Fig. 3.

Prosensory markers are up-regulated in E12.5 ears via a trigenic Tet-On system to activate Notch. (A) Three alleles are required in this system: (i) Cre recombinase, driven by the Col2a1 promoter; (ii) reverse tetracycline transactivator (rtTA), under control of the ubiquitously expressed ROSA26 promoter; and (iii) NICD allele, driven by the tetracycline inducible promoter (tetO). A floxed stop cassette is present between the ROSA26 promoter and rtTA, thus confining rtTA expression to the cells in which Cre recombinase is present. An internal ribosome entry site (IRES) followed by the EGFP gene is located downstream of rtTA, thereby allowing tracking of rtTA-expressing cells by EGFP expression. rtTA will bind to tetO in the presence of doxycycline, thereby activating expression of NICD. (B–I) SOX2, JAG1, and EGFP expression in the E12.5 cochlea from the Col2a1-Cre;ROSA-rtTA;tetO-NICD cross. (B–E) Control ears (Col2a1-Cre;ROSA-rtTA bigenic) showing EGFP expression in the dorsal nonsensory regions of the cochlea (arrowheads) that do not express SOX2 and JAG1. (F–I) Trigenic cochlea demonstrating an EGFP-positive cluster that also expresses SOX2 and JAG1. A high-power view of the boxed area is shown in the upper right corner. Note that some SOX2⁺ cells lie outside the EGFP-positive cluster. (Scale bar: 50 μm.)