Fig. 1.

Inducible recombination of the Rb gene locus. (A) Two targeted Rb alleles. Exon 19 of Rb is flanked by two unrecombined loxP sites (black triangles; Rb^lox in B Lower). Cre-mediated deletion of exon 19 results in an allele encoding a nonfunctional Rb protein (Rb⁻ in B Lower). Two combinations of PCR genotyping primer pairs were used to distinguish the Rb^lox allele (283-bp band from the green and blue pair) from the Rb⁻ allele (260-bp band from the yellow and blue pair). (B) PCR analysis, using cochlear genomic DNA of Atoh1-CreER: Rb^lox/lox mice given injections on P0. The primer combination used for each lane is indicated. The Rb⁻ band was not detected after 24 h (lane 1), but it was detected after 30 h (lane 3). The corresponding Rb^lox bands from unrecombined cells are shown as controls (lanes 2 and 4). (C–H) Cre activity within the cochlea as visualized by lacZ staining. The mice were given injections on P0 and P1 and analyzed on P6. (C and F) LacZ-positive (blue) staining of cochlear whole mounts from Atoh1-CreER: Rosa26 mice given tamoxifen injections (C) and mock injections (F). (D and G). Higher magnification reveals that Cre-activity was limited to OHCs (arrow) and IHCs (arrowhead) (blue, lacZ staining in D), whereas no lacZ staining was detected in mice given mock injections (G). (E and H) Myosin7a (green) coimmunostaining of regions in D and G reveals the presence of HCs. Note that fluorescence is masked in cells that express lacZ (E compared with H). [Scale bars: C, 0.25 mm (applies to C and F); and D, 80 μm (applies to D, E, G, and H)]. Note that ≈80% of IHCs and ≈90% of OHCs were lacZ-positive when induced at P0 and P1 under these conditions (14). (I) Illustration of HCs and their locations in different regions within a cochlear whole mount from basal (green dots) to apical (red dots). The apical tip contained the least differentiated HCs at the times analyzed in this study (P4–P6 and P9), whereas the basal HCs were more mature. Consistently, in subsequent figures (unless stated otherwise), arrows denote OHCs, and arrowheads denote IHCs.