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

One potential approach to restore hearing is to enforce regeneration of hair cells (HCs) through induction of cellular signaling pathways in supporting cells (SCs) that promote dedifferentiation and proliferation. We previously showed that the constitutive activation of ERBB2 signaling in cochlear SCs indirectly promoted SC differentiation to HCs, both in vivo and in vitro. In the current study, we aimed at identifying mechanisms and molecular pathways activated in SCs 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. The scRNA-seq analysis of supporting cells with activated ERBB2 signaling was performed using mouse line harboring an inducible “Tet-On” mutant ErbB2 transgene encoding a constitutively active ERBB2 protein (CA-ERBB2), which does not require ligand binding or heterodimerization with other ERBB partners for active signaling. To limit induction of CA-ERBB2 activation to cochlear supporting cells, CA-ERBB2 mouse line was crossed with mouse line harboring an inducible DNA Cre recombinase under control of the supporting cell gene promoter Fgfr3. Double heterozygotic mice harboring both an inducible “Tet-On” CA-ErbB2 and an Fgfr3-iCre transgene were bred to homozygous line harboring a “floxed” TA transcription factor, with an IRES-GFP to use as a lineage marker (“ROSA-rtTA-GFP”). The pups with CA-ErbB2 and Fgfr3-iCre genes (denoted here as “CA-ERBB2”) were used for analysis of transcriptome of supporting cells with activated ERBB2. Pups harboring Fgfr3-iCre were used as “Control”. Activation of iCRE was performed by two injections of tamoxifen at P0 and P1, which resulted in activation of GFP expression to label supporting cells. Induction of CA-ERBB2 expression was performed at P2 by doxycycline injection. The organs of Corti were dissected from P3 pups, and GFP+ supporting cells were purified by fluorescence-activated cell sorting (FACS). RNA cDNA libraries were prepared and sequenced from about 300 GFP+ single cells per genotype collected in three independent FACS experiments.