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Fig. 2. A wave of cell cycle exit and p27^Kip1 expression define the prosensory domain within the embryonic cochlea. (A) Cochlear whole mounts from mice labeled with BrdU at the time of sacrifice (E12.5, E13.5 and 14.5). BrdU was injected on indicated days 2 hours prior to euthanasia, and whole mounts were fixed and stained with antibody to BrdU. The base and apex of the cochlea are indicated. Dotted outlines indicate the region of the cochlear duct that is devoid of BrdU-labeled cells, indicating that cells in this region have stopped dividing prior to the time of the injection. (B) Quantification of hair cell cycle exit on E12.5, E13.5 and E14.5. Timed pregnant female mice were injected with BrdU at the indicated times (E12.5, E13.5, E14.5) and allowed to survive until E18.5, at which time hair cell differentiation along the entire length of the organ of Corti is complete. Each surface preparation is stained for BrdU, to indicate those cells in S phase of the cell cycle at the time of injection, and with antibody to Myosin VIIa, a marker for differentiated hair cells. The E18.5 preparation was divided into seven segments of equal length and myosin VIIa⁺ hair cells were counted from each segment and compared with double-labeled Myosin VIIa⁺, BrdU⁺ hair cells in the same region. The percentage of BrdU⁺ hair cells per segment is shown (error bars indicate s.d.). (C) Whole-mount preparations of embryonic cochlea at E12.5, E13.5 and E14.5 stained with anti-p27^Kip1. Antibody staining appears as a gradient with the highest intensity in the apical third of the nascent organ of Corti at E12.5. The gradient increases in length with the growing cochlear duct and the gradient of expression appears in approximately two-thirds of the length of the cochlea at E13.5. By E14.5 the p27^Kip1 gradient of expression has nearly reached the base of the cochlea, coincident with the termination of cell division within the presumptive organ of Corti at this time. (D) Reconstruction of cochlear and vestibular components of the inner ear at E14.5 depicting the level of cross-section shown in E,F. (E,F) Cross-section through an E13.5-E14.5 cochlea stained for BrdU (E) and p27^Kip1 (F). BrdU was injected three times at 2-hour intervals into a timed pregnant female mouse starting 6 hours prior to removing and fixing the embryos. The section was double-labeled to reveal both BrdU incorporation and p27^Kip1 expression. The absence of BrdU staining in the apical and middle turns indicates the presence of a zone of non-proliferating cells in the apical and middle turns (E, apex and mid bracket). BrdU-labeled cells are seen in the basal turn, indicating that the ZNPC has not yet formed in this region. p27^Kip1 staining is present in the apical and middle turns, but absent from the base (F), coincident with the apical to basal progression of cell cycle exit as revealed by the BrdU labeling (E). Scale bar: 100 µm.

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