Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea

Abstract

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Transcription Factor Reprogramming in the Inner Ear: Turning on Cell Fate Switches to Regenerate Sensory Hair Cells

“…In vivo studies employing a hair cell damage model (Pou4f3DTR) in adult mice showed that adenoviral delivery of ATOH1 and GFI1 together post hair cell ablation in the organ of Corti led to the transdifferentiation of supporting cells to give rise to hair cells at a significantly higher efficiency than ATOH1 alone (Lee S. et al, 2020).…”

“…Hence, GFI1 may play a dual role in fine-tuning hair cell differentiation by repressing non-hair cell genes (particularly neuronal genes), in addition to enabling the expression of hair cell-specific genes. In vivo studies employing a hair cell damage model ( Pou4f3DTR ) in adult mice showed that adenoviral delivery of ATOH1 and GFI1 together post hair cell ablation in the organ of Corti led to the transdifferentiation of supporting cells to give rise to hair cells at a significantly higher efficiency than ATOH1 alone (Lee S. et al, 2020 ).…”

“…One of several interventions explored is the ectopic expression of hair cell-specific transcription factors such as ATOH1 to reprogram non-sensory inner ear cells into hair cells. Studies over the past 20 years have shown that ATOH1 successfully reprograms non-sensory cells adjacent to the organ of Corti to form hair cells in the neonatal mouse cochlea, and a small number of studies have reported a similar result in older animals, although at far lower efficiency (Kelly et al, 2012 ; Lee S. et al, 2020 ). This age-dependent decline in the reprogramming ability of ATOH1 led to a search for additional transcription factors to reprogram older cochlear cells into hair cells.…”

High-resolution single cell transcriptome analysis of zebrafish sensory hair cell regeneration

“…As in the lateral line, Notch downregulation leads to proliferation in the retina, which is enhanced by lin28a (Elsaeidi et al, 2018;Romero-Carvajal et al, 2015). Other genes involved in progenitor quiescence are insm1a and the Tgfb inhibitor tgif1a (Chablais and Jazwinska, 2012;Lenkowski et al, 2013;Ramachandran et al, 2012). Likewise, some lateral line regeneration genes have also been described in fin and heart regeneration, such as inhba/b (Dogra et al, 2017;Sehring et al, 2016;Wang et al, 2020).…”

“…(Bucks et al, 2017;Burns et al, 2012;Burns and Stone, 2017;Cox et al, 2014;Du et al, 2013;Forge et al, 1993;Korrapati et al, 2013;Warchol and Corwin, 1996;White et al, 2006). One strategy to identify candidate genes and pathways that might trigger hair cell regeneration in mammals has been to identify genes involved in hair cell development (Burns et al, 2015;Cai and Groves, 2015;Jen et al, 2019;Lee et al, 2020;Sayyid et al, 2019;Scheffer et al, 2015;Valdivia et al, 2011;Zhu et al, 2019).…”