated grooves, comb plates and meridional canal endoderm may be epithelial.     

Neuronal organization of the ascidian (Urochordata) branchial basket revealed by cholinesterase activity

Summary Innervation of the ascidian branchial basket and other structures is demonstrated by staining for cholinesterase. Cholinesterase activity is not restricted to synaptic sites but is present throughout the neurons. Primary and secondary axonal bundles form a bilaterally symmetric innervation pattern around the large dorsal visceral nerve. These bundles continue to split into progressively smaller bundles as they course throughout the basket. Axons are suspended in a fibrous matrix and run within the blood sinuses on the atrial side of the basket. Stigmatal ciliated cells of the branchial basket are innervated by highly branched distal portions of neurons, whose cell bodies are located in the ganglion. Synaptic boutons, containing electron-lucent vesicles, are found at nearly all stigmatal ciliated cells. NiCl₂backfills of the visceral nerve reveal a distinct population of central neurons, some of which presumably control ciliary arrest.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.     

Alimentary tract of Kowalevskiidae (Appendicularia, Tunicata) and evolutionary implications

The alimentary tract of Kowalevskia tenuis and K. oceanica, the only species of the appendicularian family Kowalevskiidae, was studied both at the light and electron microscope levels and compared with species belonging to the other two families of the class. Kowalevskids show interesting specializations: 1) the pharynx opens on both sides through two opposing spiracles, modified into long ciliated fissures, and possesses an original filtering system of ciliated combs arranged in two pairs of opposing longitudinal rows; 2) the endostyle is absent, its place being taken by a ciliated groove without any glandular cell; 3) posterior to the esophagus, the globular stomach and rectum form a digestive nucleus comprising a few, large cells including two well-developed, specialized valves, cardiac and pyloric; 4) special apical junctions bearing characteristics of both gap and adherens junctions are diffuse along the gut epithelium; 5) the heart is absent. Our data suggest that Kowalevskiidae underwent a high degree of specialization for food filtering and are more closely related to Fritillariidae, with which they share several characters, rather than Oikopleuridae, the latter probably representing the most primitive family of appendicularians.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

A comparative study of ciliary differentiations in the branchial stigmata of ascidians

In a correlated thin sectioning and freeze-fracturing study, we have examined species belonging to the orders of the ascidian class: Stolidobranchiata (Botryllus schlosseri, Botrylloides leachi, Molgula socialis, Styela plicata), Phlebobranchiata (Ascidiella aspersa, Phallusia ingeria, Ciona intestinalis) and Aplousobranchiata (Clavelina lepadiformis). Though the branchial basket varies in the complexity and filtration efficiency in the three orders, the ciliated epithelia aroand the stigmata contain a common pattern of organization; seven rows of flattened cells, each bearing a single row of long cilia flanked by a single row of microvilli. All the species examined possess ciliary specializations represented by: (a) bridges connecting doublets number 5 and 6 as well as 9.1 and 2; (b) dense material lying between the above mentioned axonemal doublets (5-6 and 1-2) and the ciliary membrane, sometimes in the shape of longitudinal strands or often as lines of dots; (c) a fuzzy coat protruding from the ciliary membrane, consisting of tufts or scattered filaments; (d) intramembrane particles (IMPs) associated with the P-face of the membrane, often arranged in clusters and orderly alignments related to the anderlying axonemal doublets; these IMPs decorate the opposite sides of each cilium facing the adjacent cilia forming the ciliary rows of adjacent cells and are absent on the lateral sides. The stigmatal cilia propel water through the stigmata and their effective strokes follow a line at right angles to the row of cilia in each cell. The usual direction of the effective stroke is toward doublets 5-6. It is possible, therefore, to refer to structure in relation to the ciliary beat cycle. The importance of these specializations is unknown, but the structures appear to vary in the different species. A correlation between the richness of the specializations and the complexity of the branchial basket was not evidenced. It was suggested that the ciliary specializations relate to the peculiar organization of the stigmatal margin and that all are involved in the regulation of the ciliary activity.     

ESR spectra of vanadyl ion detected in branchial basket of an ascidian,ascidia ahodori

ESR spectra due to the vanadyl ion (VO²⁺, +4 oxidation state) was detected in the branchial basket of Ascidia ahodori, which is reported to contain vanadium in high amounts. The branchial basket, washed with a medium containing 1 mM EDTA, and the supernatant showed different types of vanadyl ESR spectra. On further treatment with 100 mM EDTA the branchial basket gave a characteristic ESR spectrum, indicating that the vanadyl ion binds to a high molecular weight matrix, such as proteins, which makes up the branchial basket. Judging from the relationship of the ESR parameters, g_∥ versus A_∥, the vanadyl ion is assumed to ligate with moieties such as deprotonated hydroxyl, or nitrogenous or thiolato groups from oxy- or thiolamino acid residues. The branchial basket was shown to have the ability to reduce added vanadate ion (+5 oxidation state) to the vanadyl form. On the basis of these observations, participation of the branchial basket in vanadium-accumulation by ascidians from seawater is suggested.     

JUNCTIONS BETWEEN INTIMATELY APPOSED CELL MEMBRANES IN THE VERTEBRATE BRAIN

Certain junctions between ependymal cells, between astrocytes, and between some electrically coupled neurons have heretofore been regarded as tight, pentalaminar occlusions of the intercellular cleft. These junctions are now redefined in terms of their configuration after treatment of brain tissue in uranyl acetate before dehydration. Instead of a median dense lamina, they are bisected by a median gap 20–30 A wide which is continuous with the rest of the interspace. The patency of these "gap junctions" is further demonstrated by the penetration of horseradish peroxidase or lanthanum into the median gap, the latter tracer delineating there a polygonal substructure. However, either tracer can circumvent gap junctions because they are plaque-shaped rather than complete, circumferential belts. Tight junctions, which retain a pentalaminar appearance after uranyl acetate block treatment, are restricted primarily to the endothelium of parenchymal capillaries and the epithelium of the choroid plexus. They form rows of extensive, overlapping occlusions of the interspace and are neither circumvented nor penetrated by peroxidase and lanthanum. These junctions are morphologically distinguishable from the "labile" pentalaminar appositions which appear or disappear according to the preparative method and which do not interfere with the intercellular movement of tracers. Therefore, the interspaces of the brain are generally patent, allowing intercellular movement of colloidal materials. Endothelial and epithelial tight junctions occlude the interspaces between blood and parenchyma or cerebral ventricles, thereby constituting a structural basis for the blood-brain and blood-cerebrospinal fluid barriers.     

Ultrastructural modifications of the follicular epithelium accompanying the onset of vitellogenesis in the whirligig beetle,Gyrinus natator

Summary The ultrastructure of the follicle cells during previtellogenesis and early vitellogenesis have been studied. In previtellogenesis follicle cells are columnar with numerous bundles of microtubules located along the lateral plasma membranes. Oocyte-follicle cell gap junctions are not found in this stage. At the onset of vitellogenesis, the bundles of microtubules disappear and are replaced by an apically located ring of microtubules. The modification of microtubular cytoskeleton is not followed by the development of intercellular spaces between the follicle cells. Concurrently, numerous gap junctions are formed between specialized follicle cell processes and oocyte microvilli, which are arranged in characteristic cone-shaped aggregations. It is suggested that cytoskeletal changes and formation of heterologous gap junctions, occurring at the onset of vitellogenesis, are induced by juvenile hormone.     

THE APPEARANCE AND STRUCTURE OF INTERCELLULAR CONNECTIONS DURING THE ONTOGENY OF THE RABBIT OVARIAN FOLLICLE WITH PARTICULAR REFERENCE TO GAP JUNCTIONS

Lanthanum tracer and freeze-fracture electron microscope techniques were used to study junctional complexes between granulosa cells during the differentiation of the rabbit ovarian follicle. For convenience we refer to cells encompassing the oocyte, before antrum and gap junction formation, as follicle cells. After the appearance of an antrum and gap junctions we call the cells granulosa cells. Maculae adherentes are found at the interfaces of oocyte-follicle-granulosa cells throughout folliculogenesis. Gap junctions are first detected in follicles when the antrum appears. In early antral follicles typical large gap junctions are randomly distributed between granulosa cells. In freeze-fracture replicas, they are characterized by polygonally packed 90-Å particles arranged in rows separated by nonparticulate A-face membrane. A particle-sparse zone surrounds gap junctions and is frequently occupied by small particle aggregates of closely packed intramembranous particles. The gap junctions of granulosa cells appear to increase in size with further differentiation of the follicle. The granulosa cells of large Graafian follicles are adjoined by small and large gap junctions; annular gap junctions are also present. The large gap junctions are rarely surrounded by a particle-free zone on their A-faces, but are further distinguished by particle rows displaying a higher degree of organization.     

Glial cells in peripheral nerves of the cockroach, Periplaneta americana

The ultrastructural organization and the junctional complexes of peripheral nerves have been investigated in the cockroach Periplaneta americana. Nerve 5 is surrounded by a layer of connective tissue, the neural lamella, beneath which is a layer of perineurial glial cells wrapping the axons. Adjacent perineurial cells are joined to one another by septate, gap and tight junctions. Septate and gap junctions were observed in freeze-fracture replicas of main trunk nerve 5. Septate junctions were found as rows of PF particles mainly in perineurial cell membranes. Gap junctions exhibited EF macular aggregates in perineurial and subperineurial glial cells. During incubations in vivo with extracellularly applied ionic lanthanum, the lanthanum did not penetrate beyond the perineurium. Where nerve 5 branches and contacts the muscle, lanthanum penetrated freely between the muscle fibres and the nerve branches. In small peripheral branches where the axons are surrounded by single a glial layer, lanthanum is unable to penetrate to the axolemma.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.     

Atypical gap junctions in the ciliary epithelium of the albino rabbit eye

Gap junctions are present between nonpigmented and pigmented epithelial cells layers, as well as between the adjacent pigmented cells, but not between the nonpigmented epithelial cells which face the posterior chamber. The unusual feature of these gap junctions is a dark banding which regularly appears every three rows of subunits. This dark band is the equivalent width of two rows of facets.     

The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis.

Special occluding junctions between Sertoli cells near the base of the seminiferous epithelium are the structural basis of the blood-testis permeability barrier. In micrographs of thin sections, multiple punctate pentalaminar contacts between apposed membranes are observed in the junctional regions.In freeze-fractured mature testis, the junctional membranes exhibit up to 40 parallel circumferentially oriented rows of intramembrane particles preferentially associated with the B-fracture face, but with complementary shallow grooves on the A-face. Short rows of particles may remain with the A-face resulting in discontinuities in the B-face particle rows. In addition, elongate aggregations of particles of uniform size (～70 A) arranged in one or more closely packed rows are occasionally found adjacent to the linear depressions on the A-face of the Sertoli junction. These are interpreted as atypical gap junctions.In immature testis, occluding junctions are absent but typical gap junctions are common. These gradually disappear. In the second postnatal week, linear arrays of particles appear on the B-face. Initially meandering and highly variable in direction, these gradually adopt a consistent orientation parallel to the cell base. The establishment of the blood-testis barrier appears to be correlated with this reorganization of the intramembrane particle rows. Sertoli junctions were shown to be resistant to hypertonic solutions that rapidly dissociate junctions of other epithelia.Sertoli junctions thus differ from other occluding junctions in their (1) basal location, (2) large number of parallel particle rows, (3) absence of anastomosis between rows, (4) preferential association of the particles with the B-face, (5) intercalation of atypical gap junctions, (6) unusual resistance to dissociation by hypertonic solutions.     

A novel adhering junction in the apical ciliary apparatus of the rotifer Brachionus plicatilis (Rotifera, Monogononta)

Cultures of the rotifer Brachionus plicatilis were examined with regard to their interepithelial junctions after infiltration with the extracellular tracer lanthanum, freeze-fracturing or quick-freeze deepetching. The lateral borders between ciliated cells have an unusual apical adhering junction. This apical part of their intercellular cleft looks desmosome-like, but it is characterized by unusual intramembranous E-face clusters of particles. Deep-etching reveals that these are packed together in short rows which lie parallel to one another in orderly arrays. The true membrane surface in these areas features filaments in the form of short ribbons; these are produced by projections, possibly part of the glycocalyx, emerging from the membranes, between which the electron-dense tracer lanthanum permeates. These projections appear to overlap with each other in the centre of the intercellular cleft; this would provide a particularly flexible adaptation to maintain cell-cell contact and coordination as a consequence. The filamentous ribbons may be held in position by the intramembranous particle arrays since both have a similar size and distribution. These contacts are quite different from desmosomes and appear to represent a distinct new category of adhesive cell-cell junction. Beneath these novel structures, conventional pleated septate junctions are found, exhibiting the undulating intercellular ribbons typical of this junctional type, as well as the usual parallel alignments of intramembranous rows of EF grooves and PF particles. Below these are found gap junctions as close-packed plaques of intramembranous particles on either the P-face or E-face. After freeze-fracturing, the complementary fracture face to the particles shows pits, usually on the P-face, arrayed with a very precise hexagonal pattern.     

Functional expression of connexin30 and connexin31 in the polarized human airway epithelium

Gap junctions are documented in the human airway epithelium but the functional expression and molecular identity of their protein constituents (connexins, Cx) in the polarized epithelium is not known. To address this question, we documented the expression of a family of epithelial Cx (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, and Cx43) in primary human airway epithelial cells (AEC) grown on porous supports. Under submerged conditions, AEC formed a monolayer of airway cells whereas the air-liquid interface induced within 30-60 days AEC differentiation into a polarized epithelium for up to 6-9 months. Maturation of AEC was associated with the down-regulation of Cx26 and Cx43. The well-differentiated airway epithelium exhibited gap junctional communication between ciliated and between ciliated and basal cells. Interestingly, Cx30 was mostly present between ciliated cells whereas Cx31 was found between basal cells. These results are supportive of the establishment of signal-selective gap junctions with maturation of AEC, likely contributing to support airway epithelium function. These results lay the ground for studying the role of Cx-mediated cell-cell communication during repair following AEC injury and exploring Cx-targeted interventions to modulate the healing process.