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First published online 21 June 2006
doi: 10.1242/dev.02453

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A morphogenetic wave of p27^Kip1 transcription directs cell cycle exit during organ of Corti development

Yun-Shain Lee^1,*, Feng Liu^2,* and Neil Segil^1,2,3,

¹ Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 West 3rd Street, Los Angeles, CA 90057, USA.
² Neuroscience Graduate Program, University of Southern California, Los Angeles, CA 90033, USA.
³ Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA 90033, USA.

View larger version (55K): [in a new window]   Fig. 1. Development and cellular anatomy of the mouse organ of Corti. (A-E) Cochlear epithelia from E12.5-E17.5 from mice carrying a transgenic Math1-GFP reporter (Chen et al., 2002; Lumpkin et al., 2003). Epithelia were dissected and photographed under brightfield conditions and fluorescence images of Math1/GFP expression (green) from the same preparations were superimposed to illustrate the basal to apical pattern of hair cell differentiation that initiates in the mid-basal regions of the cochlea at E14.5 (Chen et al., 2002) and spreads apically until E17.5 when the wave of differentiation is complete. Note that at E12.5 and E13.5 Math1/GFP expression is limited to the saccule where Math1-dependent hair cell differentiation begins around E10.5 (Bermingham et al., 1999). (F) Cross-section through a P1 organ of Corti illustrating the position of hair cells (green, stained with antibody to Myosin VIIa) and supporting cells (red, stained with antibody to p27Kip1). The different varieties of hair cells and supporting cells are labeled: I, inner hair cell; O1-O3, outer hair cell-row 1-3; IP, inner phalangeal.

View larger version (57K): [in a new window]   Fig. 2. A wave of cell cycle exit and p27Kip1 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-p27Kip1. 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 p27Kip1 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 p27Kip1 (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 p27Kip1 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. p27Kip1 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.

View larger version (92K): [in a new window]   Fig. 3. The apical to basal wave of p27Kip1 expression is regulated at the transcriptional level. (A) Schematic of the modified p27Kip1/GFP BAC reporter showing the position of the IRES-GFP relative to exon 1 and exon 2 of the p27Kip1 mouse gene. BAC ends are located 51 kb 5' and 78 kb 3' to the start site of p27Kip1 transcription. The first exon of the p27Kip1 gene was removed to avoid overexpression of p27Kip1. See Materials and methods for further details. (B,C) Whole-mount preparations of the developing cochlear duct from E12.5 to E14.5 were photographed to reveal the endogenous p27Kip1/GFP transgene (C, green), and then labeled with antibody to p27Kip1 (B) to demonstrate the correspondence between the pattern of gene and protein expression at each age examined. In E12.5, apex and base are marked and dotted lines indicate the region of initiation of p27Kip1 and GFP expression. (D,E) Cross-section through the approximate middle cochlea stained to reveal anti-p27Kip1 (red) and expressing native GFP reporter (green) as above, to show the coincidence of p27Kip1-GFP transgene expression with p27Kip1 protein. Scale bar: 100 µm.

View larger version (118K): [in a new window]   Fig. 4. p27Kip1 transcriptional regulation in p27Kip1-null mice is independent of cell cycle exit. (A-C) Whole-mount preparation of E13.5 cochlear epithelium from a p27Kip1-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 p27Kip1/GFP transgene expression. (D-F) Surface reconstruction and cross-section through a p27Kip1-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 p27Kip1/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 p27Kip1-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 p27Kip1. (G,H) Cross-section through the middle turn of an E14.5 cochlear duct from a p27Kip1-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 p27Kip1/GFP transgene expression (bracket). Scale bar: 100 µm.

View larger version (80K): [in a new window]   Fig. 5. Cell-type-specific transcriptional regulation of p27Kip1 in the postnatal organ of Corti and retina. (A-C) Sections through the postnatal day 1 (P1) organ of Corti triple-labeled with antibody to p27Kip1 (A), the native p27Kip1/GFP transgene (B) and the hair cell marker Myosin VIIa (C). Note high levels of p27Kip1 expression in postnatal supporting cells and the lack of staining of differentiated hair cells, indicating that p27Kip1 is downregulated in hair cells at the transcriptional level. (D-G) Cross-sections through the P21 mouse retina. D and E are the same section double-labeled with antibody to glutamine synthetase, a marker of Muller glia in the retina (D), and the native p27Kip1/GFP transgene (E). F and G are the same section double-labeled with antibody to p27Kip1 (F) and the native p27Kip1/GFP transgene (G). Note the co-linearity of staining between glutamine synthetase, the p27Kip1 protein and p27Kip1/GFP in the inner nuclear layer (INL), which is indicative of cell-type-specific transcriptional control in the postnatal retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 100 µm.

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