Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti

Abstract

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

Figure 1

Developmental expression of p27^Kip1 in the mouse organ of Corti revealed by immunocytochemistry in radial sections. The organ of Corti is composed of two sensory cell types arranged in one row of IHCs and three rows of OHCs. There are five different types of supporting cells whose nuclei lie below or adjacent to the hair-cell nuclei. These types include inner phalangeal cells (IPC), inner and outer pillar cells (PC), Deiters’ cells (DC), and Hensen’s cells (HC). Hair-cell and supporting-cell regions are indicated by arrows. Labeling with antibodies against p27^Kip1 shows that the protein is expressed only in the supporting-cell nuclei and not in the hair-cell nuclei. (A) Wild-type (p27+/+) mouse at postnatal day 0. (B) p27+/+ mouse at postnatal day 7; note the partially opened tunnel of Corti to the right of the outer PC. (C) p27+/+ adult mouse; note the fully opened tunnel of Corti. (D) The organ of Corti of the adult p27−/− animal shows no labeling; note the partially occluded tunnel of Corti. (Bar = 50 μm.)

Figure 2

Cell proliferation in the organ of Corti. (A) A whole-mount preparation of the p27+/+ organ of Corti at P11 after six BrdUrd injections between P7 and P10 to label proliferating cells. No BrdUrd-labeled cells are visible in the organ of Corti of p27+/+ control mice. (B) In p27−/− mice BrdUrd immunocytochemistry reveals labeled nuclei in the IHC (arrowhead) and OHC (open arrow) regions. (C) Higher magnification of a different region in the same animal shown in B demonstrating a BrdUrd-labeled doublet located at the luminal surface of the sensory epithelium between the pillar-cell region and the OHC region. (D) A whole-mount preparation of the p27−/− adult showing a BrdUrd-labeled doublet at the luminal surface of the sensory epithelium in the OHC region. (E) Transmission electron micrograph of a cell located between two IHCs containing condensed chromosomes in a P7 p27−/− pup. The arrow points to a region containing a centrosome and spindle microtubules. (F) The area indicated by the arrow in E here is shown at a higher magnification revealing the centrosome (arrowhead) and associated microtubules. [Bars = 50 μm (A and B), 20 μm (C and D), 10 μm (E), 0.5 μm (F).]

Figure 3

Morphological comparison of the organ of Corti of adult p27+/+ (wild-type) and p27−/− (homozygote) mice. SEMs of the apical region of p27+/+ (A) and p27−/− (B) are shown. Note that p27−/− has the fairly typical pattern of three to four rows of OHCs and one row of IHCs as seen in the p27+/+. However, additional (arrow) and abnormal-appearing hair-cells (arrowhead) are seen in p27−/−. Also note that in the region between the IHCs and first row of OHCs in p27−/− there is disorganization of the normal pattern of rectangular apices of the inner pillar cells, with a higher density of irregular apices. (C) In p27+/+ at the level of the OHC nuclei, two Deiters’-cell phalangeal processes (arrows) normally can be seen adjacent to a single OHC. (D) In the p27−/− mice a single OHC is surrounded by up to six Deiters’-cell phalangeal processes (arrows). (E) Radial sections of p27+/+ show normal pillar-cell and phalangeal-cell morphology and number, with three OHC rows and three Deiters’-cell rows (∗), with normal spaces of Nuel and tunnel of Corti (TC). (F) In radial sections of p27−/−, there is an increase in the number of Deiters’ cells (∗, six) relative to the number of OHC rows (four) present in the section. A Deiters’ cell displaying features consistent with apoptotic cell death is also visible (arrowhead). Normal ultrastructural characteristics such as large microtubule bundles can be observed within the pillar cells (arrow), although the cells display distortion that has led to partial collapse of the tunnel of Corti (TC). Also note vesiculation in OHCs. (G) SEM of the basal region of the adult p27−/− mouse cochlea showing the presence of a row of IHC stereociliary bundles but a reduced number of OHC bundles. (H) SEM of a basal turn in a postnatal day 7 (P7) p27−/− organ of Corti showing a similar supporting-cell phenotype as the adult but up to four rows of OHC bundles. [Bars = 10 μm (A, B, E–H) and 2 μm (C and D).]

Figure 4

ABR thresholds as a function of frequency in the wild-type (p27+/+), heterozygous (p27+/−), and homozygous (p27−/−) mice. Thresholds in both the p27+/+ and the p27+/− are normal but are elevated up to 30 dB at low frequencies (4–7 kHz) and up to 50 dB at higher-frequency regions in p27−/− mice. Threshold elevations are highly significant (P < 0.0001) at all frequency points as calculated by repeated-measure ANOVA using the Wolfinger method due to missing values. (Bars = SD.)