The Kölliker’s organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker’s organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker’s organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker’s organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker’s organ prior to apoptosis during the early postnatal development.

GJB2 and GJB6 are adjacent genes encoding connexin 26 (Cx26) and connexin 30 (Cx30), respectively, with overlapping expressions in the inner ear. Both genes are associated with the commonest monogenic hearing disorder, recessive isolated deafness DFNB1. Cx26 plays an important role in auditory development, while the role of Cx30 in hearing remains controversial. Previous studies found that Cx30 knockout mice had severe hearing loss along with a 90% reduction in Cx26, while another Cx30 knockout mouse model showed normal hearing with nearly half of Cx26 preserved. In this study, we used CRISPR/Cas9 technology to establish a new Cx30 knockout mouse model (Cx30−/−), which preserves approximately 70% of Cx26. We found that the 1, 3, and 6-month-old Cx30−/− mice showed mild hearing loss at full frequency. Immunofluorescence and HE staining suggested no significant differences in microstructure of the cochlea between Cx30−/− mice and wild-type mice. However, transmission electron microscopy showed slight cavity-like damage in the stria vascularis of Cx30−/− mice. And Cx30 deficiency reduced the production of endocochlear potential (EP) and the release of ATP, which may have induced hearing loss. Taken together, this study showed that lack of Cx30 can lead to hearing loss with an approximately 30% reduction of Cx26 in the present Cx30 knockout model. Hence, Cx30 may play an important rather than redundant role in hearing development.

Greater epithelial ridge cells, a transient neonatal cell group in the cochlear duct, which plays a crucial role in the functional maturation of hair cell, structural development of tectorial membrane, and refinement of audio localization before hearing. Greater epithelial ridge cells are methodologically homogeneous, while whether different cell subtypes are existence in this intriguing region and the degeneration mechanism during postnatal cochlear development are poorly understood. In the present study, single-cell RNA sequencing was performed on the cochlear duct of postnatal rats at day 1 (P1) and day 7 (P7) to identify subsets of greater epithelial ridge cell and progression. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to examine genes enriched biological processes in these clusters. We identified a total of 26 clusters at P1 and P7 rats and found that the cell number of five cell clusters decreased significantly, while four clusters had similar gene expression patterns and biological properties. The genes of these four cell populations were mainly enriched in Ribosome and P13K-Akt signal pathway. Among them, Rps16, Rpsa, Col4a2, Col6a2, Ctsk, and Jun are particularly interesting as their expression might contribute to the greater epithelial ridge cells degeneration. In conclusion, our study provides an important reference resource of greater epithelial ridge cells landscape and mechanism insights for further understanding greater epithelial ridge cells degeneration during postnatal rat cochlear development.

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2loxP/loxP; ROSA26CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker’s organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker’s organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function.