Figure 3. A gain of lateral induction disrupts the patterning and boundaries of inner ear sensory patches.

(A) Schematic of the pT2K-Hes5::Dll1 Tol2 construct used to induce an artificial gain of lateral induction. The Hes5 promoter drives expression of Dll1, along with eGFP, in transfected cells in which Notch is active. (B–D) Dissected E8 inner ears transfected with either a control Hes5::d2eGFP (B) or a Hes5::Dll1 (C–D) Tol2 construct at E2.5. In the control (B), eGFP fluorescence is detected in distinct sensory patches, in this case the basilar papilla (bp) and two cristae (arrowheads). Transfection with Hes5::Dll1 induces mild (C) or severe (D) defects in the morphogenesis of the vestibular system, and abnormal activation of eGFP expression in the dorsal regions (asterisk in D). (E–H) whole-mount preparations of E11 Hes5::Dll1 transfected cristae, immunostained with HCA and myosin 7A antibodies (in magenta) to identify hair cells. Note the formation of ectopic hair cells (E–F) or abnormal expansion of the posterior crista (arrows in G–G’) towards the macula neglecta (asterisk) compared to the contralateral untransfected posterior crista (H). (I–I’) Jag1 expression is induced (arrows) in the dorsal portion of an otocyst with strong activation of the Hes5::Dll1 construct. (J) Example of an E8 inner ear with ectopic induction of Jag1 and Hes5::Dll1 activity in the dorsal region of the vestibular system.(K–K’) Whole-mount of an E14 inner ear with widespread Hes5::Dll1 induction, immunostained with HCA and myosin 7A antibodies (in magenta). The vestibular system contains ectopic and fused sensory domains (arrows). Hair cells are also present in between the basilar papilla (bp) and the saccule-utricle region (asterisk), which appear to form a continuous sensory patch. (L–L’) Higher magnification view of the dorsal vestibular region of the sample shown in (K). Hair cell density is reduced inside the clusters of transfected cells with strongest eGFP fluorescence (arrows).