INTRODUCTION

Developmental control of morphogenesis requires that cellproliferation be coordinated with growth and differentiation.Recent studies in Drosophilahave begun to identify some ofthe developmental signals that are able to influence cell cycleprogression (Go et al., 1998; Johnston and Edgar, 1998;Weigmann et al., 1997), as well as some of their likely cellcycle targets (Edgar and Datar, 1996). In vertebrates, theexistence of analogous pathways linking development tocontrol of the cell cycle have been revealed by the occurrenceof developmental defects that result from targeted mutation ofgenes involved in cell cycle function, such as cyclin D1 andthe retinoblastoma protein family (Cobrinik et al., 1996;Sicinski et al., 1995). Nonetheless, the mechanisms that linkdevelopmental events to the cell cycle machinery that controlscell proliferation remain poorly understood.

Development of the organ of Corti, the auditory sense organof mammals, involves the differentiation of two types ofmechanosensory hair cells (inner and outer) and four types ofsupporting cells (Deiters’, Hensen’s, Claudius’ and pillar) (Fig.

1). Each of these cell types has a distinct morphology thatcontributes to the complex structural and functional propertiesof the organ of Corti. Both the auditory and vestibular senseorgans of the inner ear develop collectively from a thickeningof the epithelial sheet in the dorsolateral region of the headknown as the otic placode (Fritzsch et al., 1998). In the mouse,after its formation on embryonic day 8.5 (E8.5), the placodeinvaginates to form the otocyst on E11. By this time,specification of the regions that will give rise to sensoryneuroepithelia containing the mechanosensory hair cells andsupporting cells, as well as non- sensory epithelia, which willgive rise to tissues such as Reisner’s membrane and theendolymphatic tube and sac, have already been specified(Morsli et al., 1998). By E12, the otocyst has begun to developmorphologically into the cochlea and vestibule. The organ ofCorti is recognizable at this time as a thickened ridge in thecochlear portion of the otocyst (Fig. 2B, arrow). At E12, theprogenitors of the hair cells and supporting cells are stilldividing (Ruben, 1967).

Between E12 and E16, the cells of the primordial organ ofCorti exit the cell cycle to form a postmitotic neuroepithelial

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Strict control of cellular proliferation is required to shapethe complex structures of the developing embryo. Theorgan of Corti, the auditory neuroepithelium of the innerear in mammals, consists of two types of terminallydifferentiated mechanosensory hair cells and at least fourtypes of supporting cells arrayed precisely along the lengthof the spiral cochlea. In mice, the progenitors of greaterthan 80% of both hair cells and supporting cells undergotheir terminal division between embryonic day 13 (E13)and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal.Here we report that the correct timing of cell cyclewithdrawal in the developing organ of Corti requiresp27Kip1, a cyclin-dependent kinase inhibitor that functionsas an inhibitor of cell cycle progression. p27Kip1 expressionis induced in the primordial organ of Corti between E12and E14, correlating with the cessation of cell division ofthe progenitors of the hair cells and supporting cells. Inwild-type animals, p27Kip1 expression is downregulated

during subsequent hair cell differentiation, but it persistsat high levels in differentiated supporting cells of themature organ of Corti. In mice with a targeted deletion ofthe p27Kip1 gene, proliferation of the sensory cellprogenitors continues after E14, leading to the appearanceof supernumerary hair cells and supporting cells. In theabsence of p27Kip1, mitotically active cells are still observedin the organ of Corti of postnatal day 6 animals, suggestingthat the persistence of p27Kip1 expression in maturesupporting cells may contribute to the maintenance ofquiescence in this tissue and, possibly, to its inability toregenerate. Homozygous mutant mice are severely hearingimpaired. Thus, p27Kip1 provides a link betweendevelopmental control of cell proliferation and themorphological development of the inner ear.

Key words: p27Kip1, Organ of Corti, Inner ear, Cell cycle, Cyclin-dependent kinase inhibitor

SUMMARY

p27Kip1 links cell proliferation to morphogenesis in the developing organ of

Corti

Ping Chen 1 and Neil Segil 1,2,*1Department of Cell and Molecular Biology, House Ear Institute, 2100 West Third Street, Los Angeles, CA 90057, USA2Department of Cell and Neurobiology, University of Southern California Medical School, Los Angeles, CA 90033, USA*Author for correspondence (e-mail: nsegil@hei.org)

Accepted 10 February; published on WWW 17 March 1999

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sheet suspended in the spiral duct of the cochlea (Ruben,1967). Greater than 80% of these cells exit the cell cyclebetween E13 and E14, with a slightly increased probability ofcells located in the apex of the mature cochlea having exitedthe cell cycle earlier than those in the base (Ruben, 1967).Starting on E15, a gradient of differentiation, which radiates inboth directions from the mid-basal region of the cochlea (Sher,1971; Lim and Anniko, 1985) turns these newly postmitoticcells into the various morphologically and functionallydifferentiated cell types illustrated in Fig. 1.

These cells remain postmitotic for the life of the animal. Inmammals, the loss of auditory hair cells does not appear to leadto proliferative regeneration (Chardin and Romand, 1995, butsee Lefebvre et al., 1995) and represents the major cause ofdeafness in humans. In the mammalian vestibular system,damage to hair cells appears to lead to a very limited degreeof repair or regeneration, but whether supporting cellproliferation leads to the differentiation of new hair cells is stillcontroversial (Forge et al., 1993, 1995; Warchol et al., 1993,1995; Rubel et al., 1995). In lower vertebrates, hair cell deathleads to renewed proliferation of supporting cells, whichsubsequently leads to the differentiation of both hair cells andsupporting cells (Corwin and Cotanche, 1988; Ryals andRubel, 1988).

In higher eukaryotes, the decisions concerning whether toproceed through another round of cell division, exit the cellcycle and become quiescent, or re-enter the cell cycle fromquiescence are all regulated by a family of proteins known ascyclin-dependent kinases (CDK). Exit from the cell cycleusually occurs during G1 phase and distinct CDKs, CDK4/6and CDK2, regulate transit through G1 and entry into thefollowing S phase, respectively (see Elledge, 1996 for review).CDK activity, in turn, is regulated by multiple mechanisms,including covalent modification of the CDK catalytic subunit;the relative abundance of required positive cofactors, one of theseveral members of the cyclin family of proteins, and by theactivity of negative regulators, the cyclin-dependent kinaseinhibitors (CKI) (see Sherr and Roberts, 1995; Elledge et al.,1996; Harper and Elledge, 1996 for reviews). In vertebrates,CKIs are represented by two unrelated families of proteins, theCIP/KIP family and the INK family. These families interactwith CDKs in different ways, but both work by binding to, andinhibiting the activity of cyclin-dependent kinases (CDKs)which can lead to withdrawal from the cell cycle. In spite ofthe advances in our knowledge of the regulation of CDKactivity, little is known about how regulation of CKIs isintegrated into specific developmental programs to coordinatecell proliferation with morphogenesis.

Here we report that between E12 and E14, the cyclin-dependent kinase inhibitor (CKI) p27Kip1 is induced in theprogenitors of the primordial organ of Corti, correlating withthe time when these cells are withdrawing from the cell cycle.We also report that, in mice with a targeted deletion of thep27Kip1 gene (p27−/−) (Fero et al., 1996), the cells of thedeveloping neuroepithelium undergo a period of prolonged celldivision relative to wild-type littermates, leading to theappearance of supernumerary hair cells and supporting cells.This observation suggests that p27Kip1 is responsible fordetermining the size of the progenitor population and thus themorphology of the array of sensory hair cells. These animalsare severely hearing impaired, indicating the importance of

precise developmental control of the cell cycle to the normaldevelopment of function. Finally, we have observed thatp27Kip1 is rapidly downregulated in differentiating hair cells,although its expression persists in postmitotic supporting cellsof the mature organ of Corti, suggesting a role in maintainingthe normally quiescent state of these cells. Consistent with thishypothesis, we have observed continued proliferation of cellsin the postnatal organ of Corti of p27−/− mice.

MATERIALS AND METHODS

ImmunohistochemistryAntibody raised against p27Kip1 was purchased from NeoMarker andassayed for specificity by immunoblotting (Fig. 2A). Extracts frommouse N1E-115 neuroblastoma cells (ATCC) were preparedaccording to Kranenburg et al. (1995) before (Fig. 2A, lane 2) andafter (Fig. 2A, lane 3) differentiation and used as controls. Resultsindicated that the antibody is monospecific and recognizes a band inE14 (data not shown) and neonatal (Fig. 2A, lane 1) cochlear extracts,that comigrates with p27Kip1 from the neuroblastoma cells.Differences in abundance of p27Kip1 before and after differentiationof N1E-115 cells are consistent with previously published results(Kranenburg et al., 1995). The more slowly migrating band visible inlanes 2 and 3 most likely represents the previously reportedphosphorylated form of p27Kip1. Paraffin sections of temporalbones were prepared according to Yoho et al. (1997) andimmunohistochemistry was done according to standard procedures(Harlow and Lane, 1988). Immunohistochemistry of p27Kip1

(NeoMarkers, 1/100 dilution) included an antigen retrieval stepcarried out by boiling deparaffinized sections in 10mM Citric acidbuffer, pH 6.0 for 10 minutes. Other antibodies used in this studyincluded: polyclonal anti-myosin VIIa (courtesy of Christine Petit,Pasteur Institute) (1/2000 dilution), monoclonal anti-PCNA(NeoMarker, 1/200 dilution) and monoclonal anti-neurofilament 68(Sigma, 1/100 dilution). Antibody incubations were done overnight at4°C and binding was visualized with fluorescein- or rhodamine-conjugated secondary antibodies (1/200, Jackson ImmunoResearch).Organs of Corti from at least three wild-type and three p27Kip1 mutantanimals were examined with these antibodies at each developmentalstage described, except in the PCNA experiment shown in Fig. 6,where two mutant animals were examined.

P. Chen and N. Segil

Fig. 1.Hematoxylin-stained section through the organ of Corti of anewborn mouse. Sensory outer hair cells (OHC) and inner hair cells(IHC), as well as the non-sensory supporting cell types are indicated.Deiters’ cells surround each OHC separating them from each otherand their nuclei are located underneath the nuclei of the OHC. Thepillar cells separate the IHCs from the OHCs. Hensen’s andClaudius’ cells are external to the OHCs as indicated.

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Confocal imagingSurface preparations of organ of Corti were prepared from animalsaged P1 to 5 months old. Following fixation in paraformaldehyde,cochleae were dissected in PBS buffer, stained with rhodamine-phalloidin (Molecular Probes) and mounted on slides. Z series scanswere made on a Zeiss LSM 410 inverted laser scanning microscope.Hair cell counts were made from photographs of these surfacepreparations and analyzed with a non-parametric statistic (the Mann-Whitney U test, StatView).

AnimalsFor production of mutant mice, male (129/Sv) mice, heterozygous foran induced mutation at the p27Kip1 locus (Fero et al., 1996), weremated with female (C57BL/6NHSd) mice to produce an F1generation. p27+/− offspring were identified by polymerase chain

reaction (PCR) as described (Fero et al., 1996). Heterozygotes weremated and animals homozygous for the induced mutation wereidentified by PCR. Wild-type, p27−/− and p27+/− animals used in thisstudy were from this F2 generation. Genotyping was verified bywestern blotting of brain extracts and also by immunohistochemistryof sections taken through the cochlea, using anti-p27Kip1 antibody(NeoMarkers) (data not shown). Animal care was in accordance withinstitutional guidelines.

Auditory brainstem response (ABR)ABRs were recorded from two subcutaneous needle electrodes placedbehind the pinna and on the vertex. A third electrode was placed onthe hindleg as a ground. The Tucker Davis ABR Workstation withTDT SigGen and BioGen software were used for stimulus generationand data acquisition. Broadband clicks were delivered through an

Fig. 2.The temporal and spatialexpression of p27Kip1 in thedeveloping organ of Corti.(A) Western blot showing specificityof antibody to p27Kip1. Protein extractfrom neonatal organ of Corti (lane 1)is compared to extract fromundifferentiated neuroblastoma cells(N1E-115) (lane 2) known to expressp27Kip1 upon induction ofdifferentiation (lane 3) (Kranenburg etal., 1995). A minor protein band,probably representing phosphorylatedp27Kip1 (Vlach et al., 1997) is detectedin lanes 2 and 3 above the majorp27Kip1 band (arrow). (B) Sectionsthrough the E12 and (C) E14 otocyststained with anti-p27Kip1 to show thetime of onset of p27Kip1 expression.Arrows indicate the thickening of thedorsal region of the otocyst that givesrise to the organ of Corti.(D) Alternate section through E14otocyst stained with antibody toneurofilaments to indicate the patternof innervation of the primordial organof Corti. (E) Section through the mid-basal region of the E16 cochleadouble-labeled with p27Kip1 and(F) myosin VIIa antibodies. Singlearrow indicates the layer of p27Kip1-stained supporting cells just below thehair cells. Clustered arrows indicatethe three outer and one inner hair cells.(G) Section through the mid-basalregion of the P1 cochlea double-labeled with anti-p27Kip1 and (H) anti-myosin VIIa. Arrows are the same asin E and F. Size bar, 50 µm.

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Intelligent Hearing System Insert transducer. The broadband click wascalibrated in peak equivalent sound pressure level (peSPL), in situ fortwo wild-type animals (ER-7 probe tube microphone system). Click-evoked ABR threshold was defined as the last level (dB peSPL) atwhich an ABR waveform could be visually detected.

RESULTS

The onset of expression of p27 Kip1 in the developingorgan of CortiIn mice, greater than 80% of the cells of the developing organof Corti stop dividing between E13 and E14 of embryonicdevelopment (Ruben, 1967). At this time, the vestibular systemhas acquired its gross morphology and the cochlea has madeone and one quarter of its ultimate one and three quarter turns.Based on morphological criteria, the primordial organ of Cortidevelops along the greater epithelial ridge of the otocyst (Fig.2B, arrow). Just prior to this time, on E12, no p27Kip1 proteinexpression is observed in any region of the otocyst (Fig. 2B).However, 2 days later, correlating with the time of cessation ofcell division, p27Kip1 is strongly expressed at this site (Fig. 2C,arrow). Confirmation that this is the site of organ of Cortidevelopment was obtained by staining alternate sections witha neurofilament antibody (Fig. 2D) to visualize the nerve fibersfrom the spiral ganglion cells that are known to reach the

primordial organ of Corti before the morphologicaldifferentiation of hair cells (Pujol et al., 1998). At this time,supporting cells and hair cells are not distinguishablemorphologically or by antibodies to one of the earliest markersof hair cell differentiation, myosin VIIa (Sahly et al., 1997)

P. Chen and N. Segil

Fig. 3.Gradient of p27Kip1 downregulation and myosin VIIaexpression in hair cells along the basal-to-apical axis of the cochlea.A single section through the basal (right) and apical (left) turns of theE15.5 cochlea double-labeled with antibody to (A) myosin VIIa and(B) p27Kip1 Scale bar, 50 µm..

Fig. 4. p27Kip1 is expressed in the supporting cells of the vestibularsensory epithelia in P6 animals. Sections were labeled with antibodyto p27Kip1 as in Fig. 2. Brackets indicate the hair cell layer,arrowhead indicates the supporting cell layer. (A) Hematoxylin-stained section through sacculus. (B) Anti-p27Kip1-stained sectionthrough sacculus; (C) utriculus; (D) crista ampullaris. Notestereocilia visible on hair cells in hematoxylin-stained section (A)and unstained dark nuclei in the hair cell layer of p27Kip1-stainedsections (B-D). Size bar, 50 µm.

1585p27Kip1 and development of the inner ear

Fig. 5. A comparison of the structure of organ of Corti from p27+/+ wild-type (A-C), p27+/− heterozygous (D-F) and p27−/− homozygous mutant(G-I) mice, age P6. Loss of p27Kip1 expression due to a targeted gene deletion causes the development of supernumerary hair cells andsupporting cells in the organ of Corti. (A,D,G) Confocal images of surface preparations used to compare the overall arrangement of sensorycells in the organ of Corti. They were stained with rhodamine-conjugated phalloidin to visualize the actin-rich stereocilia of the sensory haircells. (B,E,H) Cross sections through the organ of Corti in the mid-cochlear region stained with antibody to the hair-cell-specific antigen,myosin VIIa. (C,F,I) Alternate sections stained with hematoxylin to reveal the cellular architecture of the organ of Corti. Brackets mark themultiple rows of outer hair cells, three in the p27+/+ and p27+/− animals, four in the p27−/− animals. Arrowheads point to the single row of innerhair cells present in the wild-type organ of Corti, as well as to the rows of inner hair cells containing supernumerary hair cells in the p27+/− andp27−/− mutant animals. Sections from wild-type animals contain the normal number of inner and outer hair cells (B,C). The occasional presenceof supernumerary inner hair cells in heterozygotes is illustrated in E but not in F. Note supernumerary cells present in the pillar cell region ofthe organ of Corti from p27−/− animals where normally one inner pillar and one outer pillar cells are present (compare C with I, asterisks).

Fig. 6. Anti-PCNA staining of E16organ of Corti from p27−/− (A,B) andp27+/+ embryos (C,D) reveals activelycycling cells in the mutant organ ofCorti. Sections through the mid-basalregion of the developing cochlea of E16p27−/− and p27+/+ animals were double-labeled with antibody against PCNA(A,C, red) to stain cycling cells, andmyosin VIIa (B,D, green) to staindifferentiating hair cells. The hair cellregion is indicated by brackets, whilethe region of Deiters’ cells is indicatedby clustered arrows. Note PCNA-stained nuclei (red) in a position abovethe arrows in the p27−/− organ of Corti(A) indicating the continuedproliferation at this site, as well as theabsence of PCNA staining in thecomparable region in p27+/+ animal (C).Size bar, 50 µm.

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(data not shown). The same pattern of p27Kip1 expression isstill seen 1 day later, on E15 (data not shown). Thus, p27Kip1

is one of the first molecular markers for the primordial organof Corti, allowing its identification prior to morphological orbiochemical differentiation of hair cells and supporting cells.The sharp boundaries of p27Kip1 expression within thedeveloping otocyst (Fig. 2C) reflect the precise regionalspecification of the sensory regions in the otocyst that occurredat earlier embryonic times (Morsli et al., 1998).

By E16, the differentiating sensory hair cells becomerecognizable morphologically, coincident with the appearanceof one of the earliest known hair cell markers, myosin VIIa(Sahly et al., 1997) (Fig. 2F, clustered arrows). Interestingly,p27Kip1 is no longer detected in the hair cells, althoughexpression can still be seen in the nuclei of surroundingsupporting cells of the sensory epithelium (Fig. 2E, arrow). Innewborn and adult mice (Fig. 2G,H and data not shown),p27Kip1 continues to be expressed in the differentiatedsupporting cells including Deiters’, Hensen’s, Claudius’ andpillar cells.

Based on morphological observations, it has been reportedthat, between E15 and E16, a gradient of hair celldifferentiation develops starting in the mid-basal regions of thecochlea and spreading in both the apical and basal directions(Sher, 1971; Lim and Anniko, 1985; see Rubel, 1978, forreview). To determine more precisely when p27Kip1 isdownregulated relative to hair cell differentiation and to testwhether this gradient is reflected in the pattern of myosin VIIaexpression, cross sections through a single cochlea from anembryo between E15 and E16 were stained with antibody tomyosin VIIa (Fig. 3A) and p27Kip1 (Fig. 3B). These sectionsshow both apical and basal turns and indicate the presence ofa gradient of expression of the hair cell marker myosin VIIa.In a turn of this cochlea from the basal region (Fig. 3A), wefound that inner hair cells (arrowhead) are labeled relativelystrongly with myosin VIIa (green) while outer hair cells(bracket) are more weakly labeled. In contrast, no staining ofmyosin VIIa is observed in the apical turn present in the samecochlea at this stage. p27Kip1 staining is present in thesupporting cell layer of the basal turn, but absent from all thehair cells (Fig. 3B), as it is throughout the cochlea at slightlylater embryological times. However, in the apical turn, whereno myosin VIIa staining is observed (Fig. 3A), p27Kip1 appearsto stain all of the cells in the primordial sensory epithelium.These results suggest that the inner hair cells mature before theouter hair cells and show the close temporal correlationbetween p27Kip1 downregulation and the differentiation of haircells as seen by the onset of myosin VIIa expression. They alsodemonstrate a gradient of differentiation within the cochlea,consistent with that previously reported in morphologicalstudies (cited in Rubel, 1978).

p27Kip1 is also expressed in the vestibular systemIn the vestibular sensory organs, p27Kip1 is also localized in thenon-sensory supporting cells of neonatal (P6) animals (Fig.4A-D). As shown in Fig. 4B, occasional p27Kip1-positive cellsare seen in the sensory cell layer as well, although the type andorigin of these cells is unknown. In contrast to theircounterparts in the cochlea, the cells of the vestibular sensoryorgans withdraw from the cell cycle over a longer period oftime, ranging from E14 to P3 (Ruben, 1967). Developmentally,

we observed p27Kip1 expression in subregions of the sensorycomponent of the differentiating vestibular epithelia as early asE14 (data not shown). This suggests that a similar sequence ofdevelopmental events involving p27Kip1 in exit from the cellcycle takes place in the vestibular system. However, the levelsof p27Kip1 expressed in the developing and mature vestibularsystem appear to be lower than in comparable sections of organof Corti making their analysis more difficult. Consequently, wehave not precisely correlated the expression of p27Kip1 with thecessation of cell division in the developing vestibular epithelia.

Hyperplasia of the organ of Corti in p27 Kip1 knockoutmiceThe restricted pattern of p27Kip1 expression in the developingsensory epithelium led us to examine the developmentalconsequences of an induced mutation in the p27Kip1 gene (Feroet al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). Inthe absence of p27Kip1, animals are larger than wild-typelittermates as a result of multiorgan hyperplasia (Fero et al.,1996; Kiyokawa et al., 1996; Nakayama et al., 1996). Wild-type mice normally contain a single row of inner hair cells(arrowhead) and three rows of outer hair cells (brackets, Fig.5A-C). In mice homozygous for a p27Kip1 mutation (p27−/−),supernumerary hair cells developed in both the inner and outerrows of hair cells (Fig. 5G-I). The inner row contains a partlydisorganized line of at most two hair cells (Fig. 5G-I,arrowheads), while the outer hair cells show a pattern of four,partly disorganized, lines of cells (Fig. 5G-I, brackets).Representative samples of outer and inner hair cells in the mid-basal region of the cochlea were counted and revealedsignifcantly more cells in mutant (n=4) versus wild-type (n=4)mice (P<0.05). Outer hair cell numbers in the mutant animalswere increased by a mean of 36% and inner hair cell numberswere increased by a mean of 23%. In addition, an excessnumber of supporting cells, including pillar cells separatinginner from outer hair cells and occupying the area of the tunnelof Corti, are present in p27−/− animals (Fig. 5I, asterisks).Normally, only one inner pillar cell and one outer pillar cellare present in this region (Figs 2B, 5C, asterisks). These resultsindicate that there is an increase in both hair cells andsupporting cells in the absence of p27Kip1.

In mice heterozygous for a p27Kip1 mutation (p27+/−),the outer hair cell population of the heterozygous animalsappears normal (Fig. 5D-F). However, we observed occasionalsupernumerary inner hair cells throughout the organ of Corti in 6-day-old heterozygous animals (Fig. 5D,E). Thisphenomenon was never observed in wild-type littermates (Fig.5A-C). Thus, the severity of this abnormality appears to bedose dependent in that homozygotes are more severely affectedthan heterozygotes (Fig. 5), although the degree of dosedependence has not been quantified.

On the basis of neuronal fiber staining, hair cells in P6animals appear to have a normal pattern of innervation (datanot shown). However, analysis of auditory brainstem responses(ABR) indicated that mutant animals were severely hearingimpaired. 10-week-old p27−/− animals had a significantlyelevated mean, click-evoked, ABR threshold (77 db SPL, n=3)relative to comparably aged wild-type animals (20 db SPL,n=6) (P<0.05). Homozygous mutant animals showed noobvious behavioral defects related to vestibular function suchas circling behavior or balance problems. The cause of the

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hearing deficit in homozygous animals could lie in theperipheral abnormalities that we have described or it could liein central abnormalities that result either from the p27Kip1

mutation or as a consequence of the peripheral anatomicalabnormalities. These possibilities are currently underinvestigation.

Cell proliferation in the organ of Corti of p27 Kip1

knockout miceThe abnormalities observed in the organ of Corti of p27−/−

mice suggest that p27Kip1 functions as a growth inhibitorduring development, and its ablation leads to prolonged orunscheduled proliferation of hair and supporting cellprogenitors, ultimately leading to the differentiation ofsupernumerary cells. We tested this hypothesis by comparingthe expression of Proliferating Cell Nuclear Antigen (PCNA),a marker for cells that are in, or about to enter, the DNAreplication phase of the cell cycle and thus are scheduled toundergo another round of cell division (Galand, 1989), inp27+/+ and p27−/− animals. Since the onset of p27Kip1

expression (Fig. 2C) correlates with the cessation of celldivision of organ of Corti progenitor cells on E14 (Ruben,1967), we studied the incorporation of PCNA into replicatingDNA in p27−/− and p27+/+ embryos between E15 and E16when cell division should have ceased. In p27−/− embryos,PCNA-positive cells are observed in the Deiters’ cell region(Fig. 6A, arrows) beneath the newly differentiated hair cellsstained with anti-myosin VIIa (Fig. 6B, bracket), indicatingthat proliferation continues in these animals past the normaltime of cell cycle withdrawal. In contrast, in wild-type embryosPCNA-labeled cells are absent from the Deiters’ cell region(Fig. 6C, arrows) of the organ of Corti, beneath the myosinVIIa-stained hair cells (Fig. 6D, brackets).

To test the duration of this aberrant proliferative activity, westained P6 cochleas from p27−/− and p27+/+ animals forexpression of the PCNA marker (Fig. 7). In p27−/− animals,PCNA-positive cells are no longer seen in Deiters’ cell regionas they are at E16, but appear in clusters in the region ofHensen’s cells, lateral to the outermost row of outer hair cells(Fig. 7A, arrow), as well as in the pillar cell regions separatinginner and outer hair cells (Fig. 7B, arrows). As expected, noPCNA staining is apparent in the organ of Corti (Fig. 7C,bracket and arrow) from p27+/+ animals, and no PCNA-positivecells were observed in comparable sections through the organof Corti of heterozygous animals (data not shown). Theseresults suggest that p27Kip1 is required for maintaining thesupporting cells of the mature organ of Corti in their normallyquiescent state.

DISCUSSION

While the control of cell proliferation, growth anddifferentiation are separable phenomena, coordination betweenthese phenomena is required if correct morphogeneticpatterning is to occur (see Skaer, 1998; Johnston, 1998 forreviews). By studying the effects of an induced mutation in thecell cycle regulator p27Kip1, we have identified one elementrequired for coordinating the cessation of cell division withdifferentiation and morphogenetic patterning in the developingorgan of Corti. Our results show that p27Kip1 activity is

required for the timely withdrawal from the cell cycle of thecells of the developing sensory epithelium and its absence leadsto the appearance of supernumerary cells throughout themature organ of Corti. Additional studies of the pattern ofexpression of p27Kip1 during normal development of the innerear, suggest that p27Kip1 may play a subsequent role in cellulardifferentiation and homeostasis of the various cell types in thesensory epithelium.

Coordinating cell proliferation with morphogeneticeventsThe ability of p27Kip1 to coordinate cell proliferation withmorphogenetic events has been suggested by its well-knowncapacity to block cell cycle progression by inhibiting theactivity of CDK4/6 and CDK2 (Elledge et al., 1996), by itsability to respond in vitro to a variety of known cellular growthregulators (Sherr and Roberts, 1995), and finally by thedevelopmental abnormalities that are caused by its targeteddeletion in mice (Fero et al., 1996; Kiyokawa et al., 1996;Nakayama et al., 1996). Regulation of p27Kip1 protein levels

Fig. 7.Cell division persists in the basal region of the organ of Cortiof postnatal (P6) homozygous mutant animals. (A,B) Anti-PCNAstaining of sections from early postnatal (P6) organ of Corti fromhomozygous mutant and wild-type animals and (C) wild-typecontrol. (A) Arrow indicates clusters of PCNA-positive cells inHensen’s cell region; (B) double arrow indicates positive cells in theregion of the pillar cells; (C) bracket and arrowhead indicate outerand inner hair cell regions respectively. No PCNA-positive cells areseen in the organ of Corti from wild-type animals at this stage. Scalebar, 50 µm.

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in vitro is governed at the transcriptional (Kawasaki et al.,1998), translational (Hengst and Reed, 1996; Millard et al.,1997) and, perhaps most importantly, the post-translational(Pagano et al., 1995; Vlach et al., 1997) level. However, to ourknowledge, the specific mechanisms used to regulate p27Kip1

levels in vivo have not been studied, and the molecularmachinery underlying the temporally and spatially restrictedinduction of p27Kip1 protein that we have described (Fig. 2) iscurrently unknown.

Morsli et al. (1998) have shown that the gene lunatic fringe,a modulator of the Notch signaling pathway, is expressed inthe sensory regions of the developing cochlea in a restrictedregion that appears to presage p27Kip1 expression. While it isnot clear whether the expression of this gene in the cochlea hasthe exact same boundaries as p27Kip1, it is possible that theNotch signaling pathway affects morphogenesis of the organof Corti through the regulation of cell proliferation in a mannersimilar to that shown for wing development in Drosophila(Johnston and Edgar, 1998). Although the specific stimulus forp27Kip1 induction is not known, the sharp boundaries ofp27Kip1 expression that arise in the otocyst between E12 andE14 (Fig. 2C) are likely to reflect boundaries that have beenset up by morphogenetic gradients analogous to those seenduring the patterning of other embryonic anlage and, as such,may serve as a downstream marker for the specification of theboundaries of the sensory epithelia.

Control of cell proliferation in the developing organof CortiDuring development of the organ of Corti, p27Kip1 levels riseabruptly between E12 and E14 (Fig. 2) and the majority of thecells that will make up the mature sensory epithelium exit thecell cycle over the period of 24 -48 hours that follow (E13 andE15, Ruben, 1967). In animals that lack p27Kip1, proliferationwithin the E16 sensory epithelium continues (as seen by thepresence of PCNA-positive cells in the Deiters’ cell region,Fig. 6A), even as some of the newly generated cells arebeginning to differentiate into hair cells (Fig. 6B). Thesupernumerary cells that we observe (Fig. 5) are theconsequence of this abnormal proliferation. At later times (P6,Fig. 7) PCNA-positive cells are restricted to the region ofHensen’s, Claudius’ and pillar cells and are absent from theDeiters’ cell region. The reason for this change is not known,but is likely to be related to the mechanism governing hair celland supporting cell differentiation discussed below.

The fact that both hair cells and supporting cells are able tostop dividing and differentiate in the organ of Corti of p27−/−

animals indicates that there are multiple levels of cell cyclecontrol at work. The nature of these other controls on cellproliferation in the organ of Corti is not clear. In studies ofdeveloping oligodendrocytes in vitro, it was found that theabsence of p27Kip1 leads to a prolonged period of cell division(Casaccia-Bonnefil et al., 1997; Durand et al., 1998),correlating with an increased number of cells in vivo in p27−/−

animals (Casaccia-Bonnefil et al., 1997). While p27−/−

oligodendrocytes are able to undergo cell cycle arrest anddifferentiation in vivo, under specific in vitro growthconditions, differentiation of these cells is partially impaired(Casaccia-Bonnefil et al., 1997; Tikoo et al., 1998). Theseobservations, like our own, are consistent with the overallphenotype displayed by p27−/− animals, namely the

generalized hyperplasia observed in many, if not all tissues,along with a range of abnormalities in tissue organization (Feroet al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). Inspite of the increase in cell number, the function of most tissuesis not grossly perturbed (Fero et al., 1996; Kiyokawa et al.,1996; Nakayama et al., 1996) and homozygous mutant animalsare viable. The presence in some tissues of more than one CKI(Franklin et al., 1998; Zhang et al., 1998), as well as theexistence of potential CKI-independent pathways able todownregulate CDK activity (Elledge et al., 1996), suggests thatoverlapping pathways regulating the cell cycle duringdevelopment may be the norm. However, as our resultsindicate, p27−/− animals are severely hearing impaired,probably reflecting a strict requirement for precise cellularorganization in the organ of Corti.

Specification of cell type in the organ of CortiThe primary mechanism specifying hair cell versus supportingcell differentiation remains an important question for the field.While alternative models exist, several investigators havesuggested that at the time of their terminal mitosis, the cells ofthe sensory epithelium are not determined to be either hair cellsor supporting cells, and only sometime after cell cycle exit dothey differentiate into one or the other cell type, possiblythrough a process of lateral inhibition (see Fekete, 1996 forreview). The specification of hair cells versus supporting cellsfollowing terminal mitosis is consistent with results fromrecent studies of terminal differentiation in the basilar papillaof the chicken using retroviral tracing methods (Fekete et al.,1998), as well as studies on the differentiation of sensory haircells during in vitro organ culture of the mouse organ of Corti(Kelley et al., 1993, 1995). These workers show that for alimited period of time following terminal cell division, cells ofthe sensory epithelium can be induced to becomesupernumerary hair cells, either through retinoic acid treatment(Kelley et al., 1993) or by the death of a neighboring, alreadydifferentiated hair cell (Kelley et al., 1995). This isaccomplished without additional cell division, suggesting thatthe new cells are likely to have been derived from cells thatwould otherwise have differentiated into supporting cells orfrom a population of cells that would normally have undergoneapoptosis.

If these arguments are correct, the initial induction ofp27Kip1 in the otocyst, which occurs prior to hair cell andsupporting cell differentiation, is likely to be responsible forregulating the correct number of postmitotic precursor cells onwhich these later specification processes can act. By thisargument, the supernumerary cells produced because of theabsence of p27Kip1 are the result of the normal patterningwithin an increased population of progenitors. In contrast, thesupernumerary cells that are produced in response to retinoicacid, in the absence of new cell division (Kelley et al., 1993),likely result from a more direct influence on the process ofpostmitotic differentiation.

While the very existence of both hair cells and supportingcells in p27−/− animals argues strongly against a required,positive role for p27Kip1 in the terminal differentiation of eithercell type, it leaves open the possibility of a negative role inregulating the differentiation of one or the other. In this case, therapid downregulation of p27Kip1 that occurs in differentiatinghair cells (Fig. 2) could be a necessary consequence of the

P. Chen and N. Segil

1589p27Kip1 and development of the inner ear

initiation of differentiation in these cells. We have observed thatthe induction of myosin VIIa, a marker of hair celldifferentiation, occurs after the last cell division and istemporally correlated with the downregulation of p27Kip1. Justhow tightly correlated is perhaps best seen by our demonstrationof the gradient of differentiation within the E15 cochlea, wherebasal sections reveal hair cells expressing myosin VIIa andlacking p27Kip1 expression, at the same time as postmitoticprogenitor cells in more apical regions of the cochlea have yetto begin expression of myosin VIIa and still express p27Kip1

(Fig. 3). At no time do we observe myosin VIIa and p27Kip1

double-labeled cells in our preparations. Nor do we observe cellsin the postmitotic organ of Corti that lack both markers. Thissuggests that the expression of these two markers may bemutually exclusive. Although no causative relationship has beendemonstrated, a mutually exclusive pattern of expression existsbetween one of the other CIP/Kip family members (p21Cip1), andmarkers of cell differentiation in postmitotic keratinocytes (DiCunto et al., 1998) suggesting that p21Cip1 has a direct role inthe differentiation of these cells, beyond its role in regulating cellproliferation. The downregulation of p27Kip1, which appears tobe part of the program of hair cell differentiation, could serve ananalogous function.

Quiescence and regenerationThe presence of proliferating cells in the postnatal organ ofCorti of p27−/− animals (Fig. 7), as well as the persistence ofp27Kip1 expression in mature supporting cells (Fig. 2G,H, anddata not shown), reveals a second role for p27Kip1, that ofmaintaining these cells in a quiescent state. While itspersistence may be required in mature supporting cells tomaintain this state, it may also offer a possible explanation forthe lack of regeneration in mammals. In lower vertebrates suchas chickens, the loss of hair cells due to acoustic trauma orototoxic shock leads to re-entry of supporting cells into the cellcycle and the subsequent differentiation of their progeny intohair cells (Corwin and Cotanche, 1988; Ryals and Rubel, 1988).We hypothesize that, if supporting cells in chickens also containhigh levels of a p27Kip1 homologue, it is rapidly degraded uponloss of hair cells, in order to allow them to re-enter the cell cycle.The difference between mammals and birds could lie in thelevel of p27Kip1 in mature supporting cells or the efficiency withwhich it is downregulated following injury. Alternatively, othercell cycle regulators besides p27Kip1 may be involved.Regardless, the implication is that the high levels of p27Kip1 thatpersist in supporting cells of mammals could be an impedimentto re-entry into the cell cycle following loss of hair cells.

We wish to thank Andres Collazo, Yun-Shain Lee, David Lim,Federico Kalinec, Greta Segil and Howard Worman for invaluablecomments on the manuscript and Ed Rubel for helpful discussion ofour results. Butch Welch provided expert graphics assistance and XiLin provided expert assistance with confocal imaging. CarolineAbdala generously assisted with the ABR analysis. We also thankMatthew Fero and James Roberts for the p27Kip1 knockout mice,Christine Petit (Pasteur Institut) who generously supplied the anti-myosin VIIa antibody used in this study, and Yun-Shain Lee whohelped with the surface preparation shown in Fig. 5A, D and G.

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