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

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex morphological and physiological changes resulting in the formation of the glial scar. This scar contains reactive astrocytes, activated microglia, macrophages and other myeloid cells, meningeal cells, proliferating oligodendrocyte precursor cells (OPCs), and a dense extracellular matrix. Whether the scar is beneficial or detrimental to recovery remains controversial. In the acute phase of recovery, scar-forming astrocytes limit the invasion of leukocytes and macrophages, but in the subacute and chronic phases of injury the glial scar is a physical and biochemical barrier to axonal regrowth. The signals that initiate the formation of the glial scar are unknown. Both canonical and noncanonical signaling Wnts are increased after spinal cord injury (SCI). Because Wnts are important regulators of OPC and oligodendrocyte development, we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. In adult female mice carrying an OPC-specific conditionally deleted β-catenin gene, there is reduced proliferation of OPCs after SCI, reduced accumulation of activated microglia/macrophages, and reduced astrocyte hypertrophy. Using an infraorbital optic nerve crush injury, we show that reducing β-catenin-dependent signaling in OPCs creates an environment that is permissive to axonal regeneration. Viral-induced expression of Wnt3a in the normal adult mouse spinal cord induces an injury-like response in glia. Thus canonical Wnt signaling is both necessary and sufficient to induce injury responses among glial cells. These data suggest that targeting Wnt expression after SCI may have therapeutic potential in promoting axon regeneration.

Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia. Lining the airways, brain ventricles, and oviducts, multicilia are small hair-like structures that beat in a whip-like motion to propel fluids, such as mucus, over cell surfaces. Dysfunction of multicilia arising from genetic perturbations is most prominently associated with a devastating disorder called primary ciliary dyskinesia (PCD). PCD is a rare genetic disease characterized by hydrocephalus, chronic airway infection, and infertility. Furthermore, defective airway multicilia have been implicated in several respiratory diseases, including cystic fibrosis, asthma, and chronic obstructive pulmonary disorder (COPD). While important to human health, the detailed molecular mechanisms of how multiciliated cells develop remain largely unknown. Here, we establish a new mouse model that lacks the key ciliary protein CEP164 in multiciliated cells. These mice recapitulate many symptoms of PCD patients such as hydrocephalus and infertility. We show that, in the absence of CEP164, differentiation of airway multiciliated cells is severely perturbed at multiple steps. Importantly, our data also suggest that CEP164 differentially regulates the proper recruitment of membrane-associated ciliary proteins. In summary, we have developed a powerful mouse model to study diseases affecting multicilia and shed light on novel roles of CEP164 in multiciliogenesis.

Chibby1 (Cby1) is a small, conserved coiled-coil protein that localizes to centrioles/basal bodies and plays a crucial role in the formation and function of cilia. During early stages of ciliogenesis, Cby1 is required for the efficient recruitment of small vesicles at the distal end of centrioles to facilitate basal body docking to the plasma membrane. Here, we identified family with sequence similarity 92, member A (FAM92A) and FAM92B, which harbor predicted lipid-binding BAR domains, as novel Cby1-interacting partners using tandem affinity purification and mass spectrometry. We found that in cultured cell lines, FAM92A colocalizes with Cby1 at the centrioles/basal bodies of primary cilia, while FAM92B is undetectable. In airway multiciliated cells, both FAM92A and -92B colocalize with Cby1 at the base of cilia. Notably, the centriolar localization of FAM92A and -92B depends largely on Cby1. Knockdown of FAM92A in RPE1 cells impairs ciliogenesis. Consistent with the membrane-remodeling properties of BAR domains, FAM92A and -92B in cooperation with Cby1 induce deformed membrane-like structures containing the small GTPase Rab8 in cultured cells. Our results therefore suggest that FAM92 proteins interact with Cby1 to promote ciliogenesis via regulation of membrane-remodeling processes.

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.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164^fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164^fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164^fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164^fl/fl mice. In FoxJ1-Cre;CEP164^fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164^fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility. Abstract Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164^fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164^fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164^fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164^fl/fl mice. In FoxJ1-Cre;CEP164^fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164^fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.