Morphology and quantitation of ciliated outgrowths from cultured rabbit tracheal explants

Ciliated outgrowths from cultured rabbit tracheal epithelium have been characterized with scanning and transmission electron microscopy and the ciliary frequencies measured. Outgrowth surface cells change in morphology from columnar to cuboidal to squamous shapes in their progression away from the explant. The ciliated cells retain the organization of their cilia in a cluster usually centrally on the apical cell surface. Closest to the explant the nonciliated surface of ciliated cells develops extensive microvilli. Ciliary frequencies are comparable to those observed in fresh tracheal epithelium with means of 50 cells per explant ranging from 11 to 23 beats per second. For most cultures examined no correlation exists between ciliary frequency and cell distance from the explant. The goblet cells loose their ability to synthesize the characteristic mucus granules and can only be identified by the absence of cilia. Surface cells are supported by an underlying layer of discontinuous cells and connective tissue fibers. The characteristics of an outgrowth suggest that development occurs through migration of differentiated cells from the explant rather than differentiation of cell types from migrating basal cells.     

The luminal surface of the rat trachea during ontogenesis]

To complete the hitherto existing results about the trachea epithelium, the scanning electron microscope was used to get representative statements about the apical surface of the epithelium: First of all the epithelium consists of undifferentiated round cells with cytopodia. The cells rarely carry single cilia at the same time. At the 18. day of pregnancy nearly all of the cells have a single cilium. Until the end of the intrauterine phase the single cilia are retracted again. At the 20. day ciliated cells and cells with protrusions are formed. Mucus granula are secreted into the lumen already before birth. Immediately post partum much mucus appears. Its distribution, viscosity and optical behaviour is different. For ciliated cells and goblet cells no special characteristical distribution was noticed. The building of the cells surface was detailed discussed. The question about function of the single cilia cannot yet be answered.     

Presence of a Ciliary Patch in Preoral Epithelium of Sea Urchin Plutei

Removal of the hyaline layer from sea urchin embryos at the pluteus stage discloses a densely ciliated region in the preoral area of the ectodermal epithelium. In four-armed plutei, this ciliary path is located between the anterolateral arms and in eight-armed plutei it becomes surrounded by preoral and anterolateral arms. The area of the patch and the number of cilia increase with age. This patch is covered by cilia of unusual morphology and orientation. There are more than two cilia per cell which are coiled together several times around a small cone at the apical end of the cell. These coiled cilia run parallel to the surface of the cell but do not extend beyond the hyaline layer. The ciliary axoneme consists of a "9+2" microtubular structure, but no outer or inner dynein arms are observed. Although the cells with coiled cilia are present in a cluster constituting a part of the epithelium, they have axons that project from their basal (inner) ends. The structural characteistics of the ciliary patch suggest that it possesses a sensory function.     

Ultrastructure of rabbit tracheal epithelium after saline lavage of the airways

In experiment the effect of saline lavage on the ultrastructure of the tracheal epithelium was investigated. Immediately after this procedure the trachea was lined with markedly altered pseudostratified columnar epithelium with remnants of the ciliary border. The intercellular spaces were markedly dilated, but the apical junctional complexes were left intact. 99% of the goblet cells were exhausted degenerated and were gradually expelled from the epithelium. Damage to the ciliated cells was not so generalized but some of them displayed signs of vacuolar degeneration. The ciliary border was severely impaired with only 1.5 kinocilia/microns 2, but the majority of remaining cilia were intact. Compared with controls there was only mild, but significant increase in the number of degenerating and malformed cilia. Morphological signs of impaired self-cleaning ability of the epithelium were not noticed.     

Ultrastructural study of the mucosa of the male gonoduct of Murex brandaris (Hexaplex brandaris) (Gastropoda,Prosobranchia)

Summary

The male gonoduct of Murex brandaris (Hexaplex brandarte) is covered by a columnar ciliated epithelium in which some goblet cells are located at the first level of the gonoduct. The structure of these cells suggests a transport and secretory epithelium. Some differences in ciliary organization and in cellular junctions are shown. The presence of some abnormal cilia is also detected.     

Cilia-lacking respiratory cells in ciliary aplasia

This report describes the ultrastructural alterations observed in the nasal and bronchial mucosa of an 11-yr-old male suffering from immotile cilia syndrome (ICS). The morphological features observed in this patient are consistent with a ciliary aplasia. In fact, ciliated cells appeared to be replaced by columnar cells lacking cilia and basal bodies, and bearing on their surface cilium-like projections without any internal axonemal structure. In spite of the absence of basal bodies, centrioles, and kinocilia, these cells unexpectedly showed mature striated roots and centriolar precursor material scattered throughout the apical cytoplasm. These data suggest that control over basal body assembly is distinct from control over striated root formation. The presence of the above-reported structures in cells otherwise presenting many morphological features of normal ciliated cells is discussed on the basis of current knowledge of respiratory cilia biogenesis.     

Development of the Nervous System of Sea Urchin Embryos: Formation of Ciliary Bands and the Appearance of Two Types of Ectoneural Cells in the Pluteus

An ultrastructural study of the larval integument of the sea urchin, Hemicentrotus pulcherrimus , was conducted with special emphasis on the development of the nervous system in relation to the formation of ciliary bands. In the integument of 4-armed pluteus larvae, cells associated with the ciliary band, which have 200 nm-thick projections at their apices, and cells in the squamous epithelium, which have a cilium and long, fine radiating processes in the apical region, were observed. Both cell types have axons at their basal ends that form nerve bundles beneath the ciliary bands, where the axons make contact with ectodermal effector cells with motile cilia. The cilia and other apical projections of these ectoneural cells run parallel to the surface of the cells, and are under the hyaline layer. The axoneme of the cilium has a typical "9 + 2" microtubular arrangement, but generally has no dynein arms. These ectoneural cells are more frequent on the oral surface than on the antioral surface.     

An ultrastructural study of primary cilia,abnormal cilia and ciliary knobs from the ciliated cells of the guinea-pig trachea

Summary Single primary cilia are found in developing as well as mature ciliated cells of guinea-pig tracheal epithelium. A few biciliated cells were observed, and in a rare case one cell had developed four such processes. Primary cilia are characterized by a 9 + 0 microtubular arrangement near the base, while a transition to an 8 + 1 pattern occurs at a slightly more distal position. Spokes are lacking, and dynein arms are absent or incompletely developed. The function, if any, of primary cilia remains unknown.In the population of the motile 9 + 2 cilia atypical forms are very rare, i.e. <0.1%. Of the various abnormalities cilia with supernumary microtubules are most common. Only one atypical basal body was observed. Although some of the aberrant forms undoubtedly are non-motile, their very low number suggests that they have no practical influence on the muco-ciliary clearance.Local extrusions of the ciliary membrane, here named ciliary knobs, are believed to be fixation artefacts. It is suggested that they represent circumscribed regions of the ciliary membrane which are sensitive to changes in the environmental osmotic pressure.     

Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

The ciliary apparatus of the lingual mucosa in the frog

By means of scanning and transmission microscopy it has been examined the ciliary system of the tongue mucosa. The scanning electronmicrographs of the fungiform papillae have revealed three ciliary apparatuses allocated respectively: at the papillary summit (corona ciliata and a narrow but separated paracoronal ciliary system) and on the peduncolar papillary stem. The cilia of both paracoronal and peduncolar groups have not been yet described. Also the filiform papillae are supplied with cilia but as irregularly distributed groups. The border of the tongue is a continuous and normal ciliary epithelium and finally groups of cilia are scattered also on the whole sublingual epithelium. At the transmission microscopy the cells of all the examined mucosal ciliary groups are showing a normal ultrastructural aspect.     

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity,vagina)

Summary Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.     

Histophysiological modifications of the amnion in the rat at the end of gestation]

Amniotic epithelium of the rat has been studied during the eight last days of pregnancy, by scanning electron microscope analysis. The cells of this organ progressively become larger and flatter. Changes on their surface have also been observed : density of microvilli increases but single cilia disappear. These changes are related to modifications in the exchange processes.     

Paddle cilia (discocilia) in chemosensitive structures of the gastropod mollusk Pleurobranchaea californica

Summary Scanning electron microscopy of various regions of the body of the marine gastropod Pleurobranchaea californica (McFarland) has revealed a characteristic cell type that bears cilia with dilated discoid-shaped tips. The tips of the cilia consist of an expansion of the ciliary membrane around a looped distal extension of the axoneme. These kinocilia have been observed in numerous other marine invertebrates and are generally referred to as paddle cilia (Tamarin et al. 1974) or discocilia (Heimler 1978). Although many functions have been proposed for paddle cilia, little empirical evidence supports any of the proposals. In Pleurobranchaea we have found that the distribution of this ciliated cell type corresponds exactly to areas of the body known from behavioral studies (Lee et al. 1974; Davis and Matera 1981) to mediate chemoreception. Transmission electron microscopy of the epithelium lining the rhinophores and tentacles of Pleurobranchaea revealed details of the ultrastructure of these ciliated cells and showed that they are primary receptors. These ciliated receptors lie in a yellow-brown pseudostratified columnar epithelium that superficially resembles the olfactory mucosa of vertebrates.     

Ultrastructural investigation on the cell membranes of the vomeronasal organ in the rat: A freeze-etching study

Summary The free surfaces and cell contacts in the epithelia of the vomeronasal organ of the rat were investigated by freeze-etching. The microvilli of receptor cells show a lower density of intramembranous particles (IMP) than the microvilli in the receptor-free epithelium. The ratio between the IMP on P and E-face is approximately 111 in the receptor terminals, and 3.51 in the cilia and microvilli of the receptor-free epithelium. Although atypical in length and only poorly equipped with rootlet fibers, the cilia of the receptor-free epithelium are furnished with typical ciliary necklace structures of up to 10 rows of membrane particles. Differences in the density of IMP on the P-faces of different cilia are probably due to continual ciliogenesis and also due to the different age of cilia in the receptor-free epithelium. Zonulae occludentes show different configurations in the neuroepithelium and in the receptor-free epithelium. In the former, they show a tendency to cross-link and form facet-like patterns, reflecting a constant morphology and relative stability for this apical region. In the receptor-free epithelium the junctional rows of zonulae occludentes display only loosely interconnected networks and a tendency to orient parallel to each other and to the free surface. In addition to zonulae occludentes, typical square aggregations of IMP are observed in the receptor-free epithelium. They are not exclusively restricted to the zone of intensive cell contacts by means of fine interdigitating cell processes, and their function has yet to be identified experimentally.This paper is dedicated to Dr. David G. MoultonPortions of this work are from a thesis in preparation by F.M. Supported by the Deutsche Forschungsgemeinschaft (SFB 114)     

Electron microscopic study of the intestinal epithelium of Saccoglossus mereschkowskii (Enteropneusta, Hemichordata)

Epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata) standing at the base of Chordata, has been investigated using electron microscope. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions have been characterized. The data concerning details of the organization of the ciliary apparatus and rootlets system are presented. It is justified the presence of complicated supporting construction of cilia which performs a mechanical stabilizing function and possibly also provide synchronization of ciliary movements. The presence of cilia with two centrioles is considered as an adaptation to high functional load on ciliary apparatus. Well developed bundles of myofilaments are found in the cytoplasm of the basal portions of ciliary cells that characterizes these cells as myoepithelial. The features indicating the role of ciliary cells in absorption are described. The capability of these cells to balloon-like secretion is considered. Data on the accumulation of food reserves in the form of lipid droplets and glycogen in the cell cytoplasm are presented. Ciliated cells are characterized by their function as ciliated secretory-absorptive myoepithelial cells. Based on the location of secretory granules both in the apical and basal portions of granular cells, an exocrine-endocrine function of these cells has been suggested. Typical endocrine cells in the intestinal epithelium of S. mereschkowskii are absent. Several types of granules in the nerve fibers cytoplasm are described. Junctions between the nerve fibers and basal portions of ciliary and granular epithelial cells are found. Nerve regulation of contractile and secretory functions of epithelial cells is supposed. The presence of the regulatory nerve-endocrine system that includes receptor cells of open type, secretory endocrine-like cells and nerve elements of nerve layer is supposed in the intestinal epithelium of enteropneusts.     

Differentiation of the müllerian (paramesonephric) duct epithelium in the rat

Differentiation of the Müllerian duct epithelium was studied in 15- to 21-day female rat foetuses. The proximal segment of the Müllerian duct is formed by the 15th day; it runs parallel to the Wolffian duct and the two are wrapped in a common basement membrane. On the 16th day the genital ducts are clearly separate; the Müllerian duct has a slit-like lumen and is lined with simple columnar epithelium. Throughout the whole of the given period the epithelium retains a relatively indifferent appearance. Characteristic findings from the 18th day include the apical migration of centrioles and the formation of solitary cilia.     

Immunocytochemistry of the striated rootlets associated with solitary cilia in human oviductal secretory cells

The human oviduct epithelium is a simple columnar structure that consists primarily of ciliated and secretory cells. Solitary cilia usually extend from the apical cell surface of secretory cells. By injecting crude preparations of striated rootlets into rats, we successfully obtained six monoclonal antibodies (R38, R67, R95, R149, R155, R213) that commonly labeled ciliary rootlets. Using these antibodies, proteins of 205-215 kDa were identified by immunoblotting. Using a clone, R67, we investigated the morphology of the striated rootlets associated with solitary cilia by immunocytochemistry. It was found that the shapes of the rootlets were not simple but varied considerably. The rootlets had branched, radiated, arched, and looped shapes. This is the first report of the rootlets having a variety of shapes. The 205- to 215-kDa antigens identified by the six different antibodies were mostly localized to dark bands of striations, suggesting that they are constitutive components of dark striations of the rootlet.     

Localization of the angiopoietin receptors Tie-1 and Tie-2 on the primary cilia in the female reproductive organs

Blood vessel homeostasis and endothelial cell survival depend on proper signalling through angiopoietin receptors such as the receptor tyrosine kinases Tie-1 and Tie-2. We have studied the presence and subcellular localization of these receptors in murine female reproductive organs using confocal microscopy analysis of antibody stained tissue sections of ovary and oviduct. We show that Tie-2 principally localizes to primary cilia of the surface epithelium of the ovary, bursa and extra-ovarian rete ducts as well as to plasma membranes of ovarian theca and endothelial cells. Primary cilia of follicular granulosa cells were negative. Further, Tie-1 and Tie-2 localized to motile cilia of the oviduct. Western blotting detection and immunolocalization of anti-Tie-2 in ovary and oviduct were abolished by administration of an anti-Tie-2 blocking peptide, confirming antibody specificity. In a series of immunohistochemical analysis on human ovarian tissues we also observed a unique localization of Tie-2 to the primary cilia of ovarian surface epithelium. These observations are the first to show ciliary localization of angiopoietin receptors. Our results support the hypothesis that cilia of the female reproductive organs play a novel and important sensory role in relaying physiochemical changes from the extracellular environment to epithelial cells of the oviduct, the ovary and extra-ovarian tissues.     

Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.     

Electron microscopic study of intestinal epithelium of <Emphasis Type="Italic">Saccoglossus mereschkowskii</Emphasis> (Enteropneusta,Hemichordata)

The epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata), a group located at the base of Chordata, has been studied by using electron microscopy. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions are characterized. The data on the details of the structure of the ciliary apparatus and the system of ciliary rootlets are presented. Justification is provided for the presence of a complicated construction in the ciliated cells, a supportive carcass of cilia that performs a mechanical stabilizing function, and possibly the synchronization of the ciliary movement. The existence of cilia with two centrioles is considered as adaptation to the high functional load on the ciliary apparatus. Well-developed bundles of myofilaments have been revealed in the cytoplasm of the basal parts of ciliated cells, which characterizes these cells as epitheliomuscular. Peculiarities indicating the role of ciliated cells in absorption are described, as well as the capability of these cells for balloon-like secretion. Data are presented on the accumulation of reserved nutritional substances in the cell cytoplasm in the form of lipids and glycogen. With respect to their function, ciliated cells are determined as the ciliated secretory-absorptive epitheliomuscular cells. The location of secretory granules in both apical and basal parts of granular cells indicates the exocrine-endocrine function of these cells. There are no typical endocrine cells in the intestinal epithelium of S. mereschkowskii. Several types of granules are described in the cytoplasm of nerve fibers. Junctions between nerve fibers and basal parts of ciliated and granular epithelial cells have been revealed; the neural regulation of the contractile and secretory functions of epithelial cells is assumed. The intestinal epithelium of enteropneusts is presumed to contain a regulatory neuroendocrine system composed of receptor cells of the open type, secretory endocrine-like cells, and of nerve elements of the nervous layer.