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

Abstract

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.

Fig 1. CEP164 is essential for early embryonic development and primary ciliogenesis.

(A) Comparison of control (WT or heterozygous) embryos with CEP164-knockout (KO) littermates at E7.5, E9.5, and E10.5. At E7.5, KO embryos were indistinguishable from control littermates. In contrast, E9.5 and E10.5 KO embryos displayed holoprosencephaly (arrow), an edematous pericardial sac (white arrowhead), cardiac looping defects (blue arrowhead), and a truncated posterior trunk (blue asterisk). Confocal images of neural tube sections from E9.5 control and KO embryos are presented in lower right panels. Primary cilia were labeled with the ciliary marker Arl13b (green), and nuclei are visualized with DAPI staining (blue). Asterisks indicate the lumen of the neural tube. Scale bar, 35 μm. (B) Loss of primary cilia in CEP164-KO MEFs. Mouse embryonic fibroblasts (MEFs) were prepared from E8.5 CEP164-KO or control embryos, serum-starved for 48 hours to induce primary cilia, and immunostained for Arl13b (green) and the basal body marker γ-tubulin (G-tub) (red). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Quantification is shown on the right. >200 cells were counted for each of three independent MEF preparations per genotype. Error bars represent ±SEM. **, p<0.01. (C) Schematic depiction of the localization of Cby1, FAM92, and CEP164 at the ciliary base. Basal bodies and centrioles are barrel-shaped structures composed of nine microtubule triplets. During ciliogenesis, mother centrioles transform into basal bodies by acquiring accessary structures to assemble cilia. The axoneme is the detergent-insoluble cytoskeletal structure of the cilium including microtubules and their associated proteins. (D) Serum-starved MEFs were double-labeled for CEP164, Cby1, or FAM92A (green) and the ciliary marker acetylated α-tubulin (A-tub) or G-tub (red) as indicated. Nuclei were visualized by DAPI (blue). The boxed regions are enlarged in insets, highlighting the loss of CEP164, Cby1, and FAM92A centriolar localization in CEP164-KO MEFs. Arrowheads point to primary cilia. Scale bars, 10 μm.

Fig 2. Ablation of CEP164 in the FOXJ1-positive tissues results in loss of airway and ependymal multicilia and hydrocephalus.

(A) Hematoxylin and eosin (H&E)-stained tracheal and sinus sections from control CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult mice. Scale bar, 20 μm. (B) Shown are lateral views of postnatal day 18 (P18) mice (left panels), coronal sections of adult brains (middle panels), and IF staining for A-tub (green) and β-catenin (red) in whole mounts of the adult subventricular zone (SVZ) (right panels). Asterisks denote enlarged lateral ventricular spaces indicative of hydrocephalus. Scale bar, 25 μm.

Fig 3. CEP164 plays an important role in proper development of female and male reproductive systems.

(A) H&E staining of oviduct sections from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult mice. The boxed regions are enlarged in insets. Scale bar, 10 μm. (B) IF staining of oviduct sections for A-tub (green) and DAPI (blue). Scale bar, 50 μm. (C) H&E staining of both testis and epididymis from 3-month-old CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice. Asterisk denotes seminiferous tubules lacking germ cells. Scale bars, 100 μm for low magnification and 40 μm for high magnification. (D) X-gal staining of testis sections from a control WT mouse and a mouse heterozygous for the CEP164 KO-first allele that contains a lacZ reporter. Scale bar, 40 μm.

Fig 4. Ablation of CEP164 leads to defective airway multiciliogenesis.

(A) ALId14 MTECs were stained for CEP164 (green) and A-tub (red). Nuclei were stained with DAPI (blue). Arrowheads denote CEP164-KO multiciliated cells with sparse, stubby cilia. Zoomed views of cilia are shown for the squared areas. Arrows indicate a multiciliated cell with CEP164 expression that escaped Cre-mediated recombination in FOXJ1-Cre;CEP164^fl/fl MTEC cultures. Scale bars, 10 μm and 5 μm for zoomed images. (B) Schematic model depicting the stages of multiciliated cell differentiation. See text for details. (C) Quantification of multiciliated cells at different stages (I-IV) of ciliogenesis. MTECs from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice were fixed at ALId5, d7, and d14 and immunostained for A-tub. n>225 total cells per ALI day from each of three independent MTEC preparations per genotype. (D) Quantification of fully ciliated cells. MTECs from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice were fixed at ALId14 and immunostained for A-tub. The percentages were calculated by dividing the number of fully ciliated cells with abundant cilia by total cell number. n>250 total cells from each of three independent MTEC preparations per genotype. Error bars represent ±SEM. **, p<0.01.

Fig 5. CEP164 regulates small vesicle recruitment to basal bodies in multiciliated cells.

(A) TEM images of multiciliated cells from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult tracheas. Asterisks depict multiple cytoplasmic basal bodies in FOXJ1-Cre;CEP164^fl/fl trachea. Scale bar, 500 nm. (B) TEM images of cross sections through the apical region of multiciliated cells from ALId14 MTECs derived from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl adult tracheas. Asterisks depict multiple cytoplasmic, misoriented basal bodies in FOXJ1-Cre;CEP164^fl/fl multiciliated cells. Scale bars, 500 nm. (C) TEM images of CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl P8 tracheas subjected to ex vivo culture in the presence of taxol to enrich vesicle-bound basal bodies. Arrowheads denote vesicles attached to the distal end of the cytoplasmic centriole in control CEP164^fl/fl samples. Scale bar, 200 nm.

Fig 6. CEP164 recruits Cby1 and FAM92 proteins to basal bodies in multiciliated cells.

MTECs from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice were immunostained for Cby1 (A), FAM92A (B), or FAM92B (C) (green) and G-tub (red) as indicated. DAPI staining (blue) marks nuclei in merged images. Scale bars, 5 μm.

Fig 7. CEP164 is dispensable for the basal body localization of IFT88, IFT20, and CP110 in multiciliated cells.

ALId14 MTECs from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice were immunostained for IFT components (A) or CP110 (B) (green) and A-tub (red). Nuclei were detected with DAPI (blue). All multiciliated cells were at early stage IV except for the fully differentiated cell (Fully Ciliated) imaged to show the clear basal body localization of CP110. The insets show zoomed views of the squared areas. Scale bars, 5 μm.

Fig 8. CEP164 is required for the proper targeting of ciliary membrane proteins in multiciliated cells.

(A) CEP164^fl/fl or FOXJ1-Cre;CEP164^fl/fl MTECs at ALId14 were immunostained for Rab8 or Rab11 (green) and A-tub (red) and subjected to super-resolution SIM imaging. Arrowheads point to individual CEP164-KO multiciliated cells. Scale bar, 5 μm. (B) ALId14 MTECs from CEP164^fl/fl and FOXJ1-Cre;CEP164^fl/fl mice were immunostained for Arl13b or INPP5E (green) and A-tub (red) and imaged by SIM. Arrowheads indicate individual CEP164-KO multiciliated cells. Scale bar, 5 μm.