Identification of TEKTIN1-expressing multiciliated cells during spontaneous differentiation of non-human primate embryonic stem cells.

Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.

Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain.

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENT Immature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.

Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia.

Visualization of signal transduction in live primary cilia constitutes a technical challenge owing to the organelle's submicrometer dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia, we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, high sensitivity and wide dynamic range.

TRPV4 regulates the integrity of the blood-cerebrospinal fluid barrier and modulates transepithelial protein transport.

The diffusion of materials from systemic circulation to the central nervous system (CNS) is restricted by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). Choroid plexus epithelial cells (CPECs) of the brain ventricles constitute the BCSFB and regulate the infiltration of plasma proteins as well as immune cells into the interstitium of the CNS. The barrier function is altered in pathologic conditions. However, the regulatory mechanism of BCSFB is not fully understood. Here, we investigated the function of transient receptor potential vanilloid 4 (TRPV4), a polymodally gated divalent cation channel that is highly expressed in CPECs. TRPV4 was localized broadly on the apical membrane in swine CPECs, in contrast with an intense ciliary localization found on other cell types. Treatment with the TRPV4-specific agonist, GSK1016790A (GSK; EC50 34 nM), induced a robust calcium influx and an immediate serine/threonine protein phosphorylation. The agonist treatment induced a marked decrease in the amount of filamentous actin and disintegrated the cell junctions in 10-20 minutes. In contrast, inhibition of the basal TRPV4 activity with the TRPV4-specific antagonist, HC067047 (HC; IC50 74 nM), reduced the basolateral-to-apical transport of α-2-macroglobulin (A2M). Overall, this study demonstrated a novel physiologic function of TRPV4 in the regulation of BCSFB permeability.

Structure and function of vertebrate cilia, towards a new taxonomy.

In this review, we propose a new classification of vertebrate cilia/flagella and discuss the evolution and prototype of cilia. Cilia/flagella are evolutionarily well-conserved membranous organelles in eukaryotes and serve a variety of functions, including motility and sensation. Vertebrate cilia have been traditionally classified into conventional motile cilia and sensory primary cilia. However, an avalanche of emerging evidence on the variations of cilia has made it almost impossible to classify them in a simple dichotomic manner. For example, conventional motile cilia are also involved in the sensation of bitter taste to facilitate the beating of cilia as a defense system of the respiratory system. On the other hand, the primary cilium, often regarded as a non-motile sensory organelle, has been revealed to be motile in vertebrate embryonic nodes, where they play a crucial role in the determination of left-right asymmetry of the body. Moreover, choroid plexus epithelial cells in the cerebral ventricular system exhibit multiple primary cilia on a single cell. Considering these lines of evidence on the diversity of cilia, we believe the classification of cilia should be based on their structure and function, and include more detailed criteria. Another intriguing issue is how in the evolution of cilia, their function and morphology are combined. For example, has motility been acquired from originally sensory cilia, or vice versa? Alternatively, were they originally hybrid in nature? These questions are inseparable from the classification of cilia per se. We would like to address these conundrums in this review article, principally from the standpoint of differentiation of the animal cell.

Multiple primary cilia modulate the fluid transcytosis in choroid plexus epithelium.

Functional defects in cilia are associated with various human diseases including congenital hydrocephalus. Previous studies suggested that defects in cilia not only disrupt the flow of cerebrospinal fluid (CSF) generated by motile cilia in ependyma lining the brain ventricles, but also cause increased CSF production at the choroid plexus. However, the molecular mechanisms of CSF overproduction by ciliary dysfunction remain elusive. To dissect the molecular mechanisms, choroid plexus epithelial cells (CPECs) were isolated from porcine brain. These cells expressed clusters of primary cilia on the apical surface. Deciliation of CPECs elevated the intracellular cyclic AMP (cAMP) levels and stimulated basolateral-to-apical fluid transcytosis, without detrimental effects on other morphological and physiological features. The primary cilia possessed neuropeptide FF (NPFF) receptor 2. In deciliated cells, the responsiveness to NPFF was reduced at nanomolar concentrations. Furthermore, CPECs expressed NPFF precursor along with NPFFR2. An NPFFR antagonist, BIBP3226, increased the fluid transcytosis, suggesting the presence of autocrine NPFF signaling in CPECs for a tonic inhibition of fluid transcytosis. These results suggest that the clusters of primary cilia in CPECs act as a sensitive chemosensor to regulate CSF production.

Carboxyl Terminus of HOATZ is Intrinsically Disordered and Interacts with Heat Shock Protein A Families.

BACKGROUND: Hoatz is a vertebrate-specific gene, the defects of which result in hydrocephalus and oligo-astheno-teratozoospermia in mice. It encodes a 19-kDa protein lacking any domains of known function. METHODS: To understand the protein activity, we purified the carboxyl-terminal fragment that is conserved among different species, and analyzed its structure and potential binding proteins. A soluble 9.9-kDa HOATZ fragment, including a poly-histidine tag (designated HOATZ-C), was purified to homogeneity. RESULTS: The gel filtration profile and circular dichroism spectra collectively indicated that HOATZ-C was intrinsically disordered. When HOATZ-C was mixed with cleared lysate from Hoatz-null mouse testis, several proteins, including two of ~70 kDa size, were specifically co-purified with HOATZ-C on a nickel column. CONCLUSION: Based on the peptide mass fingerprinting of these bands, two members of the heat-shock protein family A were identified. These data may indicate the role of HOATZ in stress regulation in cells characterized by motile cilia and flagella.

Ultrastructural evidence for an unusual mode of ciliogenesis in mouse multiciliated epithelia.

Multiciliogenesis is a cascading process for generating hundreds of motile cilia in single cells. In vertebrates, this process has been investigated in the ependyma of brain ventricles and the ciliated epithelia of the airway and oviduct. Although the early steps to amplify centrioles have been characterized in molecular detail, subsequent steps to establish multicilia have been relatively overlooked. Here, we focused on unusual cilia-related structures previously observed in wild-type mouse ependyma using transmission electron microscopy and analyzed their ultrastructural features and the frequency of their occurrence. In the ependyma, $\sim$5% of cilia existed as bundles; while the majority of the bundles were paired, bundles of more than three cilia were also found. Furthermore, apical protrusions harboring multiple sets of axonemes were occasionally observed (0-2 per section), suggesting an unusual mode of ciliogenesis. In trachea and oviduct epithelia, ciliary bundles were absent, but protrusions containing multiple axonemes were observed. At the base of such protrusions, certain axonemes were completely enwrapped by membranes, whereas others remained incompletely enwrapped. These data suggested that the late steps of multiciliogenesis might include a unique process underlying the development of cilia, which is distinct from the ciliogenesis of primary cilia.

Odontoblast differentiation is regulated by an interplay between primary cilia and the canonical Wnt pathway.

Primary cilium is a protruding cellular organelle that has various physiological functions, especially in sensory reception. While an avalanche of reports on primary cilia have been published, the function of primary cilia in dental cells remains to be investigated. In this study, we focused on the function of primary cilia in dentin-producing odontoblasts. Odontoblasts, like most other cell types, possess primary cilia, which disappear upon the knockdown of intraflagellar transport protein 88. In cilia-depleted cells, the expression of dentin sialoprotein, an odontoblastic marker, was elevated, while the deposition of minerals was slowed. This was recapitulated by the activation of canonical Wnt pathway, also decreased the ratio of ciliated cells. In dental pulp cells, as they differentiated into odontoblasts, the ratio of ciliated cells was increased, whereas the canonical Wnt signaling activity was repressed. Our results collectively underscore the roles of primary cilia in regulating odontoblastic differentiation through canonical Wnt signaling. This study implies the existence of a feedback loop between primary cilia and the canonical Wnt pathway.

Hypercholesterolemia negatively influences morphology and molecular markers of epithelial cells within the choroid plexus in rabbits.

BACKGROUND: Choroid plexus (CP) is an important tissue not only to produce cerebrospinal fluid (CSF) but also to regulate substances that are secreted into or absorbed from CSF through blood-cerebrospinal fluid barrier (BCSFB) formed by CP epithelial cells (CPECs). CPECs display signs of deterioration in aged and diseased people. However, whether CPECs in hypercholesterolemic animals develop such damage is not known. METHODS: We used cholesterol-fed wild-type or Watanabe hereditary hyperlipidemic (WHHL) rabbits of identical age to determine CPEC changes in terms of morphology and protein expression/localization. RESULTS: Compared with non-cholesterol-fed control rabbits, prolonged exposure to cholesterol reduced CPEC height and increased lipofuscin levels in CPECs, indicating cellular damage. Expression of aquaporin 1 on the apical membranes of CPECs was diminished in cholesterol-exposed rabbits, implying a reduced CSF-producing function in the CP. The rabbit macrophage-specific antibody (RAM11) immunoreaction became positive in CPECs adjacent to foam cells, indicating an alteration in this cell type. CONCLUSION: Cholesterol insults from the circulation (which is reflected by foam-cell accumulation in the CP) induce CPEC dysfunction, and the latter seems to be enhanced by foam cells in hypercholesterolemic rabbits.

In vitro Time-lapse Imaging of Primary Cilium in Migrating Neuroblasts.

Neuronal migration is a critical step for the development of neuronal circuits in the brain. Immature new neurons (neuroblasts) generated in the postnatal ventricular-subventricular zone (V-SVZ) show a remarkable potential to migrate for a long distance at a high speed in the postnatal mammalian brain, and are thus a powerful model to analyze the molecular and cellular mechanisms of neuronal migration. Here we describe a methodology for in vitro time-lapse imaging of the primary cilium and its related structures in migrating V-SVZ-derived neuroblasts using confocal or superresolution laser-scanning microscopy. The V-SVZ tissues are dissected from postnatal day 0-1 (P0-1) mouse brains and dissociated into single cells by trypsinization and gentle pipetting. These cells are then transduced with a plasmid(s) encoding a gene(s) of interest, aggregated by centrifugation, and cultured for 2 days in Matrigel. Time-lapse images of migratory behaviors of cultured neuroblasts and their ciliary structures, including the ciliary membrane and basal body, are acquired by confocal or superresolution laser-scanning microscopy. This method provides information about the spatiotemporal dynamics of neuroblasts' morphology and ciliary structures, and is widely applicable to various types of migrating neuronal and nonneuronal cells in various species.

Discovery of a Vertebrate-Specific Factor that Processes Flagellar Glycolytic Enolase during Motile Ciliogenesis.

Motile cilia and flagella require ATP for their formation and function. Although glycolytic enzymes are components of flagellar proteomes, how they translocate to flagella is unknown. Here we show that the expression pattern of the functionally nonannotated gene 4833427G06Rik (C11orf88), which is found only in vertebrates and is designated here as Hoatzin (Hoatz), suggests a functional association of its product with motile cilia and flagella. Hoatz knockout (KO) mice developed hydrocephalus and male infertility in an autosomal recessive manner, and the ependymal cilia frequently showed disorganized axonemes, reducing motility associated with collapsed spermatid flagella during cytodifferentiation. HOATZ was associated with certain proteins, including the flagellar glycolytic enzyme ENO4. In the testes of the Hoatz KO mice, the immature form of ENO4 accumulated in abnormal cytoplasmic puncta of developing spermatids. These data indicate that HOATZ is required for motile ciliogenesis and flagellar genesis in vertebrates by mediating the maturation of ENO4.

HSulf-1 inhibits angiogenesis and tumorigenesis in vivo.

We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1-expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1-expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA-mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate-dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate-independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal-regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis.

CFAP70 Is a Novel Axoneme-Binding Protein That Localizes at the Base of the Outer Dynein Arm and Regulates Ciliary Motility.

In the present study, we characterized CFAP70, a candidate of cilia-related protein in mice. As this protein has a cluster of tetratricopeptide repeat (TPR) domains like many components of the intraflagellar transport (IFT) complex, we investigated the domain functions of particular interest in ciliary targeting and/or localization. RT-PCR and immunohistochemistry of various mouse tissues demonstrated the association of CFAP70 with motile cilia and flagella. A stepwise extraction of proteins from swine tracheal cilia showed that CFAP70 bound tightly to the ciliary axoneme. Fluorescence microscopy of the cultured ependyma expressing fragments of CFAP70 demonstrated that the N-terminus rather than the C-terminus with the TPR domains was more important for the ciliary localization. When CFAP70 was knocked down in cultured mouse ependyma, reductions in cilia beating frequency were observed. Consistent with these observations, a Chlamydomonas mutant lacking the CFAP70 homolog, FAP70, showed defects in outer dynein arm (ODA) activity and a reduction in flagellar motility. Cryo-electron tomography revealed that the N-terminus of FAP70 resided stably at the base of the ODA. These results demonstrated that CFAP70 is a novel regulatory component of the ODA in motile cilia and flagella, and that the N-terminus is important for its ciliary localization.

Arima syndrome caused by CEP290 specific variant and accompanied with pathological cilium; clinical comparison with Joubert syndrome and its related diseases.

OBJECTIVE: Arima syndrome (AS) is a rare disease and its clinical features mimic those of Joubert syndrome or Joubert syndrome-related diseases (JSRD). Recently, we clarified the AS diagnostic criteria and its severe phenotype. However, genetic evidence of AS remains unknown. We explored causative genes of AS and compared the clinical and genetic features of AS with the other JSRD. PATIENTS AND METHODS: We performed genetic analyses of 4 AS patients of 3 families with combination of whole-exome sequencing and Sanger sequencing. Furthermore, we studied cell biology with the cultured fibroblasts of 3 AS patients. RESULTS: All patients had a specific homozygous variant (c.6012-12T>A, p.Arg2004Serfs*7) or compound heterozygous variants (c.1711+1G>A; c.6012-12T>A, p.Gly570Aspfs*19;Arg2004Serfs*7) in centrosomal protein 290 kDa (CEP290) gene. These unique variants lead to abnormal splicing and premature termination. Morphological analysis of cultured fibroblasts from AS patients revealed a marked decrease of the CEP290-positive cell number with significantly longer cilium and naked and protruded ciliary axoneme without ciliary membrane into the cytoplasm. CONCLUSION: AS resulted in cilia dysfunction from centrosome disruption. The unique variant of CEP290 could be strongly linked to AS pathology. Here, we provided AS specific genetic evidence, which steers the structure and functions of centrosome that is responsible for normal ciliogenesis. This is the first report that has demonstrated the molecular basis of Arima syndrome.

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells.

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM(1) ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.

Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage.

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9-mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22-Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 microM. A fraction of the VP22-Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22-Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY-lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL-receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22-Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.

9 + 0 and 9 + 2 cilia are randomly dispersed in the mouse node.

The initial determination of left-right asymmetry is an essential process in embryonic development. In mouse embryo, cilia in the node play an important role generating the nodal flow that subsequently triggers left-right determination in the embryo. Although nodal cilia have historically been thought to have a 9 + 0 axonemal configuration, the existence of 9 + 2 cilia has been reported so far. Because the distribution of those two types of cilia within the node has not yet been reported, we assessed the arrangement of 9 + 0 and 9 + 2 cilia in the node. In this study, we concluded that most of the nodal cilia were 9 + 0 in structure and there were much fewer 9 + 2 cilia than 9 + 0 cilia. Furthermore, the two types of cilia were randomly distributed in the node with no regularity. In addition, we studied the embryonic origin of the crown cells surrounding the node to better understand their identity.

Cilia in the choroid plexus: their roles in hydrocephalus and beyond.

Cilia are whip-like projections that are widely conserved in eukaryotes and function as a motile propeller and/or sensory platform to detect various extracellular stimuli. In vertebrates, cilia are ubiquitously found in most cells, showing structural and functional diversities depending on the cell type. In this review, we focus on the structure and function of cilia in choroid plexus epithelial cells (CPECs). CPECs form one or two dozen non-motile 9+0 cilia, which display transient acquisition of motility during development. Genetic malfunction of cilia can lead to failure of multiple organs including the brain. Especially, several groups have demonstrated that the defects in CPEC cilia cause the communicating form of hydrocephalus. In order to elucidate the molecular mechanisms underlying the hydrocephalus, we have previously demonstrated that the cilia possess an NPFF receptor for autocrine signaling to regulate transepithelial fluid transport. In this perspective, we also discuss the potential involvement of cilia in the other aspects of choroid plexus functions, such as the regulation of brain development and neuroinflammation.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report(1), the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.