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.

During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2 CreERT2/1 ::Jag1 loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1, some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1. Finally, using Fgfr3-iCreER T2 ::Jag1 loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range.

22During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important 23 role in the specification of the prosensory region, which gives rise to sound-sensing hair 24 cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in 25 SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we 26 demonstrate that JAG1 is essential for the formation and maintenance of Hensen cells 27 (HeCs), a highly specialized SC-subtype located at the edge of the auditory epithelium. 28Deletion of Jag1 at the onset of differentiation, at stage E14.5, disrupted HeC formation. 29Similar loss of HeCs was observed when Jag1 was deleted at P0/P1 and fate-mapping 30 analysis revealed that in the absence of Jag1 some HeCs die, but others convert into 31 neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved 32 in mitochondrial function and protein synthesis were downregulated in P0 cochlea lacking 33 Jag1. 34 35 106 deletion disrupted the formation or patterning of SCs and/or HCs. To label HCs, we used 107 immuno-staining against myosin VIIa, which labels both inner HCs (IHCs) and outer HCs 108 (OHCs) (Hasson et al., 1995). To visualize SCs, we immuno-stained against SOX2, which 109 at E18.5/P0 is highly expressed in the nucleus of all SC subtypes, including HeCs, and is 110 expressed at a lower level in the nucleus of IHCs and OHCs (Kempfle et al., 2016). We 111 found that Jag1 deletion had no effect on the number (density) of IHCs or OHCs (Fig. 1E, 112 F, I). Our analysis of the SC phenotype, however revealed a significant reduction in the 113 number of HeCs in Jag1 CKO mice compared to all three controls ( Fig. 1E-I). In cochlear 114 tissue of control animals, SOX2 + HeCs were located between the 3 rd row of DCs and 115 Claudius cells (CCs), with their nuclei residing in both the HC and SC layers, and with 2-116 3 HeCs sitting on top of each other (Fig. 1A, E, G). By contrast, Jag1 CKO cochlear tissue 117 contained either no or only a few scattered HeCs within the HC and SC layers (Fig. 1F, 118 H). 119In addition, DCs in Jag1 CKO mice had enlarged nuclei compared to control mice, 120 and their arrangement appeared to be disorganized, suggesting defects in DC 121 differentiation ( Fig. 1G, H). Decreased numbers of DCs in the 2 nd and 3 rd row (DC2 and 122 DC3) was observed in Jag1 CKO samples compared to the Jag1 fx/fx treated and/or 123 Jag1 fx/fx untreated control groups, but not when compared to Sox2 CreERT2/+ ::Jag1 fx/fx 124 untreated controls (Fig. 1I). A similar result was observed for IPCs and OPCs (Fig. 1I), 125 suggesting that Jag1 deficiency combined with Sox2 haploinsufficiency negatively affects 126 the differentiation of DCs and PCs. Unfortunately, we were unable to address how Jag1 127 deficiency combined with Sox2 haploinsufficiency may impact DC and PC maturation as 128 conditional deletion of Jag1 at E14.5 resulted in early postnatal lethality. In summary, our 129 analysis demonstrates that JAG1's function is ...