Inactivation of Max-interacting Protein 1 Induces Renal Cilia Disassembly through Reduction in Levels of Intraflagellar Transport 20 in Polycystic Kidney

Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.     

The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis

In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8^ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8^ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8^ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development.     

Primary cilia in murine palatal rugae development

Periodic patterning of iterative structures is a fundamental process during embryonic development, since these structures are diverse across the animal kingdom. Therefore, elucidating the molecular mechanisms in the formation of these structures promotes understanding of the process of organogenesis. Periodically patterned ridges, palatal rugae (situated on the hard palate of mammals), are an excellent experimental model to clarify the molecular mechanisms involved in the formation of periodic patterning of iterative structures. Primary cilia are involved in many biological events, including the regulation of signaling pathways such as Shh and non-canonical Wnt signaling. However, the role of primary cilia in the development of palatal rugae remains unclear. We found that primary cilia were localized to the oral cavity side of the interplacode epithelium of the palatal rugae, whereas restricted localization of primary cilia could not be detected in other regions. Next, we generated mice with a placodal conditional deletion of the primary cilia protein Ift88, using ShhCre mice (Ift88 ^fl/fl;ShhCre). Highly disorganized palatal rugae were observed in Ift88 ^fl/fl;ShhCre mice. Furthermore, by comparative in situ hybridization analysis, many Shh and non-canonical Wnt signaling-related molecules showed spatiotemporal expression patterns during palatal rugae development, including restricted expression in the epithelium (placodes and interplacodes) and mesenchyme. Some of these expression were found to be altered in Ift88 ^fl/fl;ShhCre mice. Primary cilia is thus involved in development of palatal rugae.     

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.     

CILIARY MEMBRANE DIFFERENTIATIONS IN TETRAHYMENA PYRIFORMIS : Tetrahymena Has Four Types of Cilia

We have examined thin sections and replicas of freeze-fractured cilia of Tetrahymena pyriformis. The ciliary necklace located at the base of all freeze-fractured oral and somatic cilia has been studied in thin sections. Since electron-dense linkers have been found to connect both microtubule doublets and triplets to the ciliary membrane at the level of the necklace, the linkers and the associated necklace seem to be related to the transition region between the doublets and triplets of a cilium. Plaque structures, consisting of small rectangular patches of particles located distal to the ciliary necklace, are found in strain GL, but are absent in other strains examined in this study. In freeze-cleaved material, additional structural differentiations are observed in the distal region of the ciliary membranes of somatic and oral cilia. Somatic cilia contain many randomly distributed particles within their membrane. Oral cilia can be divided into three categories on the basis of the morphology of their freeze-fractured membranes: (a) undifferentiated cilia with very few randomly distributed particles: (b) cilia with particles arranged in parallel longitudinal rows spaced at intervals of 810–1080 Å that are located on one side of the cilium; and (c) cilia with patches of particles arranged in short rows oriented obliquely to the main axis of the cilium. The latter particles, found on one side of the cilium, seem to serve as attachment sites for bristles 375–750 Å long and 100 Å wide which extend into the surrounding medium. The particles with bristles are located at the tips of cilia in the outermost membranelle and may be used to detect food particles and/or to modify currents in the oral region so that food particles are propelled more efficiently into the buccal cavity. Examination of thin-sectioned material indicates that the particles in oral cilia which form the longitudinal rows could be linked to microtubule doublets. Linkage between microtubule doublets and adjacent membrane areas on one side of the cilium could modify the form of ciliary beat by restricting the sliding of the microtubules. It is suggested that membrane-microtubule interactions may form the basis for the various forms of ciliary beat observed in different organisms.     

The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.     

NEUROID TRANSMISSION IN CILIATED EPITHELIUM

Evidence from several sources indicates that there is present in certain types of ciliated tissue a primitive form of conduction regulating the rate of beat of the cilia. (Kraft, 1890; Engelmann, 1898). A similar type of conduction has been observed by Parker (1910) in the sponge Stylotella. In the foregoing experiments a study was made of such conduction in the ciliated tissue of the gills of the clam Unio. Observations were made on the effects of temperature changes on the rate of beat of cilia adjacent to areas not themselves directly influenced. The following results were obtained. 1. The transmission through the gill of the effects of warmth applied locally is apparent through increased rate of ciliary beat on adjacent gill tissue in all directions from the region of application. Effects are not observed laterally at a distance greater than 9 to 11 mm. from the nearest edge of the stimulated area. The narrowness of the gill makes it impossible to determine the vertical limits of the transmission. 2. Effects of low temperatures are not observable beyond the limits of the region of direct application. These results differ from those of Kraft in tissue from the frog''s pharynx, where conduction was shown to take place only down a row of beating cilia. On the other hand they agree with his results in indicating that the effects of warmth only are transmitted. The phenomenon might be explained by the stimulating effect of the action current of the directly excited cilia on the neighboring relatively quiet cilia. A similar explanation has been offered by Lillie for waves of coordinated beating in the rows of swimming plates of ctenophores. Such an explanation, though in accord with the work on Unio, is inconsistent with certain of the observations of Kraft on the tissue from the frog''s pharynx.     

Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity,Density, and Beat

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.     

“Ciliophagy”: The consumption of cilia components by autophagy

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/−, map1lc3b^−/−, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/−, map1lc3b^−/−, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.     

Redescription of Licnophora chattoni Villeneuve-Brachon, 1939 (Ciliophora, Spirotrichea), associated with Zyzzyzus warreni Calder, 1988 (Cnidaria, Hydrozoa)

Licnophora chattoni, found in association with Zyzzyzus warreni, a tubulariid hydroid epizoic in sponges from São Sebastião (SP, Brazil), is redescribed and illustrated using light and electron microscopy. The ciliate has a flexible, transparent body formed by an oval anterior region linked to the posterior basal disc via a flexible neck region. Numerous cortical granules are observed scattered throughout the body and densely packed along the neck. The adoral zone is formed by about 81 external and 24 infundibular paramembranelles. The paroral membrane, formed by a row of long cilia arranged in monokinetids, extends through a groove in the body to the adhesive disc. Two dorsal kinetids are present along the right body margin and around the neck. The adhesive disc (18 μm in diameter) lacks cilia in the area above the velum. The velum covers a row of dikinetids bearing long cilia and four dikineties, two or three of which are interrupted on the ventral surface. Nine to twelve macronuclear nodules connected by isthmuses are distributed in the cytoplasm, plus two nodules located in the adhesive disc and between those there is an ovate micronucleus.     

Identification and Characterization of a Novel Allele of Caenorhabditis elegans bbs-7

Primary cilia play a role in the sensation of and response to the surrounding environment. Caenorhabditis elegans (C. elegans) have primary cilia only on the distal tips of some dendrites. In order to better understand the relationship between receptor localization to cilia, cilia structure and cilia function, we have characterized a mutation originally identified in a forward genetic screen for mutants with defective PKD-2 ciliary localization. Through behavioral assays and examination of the structure of cilia in the cil-5 (my13) mutant animals, we have found that my13 disrupts not only receptor localization, but also some cilia-mediated sensory behaviors and cilia structural integrity. We have identified the my13 lesion and found that it is a missense mutation in bbs-7, an ortholog of human BBS-7, a gene known to affect human cilia and to be involved in Bardet-Biedl syndrome. Finally, we show that bbs-7(my13) also affects the glia cells which support the cilia.     

CILIA REGENERATION IN TETRAHYMENA AND ITS INHIBITION BY COLCHICINE

The cilia of Tetrahymena were amputated by the use of a procedure in which the cells remained viable and regenerated cilia. Deciliated cells were nonmotile, and cilia regeneration was assessed by scoring the percentage of motile cells at intervals following deciliation. After a 30-min lag, the deciliated cells rapidly recovered motility until more than 90% of the cells were motile at 70 min after amputation. Cycloheximide inhibited both protein synthesis and cilia regeneration. This indicated that cilia formation in Tetrahymena was dependent on protein synthesis after amputation. Conversely, colchicine was found to inhibit cilia regeneration without affecting either RNA or protein synthesis. This observation suggested the action of colchicine to be an interference with the assembly of ciliary subunit proteins. The finding that colchicine binds to microtubule protein subunits isolated from cilia and flagella (13) supports this possibility. The potential of the colchicine-blocked cilia-regenerating system in Tetrahymena for studying the assembly of microtubule protein subunits during cilia formation and for isolating ciliary precursor proteins is discussed.     

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A^–/– PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A^–/– human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo–electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A^–/– cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1^–/– cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A^–/– cilia. However, RSPH4A^–/– cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A^–/– PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.     

On the role of sulfhydryl groups in the ATPase activity and pellet height response of Tetrahymena cilia

The effects of N-ethylmaleimide and p-hydroxymercuribenzoate on the ATPase activity of glycerinated Tetrahymena cilia, of 30 S dynein extracted from the cilia, and on the residual ATPase remaining after extraction were studied and correlated with the effects of these reagents on the pellet height response of these cilia. Simultaneous addition of N-ethylmaleimide and ATP to cilia caused a slight inhibition of ATPase activity. Preincubation of the cilia with low N-ethylmaleimide in the absence of ATP, however, enhanced the ATPase activity; the enhancement decreased with increasing time of preincubation. Preincubation of cilia with high N-ethylmaleimide caused increasing inhibition. p-Hydroxymercuribenzoate was more potent than N-ethylmaleimide, usually causing only an inhibition which increased if the cilia were preincubated with p-hydroxymercuribenzoate in the absence of ATP.The pellet height response of these cilia, which serves as a convenient assay of some events related to ciliary motility, was inhibited about 50% by high concentrations of N-ethylmaleimide in the presence of ATP. Preincubation of the cilia with low concentrations of N-ethylmaleimide led to complete loss of the pellet height response. p-Hydroxymercuribenzoate was a more potent inhibitor of the pellet height response than N-ethylmaleimide; complete inhibition was attained even in the presence of ATP, while preincubation with a low concentration of p-hydroxymercuri-benzoate caused a very rapid loss of pellet height response.The ATPase activity of the crude dynein obtained by extraction of cilia and removal of the axonemes was approximately doubled by preincubation with N-ethylmaleimide. 30 S Dynein, obtained from the crude dynein by sedimentation on a sucrose density gradient, was slightly inhibited by N-ethylmaleimide; p-hydroxy-mercuribenzoate was more potent. The residual ATPase activity remaining on the axonemes after two extractions was also only inhibited by N-ethylmaleimide and by p-hydroxymercuribenzoate.These results demonstrated that SH groups influence both the ATPase activity of dynein and the pellet height response of glycerinated cilia. The possible significance of the similarity in enhancing effect of N-ethylmaleimide on cilia ATPase and on myosin ATPase was discussed.     

The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke

Mucus propelling cilia are excitable by many stimulants, and have been shown to increase their beating frequency up to threefold, by physiological extracellular stimulants, such as adenosine-triphosphate, acetylcholine, and others. This is thought to represent the evolutionary adaptation of mucociliary systems to the need of rapid and efficient cleansing the airways of foreign particles. However, the mucus transport velocity depends not only on the beat frequency of the cilia, but on their beat pattern as well, especially in the case of mucus bearing cilia that beat in a complex, three-dimensional fashion. In this study, we directly measured the force applied by live ciliary tissues with an atomic force microscope, and found that it increases linearly with the beating frequency. This implies that the arc swept by the cilia during their effective stroke remains unchanged during frequency increase, thus leading to a linear dependence of transport velocity on the beat frequency. Combining the atomic force microscope measurements with optical measurements, we have indications that the recovery stroke is performed on a less inclined plane, leading to an effective shortening of the overall path traveled by the cilia tip during this nontransporting phase of their beat pattern. This effect is observed to be independent of the type of stimulant (temperature or chemical), chemical (adenosine-triphosphate or acetylcholine), or concentration (1 μM-100 μM), indicating that this behavior may result from internal details of the cilium mechanical structure.     

Sequential expression of Efhc1/myoclonin1 in choroid plexus and ependymal cell cilia

EFHC1 is a gene mutated in patients with idiopathic epilepsies, and encodes the myoclonin1 protein. We here report the distribution of myoclonin1 in mouse. Immunohistochemical analyses revealed that the myoclonin1 first appeared at the roof of hindbrain at embryonic day 10 (E10), and moved on to choroid plexus at E14. At E18, it moved to ventricle walls and disappeared from choroid plexus. From neonatal to adult stages, myoclonin1 was concentrated in the cilia of ependymal cells at ventricle walls. At adult stages, myoclonin1 expression was also observed at tracheal epithelial cilia in lung and at sperm flagella in testis. Specificities of these immunohistochemical signals were verified by using Efhc1-deficient mice as negative controls. Results of Efhc1 mRNA in situ hybridization were also consistent with the immunohistochemical observations. Our findings raise “choroid plexusopathy” or “ciliopathy” as intriguing candidate cascades for the molecular pathology of epilepsies caused by the EFHC1 mutations.     

OLFACTORY CILIA IN THE FROG

Olfactory epithelium from the frog was examined in the living state by light microscopy and in the fixed state by electron microscopy. Particular attention was paid to the layer of cilia and mucus which covers the surface of the epithelium. The olfactory cilia differed from typical cilia in that they (a) arose from bipolar neurons and had centrioles near their basal bodies, (b) were up to 200 microns in length, of which the greater part was a distal segment containing an atypical array of ciliary fibers, (c) were often immotile, (d) had their distal segments arranged in parallel rows near the surface of the mucus, and (e) had many vesicles along their shafts and had splits in the array of fibers in their distal segments. These specializations make the olfactory cilia similar to cilia found on other sensory cells and support the theory that they are the locus where electrical excitation in the olfactory organ is initiated by contact with odorous substances.     

Compensatory Role of Inositol 5-Phosphatase INPP5B to OCRL in Primary Cilia Formation in Oculocerebrorenal Syndrome of Lowe

Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm’s canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer’s vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.     

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.