Systematic Single Cell Rna Sequencing Analysis Reveals Unique Transcriptional Regulatory Networks of Atoh1-Mediated Hair Cell Conversion in Adult Mouse Cochleae

Abstract

Regeneration of mammalian cochlear hair cells (HCs) by modulating molecular pathways or transcription factors is a promising approach to hearing restoration; however, immaturity of the regenerated HCs in vivo remains a major challenge. Single cell RNA sequencing (scRNA-seq) datasets have been critical in identification of transcription factors which regulate the regenerating processes; however, systematic analysis of these datasets, albeit broad interest, has been challenging to many researchers. Here we adopted multiple high-throughput sequencing analytical tools (WGCNA, SCENIC, PAGA, ARACNE-AP and VIPER) and systematically re-analyzed a previously published scRNA-seq dataset during Atoh1-mediated supporting cell (SC) to HC conversion in adult mouse cochleae1. Instead of focusing on differentially expressed genes, our new analysis with WGCNA and SCENIC focuses on connections among co-expressed genes to establish independent expression modules. We independently confirmed the existence of multiple conversion stages during the Atoh1-mediated SC-to-HC conversion process in adult cochleae in vivo. Moreover, we re-analyzed the recently published scRNA-seq datasets of developing neonatal mouse cochleae2 and confirmed that the most differentiated converted HCs of the ectopic Atoh1 expression in adult cochleae resemble endogenous outer HCs (OHCs) at postnatal day 1. Furthermore, our new PAGA trajectory inference analysis revealed that the SC-to-HC conversion process is parallel, but not identical, to the pro-sensory cell-to-OHC path during normal development. Detailed analyses of the gene regulatory networks (GRNs) identified continuous regulatory dynamics for the conversion with multiple key modules and regulons (Nhlh1, Lhx3, Barhl1 and Nfia) that have not been identified previously. Finally, we used ARACNE-AP and VIPER for transcriptional interactions from gene expression data and discovered multiple key master regulators that will likely promote conversion to more mature OHCs. These new results will guide future experimental approaches to HC regeneration for hearing restoration in adult mammalian cochleae in vivo and provide an example as how to perform in-depth analysis with the large amount of scRNA-seq data.