Figure 3. Deletion of Jag1 from neonatal SCs results in the loss of Hensen's cells. A, Experimental strategy. Sox2CreERT2/+::Jag1fx/fx and Jag1fx/fx were treated with 4-hydroxy-tamoxifen at P0/P1, and their cochleae were analyzed at P5 or P7. Untreated control mice (Sox2CreERT2/+::Jag1fx/fx untreated and Jag1fx/fx, untreated) were also used. B, C, Validation of conditional Jag1 deletion. Shown is the SC nuclear layer, immunostained for JAG1 (green), in Jag1 CKO (C) and control (B, Jag1fx/fx treated) mice. D–I, SOX2 immunostaining (D–E′, blue; F–I, green) labels SC nuclei, including Hensen's cells. FABP7 (magenta) labels Hensen's cells, inner phalangeal cells, and border cells. Parvalbumin (F–I, blue) labels HCs, and CD44 (D–E′, green) labels Claudius cells and outer pillar cells. Shown are representative single-plane confocal images of the cochlear mid-turn. D–E′, Top down view of the HC (D,E) and SC nuclear layers (D′,E′) in Jag1 CKO (E,E′) and control (D,D′, Sox2CreERT2/+::Jag1fx/fx untreated) mice. Black arrows indicate the location of Hensen's cells residing within the HC (D) and SC layer (D′) in control mice. F–I, Cochlear cross-sections of Jag1 CKO (G,I) and control mice (F,H, Sox2CreERT2/+::Jag1fx/fx, untreated). White arrows indicate 2 or 3 Hensen's cell nuclei that are stacked on top of each other in control mice. Red arrowheads indicate SOX2+ Hensen's cell-like cells (G,I). Yellow arrowheads indicate dying cell (I) and missing Hensen's cell (G) in Jag1 CKO mice. J, Quantification of HC and SC subtypes in control and Jag1 CKO mice. Data are mean ± SD; n = 3/group. *p < 0.05; **p ≤ 0.001; ***p ≤ 0.0001; two-way ANOVA, with Bonferroni correction. DC1, Deiters' cell row 1; DC2, Deiters' cell row 2; DC3, Deiters' cell row 3; HeC, Hensen's cell; IPC, inner pillar cell; IPhC, inner phalageal cell; OPC, outer pillar cell. Scale bar, 20 μm.