Spontaneous Hair Cell Regeneration Is Prevented by Increased Notch Signaling in Supporting Cells

Abstract

During embryonic development, differentiation of cochlear progenitor cells into hair cells (HCs) or supporting cells (SCs) is partially controlled through Notch signaling. Many studies have shown that inhibition of Notch signaling allows SCs to convert into HCs in both normal and drug damaged neonatal mouse cochleae. This mechanism is also implicated during HC regeneration in non-mammalian vertebrates; however, the mechanism of spontaneous HC regeneration in the neonatal mouse cochlea is less understood. While inhibition of Notch signaling can force SCs to convert into HCs and increase the number of regenerated HCs, it is currently unknown whether this pathway is involved in spontaneous HC regeneration observed in vivo. Therefore, we investigated the role of Notch signaling during the spontaneous HC regeneration process using Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+ mice injected with tamoxifen at postnatal day (P) 0 and P1 to ablate HCs and stimulate spontaneous HC regeneration. Expression changes of genes in the Notch pathway were measured using immunostaining and in situ hybridization, with most changes observed in the apical one-third of the cochlea where the majority of HC regeneration occurs. Expression of the Notch target genes Hes1, Hes5, Hey1, HeyL, and Jagged1 were decreased. To investigate whether reduction of Notch signaling is involved in the spontaneous HC regeneration process, we overexpressed the Notch1 intracellular fragment (N1ICD) in cochlear SCs and other non-sensory epithelial cells in the context of HC damage. Specifically, Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+::Sox10rtTA::TetO-LacZ::TetO-N1ICD mice were injected with tamoxifen at P0/P1 to stimulate spontaneous HC regeneration and given doxycycline from P0–P7 to induce expression of N1ICD as well as LacZ for fate-mapping. We observed a 92% reduction in the number of fate-mapped regenerated HCs in mice with N1ICD overexpression compared to controls with HC damage but no manipulation of Notch signaling. Therefore, we conclude that increased Notch signaling prevents spontaneous HC regeneration from occurring in the neonatal mouse cochlea. Understanding which components of the Notch pathway regulates regenerative plasticity in the neonatal mouse cochlea will inform investigations focused on stimulating HC regeneration in mature cochlea and eventually in humans to treat hearing loss.

Hear, Hear for Notch: Control of Cell Fates in the Inner Ear by Notch Signaling

“…Thus, when N1ICD is expressed as hair cells begin to differentiate, hair cells begin to upregulate supporting cell-specific genes and the animals become functionally deaf with the hair cells dying, but if N1ICD is expressed in hair cells after they differentiate, they become largely unresponsive to Notch signaling-induced fate changes, upregulating the supporting cell genes Sox2 and Prox1, but otherwise showing no other detectable effects on hair cell maturation [95,96]. Over-expression of N1ICD in supporting cells of neonatal mice also reduces the spontaneous regeneration of hair cells that can occur in early postnatal life [97]. Loss of Notch1, Dll1, Jag2, or other Notch pathway genes during hair cell differentiation results in the generation of supernumerary hair cells (see Table 1), as does the inhibition of gamma secretases through pharmacological agents like DAPT [61,90].…”

Multiple supporting cell subtypes are capable of spontaneous hair cell regeneration in the neonatal mouse cochlea

“…Some have hypothesized that Lgr5positive SCs are the progenitor cells within the neonatal mouse cochlea (Bramhall et al, 2014;Chai et al, 2012;McLean et al, 2017;Shi et al, 2012;Shi et al, 2013;Waqas et al, 2016); however, the Lgr5-postive population includes different subtypes of SCs, and the expression of Lgr5 changes dynamically during the first postnatal week (Chai et al, 2011;Shi et al, 2012). In addition, we have recently shown that spontaneous HC regeneration in vivo can be prevented by increased Notch signaling (McGovern et al, 2018), which suggests that the SC subtypes that respond to changes in Notch signaling after HC damage contribute to spontaneous regeneration.…”

“…Recently, evidence has emerged that the neonatal mouse cochlea has a limited period in which it is capable of spontaneously regenerating HCs after they have been destroyed, both in vivo (Cox et al, 2014;Hu et al, 2016;McGovern et al, 2018) and in vitro (Bramhall et al, 2014). To demonstrate that regeneration had occurred, broad populations of SCs were fate-mapped using either Hes5 lacZ knock-in reporter mice, Lgr5 CreER ::Rosa26 tdTomato mice or Sox2 CreERT2 ::Rosa26 tdTomato mice (Bramhall et al, 2014;Cox et al, 2014).…”

“…We recently showed that, during the spontaneous HC regeneration process, PCs and DCs lose expression of Hes5, a Notch effector and inhibitor of HC fate (Abdolazimi et al, 2016;Mulvaney and Dabdoub, 2012), whereas IPhCs and BCs showed no change in Hes5 expression (McGovern et al, 2018). This may suggest that PCs and DCs have an increased ability to regenerate HCs compared with other SC subtypes, yet further investigation is needed.…”

Characterizing Adult cochlear supporting cell transcriptional diversity using single-cell RNA-Seq: Validation in the adult mouse and translational implications for the adult human cochlea

“…These Lgr5-expressing cells in the neonatal cochlea possess a latent potential to proliferate and to convert into hair cells (Cox et al 2014; Stone and Cotanche 2007). This mitotic regeneration has been explored in supporting cells as a potential avenue for hair cell regeneration with Wnt activation and Notch signaling playing roles in supporting cell conversion into hair cells (Ni et al 2016; McGovern et al 2018, 2019). Consistent with these observations, protein interaction analyses suggest potentially relevant interactions between Notch signaling and Wnt signaling pathways and cell cycle control, specifically Cdkn1b (Figure 6G).…”