High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor.

Tetsuji Yamashita,Zoe Kellard,Robert Carter,Jian Zuo,Charles Gawad,Fei Zheng,David Finkelstein,Ken Sugino,John Easton,Celeste D Rosencrance,Gregory S Barsh

doi:10.1371/journal.pgen.1007552

Abstract

In vivo direct conversion of differentiated cells holds promise for regenerative medicine; however, improving the conversion efficiency and producing functional target cells remain challenging. Ectopic Atoh1 expression in non-sensory supporting cells (SCs) in mouse cochleae induces their partial conversion to hair cells (HCs) at low efficiency. Here, we performed single-cell RNA sequencing of whole mouse sensory epithelia harvested at multiple time points after conditional overexpression of Atoh1. Pseudotemporal ordering revealed that converted HCs (cHCs) are present along a conversion continuum that correlates with both endogenous and exogenous Atoh1 expression. Bulk sequencing of isolated cell populations and single-cell qPCR confirmed 51 transcription factors, including Isl1, are differentially expressed among cHCs, SCs and HCs. In transgenic mice, co-overexpression of Atoh1 and Isl1 enhanced the HC conversion efficiency. Together, our study shows how high-resolution transcriptional profiling of direct cell conversion can identify co-reprogramming factors required for efficient conversion.

Highlights

During development, pluripotent stem cells follow lineage-specific pathways to differentiate into mature cells that can be converted back to pluripotent cells by defined transcription factors (TFs) [1]
(PCs/Deiters’ cells (DCs)) labeled with tdTomato and converted HCs (cHCs) labeled with both tdTomato and EGFP. tdTomato and EGFP expression in Fgfr3iCre+; Atoh1-HA+; Chrna9-GFP+; Rosa26-CAG-loxP-stop-loxP-tdTomato+ cochleae from mice at P33 when induced with tamoxifen at postnatal day 12 (P12) [14, 49]
To further validate the genes we found to be differentially expressed at the protein level, we found that 24 genes that had previously been shown to be differentially expressed in supporting cells (SCs), cHCs, and outer HCs (OHCs) by immunostaining showed consistent expression patterns among SCs, cHCs and OHCs [14], and such patterns were comparable to gene expression profiles determined by single-cell RNA-seq (S3G Fig)

Summary

Introduction

Pluripotent stem cells follow lineage-specific pathways to differentiate into mature cells that can be converted back to pluripotent cells by defined transcription factors (TFs) [1]. Direct lineage conversion ( termed transdifferentiation) between differentiated cells has been demonstrated in heart, pancreas, brain, and other tissues through the use of defined TFs [2,3,4,5] or pharmacologic agents [6]. Such conversions have provided a deeper mechanistic understanding of development and hold promise for regenerative medicine. A more precise understanding of the molecular events underlying Atoh1-induced HC conversion is needed to identify additional factors required for improving the efficiency and completion of the conversion

Methods

Results

Discussion

Conclusion