Electron microprobe analysis of ouabain-exposed ciliary epithelium: PE-NPE cell couplets form the functional units

Aqueous humor is secreted by the bilayered ciliary epithelium. Solutes and water enter the pigmented ciliary epithelial (PE) cell layer, cross gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, and are released into the aqueous humor. Electrical measurements suggest that heptanol reduces transepithelial ion movement by interrupting PE-NPE communication and that gap junctions may be a regulatory site of aqueous humor formation. Several lines of evidence also suggest that net ciliary epithelial transport is strongly region dependent. Divided rabbit iris-ciliary bodies were incubated in chambers under control and experimental conditions, quick-frozen, cryosectioned, and freeze-dried. Elemental intracellular contents of NPE and PE cells were determined by electron probe X-ray microanalysis. With or without heptanol, ouabain produced concentration- and time-dependent changes more markedly in anterior than in posterior epithelium. Without heptanol, there were considerable cell-to-cell variations in Na gain and K loss. However, contiguous NPE and PE cells displayed similar changes, even when nearby cell pairs were little changed by ouabain in aqueous, stromal, or both reservoirs. In contrast, with heptanol present, ouabain added to aqueous or both reservoirs produced much larger changes in NPE than in PE cells. The results indicate that 1) heptanol indeed interrupts PE-NPE junctions, providing an opportunity for electron microprobe analysis of the sidedness of modification of ciliary epithelial secretion; 2) Na and K undergo faster turnover in anterior than in posterior epithelium; and 3) PE-NPE gap junctions differ from PE-PE and NPE-NPE junctions in permitting ionic equilibration between adjoining ouabain-stressed cells. pigmented ciliary epithelial cells; nonpigmented ciliary epithelial cells; gap junctions; aqueous humor; Na⁺/K⁺ exchange pump; rabbit iris-ciliary body     

The fine structure of the hypostome and mouth of hydra

The hypostome and mouth of fresh-water Hydra were examined by scanning electron microscopy. The external surface of the hypostome possesses cnidocils, possibly sensory hairs, and small spiny protrusions surrounding the mouth; the internal surface has cylindrical microvilli, free flagella and adherent flagella. The adherent flagella are most numerous close to the mouth where they cause the cell surface to appear smooth when viewed at low magnifications. Free flagella and leaf-like microvilli increase in prominence towards the tentacles and enter on proper. The edge of the mouth has an abrupt boundary marking the apposition of epidermal and gastrodermal cells. A transitional groove occurs at the boundary and the cells underlying the groove are smaller than those on other regions of the hypostome. The transition groove may represent a site of cell loss in normal cell turnover. Some of the small underlying cells may represent nervous elements involved in regulating hypostome activity during the feeding reation.     

Gap junctions are non-randomly distributed inDrosophila wing discs

Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 m^–1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 m^–1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.     

Early development of the anterior body region of the grey widow spider Latrodectus geometricus Koch, 1841 (Theridiidae, Araneae)

We document the early morphogenesis of Latrodectus geometricus, particularly of the anterior body region. Significant changes in the development of the external prosomal structures revealed with scanning electron microscopy (SEM) images include: (1) reorganisation of each pre-cheliceral lobe by subdivision and internalisation of its central area; (2) shortening of the ventro-median bridge connecting the pre-cheliceral lobes and its eventual disappearance; (3) appearance and expansion of a prospective mouth region between the pre-cheliceral lobes with a recessed median area surrounded by lip-like borders, the anterior lip-part developing into the hypostome; (4) reduction of the mouth region to an area around the hypostome and the lip-like latero-posterior border of the mouth opening; (5) change of the position of the mouth region from anterior to the insertions of the chelicerae to posterior to them; (6) eventual shortening of the mouth opening to a slit overhung by the hypostome; (7) origination of the prosomal shield from the anterior margin of the pre-cheliceral lobes and the tergal portions of the four posterior-most prosomal segments; and (8) expansion of a ‘ventral sulcus’ from the cheliceral to the fifth opisthosomal segment separating the sides of these segments. Embryonic features are compared across the Chelicerata and discussed briefly in a phylogenetic context.     

Development of the two-part pattern during regeneration of the head in hydra

The head of a hydra is composed of two parts, a domed hypostome with a mouth at the top and a ring of tentacles below. When animals are decapitated a new head regenerates. During the process of regeneration the apical tip passes through a transient stage in which it exhibits tentacle-like characteristics before becoming a hypostome. This was determined from markers which appeared before morphogenesis took place. The first was a monoclonal antibody, TS-19, that specifically binds to the ectodermal epithelial cells of the tentacles. The second was an antiserum against the peptide Arg-Phe-amide (RFamide), which in the head of hydra is specific to the sensory cells of the hypostomal apex and the ganglion cells of the lower hypostome and tentacles. The TS-19 expression and the ganglion cells with RFamide-like immunoreactivity (RLI) arose first at the apex and spread radially. Once the tentacles began evaginating in a ring, both the TS-19 antigen and RLI+ ganglion cells gradually disappeared from the presumptive hypostome area and RLI+ sensory cells appeared at the apex. By tracking tissue movements during morphogenesis it became clear that the apical cap, in which these changes took place, did not undergo tissue turnover. The implications of this tentacle-like stage for patterning the two-part head are discussed.     

CONNEXIN EXPRESSION AND GAP JUNCTIONS IN THE MAMMARY GLAND

Gap junctional communication plays a vital role in embryogenesis, cell differentiation and the co-ordination of tissue responses. Gap junctions are formed by a family of closely-related proteins called connexins which show tissue-specific patterns of expression. The role of gap junctions in the mammary gland remains unclear. The lumena of mammary gland ducts are lined by luminal cells with an outer layer of basal cells. In rodents, the luminal cells express connexin26 only during pregnancy and lactation and the basal cells, in some reports, express connexin43. In the normal human breast the basal cells express connexin43, although human mammary epithelial cellsin vitrohave been reported to express both connexin26 and connexin43. Analysis of connexin expression at the molecular level is now bringing new insights into the structure and function of gap junctions in a range of normal and pathological cell systems.     

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium.

The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.     

Heterocellular gap junctional communication between alveolar epithelial cells

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.     

Expression and developmental regulation of the Hydra-RFamide and Hydra-LWamide preprohormone genes in Hydra: evidence for transient phases of head formation

Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, we found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head showed that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. Our head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, which they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra.     

Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid polyps

Summary Antisera to the sequence Arg-Phe-amide (RF-amide) have a high affinity to the nervous system of fixed hydroid polyps. Whole-mount incubations of several Hydra species with RFamide antisera visualize the three-dimensional structure of an ectodermal nervous system in the hypostome, tentacles, gastric region and peduncle. In the hypostome of Hydra attenuata a ganglion-like structure occurs, consisting of numerous sensory cells located in a region around the mouth opening and a dense plexus of processes which project mostly radially towards the bases of the tentacles. In Hydra oligactis an ectodermal nerve ring was observed lying at the border of hypostome and tentacle bases. This nerve ring consists of a few large ganglion cells with thick processes forming a circle around the hypostome. This is the first direct demonstration of a nerve ring in a hydroid polyp.Incubation of Hydractinia echinata gastrozooids with RFamide antisera visualizes an extremly dense plexus of neuronal processes in body and head regions. A ring of sensory cells around the mouth opening is the first group of neurons to show RFamide immunoreactivity during the development of a primary polyp. In gonozooids the oocytes and spermatophores are covered with strongly immunoreactive neurons.All examples of whole-mount incubations with RF-amide antisera clearly show that hydroid polyps have by no means a diffuse nerve net, as is often believed, and that neuronal centralization and plexus formation are common in these animals. The examples also show that treatment of intact fixed animals with RFamide antisera is a useful technique to study the anatomy or development of a principal portion of the hydroid nervous system.     

Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.     

Contacts between pigmented retina epithelial cells in culture.

The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.     

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.     

A freeze-fracture study of the papillary layer of the rat incisor enamel organ

After tooth enamel has been secreted it undergoes maturation or hardening. This process is mediated by ruffled and smooth-ended ameloblasts and associated papillary layer cells. The cells of the papillary layer are characterized by large numbers of mitochondria, coated vesicles, microvilli, and gap junctions. These features have led numerous investigators to speculate that the papillary layer is an ion-transporting epithelium. We have conducted freeze-fracture studies of the rat papillary layer in order to better characterize the surface features of these cells. The cell membranes of the papillary cells contained large numbers of intramembrane particles of various sizes ranging from 4 to 9 nm in diameter. Gap junctions were present at the cell surface and in the cytoplasm in the form of annular gap junctions. The intramembrane particles or connexons of both types of gap junctions were about 8-9 nm wide and were either packed randomly or present in the so-called 'crystallized' state. At the interface between smooth-ended ameloblasts and papillary layer cells, a well-developed zonula occludens was present along the basal surfaces of the ameloblasts and several large gap junctions were formed between the two cell types. The capillary network associated with the papillary layer was characterized by a thin endothelium containing large numbers of fenestrations.     

Intercellular contact in the unincubated chick embryo

Summary The unincubated chick blastoderm, which consists of a complete upper epithelial layer of one cell thickness (epiblast) and an incomplete lower layer (hypoblast), was examined with the electron microscope in order to define the types of cell contact present. The terminal contacts between the cells of the epiblast invariably involved several focal tight junctions, but only occasionally involved tight junctions. Desmosomes were not observed in these areas, but were encountered in various phases of development in the deeper contact regions between epiblast cells. This deeper region also showed sporadic focal tight junctions and frequent micropapillae. These micropapillae were also common on the surfaces of hypoblast cells. Intercellular spaces between epiblast and hypoblast cells and within the hypoblast were often wide, narrowing to occasional focal tight junctions. Tight junctions and desmosomes were not observed in association with hypoblast cells. Gap junctions were not observed in any region of the embryo.These observations are discussed in relation to the morphogenetic movements occurring in the forming hypoblast and also the influence of this layer on the subsequent development of the embryo. Comparisons are drawn between the contact morphology in the unincubated blastoderm and that in later stages of development.Supported by the Medical Research Council of Canada.     

Gap junctions in the differentiated neural retinae of newly hatched chickens.

Gap junctions in the neural retinae of newly hatched chickens were examined in thin section and by freeze cleaving. Unusual gap junctions containing linear arrays of intramembrane particles are found between principal and accessory cones which form a double cone at the region of the outer limiting membrane. These unusual gap junctions are often continuous with macular aggregates of hexagonally packed intramembrane particles which are characteristic of a typical gap junction. Typical gap junctions are also found in both the outer and the inner plexiform layers and in the outer nuclear layer, but are not so abundant as in the outer limiting membrane region. The sizes of intramembrane particles and their centre-to-centre spacing within the macular aggregate of a gap junction in differentiated neural retinae are slightly larger than those in undifferentiated neural retinae. Tight junctions are not found in differentiated neural retinae.     

Absence of gap junctional complexes in two established renal epithelial cell lines (LLC-PK1 and MDCK)

The type of junctions present in the membranes of the two renal epithelial cell lines, LLC-PK1 and MDCK, and of subcultured porcine aortic endothelial (PAE) cells have been studied by freeze-fracture. No gap junctions were observed in the two renal cell lines, while they were numerous in the endothelial cells. Tight junctions were abundant in LLC-PK1 and MDCK cells and varied in numbers of ridges from one to ten. ONly a few simple tight junctions unconnected with gap junctions were observed in PAE cells. The occurrence of gap junctions in these cells correlates with their ability to form intercellular communicating channels.     

Importance of molecular configuration in gap junctional permeability

Gap junctions between insect oocytes and follicular epithelial cells allow transit of elongate Ca(2+)-binding proteins Calmodulin (CaM, 17kDa) and Troponin-C (Trop-C, 18kDa), but not multi-branched dextran (10kDa) nor the Ca(2+)-binding protein Osteocalcin (Osteo, 6kDa). By microinjection of fluorescently labeled versions of each of these molecules we were able to obtain visual evidence that, despite their lesser molecular weight, molecules with greater cross-sections were unable to transit these gap junctions, while heavier but elongate molecules could. While CaM had previously been shown to pass through gap junctions from oocytes to their surrounding epithelial cells, the ability of CaM and Trop-C to transit the gap junctions between adjacent epithelial cells had not been demonstrated. Evidence shown here demonstrates that the homologous gap junctions among epithelial cells, like the heterologous gap junctions between epithelial cells and the oocyte they surround, allow transit of elongate molecules up to at least 18kDa. Furthermore, the evidence for four different molecules of differing molecular weights and configurations supports the hypothesis that it is molecular configuration, not chemical activity, that primarily determines the observed permeability of gap junctions to molecules 5-6 times larger than the molecular weight limit previously acknowledged.     

Distribution of interstitial stem cells in Hydra

The distribution of interstitial stem cells along the Hydra body column was determined using a simplified cloning assay. The assay measures stem cells as clone-forming units (CFU) in aggregates of nitrogen mustard inactivated Hydra tissue. The concentration of stem cells in the gastric region was uniform at about 0.02 CFU/epithelial cell. In both the hypostome and basal disk the concentration was 20-fold lower. A decrease in the ratio of stem cells to committed nerve and nematocyte precursors was correlated with the decrease in stem cell concentration in both hypostome and basal disk. The ratio of stem cells to committed precursors is a sensitive indicator of the rate of self-renewal in the stem cell population. From the ratio it can be estimated that <10% of stem cells self-renew in the hypostome and basal disk compared to 60% in the gastric region. Thus, the results provide an explanation for the observed depletion of stem cells in these regions. The results also suggest that differentiation and self-renewal compete for the same stem cell population.     

Structure and distribution of gap junctions in lens epithelium and fiber cells

Summary We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a double mounting method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.