The orientation of ciliary basal bodies in quail oviduct is related to the ciliary beating cycle commencement

In mature ciliated cells, the basal feet associated to the basal bodies point out in the direction of the effective stroke of the ciliary beating. In contrast, during ciliogenesis, the basal feet of the newly anchored basal bodies are randomly oriented. The reorientation of basal bodies occurs during the beginning of the coordinated beating cycle of the cilia.     

In vitro effects of benzodiazepines on ciliogenesis in the quail oviduct

Immature oviduct implants from quails stimulated by estrogen to induce ciliogenesis were submitted to the in vitro action of benzodiazepines in organotypic culture. Diazepam and medazepam were added to the culture medium for 24 or 48 hours and tissues were examined by transmission and scanning electron microscopy for alterations in ciliary differentiation. Ciliogenesis was inhibited by both diazepam and medazepam, which affected mainly the migration of the basal bodies. Assembly of basal bodies was achieved normally in the cytoplasm, but their separation from generative complexes and migration toward the apical membrane were prevented. They remained in clusters around a deuterosome or eventually anchored to the close lateral plasma membrane. Furthermore, the drugs affected mature beating cilia, which then appeared lying tangentially to the cell surface. Relation between basal bodies and cortical cytoskeleton seemed to be altered by the drugs, which implies that the bearing of cilia and probably the ciliary beating movement were modified. Microvillus development was also altered by the action of these drugs.     

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.     

Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

"Light" epithelial cells of swine and bovine oviducts

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Scanning electron microscopy of the equine oviduct and observations on ciliary currents in vitro at day 2 after ovulation

There are considerable differences between mammalian species in the distribution and activity of ciliated cells within the oviduct, and limited information is available concerning either the distribution or activity of cilia within the equine oviduct. Patterns of ciliary activity were characterized in the ampulla and isthmus of oviducts recovered at 2 d after ovulation from 10 mares, and scanning electron microscopy was used to examine regional differences in the distribution of cilia in oviducts from 3 of these mares. Based upon the motility of 15 microm latex microspheres, ciliary activity was significantly (P < 0.001) greater in the ampullar oviduct compared with that of the isthmic oviduct. The direction of ciliary beat was consistently toward the uterus in all regions of the oviduct. Scanning electron microscopy revealed ciliated and secretory cells in both regions of the oviduct at 2 d after ovulation, with no apparent differences in the proportion of ciliated versus secretory cells.     

Localization of gamma-tubulin to the basal foot associated with the basal body extending a cilium

The human oviduct epithelium primarily consists of ciliated cells and secretory cells. Solitary cilia usually extend from the apical surface of the secretory cells. We investigated the localization of -tubulin in the ciliary basal apparatus of both cell types by fluorescence immunohistochemistry and immunoelectron microscopy. In addition to basal bodies, -tubulin was identified in the lateral basal foot, especially the basal foot cap. This observation is consistent with previous observations that microtubules radiate from the basal foot and the basal foot serves as the microtubule organizing centre.     

The effect of hydroxyurea on ciliogenesis in ciliary epithelium of the mollusk Lymnaea stagnalis

Electron-microscopy study of the ciliary epithelium structure of the mollusk Lymnaea stagnalis was carried out under the action of hydroxyurea. By the method of radioautography, a high proliferative activity of the ciliary epithelium was established as the norm; a cluster distribution of cells, including the label, was noted. The presence of hydroxyurea in the mollusk organism was shown to inhibit proliferation. Scanning electron microscopy of the molluskan foot surface revealed clusters of nonciliated cells and of cells with short villi in control epithelial folds. Under hydroxyurea treatment for 24 h, such sites disappeared completely and ciliary epithelium looked uniform and was composed of cells with long cilia. By transmission electron microscopy, it was established that hydroxyurea did not affect the formation of the basal body and course of ciliogenesis. It has been suggested that hydroxyurea not only inhibits proliferative activity of epithelial cells, but also induces differentiation of unciliated into the ciliated cells.     

Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.     

The ciliary basal apparatus is adapted to the structure and mechanics of the epithelium

The basal apparatuses which anchor the gill cilia in Branchiostoma lanceolatum (Pallas) and the actinopharynx cilia in Calliactis parasitica (Couch) are similar in structure. In C. parasitica the pharynx epithelium and the basal apparatuses are flexible. The basal apparatuses, however, bend in only one direction. This mechanism may permit epithelial flexibility whilst maintaining a similar basal orientation between cilia. In B. lanceolatum the ciliated gill epithelia are mechanically stable but the epithelial surfaces are curved. The basal apparatuses may correct for this curvature, with short rootlets between the distal centrioles (basal bodies) and the cell membranes, so that their cilia also share a common orientation. A common basal orientation between cilia is important for their coordination. The degree of coordination depends upon the function of the cilia; water-propelling cilia are more precisely coordinated than mucus-propelling cilia. Much of the structural diversity of ciliary basal apparatuses in Metazoa may be due to variation in the demands of anchoring functionally different cilia to epithelia which have different structural and mechanical properties.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Relationships between cytokeratin filaments and centriolar derivatives during ciliogenesis in the quail oviduct

In the quail oviduct, the mature ciliated cells contain a well developed and polarized cytokeratin network which is bound to desmosomes and in close contact with the striated rootlets associated with basal bodies. In ovariectomized quail, the immature epithelial cells of oviduct present a rudimentary cytokeratin network associated with the centrioles of the diplosome (one of them forming a primary cilium) and with the short striated rootlets. The development of the cytokeratin network which occurs simultaneously with the ciliogenesis was observed by electron microscopy and immunocytochemistry (immunofluorescence and immunogold staining) using a prekeratin antiserum. During estrogen-induced ciliogenesis, cytokeratin intermediate filaments are always found associated with the different ciliogenic structures i.e. [dense granules, deuterosomes, procentrioles and centrioles]. In ciliogenic cells, the procentrioles and centrioles seem to be associated with the intermediate filaments by their pericentriolar material. These direct contacts decrease once the centrioles/basal bodies are anchored to the plasma membrane. Simultaneously the striated rootlets develop and associate with cytokeratin. The ciliogenic cells appear as a suitable system for studying in vivo, the possible association between centrioles and intermediate filaments and its functional meaning.     

In vivo ciliogenesis in human fetal tracheal epithelium

Development of ciliated cells (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18-19 weeks (stage II), centriologenesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22-23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P less than .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.     

Ultrastructural study of the ciliated cyst in the human uterine tube epithelium

Ciliated cysts in the human uterine tube epithelium were investigated with the transmission electron microscope. The cysts were about 3-9 microns in diameter and were provided with many ciliary apparatuses and microvilli. Degenerative changes of these cilia, such as electron-dense round or irregular bodies and amorphous substance, were observed in many cysts, but complete disappearance of ciliary structures was not detected in any ciliated cysts. The ciliated cysts were mostly observed in basal cells and were occasionally found in ciliated cells bordering the tubal lumen. In the basal cells, these cysts distended with the increase in degenerated cilia. Distended ciliated-cyst-containing cells became exposed directly to the tubal lumen. U- or reverse omega-shaped deep indentations of the apical surface of ciliated cells confirmed the opening of ciliated cysts into the lumen. It was suggested that the ciliated cysts result from the premature differentiation of basal cells or disturbed migration of centrioles in ciliogenic cells.     

Evolutionary conservation of an epitope associated with striated rootlets in different epithelial ciliated cells.

In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells, to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct.     

[Ciliogenesis in the mucous cells of the quail oviduct. I. Ultrastructural study in the laying quail]

The luminal epithelium of the oviduct (magnum) of laying quails is composed of ciliated cells and mucous cells. Ciliogenesis was observed in some of the mucous cells. Both centrioles of the diplosome migrate to the top of the cell, and one of them induces the formation of a rudimentary cilium. In some of the other cells, that are filled with mucous granules, the formation of basal bodies by an acentriolar pathway was observed. In these cells, numerous, dense fibrous masses are associated with the forming face of the Golgi apparatus. In the Golgi zone, generative complexes composed of a deuterosome and some forming procentrioles were found. Cilia develop from completed basal bodies. During ciliogenesis, the Golgi apparatus is disorganized, and generally the production of mucous granules is arrested. The nucleus is also modified: it becomes larger and the chromatin is dispersed. It is assumed that mucous cells are able to be transformed into ciliated cells in the oviduct of laying quails.     

In vitro effects of colchicine and nocodazole on ciliogenesis in quail oviduct

Oviduct implants from quails which were primarily stimulated in vivo by estrogen so as to induce ciliogenesis in some epithelial cells were cultured in vitro in the presence or absence of colchicine or nocodazole. After 24 or 48 hr of culture, implants were examined by transmission and scanning electron microscopy to determine drug-induced alterations in ciliogenesis. After 24 hr of 10(-5) M colchicine treatment, the formation of basal bodies was totally inhibited, though the precursor material of generative complexes was unchanged. The inhibitory effect was not reversed when colchicine was removed in a 24 hr recovery culture. Treatment with 10(-6) M nocodazole for 24 hr, partially inhibited the assembly of basal bodies, which exhibited altered morphology. The assembly of basal bodies was restored during the 24 hr recovery period, after removal of nocodazole. Colchicine and nocodazole did not prevent polarized migration towards the apical surface of basal bodies formed prior to drug treatment. They anchored to the plasma membrane, but the formation of cilia was strongly disturbed in the presence of the drug. Numerous cells possessed anchored basal bodies which failed to induce the formation of cilia. The elongation of cilia was inhibited, as seen by their abnormal capping structure. In the enlarged tip, microtubules diverged. In contrast, these very short cilia possessed a mature ciliary necklace which was constructed during drug treatment. Differentiation of this membrane ciliary structure appeared to be unrelated to axoneme growth.     

Patterns of basal body addition in ciliary rows in Tetrahymena

Most naked basal bodies visualized in protargol stains on the surface of Tetrahymena are new basal bodies which have not yet developed cilia. The rarity of short cilia is explained by the rapid development of the ciliary shaft once it begins to grow. The high frequency of naked basal bodies (about 50 percent) in log cultures indicates that the interval between assembly of the basal body and the initiation of the cilium is long, approximately a full cell cycle. Naked basal bodies are more frequent in the mid and posterior parts of the cell and two or more naked basal bodies may be associated with one ciliated basal body in these regions. Daughter cells produced at division are apparently asymmetric with respect to their endowment of new and old organelles.