Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT)

Centrosomal protein 164 (CEP164) is located at the edge of distal appendages in primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of CEP164 before and after BB docking in photoreceptors, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction. BBs dock to the cell cortex during postnatal day 6 (P6) and extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of a CC or outer segments (OS). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P9 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OS. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at BB/CC. Impairment of IFT may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations. Author summaryCentrosomal protein of 164 kDa (CEP164) is indispensable for docking of basal bodies to apical membranes and formation of primary cilia. Homozygous truncations of CEP164 are associated with severe ciliopathy whereas missense mutations generate milder forms of NPHP. Recessive mutations of CEP164 are associated with nephronophthisis (NPHP), Meckel-Gruber (MKS) and Bardet-Biedl syndromes (BBS) in which primary cilia fail to form, or do not function correctly. We found that deletion of CEP164 in mouse photoreceptors before docking of the basal body (BB) to the apical inner segment membrane prevents elaboration of connecting cilia (CC, equivalent to transition zones) and outer segments (OSs, modified primary cilia). We also generated mouse models in which deletion of CEP164 occurs after OS are established and observed that the BB/CC and axoneme are initially stable in the absence of CEP164. Loss of CEP164, however, diminished recruitment of IFT-B proteins to the BB/CC affecting intraflagellar transport (IFT), which is required for maintenance of cilia and OSs. We propose that, in ciliopathies caused by missense mutations (e.g., X1460WextX57 associated with LCA) impairment of IFT provides a mechanism for ciliary dysfunction and disease.