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Fig. 4. p27^Kip1 transcriptional regulation in p27^Kip1-null mice is independent of cell cycle exit. (A-C) Whole-mount preparation of E13.5 cochlear epithelium from a p27^Kip1-null embryo labeled in utero with BrdU. Timed pregnant female received three injections of BrdU every 2 hours, prior to sacrifice 6 hours after the first injection. BrdU staining (A, red) shows labeled cells throughout the epithelium, including in apical and middle regions, which are normally devoid of cycling cells in wild-type cochlea at this time (see Fig. 2). The GFP transgene (B, green) is expressed in an apical to basal gradient the extent of which matches that seen in wild-type embryos at this time (see Fig. 2). Merged image shows double-labeled cells (C, yellow) belonging to abnormally proliferating cells that are within the prosensory domain marked by p27^Kip1/GFP transgene expression. (D-F) Surface reconstruction and cross-section through a p27^Kip1-null E13.5 organ of Corti. (D) Surface reconstruction showing the approximate plane of section (red line) used in E and F. (E) Cross-section shows three turns of the E13.5 cochlea (apex, middle and base) stained for BrdU incorporation. (F) The same section as in E showing native p27^Kip1/GFP reporter. Brackets indicate the site of presumptive organ of Corti formation. Note that BrdU⁺ cells (E, red) are present at all levels (apex, middle and base) in the p27^Kip1-null embryo. GFP (F) can be seen strongly in the apical turn (bracket, apex) and weakly in the middle turn (bracket, mid) of the cochlear duct, but not in the basal turn (base), indicative of the gradient of transgene expression that occurs in spite of the failure of cell cycle exit due to the mutation of p27^Kip1. (G,H) Cross-section through the middle turn of an E14.5 cochlear duct from a p27^Kip1-null mouse, showing the presence of BrdU-labeled cells within the presumptive region of organ of Corti formation (bracket, G). (H) The same section as in G showing the overlapping pattern of p27^Kip1/GFP transgene expression (bracket). Scale bar: 100 µm.

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