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.     

Centriolar satellites: molecular characterization, ATP-dependent movement toward centrioles and possible involvement in ciliogenesis

We identified Xenopus pericentriolar material-1 (PCM-1), which had been reported to constitute pericentriolar material, cloned its cDNA, and generated a specific pAb against this molecule. Immunolabeling revealed that PCM-1 was not a pericentriolar material protein, but a specific component of centriolar satellites, morphologically characterized as electron-dense granules, approximately 70-100 nm in diameter, scattered around centrosomes. Using a GFP fusion protein with PCM-1, we found that PCM-1-containing centriolar satellites moved along microtubules toward their minus ends, i.e., toward centrosomes, in live cells, as well as in vitro reconstituted asters. These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization. Next, to understand the relationship between centriolar satellites and centriolar replication, we examined the expression and subcellular localization of PCM-1 in ciliated epithelial cells during ciliogenesis. When ciliogenesis was induced in mouse nasal respiratory epithelial cells, PCM-1 immunofluorescence was markedly elevated at the apical cytoplasm. At the electron microscopic level, anti-PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis. These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.     

[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.     

A possible cause of termination of cell growth in the cellular slime mold Dictyostelium discoideum.

The gill ctenidium growth tip of the lamellibranch mollusc Aequipecten irradians recapitulates the temporal development of ciliated gill filaments and related structures in a spatial fashion. This “meristematic” relationship has allowed a study of basal body formation and ciliogenesis in adjacent cells of gill filament papillae at stages of progressively more advanced relative development. Basal bodies appear to originate quite rapidly, subsequent to the appearance of a complex of dense granules, quite reminiscent of the “condensation forms” or “procentriole precursors” typically seen in vertebrate ciliogenesis. Unlike basal body generation in higher forms, that in Aequipecten shows no obvious organized intermediate stages. During ciliation, randomly-oriented, nearly complete procentrioles are found concomitantly with actively-functioning basal bodies. Cilia formation in more advanced, already-ciliated cells is again preceded by the presence of granular complexes. Ciliogenesis in this mollusc thus shares with certain lower forms the property of very rapid basal body formation but, like many higher forms, it is preceded by the formation of a granular precursor complex, presumably consisting of particulate microtubule protein.     

THE FORMATION OF BASAL BODIES (CENTRIOLES) IN THE RHESUS MONKEY OVIDUCT

Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mµ long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.     

Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women

Oviducts were obtained from women who elected to undergo sterilization either during a normal menstrual cycle, after the first trimester of pregnancy, or in the puerperium. The percent of ciliated cells, cell height and morphology of the fimbria and ampulla were determined and correlated with the stage of the reporductive cycle and plasma levels of the ovarian steroids. Mature ciliated and secretory cells were observed only at mid-cycle. Atrophy, deciliation and loss of secretory activity coincided with elevated levels of serum progesterone. These degenerative processes continued during pregnancy. Ciliation, hypertrophy, and restoration of secretory activity occurred when serum progesterone was essentially undetectable and estradiol relatively low. During each menstrual cycle the secretory cells were observed to undergo a complete cycle of dedifferentiation-differentiation, whereas 10--12% of the ciliated cells lost and regenerated their celia. Ciliogenic cells were frequently present in the epithelium obtained from women in the mid-follicular phase. Fibrous granules, deuterosomes, procentrioles and ciliary buds were observed in the apex of these cells. Plasma levels of estradiol were higher during periods of atrophy and deciliation than they were during periods of hypertrophy and reciliation. It appears that the serum levels of estradiol were adequate to maintain a mature epithelium at all the reproductive stages included in this study. However, progesterone, when present, blocked the growth-promoting effect of estradiol in the oviduct.     

Ultrastructure of the differentiating zygotospores of Porphyra leucosticta (Rhodophyta)

The ultrastructure of zygotosporogenesis is described for the red alga Porphyra leucosticta Thuret. Packets of eight zygotosporangia, each packet derived from a single carpogonium are interspersed among vegetative cells. Zygotospore differentiation in Porphyra can be separated into three developmental stages. (i) Young zygotospores exhibit a nucleus and a large centrally located, lobed plastid with pyrenoid. Mucilage is produced within concentric membrane structures during their dilation, thus resulting in the formation of mucilage sacs. Subsequently, these sacs release their contents, initiating the zygotospore wall formation. Straight‐profiled dictyosomes produce vesicles that also provide wall material. During the later stages of young zygotospores, starch polymerization commences, (ii) Medium‐aged zygotospores are characterized by the presence of fibrous vacuoles. These are formed from the ‘fibrous vacuole associated organelles’. The fibrous vacuoles finally discharge their contents. (iii) Mature zygotospores are recognized by the presence of numerous cored vesicles produced by dictyosomes. Cored vesicles either discharge their contents or are incorporated into the fibrous vacuoles. There is a gradual reduction of starch granules during zygotospore differentiation. Mature zygotospores are surrounded by a fibrous wall, have a large chloroplast with pyrenoid and well‐depicted phycobilisomes but are devoid of starch granules.     

Light microscopic and ultrastructural study of the development of the saccus vasculosus in the rainbow trout,Oncorhynchus mykiss

This study using light and electron microscopy indicates that the saccus vasculosus is distinguishable in 9-mm embryos and grows continuously throughout embryonic development to the adult stage. In the saccus vasculosus, epithelial mitoses are observed in all stages studied. Phases of centriologenesis, ciliogenesis, and globule formation have been characterized in developing coronet cells. During the phase of centriologenesis, new centrioles appear in association with pre-existing centrioles and not on deuterosomes. After ciliogenesis, each cilium differentiates to a globule almost at the same time as the other cilia of the coronet cell. The inner membrane system of the globules seems to derive from the ciliary plasma membrane. This membrane system often produces membrane whorls during the development. The different phases of coronet cell development have been found in the same individual and in all the stages studied except the 9-mm embryo. Cerebrospinal fluid-contacting neurons are observed in the saccus epithelium from the 12-mm embryos on and are distinguishable from coronet cells in their early formative stages. The three cell types of the saccus vasculosus increase continuously in number during development. Nerve processes are found in the saccus vasculosus of embryos, whereas differentiated synapses appear later in the fry. The significance of continued coronet cell formation is discussed in relation to a putative coronet cell and/or a globule renewal cycle in the adult.     

An ultrastructural study of morphogenesis of fibrogranular complex and centriole in ductuli efferentes of Chinese hamster.

An ultrastructural study of ciliated epithelial cells in the ductuli efferentes of young and adult hamsters has revealed that these cells possess dense granules, dense granule clusters, dense bodies and fibrogranular complexes as reservoirs or precursors for ciliogenesis. The dense granules are first seen in the centrosomal region. Later, many dense granules and dense granule clusters appear in the apical portion of the epithelial cells where, subsequently, dense bodies are also found. Finally, the fibrogranular complexes are formed in adults. Morphological evidence strongly suggests that cilia are formed from diplosomal centrioles, de novo centrioles, dense body centrioles, and fibrogranular complex centrioles. Ciliogenesis begins in the fourth day after birth and increases rapidly in the fifth day. After the sixth day, cilia appear to be generated mostly from dense bodies and the total ciliogenesis activities gradually decrease as the animal ages.     

[Ciliogenesis in the mucous cells of the quail oviduct. II. Hormonal control]

The hormonal control of ciliogenesis and transformation of mucous cells was studied in the oviduct (magnum) of ovariectomized quails. Estradiol benzoate induces ciliogenesis with doses varying from 10 mug/day to 100 mug/day after 6 days of treatment. With 100 mug/day, differentiation of some mucous cells is also induced as well as the formation of transitory "mixed cells" which are in the process of ciliogenesis and contain mucous granules. Associated with progesterone (1 mg/day), estradiol benzoate (10 mug/day) induces the differentiation of mucous cells and ciliated cells. The luminal epithelium of quails injected with this mixture is similar to the luminal epithelium observed in the oviduct of laying quails. With the same dose of progesterone (1 mg/day) and 20 mug/day of estradiol benzoate for 6 days, ciliogenesis is completely inhibited. All epithelial cells are secretory cells. Transformation of 50% of the mucous cells into ciliated cells is obtained by following the previous estradiol-progesterone treatment with the injection of estradiol benzoate (20 mug/day) for 3 days. Divisions of mucous cells were also observed. It is also possible to induce ciliogenesis in some mucous cells by withdrawing both hormones for 3 days. In this case, no cell divisions were observed.     

Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

Ultrastructure of the ovary and oogenesis in six species of patellid limpets (Gastropoda: Patellogastropoda) from South Africa

Abstract. The ultrastructural features of the ovary and oogenesis have been described in 6 species of patellid limpets from South Africa. The ovary is a complex organ that is divided radially into numerous compartments or lacunae by plate-like, blind-ended, hollow trabeculae that extend from the outer wall of the ovary to its central lumen. Trabeculae are composed of outer epithelial cells, intermittent smooth muscle bands, and extensive connective tissue. Oocytes arise within the walls of the trabeculae and progressively bulge outward into the ovarian lumen during growth while partially surrounded by squamous follicle cells. During early vitellogenesis, the follicle cells lift from the surface of the underlying oocytes and microvilli appear in the perivitelline space. Follicle cells restrict their contact with the oocytes to digitate foot processes that form desmosomes with the oolamina. When vitellogenesis is initiated, the trabecular epithelial cells hypertrophy and become proteosynthetically active. Yolk synthesis involves the direct incorporation of extraoocytic precursors from the lumen of the trabeculae (hemocoel) into yolk granules via receptor-mediated endocytosis. Lipid droplets arise de novo and Golgi complexes synthesize cortical granules that form a thin band beneath the oolamina. A fibrous jelly coat forms between the vitelline envelope and the overlying follicle cells in all species.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.     

CENTRIOLE MORPHOGENESIS IN DEVELOPING CILIATED EPITHELIUM OF THE MOUSE OVIDUCT

The differentiating mouse oviduct has been used for the study of centriole morphogenesis because its epithelium is extensively ciliated and centriole formation occurs in a brief period after birth. Proliferative elements, consisting of an extensive fibrillar meshwork encrusted with 75 mµ granules, were encountered at all ages, but were the only centriole precursors present in younger animals (2–3 days). These large aggregates were found either physically associated with a mature centriole or alone, but never associated with procentrioles. It is likely, therefore, that although proliferative elements may be derived from preexisting centrioles, they do not directly produce new centrioles. An intermediate structure, the condensation form, found primarily in older animals (4–6 days), and produced by the packing of the proliferative element material, gives rise to daughter procentrioles. This association of procentriole and condensation form has been called a generative complex. Condensation forms undergo various stages of depletion, producing hollow spheres with thin walls or small osmiophilic aggregates as procentrioles grow in length and assemble their microtubules. From these observations it is concluded that synthesis of microtubular precursor protein is mediated by the mature centriole and that this protein is packaged into many condensation forms in order to allow the rapid assembly of a large number of centrioles in a brief period of time.     

Genesis of connective matrix of Veretillum cynomorium Pall. (Cnidaria-Anthozoa). Ultrastructural and autoradiographic study (author's transl)]

Ultrastructural study of the tissues of Veretillum cynomorium shows the presence of two mesenchymatous cellular states in the mesoglea: the nongranular mesenchymatous cells and the granular mesenchymatous cells. These latter possess, besides their cytoplasmic granules, some homogeneous fibrous inclusions, very similar to the fibrous material of the mesoglea. Granules and homogeneous fibrous inclusions are also present in the cytoplasm of some ectodermic and endodermic cells. These morphological results lead us to consider that mesoglea and epithelia can be occupied by the same granular cell type. Besides this, the digestive endodermic cells are sometimes very rich in heterogeneous fibrous inclusions histochemically identified as phagosomes. An autoradiographic study indicates two possible pathways for the synthesis of the mesoglea. The first involves the endoderm which elaborates the mesoglea at a fast rate but in small amounts. The second is due to the granular cells (mesenchymatous and epithelial) which show a slow rate of synthesis leading to the formation of the homogeneous fibrous inclusions. The heterogeneous fibrous inclusions of the digestive endodermic cell support the hypothesis of the involvement of these cells in mesogleal degradation.     

Structure of the apical cells of the testis of the tenebrionid beetles: Tenebrio molitor and Zophobas rugipes

The developmental cytology of the apical tissue of the testis of Tenebrio molitor and Zophobas rugipes was studied with light and electron microscopy. In the early larvae of both species the tisue was found to be a thickened protrusion of nongerminal cells appearing at the apical end of each testis follicle following gonadal differentiation. The cells persist through pupal and adult stages in both species, becoming more prominent at these stages in Z. rugipes, despite tracheal invasion in both species. In older adults the apical tissue regresses and ultimately distintegrates. Ultrastructurally the apical cells are distinguished from adjacent germinal cells by their (1) small, rounded or oval nuclei, (2) highly convoluted plasma membrane, (3) electron-opaque cytoplasm, (4) profuse concentrically-stacked, granular endoplasmic reticulum, (5) large aggregates of glycogen-like granules, (6) numerous small, tubular mitochondria, (7) well-developed Golgi centers and (8) striking arrays of microtubules. These cells have many cytological features in common with the androgenic gland cells of crustaceans, but not with the steroidogenic cells of vertebrates. Evidence for the formation of protein granules is also lacking. As yet, experimental procedures have not indicated an endocrine function for these cells in tenebrionids. However, their cytology is consistent with secretory activity of some kind.     

Development of the taste buds in rat embryogenesis

Electron microscopic studies have been made on the developing taste buds in fungiform and vallate papillae of prenatal rats. Three stages of differentiation of these buds are described. The first stage is characterized by presence of the nervous fibers in the connective tissue of the papillae and dense granules of various size, as well as dense-cored vesicles (500-700 A in diameter) in the basal parts of some epithelial cells at the top of the papillae (16-17th days of gestation). The second stage is characterized by nerve processes entering the epithelium and by formation of afferent synaptic contacts between the differentiating epithelial cells and the nervous fibers (19th day of gestation). At the third stage, the cluster of differentiating epithelial cells attains a form which is similar to mature taste buds (21-22nd days of gestation). Thus, to the birthday of rats, differentiation of the basal parts of the taste buds takes place, whereas the apical parts of the taste buds remain undeveloped and do not communicate with the oral cavity. Peculiarities of fine structure of differentiating epithelial cells at the three stages are discussed.     

EFFECT OF ACTINOMYCIN D ON THE DIFFERENTIATION OF HATCHING GLAND CELL AND CILIA CELL IN THE FROG EMBRYO

The forehead epidermis of the stage 18–20 R. japonica embryo includes the hatching gland cell (HGC) which contains cell-specific secretory granules. The cilia cell (CC) and common epidermal cell (CEC) constitute the epidermis of the entire body surface, in addition to the forehead region.
Culture of superficial epidermal explants from various embryonic portions at various developmental stages revealed that HGCs are derived from cells localized on the neural crest in the stage 13a (early neural plate) embryo. When explants from the presumptive HGC area were treated with 1 ug/ml actinomycin D (AMD), the formation of secretory granules in HGCs was inhibited either by continuous treatment from stage 13 or by an 8-hr treatment at stage 13b. Similarly, the ciliogenesis in CCs was inhibited. The differentiation of CECs was entirely unaffected by any of the AMD treatment. After release from AMD, mucous vesicles, characteristic of the CEC, were formed in cells whose differentiation into HGC and CC had been suppressed by the antibiotic. Thread complexes and clumps of coiled strings were found in the nuclei of AMD-affected cells.
It is concluded that the DNA-dependent RNA syntheses which direct secretory granule formation in the HGC and ciliogenesis in the CC occur during a limited period at stage 13b, viz. , 20 hr before their cytodifferentiation becomes appreciable.