- Email: [email protected]
repair in the inner ear
480
Factors controlling Hinrich Staecker* Damaged
proliferation
transdifferentiation the mammalian possibly,
roles. Several
be associated insulin-like
factor
growth
factor
are important
whereas
transdifferentiation hair cells appear factors
epidermal
1 (IGF-l),
to
factor,
transforming
in the mammalian
evidence
that regeneration/repair
hair cells is possible
during
labyrinth.
growth
however,
for
for converting
in the lateral lines and inner ears of fish in the basilar papilla and vestibule of
they
have
and amphibians
Increasing
been
found
of mammals. differentiated;
to regenerate
[2], as well as in birds
in fish [l]
[3,4] (‘l’able
1).
of mammalian period
DNA-labeling
studies
of the avian
inner
ear have
sho\vn
that a spontaneous turnover of hair cells takes place in the vestibule but not in the auditory papilla [.5], However, in neonatal chick, damage to auditory hair cells
at later times.
Addresses *Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, Massachusetts 02114, USA iDepartments of Otolaryngology and Neuroscience, Kennedy Center, Room 302, Department of Otolaryngology, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York 10461, USA; e-mail: [email protected] Current Opinion in Neurobiology
ear are responsible
(e.g. sound pressure or acceleration) energy (i.e. neurotransmission). Hair
birds, and in the the cochlea and vestibule Hair cells of the inner ear are terminally
growth
the early neonatal
and may exist to a very limited degree
of the inner
mechanical energy into electrochemical cells are present and amphibians,
insulin,
for avian inner ear regeneration/repair, growth
c(, insulin, IGF-1, and IGF-2 are important
auditory
Hair cells
and, to play
process:
and fibroblast
regeneration/repair suggests
The ability to perceive sound and a sense of balance are important for orienting us within our environment.
have been found
with the regeneration/repair
growth
Introduction
are replaced
cells and
cells into hair cells. In
system,
the repair of damaged
significant
papilla
of supporting
of supporting vestibular
in the inner ear
and Thomas R Van De Watefi-
hair cells in the avian basilar
by regenerative
factors
hair-cell regeneration/repair
induced through auditory
by ototoxin or sound trauma will evoke repair hair-cell regeneration, resulting in recovery of function [6]. The source of these regenerated
hair cells is debated; some evidence suggests that many of the regenerated hair cells derive from the regenerative proliferation of the supporting cells, whereas the rest result from the transdifferentiation of supporting cells [7].
1998, 8:480-487
In mammals, spontaneous hair-cell regeneration has been observed, thus far, only in the vestibule [8]. Controversy remains as to whether the vestibular hair-cell regeneration
http://biomednet.com/elecref/0959438800800480 0 Current Biology Publications ISSN 0959-4388 Abbreviations BrdU 5-bromo-2’.deoxyuridine EGF epldermal growth factor acidic flbroblast growth factor FGF-1 basic fibroblast growth factor FGF-2 IGF insulin-like growth factor polymerase chain reaction PCR PDGF platelet-derived growth factor PDGF-AA A chain homodlmer of PDGF RT reverse transcription TGFcl transforming growth factor cx
observed in the utricle is attributable to the regenerative proliferation of supporting cells or to the transdifferentiation of supporting cells, or to the repair of damaged hair cells,
or to some
combination
of these
processes.
In this review, we compare the renewal of damaged hair cell populations within the sensory epithelium of both the auditory and vestibular receptors in avian and mammalian inner ears. IVe also discuss the possible sources
Table 1 Summary
of hair-cell regeneration/repair
in different species.
-
Ongoing production of hair cells
Neuroepithelial proliferation after damage
Spontaneous hair-cell regeneration
Regenerative proliferation
Functional recovery
Amphibian and fish/ lateral line and inner ear
f
+
+
+
Not tested
Bird/basilar papilla
0
+
+
+
+
Bird/vestibule
+
+
+
+
+
Mammal/cochlea
0
+ (postnatal)
0
0
0
Mammal/vestibule
?
+
+ (limited)
?
?
Species/sensory receptors
in the inner ear
Factors controlling hair-cell regeneration/repair
of regenerated
hair cells:
regenerative
proliferation;
Avian hair-cell
trans-
differentiation; and/or repair (see Figure 1). Finally, we report on recent studies characterizing the factors that influence the hair cell renewal process in these avian and mammalian
One
of
the
main
aims
of
regeneration
work
continues to be to identify where new hair cells come from. Evidence supports three different suggested modes of hair-cell renewal following injury (see Figure 1): regenerative proliferation of supporting cells; transdifferentiation of supporting cells; and repair of damaged hair cells. Proof of the active participation of one of these modes of hair-cell renewal does not exclude the possibility that either or both of the other modes also contributes to the hair-cell regeneration/repair following an insult to a specific hair-cell population the inner
actively process within
ear.
In sound-traumatized
chickens
regenerated
are labeled
hair cells
[9] and quails by tritiated
[lo], many thymidine,
the damaged papillae lost hair cells. After hair cells, Bhave et
nl. [l l] immunostained chick cochleae using cell-cycle markers and noted that although all the cells of the sensory epithelium left the Go phase (i.e. the quiescent phase), only those cells within the area of hair-cell loss
of hair cells
hair-cell
481
and Van De Water
regeneration
suggesting that cell division within is a major source for replacing administering otoroxin to damage
labyrinths.
Repair versus regeneration
Staecker
entered S (synthesis) phase evidence for regeneration
and underlvent mitosis. Direct of hair cells from supporting
cells has, thus far, been demonstrated lateral line neuromasts [ 121.
By using mitosis, damage take They
cytosine
arabinoside
(ara
only for amphibian
C),
an
inhibitor
place in observed
the absence this in both
of supporting cell mitosis. ototoxinand sound-trauma-
Figure 1
(a) Re-enter
AllI
Daughter cells
h
G---Ill”
-
Prollferatlon of supportIng
cells
n /I 4
I
(b) Transdifferentiatlon of supporting
cells
3
(c) Repair of injured hair cells
(e.g. oxidative
of
Adler and Raphael [13,14] found that following to the basilar papilla, hair-cell regeneration can
stress)
Schematic representation of the three different processes that may contribute to sensory ear: (a) regenerative proliferation of supportlng cells; (b) transdifferentiation of supporting HS98, Thomas R Van De Water and Hinrich Staecker, 1998.
3
TRV and HS98
hair-cell regeneration/repair within the vertebrate inner cells; and (c) repair of injured hair cells. TRV and
482
Sensory systems
damaged
basilar
papilla,
regeneration response similar. Further support
suggesting to these for these
that
the
hair-cell
pathological insults is findings comes from the
work of Roberson [15], who implanted an intracochlear canula that constantly infused tritiated thymidine into gentamicin-treated chicks. A single dose of gentamicin caused a near total the papilla; however,
loss of hair cells in the base of the regenerated hair cells were not
labeled, indicating that transdifferentiation cells contributes to hair-cell regeneration
of supporting in the avian inner
ear [15].
However,
at present,
that macrophages either regeneration [22*].
there
initiate
Factors influencing
is no direct or
modulate
evidence hair-cell
hair-cell renewal
It is important and factors
to identify and characterize the processes that can influence the hair cell renenal process,
including cellular events (e.g. re-entry of supporting cells into the cell cycle), cell interactions (e.g. migration of macrophages into the site of hair cell damage), expression of growth factor receptors in response to sensory epithelium damage (e.g. the epidermal growth factor
Mammalian The
aiuo [ll].
hair-cell
process
of
renewal/repair
hair-cell
inner ear has not yet been of regenerated hair cells
renewal
in
the
mammalian
fully characterized. Evidence labeled with S-bromo-2’-de-
oxyuridine (BrdU), or tritiated thymidine, in utricular cultures is sparse. Warchol et (I/. [Ih] have identified labeled hair cells in gcntamicin-damaged guinea pig utricular explants; however, judging from the number of regenerated hair cells present and the overall paucity of labeling, plausible
transdifferentiation source for most
utricular
explants
of supporting cells is the most of the ne\v hair cells in these
[EGF] receptor), and ear sensory epithelium Understanding these avian and mammalian
responsiveness to growth
factors and how they differ between labyrinths may provide insights into
the differences in the vertebrate inner ears. Avian hair-cell
hair cell
renewal
process
in these
regeneration
Hair-cell regeneration lvas first described in avian basilar papilla, and it remains the best understood system. llsing a laser, \\:archol and Corwin [23] lesioned hair cells in basilar papilla explants with tritiated
[16,17].
of damaged inner factors (e.g. TGFcx).
explants, thymidinc.
and then labrlcd the As discussed earlier,
An alternate mode of hair-cell renewal to explain unlabeled regenerated hair cells is the repair of damaged hair cells. Sobkowicz ettul. [ 181 have demonstrated that auditory hair cells can survive the loss of their apical cytoplasm and stereocilia bundles. Analysis of these damaged hair cells shows that repair is complete within 48 h [l&19’]. This concept of hair-cell repair is also supported by structural studies that have demonstrated the role of supporting cells
only supporting cells in close proximity to the lesion entered the S phase of the cell cycle. Supporting cells in cultures of thermolysin-separated sheets of chick utricular neuroepithelium undergo spontaneous division in defined
in scar formation strongly suggest
To study further the role of cell proliferation in hair-cell renewal, markers for immature hair cells have been sought. Stone et al. [ZS] have identified tlvo markers, calmodulin and beta tubulin, that stain regenerated hair cells selectively, as well as putative early differentiating hair cells. Recent studies show that the actin filament binding protein fimbrin localizes specifically to hair cells [26,27]. In chick basilar papilla cultures, staining for fimbrin has been observed 96 h after acoustic trauma 1271.
a capacity The
modes
after ototoxic damage [ZO]. These studies that sublethally damaged hair cells have
for self-repair. of injury
in
the
experiments
cited
above
arc either laser or mechanical disruption of the cuticular plate, so it is unclear whether other modes of injury, such as damage by ototoxins or sound trauma, would lead to similar findings. [Jsing a different approach, Kelly et al. [al] ablated hair cells in organ of Corti explants with a laser to demonstrate that the degree of supporting-cell proliferation is proportional to the degree of injury, again suggesting that contact-mediated factors are important in the initiation of regeneration/repair. hlacrophages are associated with tissue regeneration in other organs, and they produce a variety of growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor ~1 (TGFa), and basic fibroblast growth Factor (bFGF, also known as FGF-2). Localized trauma to the vestibular neuroepithelium results in recruitment of macrophages to the site of injury. This recruitment phenomenon has also been observed in the amphibian lateral line [l,?], in explants of chick basilar papilla [Z’], and in traumatized vestibular epithelium its
medium, indicating that the explantation preparation techniques (e.g. thcrmolysin trigger cell proliferation [ 2-11.
and culture digestion) can
There has also been significant interest in identifying growth factors that can potentially trigger or enhance hair-cell regeneration. \Vhen normal basilar papillae are co-cultured with aminoglycoside-damaged papillac, they demonstrate increased rates of tritiated thymidinc incorporation, suggesting that a diffusible substance is being produced by the lesioned papillae, w:hich then initiates a cell proliferation response in the normal papillae [28]. In the avian system, two growth factors have been identified as stimulators of cell proliferation: insulin and insulin-like growth factor 1 (IGF-1). Basilar papilla explants exposed to either of these factors show a significant mitogenic response (i.e. increased Brdll uptake). Treatment of explants with EGF, bombesin or TGFa do not significantly enhance Brdl! uptake, except
in areas
of nonsensory
epithelium
[2’S”].
A significant
advance in understanding the signaling cascade involved in the induction of hair-cell regeneration has come with the observation that induction of CAMP in cultures of avian basilar papillae stimulates the incorporation of BrdU in hair cells of uninjured basilar papilla explants [30]. This cAhlP-stimulated proliferation inhibitors of protein kinase A, which Mammalian
hair-cell
can be blocked by is regulated by cAhlP.
of guinea
rate of cell significantly
pig utricles.
proliferation lower than
TGFa
As discussed
earlier,
the
in these mammalian tissues is in avian tissues. More recently.
regeneration/repair of auditory hair cells has been in explants of three-day-old rat organ of Corti
reported explants
analysis
FGF-2
are present
has shown
in vice and that
on utricular
has also been
regeneration/repair
epithelial
implicated in neonatal
that IGF cells
utricular and FGF
hair cells receptors
[38”].
as a factor driving
hair-cell
organ of Corti cultures
[40].
The validity of these results have been questioned [41], but Zinc and de Ribaupierre [42”], using a different culture system, have recently replicated and extended these
renewal/repair
Hair-cell regeneration in mammals was first identified by Forge et al. [S] in gentamicin-treated guinea pigs and by Warchol et al. [16] in aminoglycoside-damaged explants
histochemical produce
483
in the inner ear Staecker and Van De Water
Factors controlling hair-cell regeneration/repair
results.
They
treated
organ of Corti explants 5-7 days of treatment
with with
three-day-old
postnatal
rat
1 mhl neomycin followed by either TGFcx or EGF, or a
combination of the two. Apical turns of the organ of Corti explants were removed to prevent inadvertent analysis of spared apical hair cells. Cultures treated with either TGFa or EGF, or a combination of both, showed a significant number
of regenerated/repaired
hair cells
displaying
the
damaged by administration of neomycin and then treated with retinoic acid and fetal calf serum [31]. Kelly r~
surface features of immature hair cells (see Figure ‘liitiated thymidine labeling showed cell proliferation the inner sulcus area of EGF-treated explants but
al. [Zl] have demonstrated that embryonic and neonatal cochleae replace hair cells destroyed by laser irradiation, but renewal is greatly attenuated in postnatal explants.
labeled hair cells, lending credence to the theory that these cells are derived from differentiation of a precursor cell, or transdiffcrentiation of existing supporting cells (or
Spontaneous regeneration of hair cells in the cristae of adult chinchilla has also been reported [32]. Interestingly, there is a notable difference between the ability of type I
to the repair hair cells).
and type
Regeneration/repair
II hair cells
to regenerate
[33].
Recently, cells carrying immature appearing stereocilia have been identified in the undamaged utricles of adult guinea pigs [34*]. These cells appear to represent 0.7% of the utricular hair-cell population, suggesting that some degree of hair cell replacement may be an active process in the vestibule of adult mammals [34*]. Lambert [35] has demonstrated that hair-cell regeneration takes place in adult mouse cristae and that cultures of adult mouse utricles pretreated with neomycin show an increased r3te of cell proliferation after treatment with TGFa. In combination with insulin, TGFa has also been shown to increase proliferation of cells in extrasensory epithelia and supporting cells in adult rat utricles [36]. Other studies have sho\vn that IGF-1, IGF-2, TGFa and EGF increase proliferation of utricular epithelial cells [37,38**,39]. This effect on proliferation can be inhibited by neutralizing antibodies against FGF-2 and IGF-1, suggesting that these factors may be part of the hair-cell regeneration cascade. Growth factor treatment can also enhance the process of vcstibular hair-cell regeneration/repair within the utricles of adult guinea pig inner ears exposed to vestibulotoxic levels of gentamicin (R Kopke et al., Sot h’turo.b Abs~-rr 1996, 22636.3). Infusion of a combination of TGFa, IGF-1, and retinoic acid into the perilymphatic space of the guinea pig vestibule one week post-gentamicin administration results in a significant enhancement of the regeneration/repair of the vestibular hair cells (i.e. 3- to lo-fold; the variability is site specific) and stimulates the maturation of their sensory hair bundles. Immuno-
of sublethal
damage
2). in no
to the ototoxin-damaged
of hair cells in the ototoxin-damaged,
EGF-treated explants WAS more vigorous at the middle turn, leading Zine and de Ribaupierre [42**] to speculate that more ‘undifferentiated’ precursors are present at the middle turn compared to at the basal turn. In a follo\v-up study using ELI%, these investigators (A Zinc, F de Ribaupierre, personal communication) reported an upregulation of EGF receptor expression in organ of Corti explants in response to ototoxic damage. ‘I’hus far, the regeneration/repair of Corti appears to be limited
phenomenon in the organ to in vitro studies and has
not been demonstrated in organ of Corti explants excised from rdt pups after postnatal day 5. Some evidence of a limited attempt at regeneration/repair is provided by the experiments of Lenoir and \‘ago [43.34*], who injected nine-day-old rdt pups with amikacin for seven consecutive days and then sacrificed the animals at intervals to 90 days post initiation of ototoxin injections. At 21 and 35 days after ototoxin treatment, atypical cells bearing tufts of microvilli and resembling immature hair cells were observed only in the area previously occupied by the apical outer hair cells. In the inner hair-cell area of these animals, Lenoir and Vago observed cells that had both efferent and afferent innervations but no stereocilia or cuticular plates. They hypothesize that the apical region of the rat cochlea does not reach adult morphology until after postnatal day 21 and, therefore, only a limited attempt at regeneration/repair is possible. One approach in growth factor studies has been to study cultures of supporting cells and to determine which factors
484
Sensory systems
Figure 2
Scanning a control
electron micrographs of the auditory hair-cell area of neonatal rat organ of Corti explants after 10 days in vitro. (a) Surface view of culture after 10 days in vitro, displaying well-defined rows of inner and outer hair cells, as well as normal stereociliary arrays. (b) An
ototoxin-damaged culture hair cells from the organ
(exposed for 24 h to 1 mM neomycin and then placed for 7 days in normal medium) showing complete loss of auditory of Corti. (c) Area of replacement hair cells in ototoxin-damaged cultures treated for 7 days with TGFa. Numerous
immature stereocilia bundles are visible on the apical surfaces of the replacement hair cells. Kinocilia of these replacement hair cells are found In an eccentric position at the external border of the cell (arrow). The surface of the Corti’s organ is partially hidden by the re-grown tectorial membrane (TM). (d) Higher magnification of the apical surface of a single replacement hair cell shown in (c). Hair cells like this one, with a circular bundle of closely packed immature, uniformly sized stereocilia and a longer ear of the rat. Bars=2 PM in all panels. Reproduced with permission from [42”1.
can potentially initiate their differentiation into hair cells. Initial research has focused on determining what markers, aside from the presence of actin-containing stereocilia, are specific for hair cells. A recent study by Zheng and Gao [45*] confirms that the calcium-binding protein calretinin is present in immature hair cells just after their terminal mitosis and before stereocilia bundle formation. They then set out to test if any mitotic cells in rat utricular epithelium cultures co-label with BrdU and calretinin. No double labeling U’ZISseen in partially dissociated utricular epithelium, indicating that spontaneous mitosis occurring after dissociation does not produce early hair-cell progenitors. Cultures of dissociated epithelial sheets treated with gentamicin for 2 days followed by BrdU labeling ifi vitru for 7-11 days contained a few cells that were double
kinocilium
(arrow),
are normally
found
in the developing
inner
labeled with both anti-BrdU and anti-calretinin. Control cultures never showed any double labeling, negating the possibility of dissociation-induced damage and repair of hair-cell DNA. ‘Ib study further this effect, Zheng, Lewis and Gao [46] produced conditionally immortalized utricular cpithelial cell lines that possess the morphologic characteristics of supporting cells. These immortalized utricular ccl1 lines express the same growth factor receptors found on supporting cells (i.e. EGF and FGF receptors). When cultured at a nonpermissive temperature in the prescncc of FGF-2, these cells stop proliferating, and express both calretinin and calmodulin [46]. Immortalized hair cells have also been produced from embryonic inner ear cells of
Factors controlling hair-cell regeneration/repair
the ImortomouseB for hair cells; appears
[47]. These
however,
Another
approach
are involved the cDNAs normal
auditory
involved
Growth factors and receptors organ of Cot%*.
transcription noise-damaged fication
growth
and
lesioned
auditory
for the
EGF
two-week-old performed
receptor,
fibroblast
growth
factor
chicks reverse
FGF
receptor,
(FGF-1)
mRNA
is
upregulated after basilar papilla damage. The Eph family tyrosine kinase receptor &k-l0 is also localized in the sensory epithelium [@I. A differential display PCR study of noise-damaged basilar papillae idencitied four genes that are dysregulatcd in response to this trauma [SO]: genes lated protein, GTP-binding gene. These signaling and regeneration.
that encode for parathyroid hormone reCa?+/calmodulin-regulated protein kinase II, protein CDC32 and a novel uncharacterized results suggest that CaZ+/calmodulin-related GTPase cascades may play a role in hair-cell
The use of PCR amplification of reverse transcribed mRNAs has confirmed that the FGF family of growth factors plays an important role in avian hair cell regeneration/repair, In addition, these gene amplification results have identified several other candidate factors potentially contribute to the regeneration/repair Genes
XI‘-PCR
present
in mammalian
analysis
of control
that may response.
hair cells
and
FGF-2,
IGF-1,
IGF-2,
PDGF-AA,
PDGF-BB
factor receptors EGF-R, FGF-R2, FGF-R4, IGF-1 R, PDGF-Rc(,
PDGF-Rfi
‘Modlfled
homodimer
renewal
normal papillae. Interestingly, the expression pattern of the FGF receptor changed position after noise damage: localization of immunostaining changed from the hair-cell stereocilia to the apical areas of surrounding supporting RT-PCR was used to show cells. Semi-quantitative acidic
in the mammalian
Growth
from
[51].
PDGF-BB,
factors
fundamental
regeneration/repair
IGF receptor, insulin receptor. rctinoic acid receptors B and CL,and FGF-2 are present in both noise-damaged and
that
identified
B
chain
of
PDGF;
-R, receptor.
regeneration/repair.
(RT) PCR on RNA extracted from both and normal chick basilar papillae. Ampli-
products
485
factors
has been to identify and their receptors in
Cotanche [&I exposed trauma and subsequently
sound
and Van De Water
Table 2
markers
EGF, TGFc(, FGF-I,
which
epithelium
in avian hair-cell
ear Staecker
Growth factors
to determining
undergoing
I ,ee and to
some
differentiation
studies
in regeneration/repair of growth factors
epithclium Genes
cells express morphological
to be aberrant.
Gene amplification
both
their
in the inner
neomycin-damaged
process
Several growth regeneration/repair
and modulate
auditory
response to growth IGF-1 and insulin.
the hair-cell
1).
factors that appear process have been
preliminary overview between the avian as between
that initiate
(Table
to modulate the identified, and a
indicates that there are differences and mammalian systems, as well and factors
vestibular such
hair
as FGF-2,
cells,
in their
TGFcx,
EGF,
‘I‘here also appear to be three mechanisms by which hair cells can be replaced following damage to the inner ear (Figure 1). The avian system replaces lost hair cells by both regenerative proliferation of supporting cells (with subsequent differentiation of the daughter cells into both hair cells and supporting cells), and by transdifferentiation of mature supporting cells into hair cells. Repair of damaged hair cells may also play a role in hair-cell renewal in this system, but, at present, there is no evidence to support such a repair process in the damaged avian inner ear. In mammals, hair-cell regeneration/repair processes include both transdifferentiation of mature and immature supporting cells into hair cells and self-repair of sublethal damage to the hair cells. Regeneration/repair of mammalian auditory hair cells, at present, is limited to the neonatal period, with some evidence of an unsuccessful attempt at regeneration/repair (i.e. atypical cells) at a slightly later stage of postnatal development in the cochlea of the rat. Currently, there are no experimental findings that support the participation of regenerative cell proliferation as a major participant in the replacement of lost hair cells from either the auditory or vestibular receptors of mammals.
rat
utricles has shown that receptors for IGF-1, FGF-2, EGF and PDGF-AA are present, and also suggest that the PDGF-AA and IGF-1 receptors may localize to the hair cells [jl]. The presence of mRNA for a wide variety of growth factors and growth factor receptors have also been identified in the postnatal rat cochlea (see Table 2) [52].
Conclusions Hair-cell regeneration is a rapidly developing field that has used four model systems over the past ten years-avian basilar papilla. avian utricle, mammalian organ of Corti, and mammalian utricle - in an attempt to characterize the
The challenge for future investigations will be to identify the mechanism that initiate the transdifferentiation and repair processes, and to determine the growth factors and other factors that are involved in these processes. In addition, the identification and characterization of underlying molecular changes that prevent auditory haircell regeneration/repair in the auditory system of adult mammals should prove a fruitful area of research. One final challenge to the biologists working in the area of hair-cell regeneration/repair will be to identify the differences between the hair-cell populations of the avian and mammalian inner ears that prevent regenerative proliferation
486
Sensory
(c.g.
systems
cell cycle
control
process of hair-cell mammals.
factors)
from
regeneration/repair
participating in the inner
in the ear of
Acknowledgements ‘1‘11~ prcp~rar~on of[his rcvic\v
\vas supported by the Shulsky Hearing Kcbcarch Foundation of the hlontcfiorc hlcdical Ccntcr (‘1X VXI Dc b\‘ater). ‘l‘hc ;~uthor\ thank A Chcng, a fourth year medical student, for his help in preparing Figure I, and A %inc and 1; dc Kibaupicrrc for the USC of rhc illusmtrron in Frgurc ?. LVe &o thank A Zinc. 1; de kibaupicrrc and I’ I,cfcbvre for their critic;11 commcncs. and K Impcrati for \vord processing of thus manuscripr.
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19. .
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Leonova EV, Raphael Y: Organization of cell junctions and cytoskeleton in the reticular lamina in normal and ototoxically damaged organ of Corti. Hear Res 1997, 113:14-28.
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McFadden EA, Saunders JC: Recovery of auditory function following intense sound exposure in the neonatal chick. Hear Res 1989, 41:205-2 15.
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Cotanche DA: Hair cell regeneration in the avian cochlea. Otol Rhmol Laryngol 1997, 168(suppl):Q-15.
8.
Forge A, Li L, Corv-in JT, Nevill G: Ultrastructural evidence for hair cell regeneration in the mammalian inner ear. Science 1993, 259:1616-l 619.
9.
Cowin JT, Cotanche DA: Regeneration of sensory hair cells after acoustic trauma. Science 1988, 240:1772-l 774.
10.
Ryals EM, Rubel EW: Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 1988, 240:1774-l 776.
Il.
Bhave SA, Stone JS, Rubel EW, Coltrera MD: Cell cycle progression in gentamicin-damaged avian cochleas. J Neurosci 1995, 15:4618-4628.
30.
Navaratnam DS, Su HS, Scott SP, Oberholtzer JC: Proliferation in the auditory receptor epithelium mediated by a cyclic AMP dependent signaling pathway. Nat Med 1996, 2:l 136-I 139.
12.
Jones JE, Corwin JT: Regeneration of sensory cells after laser ablation in the lateral line system: hair cell lineage and macrophage behavior revealed by time lapse microscopy. J Neurosci 1996, 16:649-662.
31.
Lefebvre P, Malgrange B, Staecker H, Moonen G, Van De Water TR: Retinoic acid stimulates regeneration of mammalian auditory hair cells. Science 1993, 260:692-695.
32.
13.
Adler H, Raphael Y: New hair cells arise from supporting cell conversion in the acoustically damaged chick inner ear. Neurosci Lett 1996, 205:17-20.
Tanyeri H, Lopez I, Honrubia V: Histological evidence for hair cell regeneration after ototoxic cell destruction with local application of gentamicin in the chinchilla crista ampullaris. Hear Res 1995, 89:194-202.
14.
Adler H: Further evidence for supporting cell conversion in the damaged avian basilar papilla. Int J Dev Neurosci 1997, 15:375-385.
33.
15.
Roberson D: Light microscopic evidence that direct transdifferentiation gives rise to new hair cells in regenerating avian auditory epithelium. Auditory Neurosci 1996, 2:195-205.
Lopez I, Honrubia V, Lee SC, Schoeman G, Beykirch K: Quantification of the process of hair cell loss and recovery in the chinchilla crista ampullaris after gentamicin treatment Inf J Dev Neurosci 1997, 15:447-461.
DA: Regeneration of hair cell stereociliary bundles severe acoustic trauma. Hear Res 1987, 30:181-i 95.
Ann
16.
Warchol ME, Lambert PR, Goldstein BJ, Forge A, Corwin JT: Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans. Science 1993, 259:161 Q-l 622.
1 7.
Li L, Forge A: Morphological evidence for supporting cell to hair cell conversion in the mammalian utricular macula. Int J Dev Neurosci 1997, 15:433-446.
18.
Sobkowicz HM, August BK, Slapnick SM: Post traumatic survival and recovery of the auditory sensory cells in culture. Acfa Otolaryngol (Sfockh) 1996, 16:257-262.
of the hair cell specific in the chicken cochlea.
29. ..
Oesterle EC, Tsue TT, Rubel EW: Induction of cell proliferation in avian inner ear sensory epithelia by insulin like growth factor 1 and insulin. J Comp Neural 1997, 380:262-274. This study convincingly demonstrates that both insulin and insulin-like growth factor type I can evoke a cell proliferation response from normal mature vestibular sensory epithelium explanted from 8- to 1 E-day-old chicks. This paper has an excellent discussion of the differences between growth-factorelicited cell proliferation responses by avian and mammalian vestibular sensory epithelp
34.
Lambert PR, Gu R, Corwin JT: Analysis of small hair bundles in the utricles of mature guinea pigs. Am J Ofol 1997, 18:637-643. inalysis of variations in the maturity of stereociliary bundle morphology In the utricles of mature guinea pigs formed the basis for these authors to put forth the intrrguing hypothesis that there is a low level of ongoing hair-cell prolrferation to replace hair cells lost to normal processes. This phenomena has been demonstrated in the vestibule of adult birds but until now has not been thought to occur actively in the vestibule of mammals, This is a potentially important observation that needs confirmation by other methodologies, 35.
Lambert PR: Inner ear hair cell regeneration in a mammal: identification of a trigger factor. Laryngoscope 1994, 104:701718.
Factors
36.
37.
controlling
Kuntz AL, Oesterle EC: TGF alpha with insulin induce proliferation in rat vestibular epithelia. Arch Otolaryngol Neck Surg 1998, in press.
hair-cell
Head
Yamashlta H, Oesterle EC: Induction of cell proliferation in mammalian inner ear sensory epithelia by transforming growth factor alpha and epidermal growth factor. Proc Nat/ Acad Sci USA 1995, 92:3152-3155.
38. ..
Zheng JL, Helbig C, Gao WQ: Induction of cell proliferation by fibroblast and insulin like growth factors in pure rat inner ear epithelial cell cultures. J Neurosci 1997, 17:216-226. This novel approach used partially dissociated sheets of early postnatal rat utricular epithelium to screen a large number of different growth factors for their ability to initiate a cell proliferation response. Several growth factors from different families were identified as mitogens for utricular sensory epithelium in this system, with some growth factor combinations demonstrating an additive effect on the cell proliferation response of this tissue. This is a well designed and executed experimental series using blocking antibodies to confirm the growth factor effects on mitogenesis in vitro. 39.
Yamane H, Nakagawa T, lguchi H, Shibata S, Takayama M, Sunami K, Nakai Y: Triggers of hair cell regeneration in the avian inner ear. Auris Nasus Larynx 1997, 24:221-225.
40.
Staecker H, Lefebvre P, Malgrange B, Moonen G, Van De Water TR: Response to: Regeneration and mammalian auditory hair cells: technical comment Science 1995, 267:709711.
41.
Chardin S, Romand R: Regeneration and mammalian auditory hair cells: technical comment Science 1995, 267:707-709.
Zine A, de Ribaupierre F: Replacement of mammalian auditory hair cells. Neurorepoti 1998, 9:263-268. Gis paper provides a convincing demonstration of hair-cell renewal in early postnatal rat organ of Corti explants following aminoglycoside damage to the auditory hair cells. The results suggest that the EGF receptor plays a role in the hair-cell renewal response by the damaged organ of Corti, as either TGFa or EGF can initiate this hair-cell renewal response in the aminoglycoside-damaged explants. The results suggest that the damaged halr-cell population is renewed via transdlfferentiation of either supporting cells or latent hair-cell precursors and/or repair of damaged hair cells, as none of the renewed hair-cell population labeled for thymidine incorporation.
regeneration/repair
44. .
Lenoir M, Vago P: Morphological indications of hair cell neodifferentiation in the organ of Corti of amikacin treated pups. C R Acad Sci (Paris) 1996, 319:269-276. Lenoir M, Vago P: Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication pups? Int I Dev Neurosci 1997, 15:487-495.
in rat
ear Staecker
and Van De Water
407
This study characterizes a transient attempt at hair-cell renewal in situ at the level of the outer hair cells in the aminoglycoside-damaged cochleae of young rats. Atypical cells that have similarities in surface morphology to developing hair cells appear and then disappear at the junction between damaged and undamaged hair cells within the organ of Cotii. These results may represent an unsuccessful attempt at repair or unsuccessful hair-cell neodifferentiation, as suggested by the authors. Their observation is important because it is the first evidence of an attempt at hair-cell renewal in situ in the mammalian cochlea. 45. .
Zheng JL, Gao WQ: Analysis of rat vestibular hair cell development and regeneration using calretinin as an early marker. J Neurosci 1997, 17:8270-8282. This study confirms calretinin’s usefulness as an early marker of hair-cell differentiation and Its appiication to mammalian vestibular hair-cell regeneration research. The authors used antibodles raised against calretinin to track damaged vestibular hair cells that were no longer identifiable by their characterestic morphology following ototoxic damage, and the consequent loss of their identlfylng sensory hair bundles. This type of approach may prove to be useful for sorting out the relative contnbutions of transdifferentiation and repair to the hair-cell renewal process. 46.
Zheng JL, Lewis A, Gao WQ: Establishment of conditionally immortalized rat utricular epithelial cell lines using a retrovirus mediated gene transfer technique. Hear Res 1998, 117:13-23.
47.
Holey MC, Nishida Y, Grix N: Conditional immortalization of hair cells from the inner ear. Int J Dev Neurosci 1997, 15:541-552.
48.
Lee KH, Cotanche DA: Potential role of bFGF and retinoic acid in the regeneration of chicken cochlear hair cells. Hear Res 1996, 94:1-13.
49.
Pickles JO, van Heumen WR: The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochlea. Int J Dev Neurosci 1997, 19:476-487.
50.
Gong TW, Hegeman AD, Shin JJ, Adler HJ, Raphael Y, Lomax Ml: Identification of genes expressed after noise exposure in the chick basilar papilla. Hear Res 1996, 96:20-32.
51.
Saffer L, Gu R, Corwin JT: An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles. Hear Res 1996, 94:14-23.
52.
Malgrange B, Rogister B, Lefebvre P?, Mazg-Servais C, Welcher AA, Bonnet C, Hsu R-Y, Rigo J-M, Van De Water TR, Moonen G: Expression of growth factors and their receptors in the postnatal rat cochlea. Neurochem Res 1998, 23:l 135-I 140.
42.
43.
in the inner
rat
Factors controlling Hinrich Staecker* Damaged
proliferation
transdifferentiation the mammalian possibly,
roles. Several
be associated insulin-like
factor
growth
factor
are important
whereas
transdifferentiation hair cells appear factors
epidermal
1 (IGF-l),
to
factor,
transforming
in the mammalian
evidence
that regeneration/repair
hair cells is possible
during
labyrinth.
growth
however,
for
for converting
in the lateral lines and inner ears of fish in the basilar papilla and vestibule of
they
have
and amphibians
Increasing
been
found
of mammals. differentiated;
to regenerate
[2], as well as in birds
in fish [l]
[3,4] (‘l’able
1).
of mammalian period
DNA-labeling
studies
of the avian
inner
ear have
sho\vn
that a spontaneous turnover of hair cells takes place in the vestibule but not in the auditory papilla [.5], However, in neonatal chick, damage to auditory hair cells
at later times.
Addresses *Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, Massachusetts 02114, USA iDepartments of Otolaryngology and Neuroscience, Kennedy Center, Room 302, Department of Otolaryngology, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York 10461, USA; e-mail: [email protected] Current Opinion in Neurobiology
ear are responsible
(e.g. sound pressure or acceleration) energy (i.e. neurotransmission). Hair
birds, and in the the cochlea and vestibule Hair cells of the inner ear are terminally
growth
the early neonatal
and may exist to a very limited degree
of the inner
mechanical energy into electrochemical cells are present and amphibians,
insulin,
for avian inner ear regeneration/repair, growth
c(, insulin, IGF-1, and IGF-2 are important
auditory
Hair cells
and, to play
process:
and fibroblast
regeneration/repair suggests
The ability to perceive sound and a sense of balance are important for orienting us within our environment.
have been found
with the regeneration/repair
growth
Introduction
are replaced
cells and
cells into hair cells. In
system,
the repair of damaged
significant
papilla
of supporting
of supporting vestibular
in the inner ear
and Thomas R Van De Watefi-
hair cells in the avian basilar
by regenerative
factors
hair-cell regeneration/repair
induced through auditory
by ototoxin or sound trauma will evoke repair hair-cell regeneration, resulting in recovery of function [6]. The source of these regenerated
hair cells is debated; some evidence suggests that many of the regenerated hair cells derive from the regenerative proliferation of the supporting cells, whereas the rest result from the transdifferentiation of supporting cells [7].
1998, 8:480-487
In mammals, spontaneous hair-cell regeneration has been observed, thus far, only in the vestibule [8]. Controversy remains as to whether the vestibular hair-cell regeneration
http://biomednet.com/elecref/0959438800800480 0 Current Biology Publications ISSN 0959-4388 Abbreviations BrdU 5-bromo-2’.deoxyuridine EGF epldermal growth factor acidic flbroblast growth factor FGF-1 basic fibroblast growth factor FGF-2 IGF insulin-like growth factor polymerase chain reaction PCR PDGF platelet-derived growth factor PDGF-AA A chain homodlmer of PDGF RT reverse transcription TGFcl transforming growth factor cx
observed in the utricle is attributable to the regenerative proliferation of supporting cells or to the transdifferentiation of supporting cells, or to the repair of damaged hair cells,
or to some
combination
of these
processes.
In this review, we compare the renewal of damaged hair cell populations within the sensory epithelium of both the auditory and vestibular receptors in avian and mammalian inner ears. IVe also discuss the possible sources
Table 1 Summary
of hair-cell regeneration/repair
in different species.
-
Ongoing production of hair cells
Neuroepithelial proliferation after damage
Spontaneous hair-cell regeneration
Regenerative proliferation
Functional recovery
Amphibian and fish/ lateral line and inner ear
f
+
+
+
Not tested
Bird/basilar papilla
0
+
+
+
+
Bird/vestibule
+
+
+
+
+
Mammal/cochlea
0
+ (postnatal)
0
0
0
Mammal/vestibule
?
+
+ (limited)
?
?
Species/sensory receptors
in the inner ear
Factors controlling hair-cell regeneration/repair
of regenerated
hair cells:
regenerative
proliferation;
Avian hair-cell
trans-
differentiation; and/or repair (see Figure 1). Finally, we report on recent studies characterizing the factors that influence the hair cell renewal process in these avian and mammalian
One
of
the
main
aims
of
regeneration
work
continues to be to identify where new hair cells come from. Evidence supports three different suggested modes of hair-cell renewal following injury (see Figure 1): regenerative proliferation of supporting cells; transdifferentiation of supporting cells; and repair of damaged hair cells. Proof of the active participation of one of these modes of hair-cell renewal does not exclude the possibility that either or both of the other modes also contributes to the hair-cell regeneration/repair following an insult to a specific hair-cell population the inner
actively process within
ear.
In sound-traumatized
chickens
regenerated
are labeled
hair cells
[9] and quails by tritiated
[lo], many thymidine,
the damaged papillae lost hair cells. After hair cells, Bhave et
nl. [l l] immunostained chick cochleae using cell-cycle markers and noted that although all the cells of the sensory epithelium left the Go phase (i.e. the quiescent phase), only those cells within the area of hair-cell loss
of hair cells
hair-cell
481
and Van De Water
regeneration
suggesting that cell division within is a major source for replacing administering otoroxin to damage
labyrinths.
Repair versus regeneration
Staecker
entered S (synthesis) phase evidence for regeneration
and underlvent mitosis. Direct of hair cells from supporting
cells has, thus far, been demonstrated lateral line neuromasts [ 121.
By using mitosis, damage take They
cytosine
arabinoside
(ara
only for amphibian
C),
an
inhibitor
place in observed
the absence this in both
of supporting cell mitosis. ototoxinand sound-trauma-
Figure 1
(a) Re-enter
AllI
Daughter cells
h
G---Ill”
-
Prollferatlon of supportIng
cells
n /I 4
I
(b) Transdifferentiatlon of supporting
cells
3
(c) Repair of injured hair cells
(e.g. oxidative
of
Adler and Raphael [13,14] found that following to the basilar papilla, hair-cell regeneration can
stress)
Schematic representation of the three different processes that may contribute to sensory ear: (a) regenerative proliferation of supportlng cells; (b) transdifferentiation of supporting HS98, Thomas R Van De Water and Hinrich Staecker, 1998.
3
TRV and HS98
hair-cell regeneration/repair within the vertebrate inner cells; and (c) repair of injured hair cells. TRV and
482
Sensory systems
damaged
basilar
papilla,
regeneration response similar. Further support
suggesting to these for these
that
the
hair-cell
pathological insults is findings comes from the
work of Roberson [15], who implanted an intracochlear canula that constantly infused tritiated thymidine into gentamicin-treated chicks. A single dose of gentamicin caused a near total the papilla; however,
loss of hair cells in the base of the regenerated hair cells were not
labeled, indicating that transdifferentiation cells contributes to hair-cell regeneration
of supporting in the avian inner
ear [15].
However,
at present,
that macrophages either regeneration [22*].
there
initiate
Factors influencing
is no direct or
modulate
evidence hair-cell
hair-cell renewal
It is important and factors
to identify and characterize the processes that can influence the hair cell renenal process,
including cellular events (e.g. re-entry of supporting cells into the cell cycle), cell interactions (e.g. migration of macrophages into the site of hair cell damage), expression of growth factor receptors in response to sensory epithelium damage (e.g. the epidermal growth factor
Mammalian The
aiuo [ll].
hair-cell
process
of
renewal/repair
hair-cell
inner ear has not yet been of regenerated hair cells
renewal
in
the
mammalian
fully characterized. Evidence labeled with S-bromo-2’-de-
oxyuridine (BrdU), or tritiated thymidine, in utricular cultures is sparse. Warchol et (I/. [Ih] have identified labeled hair cells in gcntamicin-damaged guinea pig utricular explants; however, judging from the number of regenerated hair cells present and the overall paucity of labeling, plausible
transdifferentiation source for most
utricular
explants
of supporting cells is the most of the ne\v hair cells in these
[EGF] receptor), and ear sensory epithelium Understanding these avian and mammalian
responsiveness to growth
factors and how they differ between labyrinths may provide insights into
the differences in the vertebrate inner ears. Avian hair-cell
hair cell
renewal
process
in these
regeneration
Hair-cell regeneration lvas first described in avian basilar papilla, and it remains the best understood system. llsing a laser, \\:archol and Corwin [23] lesioned hair cells in basilar papilla explants with tritiated
[16,17].
of damaged inner factors (e.g. TGFcx).
explants, thymidinc.
and then labrlcd the As discussed earlier,
An alternate mode of hair-cell renewal to explain unlabeled regenerated hair cells is the repair of damaged hair cells. Sobkowicz ettul. [ 181 have demonstrated that auditory hair cells can survive the loss of their apical cytoplasm and stereocilia bundles. Analysis of these damaged hair cells shows that repair is complete within 48 h [l&19’]. This concept of hair-cell repair is also supported by structural studies that have demonstrated the role of supporting cells
only supporting cells in close proximity to the lesion entered the S phase of the cell cycle. Supporting cells in cultures of thermolysin-separated sheets of chick utricular neuroepithelium undergo spontaneous division in defined
in scar formation strongly suggest
To study further the role of cell proliferation in hair-cell renewal, markers for immature hair cells have been sought. Stone et al. [ZS] have identified tlvo markers, calmodulin and beta tubulin, that stain regenerated hair cells selectively, as well as putative early differentiating hair cells. Recent studies show that the actin filament binding protein fimbrin localizes specifically to hair cells [26,27]. In chick basilar papilla cultures, staining for fimbrin has been observed 96 h after acoustic trauma 1271.
a capacity The
modes
after ototoxic damage [ZO]. These studies that sublethally damaged hair cells have
for self-repair. of injury
in
the
experiments
cited
above
arc either laser or mechanical disruption of the cuticular plate, so it is unclear whether other modes of injury, such as damage by ototoxins or sound trauma, would lead to similar findings. [Jsing a different approach, Kelly et al. [al] ablated hair cells in organ of Corti explants with a laser to demonstrate that the degree of supporting-cell proliferation is proportional to the degree of injury, again suggesting that contact-mediated factors are important in the initiation of regeneration/repair. hlacrophages are associated with tissue regeneration in other organs, and they produce a variety of growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor ~1 (TGFa), and basic fibroblast growth Factor (bFGF, also known as FGF-2). Localized trauma to the vestibular neuroepithelium results in recruitment of macrophages to the site of injury. This recruitment phenomenon has also been observed in the amphibian lateral line [l,?], in explants of chick basilar papilla [Z’], and in traumatized vestibular epithelium its
medium, indicating that the explantation preparation techniques (e.g. thcrmolysin trigger cell proliferation [ 2-11.
and culture digestion) can
There has also been significant interest in identifying growth factors that can potentially trigger or enhance hair-cell regeneration. \Vhen normal basilar papillae are co-cultured with aminoglycoside-damaged papillac, they demonstrate increased rates of tritiated thymidinc incorporation, suggesting that a diffusible substance is being produced by the lesioned papillae, w:hich then initiates a cell proliferation response in the normal papillae [28]. In the avian system, two growth factors have been identified as stimulators of cell proliferation: insulin and insulin-like growth factor 1 (IGF-1). Basilar papilla explants exposed to either of these factors show a significant mitogenic response (i.e. increased Brdll uptake). Treatment of explants with EGF, bombesin or TGFa do not significantly enhance Brdl! uptake, except
in areas
of nonsensory
epithelium
[2’S”].
A significant
advance in understanding the signaling cascade involved in the induction of hair-cell regeneration has come with the observation that induction of CAMP in cultures of avian basilar papillae stimulates the incorporation of BrdU in hair cells of uninjured basilar papilla explants [30]. This cAhlP-stimulated proliferation inhibitors of protein kinase A, which Mammalian
hair-cell
can be blocked by is regulated by cAhlP.
of guinea
rate of cell significantly
pig utricles.
proliferation lower than
TGFa
As discussed
earlier,
the
in these mammalian tissues is in avian tissues. More recently.
regeneration/repair of auditory hair cells has been in explants of three-day-old rat organ of Corti
reported explants
analysis
FGF-2
are present
has shown
in vice and that
on utricular
has also been
regeneration/repair
epithelial
implicated in neonatal
that IGF cells
utricular and FGF
hair cells receptors
[38”].
as a factor driving
hair-cell
organ of Corti cultures
[40].
The validity of these results have been questioned [41], but Zinc and de Ribaupierre [42”], using a different culture system, have recently replicated and extended these
renewal/repair
Hair-cell regeneration in mammals was first identified by Forge et al. [S] in gentamicin-treated guinea pigs and by Warchol et al. [16] in aminoglycoside-damaged explants
histochemical produce
483
in the inner ear Staecker and Van De Water
Factors controlling hair-cell regeneration/repair
results.
They
treated
organ of Corti explants 5-7 days of treatment
with with
three-day-old
postnatal
rat
1 mhl neomycin followed by either TGFcx or EGF, or a
combination of the two. Apical turns of the organ of Corti explants were removed to prevent inadvertent analysis of spared apical hair cells. Cultures treated with either TGFa or EGF, or a combination of both, showed a significant number
of regenerated/repaired
hair cells
displaying
the
damaged by administration of neomycin and then treated with retinoic acid and fetal calf serum [31]. Kelly r~
surface features of immature hair cells (see Figure ‘liitiated thymidine labeling showed cell proliferation the inner sulcus area of EGF-treated explants but
al. [Zl] have demonstrated that embryonic and neonatal cochleae replace hair cells destroyed by laser irradiation, but renewal is greatly attenuated in postnatal explants.
labeled hair cells, lending credence to the theory that these cells are derived from differentiation of a precursor cell, or transdiffcrentiation of existing supporting cells (or
Spontaneous regeneration of hair cells in the cristae of adult chinchilla has also been reported [32]. Interestingly, there is a notable difference between the ability of type I
to the repair hair cells).
and type
Regeneration/repair
II hair cells
to regenerate
[33].
Recently, cells carrying immature appearing stereocilia have been identified in the undamaged utricles of adult guinea pigs [34*]. These cells appear to represent 0.7% of the utricular hair-cell population, suggesting that some degree of hair cell replacement may be an active process in the vestibule of adult mammals [34*]. Lambert [35] has demonstrated that hair-cell regeneration takes place in adult mouse cristae and that cultures of adult mouse utricles pretreated with neomycin show an increased r3te of cell proliferation after treatment with TGFa. In combination with insulin, TGFa has also been shown to increase proliferation of cells in extrasensory epithelia and supporting cells in adult rat utricles [36]. Other studies have sho\vn that IGF-1, IGF-2, TGFa and EGF increase proliferation of utricular epithelial cells [37,38**,39]. This effect on proliferation can be inhibited by neutralizing antibodies against FGF-2 and IGF-1, suggesting that these factors may be part of the hair-cell regeneration cascade. Growth factor treatment can also enhance the process of vcstibular hair-cell regeneration/repair within the utricles of adult guinea pig inner ears exposed to vestibulotoxic levels of gentamicin (R Kopke et al., Sot h’turo.b Abs~-rr 1996, 22636.3). Infusion of a combination of TGFa, IGF-1, and retinoic acid into the perilymphatic space of the guinea pig vestibule one week post-gentamicin administration results in a significant enhancement of the regeneration/repair of the vestibular hair cells (i.e. 3- to lo-fold; the variability is site specific) and stimulates the maturation of their sensory hair bundles. Immuno-
of sublethal
damage
2). in no
to the ototoxin-damaged
of hair cells in the ototoxin-damaged,
EGF-treated explants WAS more vigorous at the middle turn, leading Zine and de Ribaupierre [42**] to speculate that more ‘undifferentiated’ precursors are present at the middle turn compared to at the basal turn. In a follo\v-up study using ELI%, these investigators (A Zinc, F de Ribaupierre, personal communication) reported an upregulation of EGF receptor expression in organ of Corti explants in response to ototoxic damage. ‘I’hus far, the regeneration/repair of Corti appears to be limited
phenomenon in the organ to in vitro studies and has
not been demonstrated in organ of Corti explants excised from rdt pups after postnatal day 5. Some evidence of a limited attempt at regeneration/repair is provided by the experiments of Lenoir and \‘ago [43.34*], who injected nine-day-old rdt pups with amikacin for seven consecutive days and then sacrificed the animals at intervals to 90 days post initiation of ototoxin injections. At 21 and 35 days after ototoxin treatment, atypical cells bearing tufts of microvilli and resembling immature hair cells were observed only in the area previously occupied by the apical outer hair cells. In the inner hair-cell area of these animals, Lenoir and Vago observed cells that had both efferent and afferent innervations but no stereocilia or cuticular plates. They hypothesize that the apical region of the rat cochlea does not reach adult morphology until after postnatal day 21 and, therefore, only a limited attempt at regeneration/repair is possible. One approach in growth factor studies has been to study cultures of supporting cells and to determine which factors
484
Sensory systems
Figure 2
Scanning a control
electron micrographs of the auditory hair-cell area of neonatal rat organ of Corti explants after 10 days in vitro. (a) Surface view of culture after 10 days in vitro, displaying well-defined rows of inner and outer hair cells, as well as normal stereociliary arrays. (b) An
ototoxin-damaged culture hair cells from the organ
(exposed for 24 h to 1 mM neomycin and then placed for 7 days in normal medium) showing complete loss of auditory of Corti. (c) Area of replacement hair cells in ototoxin-damaged cultures treated for 7 days with TGFa. Numerous
immature stereocilia bundles are visible on the apical surfaces of the replacement hair cells. Kinocilia of these replacement hair cells are found In an eccentric position at the external border of the cell (arrow). The surface of the Corti’s organ is partially hidden by the re-grown tectorial membrane (TM). (d) Higher magnification of the apical surface of a single replacement hair cell shown in (c). Hair cells like this one, with a circular bundle of closely packed immature, uniformly sized stereocilia and a longer ear of the rat. Bars=2 PM in all panels. Reproduced with permission from [42”1.
can potentially initiate their differentiation into hair cells. Initial research has focused on determining what markers, aside from the presence of actin-containing stereocilia, are specific for hair cells. A recent study by Zheng and Gao [45*] confirms that the calcium-binding protein calretinin is present in immature hair cells just after their terminal mitosis and before stereocilia bundle formation. They then set out to test if any mitotic cells in rat utricular epithelium cultures co-label with BrdU and calretinin. No double labeling U’ZISseen in partially dissociated utricular epithelium, indicating that spontaneous mitosis occurring after dissociation does not produce early hair-cell progenitors. Cultures of dissociated epithelial sheets treated with gentamicin for 2 days followed by BrdU labeling ifi vitru for 7-11 days contained a few cells that were double
kinocilium
(arrow),
are normally
found
in the developing
inner
labeled with both anti-BrdU and anti-calretinin. Control cultures never showed any double labeling, negating the possibility of dissociation-induced damage and repair of hair-cell DNA. ‘Ib study further this effect, Zheng, Lewis and Gao [46] produced conditionally immortalized utricular cpithelial cell lines that possess the morphologic characteristics of supporting cells. These immortalized utricular ccl1 lines express the same growth factor receptors found on supporting cells (i.e. EGF and FGF receptors). When cultured at a nonpermissive temperature in the prescncc of FGF-2, these cells stop proliferating, and express both calretinin and calmodulin [46]. Immortalized hair cells have also been produced from embryonic inner ear cells of
Factors controlling hair-cell regeneration/repair
the ImortomouseB for hair cells; appears
[47]. These
however,
Another
approach
are involved the cDNAs normal
auditory
involved
Growth factors and receptors organ of Cot%*.
transcription noise-damaged fication
growth
and
lesioned
auditory
for the
EGF
two-week-old performed
receptor,
fibroblast
growth
factor
chicks reverse
FGF
receptor,
(FGF-1)
mRNA
is
upregulated after basilar papilla damage. The Eph family tyrosine kinase receptor &k-l0 is also localized in the sensory epithelium [@I. A differential display PCR study of noise-damaged basilar papillae idencitied four genes that are dysregulatcd in response to this trauma [SO]: genes lated protein, GTP-binding gene. These signaling and regeneration.
that encode for parathyroid hormone reCa?+/calmodulin-regulated protein kinase II, protein CDC32 and a novel uncharacterized results suggest that CaZ+/calmodulin-related GTPase cascades may play a role in hair-cell
The use of PCR amplification of reverse transcribed mRNAs has confirmed that the FGF family of growth factors plays an important role in avian hair cell regeneration/repair, In addition, these gene amplification results have identified several other candidate factors potentially contribute to the regeneration/repair Genes
XI‘-PCR
present
in mammalian
analysis
of control
that may response.
hair cells
and
FGF-2,
IGF-1,
IGF-2,
PDGF-AA,
PDGF-BB
factor receptors EGF-R, FGF-R2, FGF-R4, IGF-1 R, PDGF-Rc(,
PDGF-Rfi
‘Modlfled
homodimer
renewal
normal papillae. Interestingly, the expression pattern of the FGF receptor changed position after noise damage: localization of immunostaining changed from the hair-cell stereocilia to the apical areas of surrounding supporting RT-PCR was used to show cells. Semi-quantitative acidic
in the mammalian
Growth
from
[51].
PDGF-BB,
factors
fundamental
regeneration/repair
IGF receptor, insulin receptor. rctinoic acid receptors B and CL,and FGF-2 are present in both noise-damaged and
that
identified
B
chain
of
PDGF;
-R, receptor.
regeneration/repair.
(RT) PCR on RNA extracted from both and normal chick basilar papillae. Ampli-
products
485
factors
has been to identify and their receptors in
Cotanche [&I exposed trauma and subsequently
sound
and Van De Water
Table 2
markers
EGF, TGFc(, FGF-I,
which
epithelium
in avian hair-cell
ear Staecker
Growth factors
to determining
undergoing
I ,ee and to
some
differentiation
studies
in regeneration/repair of growth factors
epithclium Genes
cells express morphological
to be aberrant.
Gene amplification
both
their
in the inner
neomycin-damaged
process
Several growth regeneration/repair
and modulate
auditory
response to growth IGF-1 and insulin.
the hair-cell
1).
factors that appear process have been
preliminary overview between the avian as between
that initiate
(Table
to modulate the identified, and a
indicates that there are differences and mammalian systems, as well and factors
vestibular such
hair
as FGF-2,
cells,
in their
TGFcx,
EGF,
‘I‘here also appear to be three mechanisms by which hair cells can be replaced following damage to the inner ear (Figure 1). The avian system replaces lost hair cells by both regenerative proliferation of supporting cells (with subsequent differentiation of the daughter cells into both hair cells and supporting cells), and by transdifferentiation of mature supporting cells into hair cells. Repair of damaged hair cells may also play a role in hair-cell renewal in this system, but, at present, there is no evidence to support such a repair process in the damaged avian inner ear. In mammals, hair-cell regeneration/repair processes include both transdifferentiation of mature and immature supporting cells into hair cells and self-repair of sublethal damage to the hair cells. Regeneration/repair of mammalian auditory hair cells, at present, is limited to the neonatal period, with some evidence of an unsuccessful attempt at regeneration/repair (i.e. atypical cells) at a slightly later stage of postnatal development in the cochlea of the rat. Currently, there are no experimental findings that support the participation of regenerative cell proliferation as a major participant in the replacement of lost hair cells from either the auditory or vestibular receptors of mammals.
rat
utricles has shown that receptors for IGF-1, FGF-2, EGF and PDGF-AA are present, and also suggest that the PDGF-AA and IGF-1 receptors may localize to the hair cells [jl]. The presence of mRNA for a wide variety of growth factors and growth factor receptors have also been identified in the postnatal rat cochlea (see Table 2) [52].
Conclusions Hair-cell regeneration is a rapidly developing field that has used four model systems over the past ten years-avian basilar papilla. avian utricle, mammalian organ of Corti, and mammalian utricle - in an attempt to characterize the
The challenge for future investigations will be to identify the mechanism that initiate the transdifferentiation and repair processes, and to determine the growth factors and other factors that are involved in these processes. In addition, the identification and characterization of underlying molecular changes that prevent auditory haircell regeneration/repair in the auditory system of adult mammals should prove a fruitful area of research. One final challenge to the biologists working in the area of hair-cell regeneration/repair will be to identify the differences between the hair-cell populations of the avian and mammalian inner ears that prevent regenerative proliferation
486
Sensory
(c.g.
systems
cell cycle
control
process of hair-cell mammals.
factors)
from
regeneration/repair
participating in the inner
in the ear of
Acknowledgements ‘1‘11~ prcp~rar~on of[his rcvic\v
\vas supported by the Shulsky Hearing Kcbcarch Foundation of the hlontcfiorc hlcdical Ccntcr (‘1X VXI Dc b\‘ater). ‘l‘hc ;~uthor\ thank A Chcng, a fourth year medical student, for his help in preparing Figure I, and A %inc and 1; dc Kibaupicrrc for the USC of rhc illusmtrron in Frgurc ?. LVe &o thank A Zinc. 1; de kibaupicrrc and I’ I,cfcbvre for their critic;11 commcncs. and K Impcrati for \vord processing of thus manuscripr.
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407
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42.
43.
in the inner
rat