Conditional knockout mice for the distal appendage protein CEP164 reveal its essential roles in airway multiciliated cell differentiation

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.
Author summary 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.