Defective primary cilia cause a range of diseases known as ciliopathies, including hearing loss. The etiology of hearing loss in ciliopathies, however, remains unclear. We analyzed cochleae from three ciliopathy mouse models exhibiting different ciliogenesis defects: Intraflagellar transport 88 (Ift88), Tbc1d32 (a.k.a. bromi), and Cilk1 (a.k.a. Ick) mutants. These mutants showed multiple developmental defects including shortened cochlear duct and abnormal apical patterning of the organ of Corti. Although ciliogenic defects in cochlear hair cells such as misalignment of the kinocilium are often associated with the planar cell polarity pathway, our results showed that inner ear defects in these mutants are primarily due to loss of sonic hedgehog signaling. Furthermore, an inner ear-specific deletion of Cilk1 elicits low-frequency hearing loss attributable to cellular changes in apical cochlear identity that is dedicated to low-frequency sound detection. This type of hearing loss may account for hearing deficits in some patients with ciliopathies.

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The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1 mice with Exoc5 mice for hair cell-specific deletion (Gfi1;Exoc5) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5). Gfi1;Exoc5 mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5 mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.

Primary cilia have essential roles as signaling centers during development and adult homeostasis. Disruption of ciliary structure or function causes congenital human disorders called ciliopathies. Centriolar distal appendage (DAP) proteins are important for anchoring cilia to the membrane. However, the exact functions of DAP during in vivo ciliogenesis and animal development remain poorly understood. Here, we showed that the DAP component sodium channel and clathrin linker 1 (Sclt1) mutant mice had abnormal craniofacial and limb development with postnatal lethality. In mutant embryos, most of the affected tissues had defects in DAP recruitment to the basal body and docking to the membrane that resulted in reduced ciliogenesis and disrupted hedgehog (Hh) signaling in limb bud mesenchymal cells. However, limb digit formation and ciliogenesis in Sclt1 mutant mice were differentially affected between the fore- and hindlimb buds. The forelimbs developed normally in Sclt1 mutants, but the hindlimbs had preaxial polydactyly. Heterozygous loss of Cep83, another core DAP component, in Sclt1 mutant mice, caused forelimb and hindlimb polydactyly. These findings revealed the tissue-specific differential requirement of DAPs. Taken together, these results indicated that during limb development the ciliary base components, DAPs, play an essential role in ciliogenesis and Hh signaling in vivo in a position-dependent manner.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). While mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; Ift88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix (ECM), mesenchymal-epithelial transition, canonical Hh, and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.