Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor, a candidate mediator of cochlear regeneration mechanisms

Abstract

Hearing loss is generally caused by loss of the hair cells (HCs) in the organ of Corti due to noise trauma or ototoxic medications. While in birds, amphibians, and reptiles HCs are regenerated from supporting cells (SCs), mammals show only limited regenerative capacity at the neonatal stage, which quickly declines with age. One potential approach to restore hearing is to enforce regeneration of HCs through induction of cellular signaling pathways in SCs that promote dedifferentiation and proliferation. In a previous study, we showed that the constitutive activation of ERBB2 signaling in cochlear supporting cells indirectly promoted supporting cell differentiation to hair cells, both in vivo and in vitro. In the current study, we aimed at identifying mechanisms and molecular pathways activated in supporting cells with constitutive ERBB2 signaling. To this end, we used single cell RNA sequencing (scRNA-seq) to characterize the transcriptomes of individual neonatal mouse cochlear SCs that were induced to express ERBB2. We found that induction of ERBB2 in vivo resulted in generation of a new distinct cluster of cells with unique transcriptome. This population has de novo expression of two members of the small integrin-binding ligand n-linked glycoproteins (SIBLING) family and their regulators. We confirm expression of the SIBLING Secreted phosphoprotein 1 (SPP1) as one of the most up-regulated genes in response to ERBB2 activation. SPP1 mediates signaling through the CD44 receptor, promoting survival, proliferation, and differentiation of osteoblast lineage cells. Histological analyses of cochlear samples collected from young adult mice confirmed that induction of ERBB2 after noise exposure resulted in up-regulation of both SPP1 and its receptor CD44, and the formation of mitotic sensory stem-like cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2-signaling in young adult mice secondarily promotes SPP1/CD44 signaling, possibly altering the microenvironment of the organ of Corti.