Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Role of ciliopathy protein TMEM107 in eye development: insights from a mouse model and retinal organoid.

Primary cilia are cellular surface projections enriched in receptors and signaling molecules, acting as signaling hubs that respond to stimuli. Malfunctions in primary cilia have been linked to human diseases, including retinopathies and ocular defects. Here, we focus on TMEM107, a protein localized to the transition zone of primary cilia. TMEM107 mutations were found in patients with Joubert and Meckel-Gruber syndromes. A mouse model lacking Tmem107 exhibited eye defects such as anophthalmia and microphthalmia, affecting retina differentiation. Tmem107 expression during prenatal mouse development correlated with phenotype occurrence, with enhanced expression in differentiating retina and optic stalk. TMEM107 deficiency in retinal organoids resulted in the loss of primary cilia, down-regulation of retina-specific genes, and cyst formation. Knocking out TMEM107 in human ARPE-19 cells prevented primary cilia formation and impaired response to Smoothened agonist treatment because of ectopic activation of the SHH pathway. Our data suggest TMEM107 plays a crucial role in early vertebrate eye development and ciliogenesis in the differentiating retina.

Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities.

PURPOSE: The clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. METHODS: Clinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. RESULTS: In this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. CONCLUSION: Our data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Airway Cells on Vitrigel Membrane into Rat Nasal Cavity.

The nasal mucosa functions as a frontline biological defense against various foreign substances and pathogens. Maintaining homeostasis of the nasal epithelium is necessary to promote good health. Nasal epithelia are constantly replaced under normal conditions. However, hereditary diseases, including primary ciliary dyskinesia and cystic fibrosis, can result in intractable dysfunction of the nasal mucosa. Since there is no treatment for this underlying condition, extrinsic manipulation is necessary to recover and maintain nasal epithelia in cases of hereditary diseases. In this study, we explored the use of airway epithelial cells (AECs), including multiciliated airway cells, derived from human induced pluripotent stem cells (iPSCs) on porcine atelocollagen vitrigel membranes, as a candidate of a therapeutic method for irreversible nasal epithelial disorders. To confirm the regenerative capacity of iPSC-derived AECs, we transplanted them into nasal cavities of nude rats. Although the transplanted cells were found within cysts isolated from the recipient nasal respiratory epithelia, they survived in some rats. Furthermore, the surviving cells were composed of multiple cell types similar to the human airway epithelia. The results could contribute to the development of novel transplantation-related technologies for the treatment of severe irreversible nasal epithelial disorders. Impact Statement Nasal respiratory epithelia are important for the functions of nasal cavity, including humidifying the air and filtering various toxic substances. However, hereditary diseases, including primary ciliary dyskinesia and cystic fibrosis, can result in intractable dysfunction of the nasal mucosa. Our novel method to transplant airway epithelial cells derived from human induced pluripotent stem cells will be a candidate method to replace malfunctioned nasal respiratory epithelia in such a situation. To secure our method's safety, we used porcine atelocollagen vitrigel membranes, which prevent the immune response and bovine spongiform encephalopathy, as a scaffold.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules.

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

Current and Future Treatments in Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare genetic ciliopathy in which mucociliary clearance is disturbed by the abnormal motion of cilia or there is a severe reduction in the generation of multiple motile cilia. Lung damage ensues due to recurrent airway infections, sometimes even resulting in respiratory failure. So far, no causative treatment is available and treatment efforts are primarily aimed at improving mucociliary clearance and early treatment of bacterial airway infections. Treatment guidelines are largely based on cystic fibrosis (CF) guidelines, as few studies have been performed on PCD. In this review, we give a detailed overview of the clinical studies performed investigating PCD to date, including three trials and several case reports. In addition, we explore precision medicine approaches in PCD, including gene therapy, mRNA transcript and read-through therapy.

Emerging Genotype-Phenotype Relationships in Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare inherited condition affecting motile cilia and leading to organ laterality defects, recurrent sino-pulmonary infections, bronchiectasis, and severe lung disease. Research over the past twenty years has revealed variability in clinical presentations, ranging from mild to more severe phenotypes. Genotype and phenotype relationships have emerged. The increasing availability of genetic panels for PCD continue to redefine these genotype-phenotype relationships and reveal milder forms of disease that had previously gone unrecognized.

The Antimicrobial Activity of Peripheral Blood Neutrophils Is Altered in Patients with Primary Ciliary Dyskinesia.

The airways of patients with primary ciliary dyskinesia (PCD) contain persistently elevated neutrophil numbers and CXCL8 levels. Despite their abundance, neutrophils fail to clear the airways from bacterial infections. We investigated whether neutrophil functions are altered in patients with PCD. Neutrophils from patients and healthy controls (HC) were isolated from peripheral blood and exposed to various bacterial stimuli or cytokines. Neutrophils from patients with PCD were less responsive to low levels of fMLF in three different chemotaxis assays (p < 0.05), but expression of the fMLF receptors was unaltered. PCD neutrophils showed normal phagocytic function and expression of adhesion molecules. However, PCD neutrophils produced less reactive oxygen species upon stimulation with bacterial products or cytokines compared to HC neutrophils (p < 0.05). Finally, the capacity to release DNA, as observed during neutrophil extracellular trap formation, seemed to be reduced in patients with PCD compared to HC (p = 0.066). These results suggest that peripheral blood neutrophils from patients with PCD, in contrast to those of patients with cystic fibrosis or COPD, do not show features of over-activation, neither on baseline nor after stimulation. If these findings extend to lung-resident neutrophils, the reduced neutrophil activity could possibly contribute to the recurrent respiratory infections in patients with PCD.

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.

Transcript analysis for variant classification resolution in a child with primary ciliary dyskinesia.

Transcriptional analysis can be utilized to reconcile variants of uncertain significance, particularly those predicted to impact splicing. Laboratory analysis of the predicted mRNA transcript may allow inference of the in vivo impact of the variant and aid prediction of its clinical significance. We present a patient with classical features of primary ciliary dyskinesia (PCD) who was identified to have compound heterozygous variants in the DNAH11 gene (c.10691 + 2T > C, c.13523_13543dup21) via trio whole-exome sequencing in 2013. These variants were originally classified as Mutation and Likely Mutation. However, these variants were downgraded to variants of uncertain significance (VUSs) during reanalysis in 2016 because of uncertainty that they caused a loss of function of the gene. c.10691 + 2T > C is predicted to abrogate the canonical splice site and lead to the skipping of exon 65, but the adjoining of exon 64 and exon 66 in the DNAH11 transcript preserves the reading frame of the resultant protein. c.13523_13543dup21 is located in the last exon of the DNAH11 coding sequence, upstream of the canonical stop codon, which suggests a reduced likelihood to trigger nonsense-mediated decay (NMD). Transcriptional analysis was performed to characterize the impact of the variants, resulting in reclassification of c.10691 + 2T > C to Likely Pathogenic by providing evidence that it results in a deleterious effect and subsequent downstream reclassification of c.13523_13543dup21 to Likely Pathogenic as well. Our case illustrates the potential impact of transcriptional analysis on variant resolution, supporting its usage on variants that exert an unpredictable effect on splicing.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

Motile cilia and airway disease.

A finely regulated system of airway epithelial development governs the differentiation of motile ciliated cells of the human respiratory tract, conferring the body's mucociliary clearance defence system. Human cilia dysfunction can arise through genetic mutations and this is a cause of debilitating disease morbidities that confer a greatly reduced quality of life. The inherited human motile ciliopathy disorder, primary ciliary dyskinesia (PCD), can arise from mutations in genes affecting various aspects of motile cilia structure and function through deficient production, transport and assembly of cilia motility components or through defective multiciliogenesis. Our understanding about the development of the respiratory epithelium, motile cilia biology and the implications for human pathology has expanded greatly over the past 20 years since isolation of the first PCD gene, rising to now nearly 50 genes. Systems level insights about cilia motility in health and disease have been made possible through intensive molecular and omics (genomics, transcriptomics, proteomics) research, applied in ciliate organisms and in animal and human disease modelling. Here, we review ciliated airway development and the genetic stratification that underlies PCD, for which the underlying genotype can increasingly be connected to biological mechanism and disease prognostics. Progress in this field can facilitate clinical translation of research advances, with potential for great medical impact, e.g. through improvements in ciliopathy disease diagnosis, management, family counselling and by enhancing the potential for future genetically tailored approaches to disease therapeutics.

Ciliopathies and the Kidney: A Review.

Primary cilia are specialized sensory organelles that protrude from the apical surface of most cell types. During the past 2 decades, they have been found to play important roles in tissue development and signal transduction, with mutations in ciliary-associated proteins resulting in a group of diseases collectively known as ciliopathies. Many of these mutations manifest as renal ciliopathies, characterized by kidney dysfunction resulting from aberrant cilia or ciliary functions. This group of overlapping and genetically heterogeneous diseases includes polycystic kidney disease, nephronophthisis, and Bardet-Biedl syndrome as the main focus of this review. Renal ciliopathies are characterized by the presence of kidney cysts that develop due to uncontrolled epithelial cell proliferation, growth, and polarity, downstream of dysregulated ciliary-dependent signaling. Due to cystic-associated kidney injury and systemic inflammation, cases result in kidney failure requiring dialysis and transplantation. Of the handful of pharmacologic treatments available, none are curative. It is important to determine the molecular mechanisms that underlie the involvement of the primary cilium in cyst initiation, expansion, and progression for the development of novel and efficacious treatments. This review updates research progress in defining key genes and molecules central to ciliogenesis and renal ciliopathies.

Novel dynein axonemal assembly factor 1 mutations identified using whole­exome sequencing in patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder caused by dysfunction of the cilia and flagella; however, causative genetic defects have not been detected in all patients with PCD. Seven Chinese Han patients with Kartagener syndrome were enrolled onto the present study. Transmission electron microscopy (TEM) was performed to evaluate the cilial defects and whole­exome sequencing was used to analyze relevant genetic variations in all patients. In two of the seven patients with PCD, four novel dynein axonemal assembly factor 1 (DNAAF1) mutations were identified (NM_178452.6:c.3G>A, c.124+1G>C, c.509delG and c.943A>T) in three alleles. Both of these patients had long­standing infertility. Their chest computed tomography results showed bronchiectasis, lung infections and situs inversus, and paranasal computed tomography revealed sinusitis. Semen analysis of the male patient showed poor sperm motility. TEM showed defects in the inner and outer dynein arms in both patients. The DNAAF1 sequences of family members were then analyzed. Bioinformatics analysis indicated that these mutations may be the cause of the cilial defects in these two probands. Thus, the present study identified novel PCD­causing mutations in DNAAF1 in two patients with PCD. These genetic variations were predicted to alter DNAAF1 amino acid residues and lead to loss of function, thereby inhibiting cilia­mediated motility. Accordingly, the two probands had PCD symptoms, and one of them died due to PCD­associated complications.

Two novel mutations in the DNAH11 gene in primary ciliary dyskinesia (CILD7) with considerable variety in the clinical and beating cilia phenotype.

BACKGROUND: Diagnosis of primary ciliary dyskinesia (PCD) still remains a challenge, especially with mutations in the Dynein Arm Heavy Chain 11 (DNAH11) gene. Classical diagnostic measures like Transmission Electron Microscopy (TEM) are not applicable for mutations in the DNAH11 gene since ultrastructural defects of the ciliary apparatus are absent. Novel mutations encoding for PCD appear all the time with considerable variation in the clinical picture, making it necessary to update data bases and guidelines for PCD diagnostics. METHODS: In this study we examined two unrelated, Finnish families with symptoms of PCD applying the clinical scoring system: Primary ciliary dyskinesia Rule (PICADAR), high speed video microscopy analysis (HSVMA) for ciliary movement, a commercially available gene panel analysis and nasal Nitric Oxide (nNO) measurements if applicable. RESULTS: Two, likely pathogenic variants in the DNAH11 gene (c.2341G > A, p. (Glu781Lys) ja c.7645 + 5G > A) were detected. In the first family, compound heterozygous mutations led to disease manifestation in two of 4 children, which showed a similar phenotype of cilia beating pattern but marked differences in disease severity. In the second family, all three children were homozygotes for the c.2341G > A p.(Glu781Lys) mutation and showed a similar degree of disease severity. However, the phenotype of cilia beating pattern was different ranging from stiff, static cilia to a hyperkinetic movement in one of these children. CONCLUSIONS: In this study we describe two Finnish families with PCD, revealing two novel mutations in the DNAH11 gene which show considerable variety in the clinical and beating cilia phenotype. The results of this study show the clinician that PCD can be much milder than generally expected and diagnosis demands a combination of measures which are only successful in experienced hands. Chronic and repeatedly treated wet cough should raise suspicion of PCD, referring the patient for further diagnostics to a specialised PCD centre.

Comparative Super-Resolution Mapping of Basal Feet Reveals a Modular but Distinct Architecture in Primary and Motile Cilia.

In situ molecular architecture analysis of organelles and protein assemblies is essential to understanding the role of individual components and their cellular function, and to engineering new molecular functionalities. Through a super-resolution-driven approach, here we characterize the organization of the ciliary basal foot, an appendage of basal bodies whose main role is to provide a point of anchoring to the microtubule cytoskeleton. Quantitative image analysis shows that the basal foot is organized into three main regions linked by elongated coiled-coil proteins, revealing a conserved modular architecture in primary and motile cilia, but showing distinct features reflecting its specialized functions. Using domain-specific BioID proximity labeling and super-resolution imaging, we identify CEP112 as a basal foot protein and other candidate components of this assembly, aiding future investigations on the role of basal foot across different cilia systems.

Phenotypic features of ciliary dyskinesia among patients with congenital cardiovascular malformations.

BACKGROUND: Cilia are cell membrane-bound organelles responsible for airway mucus clearance, establishment of left-right organ asymmetry, cardiogenesis, and many other functions in utero. Phenotypic features suggestive of respiratory ciliary dyskinesia among patients with heterotaxy syndrome, defined as complex cardiovascular malformations (CVM) and situs ambiguus (SA), has not been adequately explored. OBJECTIVES: We hypothesized that there is a greater incidence of phenotypic features consistent with ciliary dyskinesia among patients with heterotaxy syndrome compared to patients with other CVM and laterality defects without heterotaxy syndrome. METHODS: Thirty six subjects were identified by medical record search and divided into four groups based on situs status and type of CVM as follows: SA and complex CVM (group 1); SA and simple CVM (group 2); situs solitus and complex CVM (group 3); and situs solitus and simple CVM (group 4). Phenotype was assessed with a clinical questionnaire, nasal nitric oxide (NO) level, and pulmonary function testing. Those with complex CVM underwent additional testing for variants in genes involved in ciliary structure and function. RESULTS: The mean nasal NO level was significantly lower among all subjects with complex CVM regardless of situs anomalies (groups 1 and 3). There was no significant difference in respiratory symptoms or lung function among the four groups. No bi-allelic genetic mutations were detected among patients with complex CVM. CONCLUSIONS: This study identified a relatively lower mean nasal NO level, suggestive of relative ciliary dyskinesia, among subjects with complex CVM. Pulmonary function and clinical symptoms did not reflect significant pulmonary disease among those with complex CVM.

CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia.

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.

Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia.

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.