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.

K201 (JTV-519) may prevent abnormal Ca 21 leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca

gel matrix). The mathematical model establishes a closed feedback loop between the calcium system and the extracellular environment that gives rise to the self-regulation we observed. We ran extensive simulations where parameters associated with the influx and efflux of Ca 2þ were modulated such that they are either up-regulated or down-regulated by NO. In silico results are filtered out to qualitatively match cell-in-gel in vitro results. The filtering process is based on measures that capture multiple properties of calcium profiles. Conclusion: Our approach of identification hints that the upregulation of NO has the effect of simultaneously modulating multiple parameters of the Ca 2þ handling pathway. Of the modulated parameters, the L-type current amplitude has to be consistently increased. Coupled with the increase in L-type current, parameters associated with release and uptake of calcium by the SR have to be modulated in opposite directions.

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 ...

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