1 Institute of Biotechnology, University of Helsinki, 00014 Helsinki,
Finland
2 Departments of Medicine, Laboratory Medicine and Pathobiology and Medical
Biophysics, Division of Cell and Molecular Biology, Toronto General Research
Institute, University Health Network, University of Toronto, Toronto, Ontario
M5G-2M1, Canada
3 Creighton University, Department of Biomedical Sciences, Omaha, NE 68178,
USA
* Author for correspondence (e-mail: ulla.pirvola{at}helsinki.fi)
Accepted 23 March 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Inner ear, Cochlea, Vestibular organ, Hair cell, Proliferation, Differentiation, Apoptosis, Cell cycle, Mitosis, Polyploidy, Rb (Rb1), p21 (Cdkn1a), Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A recent study by Chen et al. (Chen et
al., 2003) showed that targeted disruption of the gene encoding a
member of the Ink4 family of CKIs, p19 (Cdkn2d Mouse
Genome Informatics), leads to abnormal DNA synthesis in postnatal cochlear
HCs. Bromodeoxyuridine incorporation was shown to occur at a low rate.
Aberrant proliferation was accompanied by apoptosis and resulted in
progressive hearing loss. In contrast to postnatal HCs, p19
inactivation did not affect the antiproliferative state of HCs during
late-embryogenesis, although p19 was reported to be expressed in the
embryonic organ of Corti (Chen et al.,
2003
). These data suggest that additional CKIs compensate for
p19 deficiency in developing cochlear HCs. Cell cycle regulation
downstream of CKIs has not been reported in the cochlea. Furthermore, the
mechanisms underlying cell cycle arrest of vestibular HCs have not been
explored.
A key regulator of the cell cycle is pRb, the protein product of the
Rb (Rb1 Mouse Genome Informatics) tumour suppressor
gene (Weinberg, 1995). pRb is
the prototypical member of the pocket protein family, which also comprises
p107 and p130. Pocket proteins have both unique and overlapping functions in
cell cycle control, in regulation of cell differentiation and survival, and in
inhibition of oncogenic transformation
(Classon and Harlow, 2002
).
pRb is a nuclear phosphoprotein. It binds members of the E2f transcription
factor family during G1 and represses genes required for G1 to S-transition.
In response to mitogens, pRb becomes phosphorylated (inactivated) by
cyclin/cyclin dependent kinase (CDK) complexes, resulting in the release of
bound E2fs, transcriptional de-repression and cell cycle progression.
Mitogenic signals induce the cell cycle machinery at the level of cyclins and
CDKs (Murray, 2004
). CDK
activation is regulated by various mechanisms, a particularly important one
being the inhibition by CKIs (Vidal and
Koff, 2000
). Inhibition of CDK activity by CKIs maintains pRb in a
hypophosphorylated (active) state. There are two families of CKIs, the Ink4
family (p15, p16, p18, p19) and the Cip/Kip family (p21, p27, p57).
Analyses of loss-of-function mutant mice have demonstrated the essential
role of Rb as a repressor of cell cycle progression during
embryogenesis. Rb knockouts die in midgestation, between embryonic
day 13 (E13) and E14. In addition to ectopic cell cycles, development of the
nervous system, skeletal muscles, lens and haematopoietic cells of the mutants
is characterized by aberrant differentiation and extensive apoptosis
(Clarke et al., 1992;
Jacks et al., 1992
; Lee et
al., 1993; Morgenbesser et al.,
1994
; Zacksenhaus et al.,
1996
). Consistently, Rb is prominently expressed in these
tissues (Jiang et al., 1997
).
Recent conditional mutagenesis and placental rescue indicate that the
apoptotic phenotype of whole-embryo Rb knockouts is not caused by
cell-autonomous mechanisms in all tissues: apoptosis in the brain largely
occurs secondarily to other embryonic defects, whereas apoptosis in skeletal
muscles, retina and lens appears to be a direct consequence of Rb
inactivation (Ferguson et al.,
2002
; de Bruin et al.,
2003
; MacPherson et al.,
2003
; MacPherson et al.,
2004
; Wu et al.,
2003
; Chen et al.,
2004
; Zhang et al.,
2004
).
How Rb regulates cell differentiation is in general poorly
understood. The role of Rb in differentiation appears to be more
versatile than merely indirectly stimulating this process through the
inhibition of cell cycle progression. In some cases, it has been possible to
separate the effects of Rb on cell proliferation and differentiation
(Sellers et al., 1998;
Liu and Zacksenhaus, 2000
;
Takahashi et al., 2003
;
Zhang et al., 2004
). In
skeletal muscles, Rb transcriptionally upregulates genes involved in
the late stages of differentiation through the bHLH gene myogenin
(Gu et al., 1993
;
Novitch et al., 1996
;
Novitch et al., 1999
).
Similarly, adipocyte differentiation is induced by the positive effect of
Rb on the transcriptional activity of CCAAT/enhancer-binding proteins
(Chen et al., 1996
).
We show here that pRb is expressed in inner ear HCs. The lethality of
Rb knockouts at the stage when part of HCs have not yet started to
differentiate precludes the use of these mutants in our studies. To
genetically dissect the Rb pathway and unravel the requirement for
pRb during HC development, we have analysed the inner ear sensory epithelia of
mgRb:Rb-/- mutants, which are rescued to birth by a
hypomorphic Rb transgene
(Zacksenhaus et al., 1996).
The transgene is expressed in the nervous system, but not in non-neuronal
tissues (Jiang et al., 2001
),
including the inner ear HCs (this study). Our results suggest that pRb
regulates HC quiescence and that, during development, p21 may act
co-operatively with other CKI(s) as an upstream effector of pRb activity.
Rb loss induced aberrant HC proliferation, but these cells also
showed pathological features, including mitotic abnormalities and signs of
apoptosis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histology, immunohistochemistry and TUNEL staining
Whole heads of E12.5, E13.5, E14.5, E15.5 and E16.5 embryos, and dissected
inner ears of E17.5 and E18.5 embryos and of postnatal day (PN) 2 and 6 pups
were fixed overnight in 4% paraformaldehyde (PFA), embedded in paraffin and
cut to 5-µm sections. Inner ears of 8-week-old mice were perilymphatically
fixed with PFA, immersed in this fixative overnight, decalcified with 0.5 M
EDTA and embedded in paraffin wax. Following antibodies were used: monoclonal
Rb (BD Biosciences), polyclonal myosins VI and VIIa
(Hasson et al., 1997;
Pirvola et al., 2004
),
monoclonal calretinin and calbindin (Swant), polyclonal phospho-histone H3
(Ser10, Cell Signaling Technology)
(Pirvola et al., 2004
),
polyclonal espin (Zheng et al.,
2000
), polyclonal p75 neurotrophin receptor (p75NTR)
(Pirvola et al., 2002
),
monoclonal p27 (Neomarkers), and monoclonal (rabbit) cleaved caspase 3 (Cell
Signaling Technology). Detection was carried out with the Vectastain Elite ABC
kit or the Vectastain Mouse-On-Mouse kit and the diaminobenzidine substrate
(Vector Laboratories). Methyl Green was used for counterstaining. For
double-labelling experiments, PFA-fixed inner ears of E18.5 embryos were
cryosectioned, and phospho-histone H3 and calretinin or calbindin were used as
primary antibodies. Binding was visualized by fluorochrome-conjugated
secondary antibodies (Alexa Fluor 488 and 568, Molecular Probes). In addition
to cleaved caspase 3 immunostaining, TUNEL method-based Fluorescein In Situ
Cell Death Detection Kit (Roche) was used to detect apoptotic cells.
Semi-thin sections
Inner ears of mgRb:Rb-/- mice and control littermates
were dissected at E18.5 and fixed overnight in 2.5% glutaraldehyde, postfixed
in 1% osmium tetroxide and embedded in Epon. Sections (0.5 µm) were cut in
transverse (midmodiolar) plane and stained with 2% Toluidine Blue.
In situ hybridization
In situ hybridization was performed with 35S-labelled riboprobes
on PFA-fixed paraffin wax sections according to the protocol by Wilkinson and
Green (Wilkinson and Green,
1991). Rb, p107 (Rbl1 Mouse Genome
Informatics), p130 (Rbl2 Mouse Genome Informatics),
p21 (Cdkn1a Mouse Genome Informatics), Math1,
fibroblast growth factor 8 (Fgf8), Fgf10, Brn3c
(Pou4f3 Mouse Genome Informatics) and brain-derived
neurotrophic factor (Bdnf) cDNAs were used.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Hyperplasia of the developing auditory sensory epithelium in the absence of Rb
Global inner ear morphology of mgRb:Rb-/- mutants at
E17.5 (n=8 ears) and E18.5 (n=15 ears) was comparable with
controls, but distinct differences were seen in the sensory epithelia (Figs
2 and
3). Only minor variations were
observed between individual mutants, both at the level of vestibular and
auditory sensory epithelia. The normal organ of Corti shows one inner HC and
three outer HCs in a midmodiolar, radially sectioned cochlear duct. This was
apparent from histology and from the positive staining with myosin VI, a
cytoplasmic marker (Fig. 2A).
One layer of supporting cells is normally located below HCs on the basilar
membrane. The organ of Corti of mgRb:Rb-/- mutants was
hyperplastic because of excessive HC formation
(Fig. 2A').
Hyperplasticity was most prominent in the basal half of the cochlea, where
cellular differentiation is more advanced than in the apical half. When
analysing transverse sections from the upper basal turn of E18.5 cochleas,
myosin VI-positive area showed sixfold increase in the numbers of HC nuclei
(n=6 mutant cochleas, mean±s.d. 23.2±4.3, contrasting
with controls showing four HC nuclei in the same view). We first asked whether
supernumerary HCs were generated at the expense of supporting cells. We used
the CKI p27 (Fig. 2B,B') and p75NTR (Fig. 2C,C')
as markers for differentiating supporting cells, the former one being
expressed in the nuclei of several supporting cell populations, including
pillar, Deiters', Hensen's and Claudius' cells, and the latter in the
cytoplasm of pillar and Hensen's cells. p75NTR (Ngfr Mouse Genome
Informatics) was also expressed in the cochlear ganglion neurons. At E17.5 and
E18.5, supporting cell numbers were not decreased in the mutants, rather,
their numbers were increased, but not to the extent of HC overproduction.
Based on the analysis of transverse sections from the upper basal turn of
E18.5 cochleas, a threefold increase in the numbers of p27-positive Deiters'
plus pillar cells was found below HCs (n=6 mutant cochleas,
mean±s.d. 14.5±3.1, contrasting with controls with five of these
cells in a corresponding view).
|
|
To determine the developmental time window of aberrant mitoses in the
cochleas of mgRb:Rb-/- mutants, we next focused on earlier
developmental stages. The bHLH gene Math1 is one of the earliest
markers for the HC lineage, being first expressed in the cochlea at E14.5
(Chen et al., 2002). This is
followed by the induction of myosin VI at E15.5. At E13.5 (n=4 ears
of mutants and controls each) and E14.5 (n=4 ears of mutants and
controls each), the caudal wall of the cochlear duct, which houses precursor
cells, showed comparable structure in mgRb:Rb-/- mutants
and controls. At these stages, there was a slight increase in the numbers of
phospho-histone 3-positive cells at the site of future organ of Corti in the
mutants, but the increase was not statistically significant
(Fig. 2F,F', data not
shown). By contrast, the adjacent mesenchyme of the mutants showed a clear
increase in the amount of dividing cells, this being in line with the
knowledge that Rb is expressed in the embryonic mesenchyme
(Leezer et al., 2002
). In the
organ of Corti of the mutants, increased mitotic activity and overproduction
of myosin VI-positive HCs became clear at E15.5 (n=8 ears of mutants
and controls each), at the stage when pRb expression ensues in cochlear HCs of
normal animals.
Available data show that in addition to a role as a repressor of cell cycle
progression, depending on the cell context, Rb positively regulates
differentiation by inducing expression of cell type-specific genes
(Chen et al., 1996;
Novitch et al., 1996
;
Novitch et al., 1999
). To find
out the possible role of Rb on cochlear HC differentiation, we
studied the expression of a panel of early (Math1, myosins VI and
VIIa, calbindin, Fgf8) and late (Brn3c) differentiation
markers in mgRb:Rb-/- mutants at E17.5 and E18.5.
Math1 (Fig.
2G,G') and the other markers (data not shown) were expressed
in Rb-null cochlear HCs, similar to controls, although consistent
with the expansion of HCs the signal covered a correspondingly wider zone. In
normal animals, Fgf8 is expressed in inner HCs, but not in outer HCs
(Pirvola et al., 2002
).
Likewise, in the mutants, Fgf8 expression was restricted to
supernumerary inner HCs, indicating that cochlear HCs were subtyped despite
their overproduction (data not shown). Thus, despite the altered
cytoarchitecture of the organ of Corti, cochlear HCs of the mutants showed a
normal profile of molecules involved in differentiation and maturation.
In addition, the greater epithelial ridge, the epithelial domain located
medially to the organ of Corti, was hyperplastic in
mgRb:Rb-/- mutants at birth
(Fig. 2). Earlier studies have
shown that Fgf10 is expressed in the greater epithelial ridge
(Pirvola et al., 2000;
Pauley et al., 2003
)
(Fig. 2H) and this structure
has been suggested to contain sensory precursor-like cells
(Zheng and Gao, 2000
;
Woods et al., 2004
). At birth,
Fgf10 was expressed throughout the abnormally thick greater
epithelial ridge of the mutants, suggesting that ectopic cells in this region
have characteristics of sensory precursor cells
(Fig. 2H,H').
Differentiating vestibular hair cells mitose in the absence of Rb
In addition to cochlear HCs, pRb was expressed in vestibular HCs
(Fig. 1). We therefore studied
the consequences of Rb loss in the vestibular sensory epithelia of
mgRb:Rb-/- mice at E17.5 (n=8 ears) and E18.5
(n=15 ears). Prominent abnormalities were found
(Fig. 3). Similar to the organ
of Corti, Rb inactivation led to increase in the thickness of the
vestibular sensory epithelia and to overproduction of myosin VI-positive HCs
(Fig. 3A-C'). In normal
sensory epithelia, HCs are situated lumenally and supporting cells basally. In
the mutants, HCs were intermixed with supporting cells in the middle layers
and the basal part of the epithelium was filled with supernumerary HCs.
Moreover, some myosin VI-stained HCs were dislocated through the basal lamina
into the mesenchyme (Fig.
3A',C'). Mitotic figures were found in rounded, myosin
VI-positive cells, demonstrating divisions of differentiating vestibular HCs
(Fig. 3D,E), similar to
Rb-null cochlear HCs (Fig.
2D). Most mitotic vestibular HCs were located at the lumenal
surface, but some were seen at deeper epithelial levels
(Fig. 3E) and in the
mesenchyme. Accordingly, high numbers of phospho-histone H3-positive cells
were observed in the vestibular sensory epithelia of the mutants, the majority
of them at the lumenal surface, but some in deeper layers. By contrast, only a
few dividing cells were found in control specimens
(Fig. 3F-H').
Double-labelling experiments showed co-expression of phospho-histone H3 and
calretinin, a marker for vestibular HCs, these data confirming that vestibular
HCs of mgRb:Rb-/- mice undergo mitosis (data not shown).
Similar to the situation at birth, at E14.5, high numbers of dividing HCs were
found in the early-differentiating vestibular sensory epithelia of the
mutants, in contrast to controls (n=6 ears of mutants and controls
each) (Fig. 3I,I').
Owing to differences in the timing of onset of differentiation, vestibular HCs at birth represent a more mature HC status when compared with cochlear HCs. Therefore, we also focused on the development of HC stereocilia in the vestibular organs. Math1, myosins VI and VIIa, calretinin, Fgf8, Bdnf and Brn3c were expressed in vestibular HCs of the mutants, similar to controls (data not shown). The fact that the neurotrophic factor Bdnf was expressed in supernumerary HCs suggests that these cells can attract neuronal endings for the establishment of synaptic contacts. Together, molecular differentiation of Rb-deficient vestibular HCs appears well advanced, similar to Rb-null auditory HCs. However, HCs of the mutants showed abnormalities in the stereociliary bundle development, as evidenced by espin staining (Fig. 4A-H). Controls showed espin expression exclusively in stereocilia (Fig. 4A,E,G). In the mutants, espin-staining showed abnormal stereociliary bundle morphologies, particularly in utricles (Fig. 4B-F), but near-normal bundles were also seen, often in ampullae (Fig. 4G,H). Interestingly, supernumerary HCs in the deeper epithelial layers and in the mesenchyme showed espin-positive, disorganized cilia-like protusions and staining along cell membrane, indicating that the apicobasal polarity of these cells was lost (Fig. 4B-F).
|
|
Rb loss induces hair cell multinucleation
To further study the developmental status of supernumerary HCs of
mgRb:Rb-/- mutants at birth, we analysed 0.5 µm
Toluidine Blue-stained plastic sections. Intermixture of HCs and supporting
cells within the hyperplastic vestibular sensory epithelia was readily
observed in the absence of Rb
(Fig. 6A-H). In contrast to
controls (Fig. 6A,G), the
mutant sensory epithelia contained large numbers of HCs with a single,
bizarre-shaped nucleus or with two (occasionally three or four) nuclei
(Fig. 6B-E,H). Most of these
HCs showed decondensed DNA. The occurrence of multinucleated HCs suggests that
nuclear divisions had occurred without cytokinesis. Despite these distinct
nuclear abnormalities, many of the lumenally located HCs showed near-normal
stereociliary bundles (Fig.
6B,E,H). Many multinucleated HCs seemed to have a giant size,
extending from the lumenal surface to the deeper epithelial strata
(Fig. 6B,C,E). In addition to
HCs with aberrant nuclear morphologies, small and rounded cells with mitotic
figures in a single nucleus were seen, most of them at the epithelial surface
(Fig. 6C-E,H). Many of these
mitotic cells were identified as HCs, based on their immature stereociliary
bundles and, in paraffin sections, on myosin VI expression
(Fig. 3E). Thus, the
hyperplastic phenotype of the inner ear sensory epithelia of
mgRb:Rb-/- mutants seems to be caused by increase both in
numbers and size of HCs. The relationship between the two types of
Rb-null HCs is unclear, although in some cases HCs undergoing mitosis
appeared to be derived from multinucleated HCs (data not shown). Based on
observations made in some semi-thin sections, we cannot exclude the
possibility that some supporting cells normally located basally in the sensory
epithelia translocate to the lumenal surface and divide there (the mitotic
cell marked with arrowhead in Fig.
6C). Consistent with an increase in apoptosis, as revealed by
TUNEL- and cleaved caspase 3 staining (Fig.
5), semi-thin sections revealed scattered apoptotic profiles in
the inner ear sensory epithelia of the mutants
(Fig. 6D,F). The cytoplasm of
some mitotic HCs exhibited signs of degeneration and, in a some cases,
appeared apoptotic with condensed and fragmented nuclei
(Fig. 6E,F). The organ of Corti
of the mutants (Fig. 6I,J)
showed similar pathological features to the vestibular sensory epithelia.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In several tissues, precursor cells permanently withdraw from the cell
cycle before the onset of differentiation. This has also been shown to be the
case in the embryonic cochlea (Chen et al.,
2002). Although we could not see a clear effect of
Rb-inactivation on the size of the precursor cell pool at E13.5 and
E14.5, other observations suggest that pRb regulates the timing of their
terminal mitoses. First, at these stages, there was a slight increase in the
amount of phospho-histone 3-positive cells at the site of presumptive organ of
Corti in mgRb:Rb-/- mice. Second, the greater epithelial
ridge, which harbours precursor-like cells that can develop into HCs (Zhang et
al., 2000; Woods et al.,
2004
), contained aberrantly cycling cells in the late-embryonic
mutants. Third, in the cochleas of E18.5 mutants, the supporting cell
population was to some extent expanded. As mitoses were not detected in the
supporting cell layer of mutant cochleas, elevated numbers of precursor cells
might contribute to this expansion. Based on our studies on the expression of
members of the pocket protein family in the midgestational inner ear (U.P. and
J.M., unpublished), the role of pRb on precursor cells appears to be to some
extent redundant with other pocket proteins.
The present data show that HCs differentiate despite abnormal cell cycling,
suggesting that HC proliferation and differentiation are uncoupled processes.
Supernumerary HCs expressed a wide panel of markers characteristic for
differentiating (and mature) HCs. During skeletal myogenesis, Rb
stimulates the expression of differentiation markers by influencing
Myod1 activity (Gu et al.,
1993; Novitch et al.,
1996
; Novitch et al.,
1999
; Zacksenhaus et al.,
1996
). A similar mechanism might occur in the developing inner
ear, but as the interacting genes of the equivalent bHLH gene (Math1)
in HCs have not been characterized, this possibility remains to be explored.
Rb-null HCs at birth showed molecular evidence of differentiation,
including expression of integral molecular constituents of stereocilia.
However, supernumerary HCs situated in the deeper epithelial strata showed
distinctly abnormal stereociliary bundles. At the lumenal surface, where
stereociliary development normally occurs, large numbers of nascent bundles
were seen. In many cases, this immaturity could be linked with HCs undergoing
mitosis. Thus, the aberrant stereociliary development seems to be a
consequence of deregulated cell cycles. The fact that stereociliary bundles of
some of the supernumerary HCs appeared histologically almost comparable with
controls implies they are functional in terms of mechanotransduction
(Sage et al., 2005
).
Using a phospho-histone H3 antibody, we were able to determine the kinetics
of dividing HCs. Phospho-histone H3 is an M-phase marker, based on the fact
that histone H3 Ser10 phosphorylation coincides with chromosome condensation
of mitotic cells. Initiation of this phosphorylation is associated with
heterochromatin of late G2 phase, and, thus, this antibody is not completely
M-phase specific (Hendzel et al.,
1997; Van Hooser et al.,
1998
). The present results demonstrate, in controls and mutants,
the difference between the strong staining of condensed chromatin of mitotic
cells and the `patchy' staining of heterochromatin of G2-phase cells. In the
inner ear sensory epithelia of normal embryos, at the stages before HC
differentiation, mitotic nuclei were invariably located to the lumenal
surface. The majority of late G2-phase (this study) and S-phase cells
(bromodeoxyuridine-positive) (e.g. Pirvola
et al., 2002
) are situated deeper in the sensory epithelia. This
kind of relationship between nuclear position and the phase of cell cycle,
termed as interkinetic nuclear migration, has been found in several types of
developing epithelia, including the traumatized inner ear sensory epithelia of
birds (Bhave et al., 1995
).
Also in mgRb:Rb-/- mutants, most mitotic HCs were located
to the epithelial surface. However, HCs undergoing mitosis were also seen
deeper in the epithelia, indicating that translocation of nucleus or cell body
to the surface is not a prerequisite for mitosis.
Our results suggest that postmitotic HCs retain the potential for cell
cycle entry, but the pRb pathway acts as a guard to prohibit proliferation.
The activity of pRb is probably kept in check by post-translational
modifications, such as phosphorylation. Mitogens activate CDKs through
upregulation of cyclins. A primary mechanism inhibiting CDK activity is the
binding of CKIs to these kinases. CKIs efficiently inhibit CDKs even in the
presence of cyclins (Olson et al.,
2000). Indirect evidence for the importance of CDK regulation by
CKIs in HCs comes from the fact that HCs do not proliferate in response to
serum or mitogenic growth factors (Ryan,
2003
).
Our data show that the CKI p21 is expressed in the differentiating
cochlear and vestibular HCs, and that the expression is induced at the
initiation of HC differentiation. In the auditory sensory epithelium,
p21 expression was initiated at E14.5, at the stage when
Math1 expression has been first detected
(Chen et al., 2002). It is
possible that p21 induction in HCs is regulated by Math1, in
analogy to the positive role of bHLH proteins such as Myod1 and myogenin in
skeletal myogenesis (Halevy et al.,
1995
; Guo et al.,
1995
). Thereafter, p21 together with other CKI(s) (see below)
might have an active role in keeping pRb in a hypophosphorylated form. Thus,
negative regulation at the level of both pRb and CKIs seems to be responsible
for the maintenance of HC quiescence.
We did not find phenotypic alterations or aberrant mitoses in the inner
ears of developing or adult p21-/- mice
(Fig. 7). Interestingly, in
addition to p21, another CKI, p19, has been shown to be
expressed in the late-embryonic organ of Corti, but its inactivation does not
result in developmental abnormalities (Chen
et al., 2003). Thus, functional redundancy may exist between
p21 and p19 in developing cochlear HCs. In addition,
developing vestibular HCs express p21, but do not show phenotypic
changes following targeted gene disruption, most probably owing to functional
compensation. The identity of the CKI that may cooperate with p21 in
vestibular HCs remains to be identified, as p19 expression and the
consequences of p19 inactivation have not been reported in vestibular
organs.
In contrast to developmental stages, we did not detect p21
expression in adult HCs. Consistent with these observations are the data that
cochlear HCs of p19-null mice show aberrant S-phase entry only during
postnatal life (Chen et al.,
2003). Interestingly, in the mature cochlea, p19
inactivation was shown to have a stronger effect on inner HCs when compared
with outer HCs (Chen et al.,
2003
), suggesting differences in the regulation of postmitotic
state between the two cochlear HC subtypes. There might be differences in the
expression of p19 and/or other CKIs in these cells (postnatal
expression of p19 was not shown by Chen et al.) or cell cycle
regulation downstream of CKIs might be different. The latter possibility is
supported by the present data showing detectable expression of pRb in inner,
but not outer, HCs of the adult cochlea. Interestingly, also in adult
vestibular organs, pRb and Rb were expressed in a subset of HCs.
Although non-labelled HCs may express pRb levels that are below the detection
limit of the methods used, it remains to be determined whether this
non-homogenous expression is linked to the morphological classification into
type I and type II vestibular HCs
(Wersäll and
Bagger-Sjöbäck, 1974
).
Existing data provide evidence for the sensitization of cells to apoptosis
because of Rb loss-induced unscheduled proliferation at ectopic
sites, although there is also evidence that pRb harbours inherent
anti-apoptotic functions (Chau and Wang,
2003). We observed low-level apoptosis in the inner ear sensory
epithelia of mgRb:Rb-/- mice at birth. The extent of this
death did not compensate for the prominent hyperplasia of these epithelia, in
marked contrast to several other Rb-deficient tissues showing massive
cell-autonomous apoptosis in conjunction with ectopic proliferation
(Zacksenhaus et al., 1996
;
Guo et al., 2001
;
Chen et al., 2004
;
MacPherson et al., 2004
;
Zhang et al., 2004
). Thus, at
least during development, the extent of apoptosis associated with Rb
loss appears to be context dependent. The present data raise the issue of the
fate of Rb-null HCs. Intriguingly, Rb loss induced HC
cycling, but a large part of supernumerary HCs had defects in the completion
of the cell cycle, with apparent failures in cytokinesis. The formation of
multinucleated HCs, most of which appeared binucleated, implies for
polyploidy. Based on the knowledge that polyploidy induced by manipulation of
the cell cycle machinery often triggers cell death
(Storchova and Pellman, 2004
),
the rate of apoptosis of supernumerary HCs may accelerate during postnatal
life, an issue that can not be studied in the mgRb:Rb-/-
mice because of their lethality at birth. The data that supernumerary HCs of
adult p19 knockout mice, which are generated postnatally rather than
during embryogenesis, apoptose (Chen et
al., 2003
) support the possibility that Rb-deficient HCs
may ultimately be lost. However, the aberrant proliferation, polyploidy and
infiltration of Rb-null HCs into the mesenchyme may lead to
neoplastic transformation.
In conclusion, the present work reveals unexpected plasticity of
differentiating HCs. We show that the normally quiescent HCs proliferate in
response to Rb loss and that these divisions are to a large extent
tolerated, as analysed at birth. Continued Rb and pRb expression in
mature HCs speaks for the role of this tumour suppressor as a guard against
mitoses during adulthood as well. Our results point to the importance of
upstream effectors of the CKI family in modulating pRb activity. While our
work was under review, Sage et al. showed, in agreement with our results, that
Rb inactivation induces HC proliferation
(Sage et al., 2005). Similar
to our data, they also showed that stereociliary bundles of Rb-null
HCs were disoriented, but they also demonstrated that supernumerary HCs can
function as mechanoelectric transducers. Our findings on the pathology of
Rb-null HCs, including apoptosis, polyploidy and disorganization
within and outside the inner ear sensory epithelia, were not reported by Sage
et al. but suggest that HC quiescence is essential for the maintenance of
coordinated development of these epithelia. Our results suggest that forced HC
proliferation may induce tumour cell-like and death-prone phenotypes. These
data are likely to have implications in the design of future therapies to
induce HC re-growth.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bermingham, N. A., Hassan, B. A., Price, S. D., Vollrath, M. A.,
Ben-Arie, N., Eatock, R. A., Bellen, H. J., Lysakowski, A. and Zoghbi, H.
Y. (1999). Math1: an essential gene for the generation of
inner ear hair cells. Science
284,1837
-1841.
Bhave, S. A., Stone, J. S., Rubel, E. W. and Coltrera, M. D. (1995). Cell cycle progression in gentamicin-damaged avian cochleas. J. Neurosci. 16,4618 -4628.
Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D., Jacks, T. and Hannon, G. J. (1995). Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377,552 -557.[CrossRef][Medline]
Chau, B. N. and Wang, J. Y. (2003). Coordinated regulation of life and death by RB. Nat. Rev. Cancer 3, 130-138.[CrossRef][Medline]
Chen, P. L., Riley, D. J., Chen, Y. and Lee, W. H. (1996). Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs. Genes Dev. 10,2794 -2804.[Abstract]
Chen, P., Johnson, J. E., Zoghbi, H. Y. and Segil, N. (2002). The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination. Development 129,2495 -2505.[Medline]
Chen, P., Zindy, F., Abdala, C., Liu, F., Li, X., Roussel, M. F. and Segil, N. (2003). Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d. Nat. Cell Biol. 5,422 -426.[CrossRef][Medline]
Chen, D., Livne-bar, I., Vanderluit, J. L., Slack, R. S., Agochiya, M. and Bremner, R. (2004). Cell-specific effects of RB or RB/p107 loss on retinal development implicate an intrinsically death-resistant cell-of-origin in retinoblastoma. Cancer Cell 5,539 -551.[CrossRef][Medline]
Clarke, A. R., Maandag, E. R., van Roon, M., van der Lugt, N. M., van der Valk, M., Hooper, M. L., Berns, A. and te Riele, H. (1992). Requirement for a functional Rb-1 gene in murine development. Nature 359,328 -330.[CrossRef][Medline]
Classon, M. and Harlow, E. (2002). The retinoblastoma tumour suppressor in development and cancer. Nat. Rev. Cancer 2,910 -917.[CrossRef][Medline]
de Bruin A., Wu, L., Saavedra, H. I., Wilson, P., Yang, Y.,
Rosol, T. J., Weinstein, M., Robinson, M. L. and Leone, G.
(2003). Rb function in extraembryonic lineages suppresses
apoptosis in the CNS of Rb-deficient mice. Proc. Natl. Acad. Sci.
USA 100,6546
-6551.
Deng, C., Zhang, P., Harper, J. W., Elledge, S. J. and Leder, P. (1995). Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82,675 -684.[CrossRef][Medline]
Fekete, D. M., Muthukumar, S. and Karagogeos, D.
(1998). Hair cells and supporting cells share a common progenitor
in the avian inner ear. J. Neurosci.
18,7811
-7821.
Ferguson, K. L., Vanderluit, J. L., Hebert, J. M., McIntosh, W.
C., Tibbo, E., MacLaurin, J. G., Park, D. S., Wallace, V. A., Vooijs, M.,
McConnell, S. K. and Slack, R. S. (2002).
Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and
abnormal cortical development. EMBO J.
21,3337
-3346.
Field, S. J., Tsai, F. Y., Kuo, F., Zubiaga, A. M., Kaelin, W. G. Jr, Livingston, D. M., Orkin, S. H. and Greenberg, M. E. (1996). E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85,549 -561.[CrossRef][Medline]
Fritzsch, B., Matei, V. A., Nichols, D. H., Bermingham, N., Jones, K., Beisel, K. W. and Wang, V. Y. (2005). Atoh1 null mutants show directed afferent fiber growth to undifferentiated ear sensory epithelia followed by incomplete fiber retention. Dev. Dyn. (in press).
Gu, W., Schneider, J. W., Condorelli, G., Kaushal, S., Mahdavi, V. and Nadal-Ginard, B. (1993). Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell 72,309 -324.[CrossRef][Medline]
Guo, K., Wang, J., Andres, V., Smith, R. C. and Walsh, K. (1995). MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation. Mol. Cell. Biol. 15,3823 -3829.[Abstract]
Guo, Z., Yikang, S., Yoshida, H., Mak, T. W. and Zacksenhaus,
E. (2001). Inactivation of the retinoblastoma tumor
suppressor induces apoptosis protease-activating factor-1 dependent and
independent apoptotic pathways during embryogenesis. Cancer
Res. 61,8395
-8400.
Halevy, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J., Hannon, G. J., Beach, D. and Lassar, A. B. (1995). Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 267,1018 -1021.[Medline]
Hasson, T., Gillespie, P. G., Garcia, J. A., MacDonald, R. B.,
Zhao, Y., Yee, A. G., Mooseker, M. S. and Corey, D. P.
(1997). Unconventional myosins in inner-ear sensory epithelia.
J. Cell Biol. 137,1287
-1307.
Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106,348 -360.[CrossRef][Medline]
Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A. and Weinberg, R. A. (1992). Effects of an Rb mutation in the mouse. Nature 359,295 -300.[CrossRef][Medline]
Jiang, Z., Zacksenhaus, E., Gallie, B. L. and Phillips, R. A. (1997). The retinoblastoma gene family is differentially expressed during embryogenesis. Oncogene 14,1789 -1797.[CrossRef][Medline]
Jiang, Z., Liang, P., Leng, R., Guo, Z., Liu, Y., Liu, X., Bubnic, S., Keating, A., Murray, D., Goss, P. and Zacksenhaus, E. (2000). E2F1 and p53 are dispensable, whereas p21(Waf1/Cip1) cooperates with Rb to restrict endoreduplication and apoptosis during skeletal myogenesis. Dev. Biol. 227, 8-41.[Medline]
Jiang, Z., Guo, Z., Saad, F. A., Ellis, J. and Zacksenhaus,
E. (2001). Retinoblastoma gene promoter directs transgene
expression exclusively to the nervous system. J. Biol.
Chem. 276,593
-600.
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. C., Lai, C. C., Herrup, K., Lee, W. H. and Bradley, A. (1992). Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359,288 -294.[CrossRef][Medline]
Leezer, J. L., Hackmiller, R. C., Greene, R. M. and Pisano, M. M. (2002). Expression of the retinoblastoma family of tumor suppressors during murine embryonic orofacial development. Orthod. Craniofacial Res. 6,32 -47.
Lipinski, M. M., Macleod, K. F., Williams, B. O., Mullaney, T.
L., Crowley, D. and Jacks, T. (2001). Cell-autonomous and
non-cell-autonomous functions of the Rb tumor suppressor in developing central
nervous system. EMBO J.
20,3402
-3413.
Liu, Y. and Zacksenhaus, E. (2000). E2F1 mediates ectopic proliferation and stage-specific p53-dependent apoptosis but not aberrant differentiation in the ocular lens of Rb deficient fetuses. Oncogene 19,6065 -6073.[CrossRef][Medline]
Macleod, K. F., Hu, Y. and Jacks, T. (1996). Loss of Rb activates both p53-dependent and independent cell death pathways in the developing mouse nervous system. EMBO J. 15,6178 -6188.[Abstract]
MacPherson, D., Sage, J., Crowley, D., Trumpp, A., Bronson, R. T. and Jacks, T. (2003). Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system. Mol. Cell. Biol. 3,1044 -1053.[CrossRef]
MacPherson, D., Sage, J., Kim, T., Ho, D., McLaughlin, M. E. and
Jacks, T. (2004). Cell type-specific effects of Rb deletion
in the murine retina. Genes Dev.
18,1681
-1694.
Morgenbesser, S. D., Williams, B. O., Jacks, T. and DePinho, R. A. (1994). p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature 371, 72-74.[CrossRef][Medline]
Murray, A. W. (2004). Recycling the cell cycle: cyclins revisited. Cell 116,221 -234.[CrossRef][Medline]
Novitch, B. G., Mulligan, G. J., Jacks, T. and Lassar, A. B. (1996). Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle. J. Cell Biol. 135,441 -456.[Abstract]
Novitch, B. G., Spicer, D. B., Kim, P. S., Cheung, W. L. and Lassar, A. B. (1999). pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation. Curr. Biol. 9, 449-459.[CrossRef][Medline]
Olson, N. E., Kozlowski, J. and Reidy, M. A.
(2000). Proliferation of intimal smooth muscle cells. Attenuation
of basic fibroblast growth factor 2-stimulated proliferation is associated
with increased expression of cell cycle inhibitors. J. Biol.
Chem. 275,11270
-11277.
Pauley, S., Beisel, K., Wright, T., Pirvola, U., Ornitz, D. and Fritzsch, B. (2003). Expression and function of FGF10 in mammalian inner ear development. Dev. Dyn. 227,203 -215.[CrossRef][Medline]
Pirvola, U., Spencer-Dene, B., Xing-Qun, L., Kettunen, P.,
Thesleff, I., Fritzsch, B., Dickson, C. and Ylikoski, J.
(2000). FGF/FGFR-2(IIIb) signaling is essential for inner ear
morphogenesis. J. Neurosci.
20,6125
-6134.
Pirvola, U., Ylikoski, J., Trokovic, R., Hebert, J. M., McConnell, S. K. and Partanen, J. (2002). FGFR1 is required for the development of the auditory sensory epithelium. Neuron 35,671 -680.[CrossRef][Medline]
Pirvola, U., Zhang, X., Mantela, J., Ornitz, D. M. and Ylikoski, J. (2004). Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions. Dev. Biol. 273,350 -360.[CrossRef][Medline]
Ryan, A. F. (2003). The cell cycle and the development and regeneration of hair cells. Curr. Top. Dev. Biol. 57,449 -466.[Medline]
Ruben, R. J. (1967). Development of the inner ear of the mouse: a radioautographic study of terminal mitoses. Acta Otolaryngol. Suppl. 220, 1-44.
Sage, C., Huang, M., Karimi, K., Gutierrez, G., Vollrath, M. A.,
Zhang, D. S., Garcia-Anoveros, J., Hinds, P. W., Corwin, J. T., Corey, D. P.
and Chen, Z. Y. (2005). Proliferation of functional hair
cells in vivo in the absence of the retinoblastoma protein.
Science 307,1114
-1118.
Sellers, W. R., Novitch, B. G., Miyake, S., Heith, A., Otterson,
G. A., Kaye, F. J., Lassar, A. B. and Kaelin, W. G. Jr
(1998). Stable binding to E2F is not required for the
retinoblastoma protein to activate transcription, promote differentiation, and
suppress tumor cell growth. Genes Dev.
12, 95-106.
Storchova, Z. and Pellman, D. (2004). From polyploidy to aneuploidy, genome instability and cancer. Nature Rev. 5,45 -54.
Takahashi, C., Bronson, R. T., Socolovsky, M., Contreras, B.,
Lee, K. Y., Jacks, T., Noda, M., Kucherlapati, R. and Ewen, M. E.
(2003). Rb and N-ras function together to control differentiation
in the mouse. Mol. Cell. Biol.
23,5256
-5268.
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L. and Jacks, T. (1998). Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends survival of Rb-deficient mouse embryos. Mol. Cell 3,293 -304.[CrossRef]
Van Hooser, A., Goodrich, D. W., Allis, C. D., Brinkley, B. R.
and Mancini, M. A. (1998). Histone H3 phosphorylation is
required for the initiation, but not maintenance, of mammalian chromosome
condensation. J. Cell Sci.
111,3497
-3506.
Vidal, A. and Koff, A. (2000). Cell-cycle inhibitors: three families united by a common cause. Gene 247,1 -15.[CrossRef][Medline]
Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81,323 -330.[CrossRef][Medline]
Wersäll, J. and Bagger-Sjöbäck, D. (1974). Morphology of the vestibular sense organ. In: Handbook of Sensory Physiology. Vestibular System. Basic Mechanisms (ed. H. H. Kornhuber), pp.123 -170. New York: Springer.
Wilkinson, D. G and Green, J. (1991). In situ hybridization and the three-dimensional construction of serial sections. In: Postimplantation Mammalian Embryos (ed.. A. J. Copp and D. L. Cockroft), pp. 155-171. Oxford, UK: IRL Press.
Woods, C., Montcouquiol, M. and Kelley M. W. (2004). Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat. Neurosci. 7,1310 -1318.[CrossRef][Medline]
Wu, L., de Bruin, A., Saavedra, H. I., Starovic, M., Trimboli, A., Yang, Y., Opavska, J., Wilson, P., Thompson, J. C., Ostrowski, M. C. et al. (2003). Extra-embryonic function of Rb is essential for embryonic development and viability. Nature 421,942 -947.[CrossRef][Medline]
Yoshida, H., Kong, Y. Y., Yoshida, R., Elia, A. J., Hakem, A., Hakem, R., Penninger, J. M. and Mak, T. W. (1998). Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94,739 -750.[CrossRef][Medline]
Zacksenhaus, E., Jiang, Z., Chung, D., Marth, J. D., Phillips, R. A. and Gallie, B. L. (1996). pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis. Genes Dev. 10,3051 -3064.[Abstract]
Zhang, J., Gray, J., Wu, L., Leone, G., Rowan, S., Cepko, C. L., Zhu, X., Craft, C. M. and Dyer, M. A. (2004). Rb regulates proliferation and rod photoreceptor development in the mouse retina. Nat. Genet. 36,351 -360.[CrossRef][Medline]
Zheng, J. L. and Gao, W. O. (2000). Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat. Neurosci. 3, 580-586.[CrossRef][Medline]
Zheng, L., Sekerkova, G., Vranich, K., Tilney, L. G., Mugnaini, E. and Bartles, J. R. (2000). The deaf jerker mouse has a mutation in the gene encoding the espin actin-bundling proteins of hair cell stereocilia and lacks espins. Cell 102,377 -385.[CrossRef][Medline]