Airway cilia provide the coordinated motive force for mucociliary transport, which prevents the accumulation of mucus, debris, pollutants, and bacteria in our respiratory tracts. As airway cilia are constantly exposed to the environment and, hence, are an integral component of the pathogenesis of several congenital and chronic pulmonary disorders, it is necessary to understand the molecular mechanisms that control ciliated cell differentiation and ciliogenesis. We have previously reported that loss of the basal body protein Chibby (Cby) results in chronic upper airway infection in mice due to a significant reduction in the number of airway cilia. In the present work, we demonstrate that Cby is required for normal ciliary structure and proper distribution of proteins involved in the bidirectional intraflagellar transport (IFT) system, which consists of 2 distinct sub-complexes, IFT-A and IFT-B, and is essential for ciliary biogenesis and maintenance. In fully differentiated ciliated cells, abnormal paddle-like cilia with dilated ciliary tips are observed in Cby-/- airways and primary cultures of mouse tracheal epithelial cells (MTECs). In addition, IFT88, an IFT-B sub-complex protein, robustly accumulates within the dilated tips of both multicilia in Cby-/- MTECs and primary cilia in Cby-/- mouse embryonic fibroblasts (MEFs). Furthermore, we show that only IFT-B components, including IFT20 and IFT57, but not IFT-A and Bardet-Biedl syndrome (BBS) proteins, amass with IFT88 in these distended tips in Cby-/- ciliated cells. Taken together, our findings suggest that Cby plays a role in the proper distribution of IFT particles to preserve normal ciliary morphology in airway ciliated cells.
Centrosomal protein 164 (CEP164) is located at the edge of distal appendages in primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of CEP164 before and after BB docking in photoreceptors, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction. BBs dock to the cell cortex during postnatal day 6 (P6) and extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (ret Cep164 -/-) are unable to dock BBs, thereby preventing formation of a CC or outer segments (OS). In rodspecific knockouts (rod Cep164 -/-), Cre expression starts after P9 and CC/OS form. P16 rod Cep164 -/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rod Cep164 -/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rod Cep164 -/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1 ETCre; Cep164 F/F (tam Cep164 -/-) adult mice affected maintenance of both rod and cone OS. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at BB/CC. Impairment of IFT may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.
Chibby (Cby) was originally identified as an antagonist of the Wnt/β-catenin signaling pathway. It physically interacts with the key co-activator β-catenin and inhibits β-catenin-mediated transcriptional activation. More recently, we demonstrated that Cby protein localizes to the base of motile cilia and is required for ciliogenesis in the respiratory epithelium of mice. To gain further insight into the physiological function of Cby, we developed mouse monoclonal antibodies (MAbs) against human Cby protein and characterized two Cby MAbs, designated 8-2 and 27-11, in depth. Western blot analysis revealed that 8-2 reacts with both human and mouse Cby proteins, whereas 27-11 is specific to human Cby. The epitopes of 8-2 and 27-11 were narrowed down to the middle portion (aa 49–63) and N-terminal region (aa 1–31) of the protein, respectively. We also determined their isotypes and found that 8-2 and 27-11 belong to IgG2a and IgG1 with κ light chains, respectively. Both MAbs can be employed for immunoprecipitation assays. Moreover, 8-2 detects endogenous Cby protein on Western blots, and marks the ciliary base of motile cilia in the murine lung and trachea as shown by immunofluorescence staining. These Cby MAbs therefore hold promise as useful tools for the investigation of Wnt signaling and ciliogenesis.
The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20–30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the β-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of β1 and β3integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide −54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3β (GSK-3β) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells. The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20–30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the β-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of β1 and β3integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide −54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3β (GSK-3β) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells. The cyclin D1 gene encodes a regulatory subunit of a serine-threonine kinase that phosphorylates and inactivates the tumor suppressor pRB (1Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4326) Google Scholar). The abundance of cyclin D1 was shown to be rate-limiting in cellular proliferation induced by diverse signaling pathways in fibroblasts and breast epithelial cells, including MCF7 cells (2Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (321) Google Scholar, 3Lukas J. Bartkova J. Bartek J. Mol. 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The abundance of cyclin D1 is increased in more than 30% of human breast tumors, and overexpression of cyclin D1 under control of the MMTV 1MMTV, mouse mammary tumor virus; CREB, cAMP-responsive element-binding protein; TCF, T cell factor; LEF, lymphoid enhancer factor; PI, phosphatidylinositol; UAS, upstream activating sequence; JNK, c-Jun NH2-terminal kinase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; ILK, integrin-linked kinase; EMSA, electrophoretic mobility shift assay; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; PKB, protein kinase B; PKA, protein kinase A; PCR, polymerase chain reaction; GSK-3β, glycogen synthase kinase-3β; CRE, cAMP response element; GDI, guanine nucleotide dissociation inhibitor; JIP-1, JNK-interacting protein-1. promoter in transgenic mice induces mammary adenocarcinoma (7Wang T.C. Cardiff R.D. Zukerberg L. Lees E. Arnold A. Schmidt E.V. 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ILK enhanced both CREB transactivation and binding of CREB to the cyclin D1 promoter CRE. GSK-3β inhibited ILK-induced cyclin D1 promoter activity and CREB binding to the cyclin D1 CRE. The identification of the cyclin D1 promoter as a direct target of ILK underscores a likely mechanism by which integrin signaling may enhance DNA synthesis. The human cyclin D1 promoter reporter constructions (9Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar) PALUC, which contains 7 kilobase pairs of the human cyclin A promoter sequence (36Henglein B. Chenivesse X. Wang J. Eick D. Brechot C. Proc. Natl. Acad. Sci. U. S. 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The subtracted cDNA library was subcloned with the pGEM-T Easy Vector Systems (Promega, Madison, WI) for further analysis. For Northern blot analysis, total RNA was isolated by guanidium thiocyanate-phenol-chloroform using a single-step extraction (45Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNA samples were resolved on a 1% agarose, 3% formaldehyde gel. Transfer, hybridization, and washing were performed as described by Sambrooket al. (46Sambrook T. Fritsch E.R. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Probes were labeled with [α-32P]dCTP by use of a random priming kit (Ambion, Austin, TX). Cell culture, DNA transfection, and luciferase assays were performed as described previously (10Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar). The MCF7 cells and human embryonic kidney cells (HEK-293; obtained from M. Moran, University of Toronto, Toronto, Ontario, Canada) (32Troussard A.E. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar) were maintained in Dulbecco's modified Eagle's medium with 10% (v/v) calf serum and 1% penicillin/streptomycin. In transient expression studies, cells were transfected either by calcium phosphate precipitation or the use of Superfect transfection reagent (Qiagen, Valencia, CA) as described by the manufacturer. The medium was changed after 6 h and luciferase activity determined after an additional 24 h. The effect of an expression vector was compared with the effect of an equal amount of vector cassette. Treatments with the MAP kinase/ERK kinase inhibitor PD98059 (10–20 μm) (47Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. 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Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Statistical analyses were performed using the Mann-Whitney Utest with significant differences established as p < 0.05. The wild type CRE/ATF site of the cyclin D1 promoter, CD1CREwt, was synthesized as complementary oligodeoxyribonucleotide strands for EMSA (6Brown J.R. Nigh E. Lee R.J. Ye H. Thompson M.A. Saudou F. Pestell R.G. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 5609-5619Crossref PubMed Scopus (211) Google Scholar). The sequence of the cyclin D1 promoter CRE/ATF site oligodeoxyribonucleotide (CD1CREwt) was 5′-AAC AAC AGTAACGTCACA CGG AC-3′. EMSA were performed using nuclear extracts as described previously (32Troussard A.E. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). Cells were washed with ice-cold PBS and harvested by scraping in 1.5 ml of PBS. Cells were pelleted and resuspended in 400 μl of buffer A (10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 0.2 mm PMSF). After 10 min of incubation on ice, nuclei were pelleted and then resuspended in 50 μl of buffer B (20 mm HEPES-KOH (pH 7.9), 25% glycerol, 420 mm sodium chloride, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm DTT, 0.2 mmPMSF). Proteins were incubated for 20 min on ice and then centrifuged to clear cellular debris. Gel shift assays were performed by incubating 2 μg of the nuclear extracts for 20 min at room temperature with the γ-32P-labeled oligonucleotides (50 fmol, 100,000 counts/min). The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel, with 0.5× Tris borate, EDTA buffer (TBE: 0.045 m Tris borate, 0.001 m EDTA) and 3.5% glycerol at 180 V for 3–4 h. The specificity of the DNA-protein interaction was established by competition experiment and supershift assays as described (8Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines III, G.K. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The gels were dried and exposed to autoradiographic film (Labscientific Inc., Livingston, NJ). The genotype of Wnt-1 transgenic mice was determined by isolating tail DNA and Southern blot hybridization with Wnt-1-labeled DNA probes as described previously (50Tsukamoto A.S. Grosschedl R. Guzman R.C. Parslow T. Varmus H.E. Cell. 1988; 18: 619-625Abstract Full Text PDF Scopus (592) Google Scholar, 51Donehower L.A. Godley L.A. Aldaz C.M. Pyle R. Shi Y.P. Pinkel D. Gray J. Bradley A. Medina D. Varmus H.E. Genes Dev. 1995; 9: 882-895Crossref PubMed Scopus (247) Google Scholar). The ILK kinase assays were performed as described previously (29Wu C. Keightley S.Y. Leung-Hagesteijn C. Radeva G. Coppolino M. Goicoechea S. McDonald J.A. Dedhar S. J. Biol. Chem. 1998; 273: 528-536Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) using a rabbit immunoaffinity-purified ILK antibody (06-592) (Upstate Biotechnology, Lake Placid, NY) and myelin basic protein as substrate. Tissues from MMTV-Wnt-1 transgenic mice were homogenized by Dounce in lysis buffer (150 mm NaCl, 50 mm HEPES (pH 7.2), 1 mm EDTA, 1 mm EGTA, 1 mmDTT, 0.1% Tween 20, 0.1 mm PMSF, 2.5 μg/ml leupeptin, and 0.1 mm sodium orthovanadate (Sigma)), at 4 °C. Lysates were centrifuged at 10,000 × g for 5 min, and protein concentrations were determined using a modified Bradford assay protocol (Bio-Rad). The supernatants (100 μg) were precipitated for 12 h at 4 °C with protein A-agarose beads precoated with saturating amounts of the antibody. Immunoprecipitated proteins on beads were washed twice with 1 ml of lysis buffer and twice with kinase buffer (50 mm HEPES (pH 7.0), 10 mmMgCl2, 5 mm MnCl2, 1 mmDTT). The beads were then resuspended in 40 μl of kinase buffer containing the protein substrate (2 μg of myelin basic protein), 10 mm ATP, and 5 mCi of [γ-32P]ATP (6000 Ci/mmol; 1 Ci = 37 GBq, Amersham Pharmacia Biotech). The samples were incubated for 30 min at 30 °C with occasional mixing. The samples were boiled in polyacrylamide gel sample buffer containing sodium dodecyl sulfate and separated by electrophoresis. Phosphorylated proteins were quantified after exposure to autoradiographic film (Labscientific, Inc., Livingston, NJ) by densitometry using ImageQuant version 1.11 (Molecular Dynamics Computing Densitometer, Sunnyvale, CA). The abundance of cyclin D1 protein was determined by Western analysis as described previously (9Watanabe G. Lee R.J. Albanese C. Rainey W.E. Batlle D. Pestell R.G. J. Biol. Chem. 1996; 271: 22570-22577Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar), using a monoclonal cyclin D1 antibody DCS-6 (NeoMarkers, Fremont, CA) and a guanine nucleotide dissociation inhibitor (GDI) antibody (a generous gift from Dr. Perry Bickel, Washington University, St. Louis, MO) (52Lee R.J. Albanese C. Fu M. D'Amico M. Lin B. Watanabe G. Haines G.K.I. Siegel P.M. Hung M.C. Yarden Y. Horowitz J.M. Muller W.J. Pestell R.G. Mol. Cell. Biol. 2000; 20: 672-683Crossref PubMed Scopus (311) Google Scholar) as internal control for protein abundance. Cell homogenates (50 μg) were electrophoresed in an 12% SDS-polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane (Micron Separations Inc., Westborough, MA). After transfer, the gel was stained with Coomassie Blue as a control for blotting efficiency. The blotting membrane was incubated for 2 h at 25 °C in T-PBS buffer supplemented with 5% (w/v) dry milk to block nonspecific binding sites. Following a 6 h incubation with primary antibody at a 1:1000 dilution (cyclin D1) or 1:2500 (GDI) in T-PBS buffer containing 0.05% (v/v) Tween 20, the membrane was washed with the same buffer. For detection of cyclin D1, the membrane was incubated with goat anti-mouse horseradish peroxidase secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and washed again. The cyclin D1 protein was visualized by the enhanced chemiluminescence system (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Previous studies have shown that overexpression of ILK induces anchorage-independent growth and elevates cyclin D1 levels (33Radeva G. Petrocelli T. Behrend E. Leung-Hagesteijn C. Filmus J. Slingerland J. Dedhar S. J. Biol. Chem. 1997; 272: 13937-13944Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). To determine whether ILK overexpression induces the cyclin D1 gene, transient expression studies were conducted in MCF7 cells. ILK overexpression induced the full-length cyclin D1 promoter linked to the luciferase reporter (−1745CD1LUC) by 4.5-fold (Fig. 1 A). Recent studies have shown GSK-3β is inhibited by induction of ILK signaling (32Troussard A.E. Tan C. Yoganathan T.N. Dedhar S. Mol. Cell. Biol. 1999; 19: 7420-7427Crossref PubMed Scopus (140) Google Scholar). Wild type ILK activates protein kinases B (PKB/AKT) and inhibits GSK-3β activity in a PI 3-kinase-dependent manner (31Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar). GSK-3β regulates several downstream signaling events, including the function of the CREB transcription factor (23Bullock B.P. Habener J.F. Biochemistry. 1998; 37: 3795-3809Crossref PubMed Scopus (136) Google Scholar, 53Fiol C.J. Williams J.S. Chou C.H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar). Since the cyclin D1 promoter contains a CREB binding site (37Watanabe G. Howe A. Lee R.J. Albanese C. Shu I.-W. Karnezis A.N. Zon L. Kyriakis J. Rundell K. Pestell R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12861-12866Crossref PubMed Scopus (197) Google Scholar), we examined the effect of wild type ILK on a cyclin D1 promoter containing a point mutation in the CRE/ATF binding site. This point mutation, shown to abolish binding of CREB/ATF-2 to the cyclin D1 promoter sequence (8Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines III, G.K. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) (−1745CD1LUC CREmut), was not induced by ILK (Fig. 1 A). Point mutation of the TCF site did not affect regulation of the cyclin D1 promoter by wild type ILK (data not shown). The activation of the cyclin D1 promoter by ILK was specific as reporter plasmids containing the Rous sarcoma virus, cyclin A, and MMTV promoters, and the empty luciferase vector pA3LUC, were not induced by ILK overexpression (Fig.1 B). These results suggest that the CREB/ATF-2 binding site of the cyclin D1 promoter is required for its induction by ILK. To determine the intracellular signaling pathway(s) by which ILK induced cyclin D1 promoter activity, several chemical inhibitors and dominant negative expression vectors were employed. In these studies, the level of the cyclin D1 promoter activity in the presence of ILK was set as 100% (Fig. 2 A). The activity of ILK was previously linked to the PI 3-kinase pathway (31Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar), and ILK contains a phosphoinositide binding motif (30Dedhar S. Williams B. Hannigan G. Trends Cell Biol. 1999; 9: 319-323Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The addition of the PI 3-kinase inhibitor LY294002 to cells transfected with ILK and the cyclin D1 promoter reporter reduced the ILK-induced cyclin D1 promoter activity by 35% (Fig. 2 A). In contrast, the basal activity of the cyclin D1 promoter in the absence of ILK expression was not inhibited by LY294002 (Fig. 2 B). These results are consistent with previous observations showing that the basal activity of the cyclin D1 promoter is
Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.
New neurons are continually born throughout adulthood in the subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus in the hippocampus. The sequential synaptic integration of adult-born neurons has been widely examined in rodents, but the mechanisms regulating the integration remain largely unknown. The primary cilium, a microtubule–based signaling center, plays essential roles in vertebrate development, including the development of the central nervous system. We examined the assembly and function of the primary cilium in the synaptic integration of adult-born hippocampal neurons. Strikingly, primary cilia are absent in young adult-born neurons but assemble precisely at the stage when newborn neurons approach their final destination, further extend dendrites and form synapses with entorhinal cortical projections. Conditional cilia deletion from adult-born neurons induced severe defects in dendritic refinement and synapse formation. Primary cilia deletion leads to enhanced Wnt/beta-catenin signaling which may account for these developmental defects. Taken together, our study identifies the assembly of primary cilia as a critical regulatory event in the dendritic refinement and synaptic integration of adult-born neurons.
