Regulated Reprogramming in the Regeneration
of Sensory Receptor Cells
Olivia Bermingham-McDonogh1,* and Thomas A. Reh1,*
1Department of Biological Structure, Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA
*Correspondence: email@example.com (O.B.-M.), firstname.lastname@example.org (T.A.R.)
Vision, olfaction, hearing, and balance are mediated by receptors that reside in specialized sensory epithelial
organs. Age-related degeneration of the photoreceptors in the retina and the hair cells in the cochlea, caused
many nonmammalian vertebrates, these sensory epithelia show remarkable regenerative potential. We
summarize the current state of knowledge of regeneration in the specialized sense organs in both nonmam-
malian vertebrates and mammals and discuss possible areas where new advances in regenerative medicine
might provide approaches to successfully stimulate sensory receptor cell regeneration. The field of regener-
ative medicine isstill inits infancy, butnew approaches usingstem cells and reprogrammingsuggest ways in
which the potential for regeneration may be restored in individuals suffering from sensory loss.
Our special senses, vision, olfaction, taste, hearing, and balance
are mediated by receptors that reside in specialized epithelial
organs. To best capture the physical stimuli required for their
function these receptors are ‘‘exposed’’ to the environment
and subject to excesses in the very stimuli they are optimized
to detect. Olfactory receptor cells have an average lifetime of
a few months. Excessive noise leads to the degeneration of
auditory hair cells; constant high levels of illumination can cause
retinal photoreceptor loss. In addition, sensory receptor cells
have many specialized proteins that are not present in other
tissues; mutations in the genes coding for these proteins are
often not lethal due to their very specific expression but can
cause sensory receptor degeneration, leading to devastating
syndromes in humans. Individuals with Usher’s syndrome, for
example, in which both the photoreceptors in the retina and
the hair cells in the cochlea degenerate, ultimately become
both blind and deaf. While thankfully these disorders are rare,
more common degenerative disorders of the retina and cochlea,
such as macular degeneration and most acquired sensorineural
viduals as the aged human population increases. It is estimated
that over 50% of the individuals over 60 have significant hearing
and at least some part of this decline may be related to a re-
duction in receptor neurons; estimates of olfactory impairment
range from 50% to 75% of people over the age of 65 (Doty
et al., 1984). Although there are focused efforts in medical and
gene therapy to treat these conditions and slow the degenera-
tion of sensory receptor cells, there are many millions of individ-
uals with varying degrees of impairment already. Moreover,
of the sensory receptors have already degenerated. For these
patients, prosthetic devices or regenerative medical approaches
may be the only options.
What hope have we for stimulating the functional regeneration
of sensory epithelial receptor cells in the human retina and inner
ear? The field of regenerative medicine is still in its infancy, but it
is rapidly developing. New approaches using stem cells and
reprogramming have provided insights into the plasticity of cell
identity, suggesting new ways in which the potential for regener-
ation may be restored. Moreover, although sensory receptor
cells in the mammalian retina and inner ear show only limited
or no regeneration, in many nonmammalian vertebrates, these
sensory epithelia show remarkable regenerative potential. In
newts, for example, most parts of the eye regenerate. In birds,
the sensory receptors in the auditory and vestibular (balance)
organs regenerate almost completely after various types of
injury. In this review, we will summarize the current state of
knowledge for regeneration in the specialized sense organs in
both nonmammalian vertebrates and mammals and discuss
possible areas where new advances in regenerative medicine
might provide approaches to successfully stimulate sensory
receptor cell regeneration in patients.
Functional and Structural Features of Sensory Epithelia
for their regeneration are the olfactory epithelium, the auditory
and vestibular epithelia of the inner ear, and the retina of the
eye. The details of the structure and function of these organs
are beyond the scope of this review, but a brief description of
their common features and their differences will place the
research on their regeneration in context.
The olfactory epithelium is contained within the nasal cavity
(Figure 1A). Most of the studies on regeneration have been
done in the main olfactory epithelium, but many vertebrates
also have additional sensory regions, like the vomeronasal
organ. The olfactory receptor neurons have a single dendrite
that extends to the apical surface of the epithelium and ends in
a terminal knob, which has many small cilia extending into the
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
mucosa. A single axon projects through the basal side of the
epithelium through the lamina cribosa to terminate in the olfac-
of over 1000 olfactory receptor proteins, G protein-coupled
receptor molecules,in theircilia(Kaupp, 2010)forrecentreview).
The neurons are surrounded by glial-like cells, called sustentac-
ular cells. Other cells in the epithelium contribute to the continual
production of the new receptor neurons and will be described
later in the review.
The vestibular and auditory epithelia in vertebrates have some
structural similarities to the olfactory epithelia (Figure 1B). The
mechanosensory receptor cells in these organs are called hair
cells. There are five distinct regions of vestibular epithelia in
the inner ear: the three cristae and the maculae of the utricle
and saccule. Like the olfactory receptor neurons, the hair cells
are surrounded on all sides by glial-like support cells but are
organized in a more regular mosaic than the olfactory receptor
cells. In addition to the inner ear sensory epithelia, aquatic
amphibians and fish have small mechanoreceptor organs
distributed along the body, called the lateral line organs. All
hair cells contain a mechanosensitive structure at their apical
surface called the hair bundle, consisting of a group of approxi-
mately 100 actin-containing sterocilia and a microtubule con-
taining kinocilium (for recent review, see Gillespie and Mu ¨ller,
In the auditory sensory epithelium of nonmammalian verte-
brates (the basilar papilla; BP), the hair cell and support cells
have a similar organization to that in the vestibular organs, with
alternating hair cells and support cells. However, in the mamma-
lian auditory sense organ (the cochlea) the hair cells are orga-
nized in a striking pattern, with a single row of ‘‘inner’’ hair cells
and three rows of ‘‘outer’’ hair cells, while the support cells
assume a variety of specialized morphologies. The inner hair
cells are the primary sensory receptors, while the outer hair cells
act to amplify sound at least in part through regulation of
ized support cells, the inner phalangeal cells. Lining the space
between the inner and outer hair cells, the tunnel of Corti, are
the pillar cells, which provide rigidity and structure to the epithe-
lium. Finally, the support cells associated with the outer hair cells
that reaches up around the outer hair cell and forms a contact
with its apical surface. It is thought that the development of the
tunnel of Corti and specializations of the cells may be an adapta-
tion necessary for higher frequency hearing (Dallos and Harris,
1978; Hudspeth, 1985).
The sensory receptors for visual information, the rod and cone
photoreceptor cells, arecontained in apart of the CNS called the
retina (Figure 1C). The retina is quite different in its embryology
from the olfactory and inner ear sensory epithelia in that the
former is derived from the neural plate with the rest of the
CNS, while the latter two are derived from ectodermal placodes
(Schlosser, 2010). There are several different types of cone
photoreceptors, and the different types are most sensitive to
aparticular wavelength. Inhumans,cones with peak sensitivities
to three different wavelengths (short, middle, and long) provide
us with trichromatic vision. Rods are specialized for high sensi-
tivity at low light levels and are responsible for nighttime vision.
All vertebrate retinas contain both rods and cones. The sensory
receptors are concentrated at the apical surface of the retinal
epithelium, organized in regular arrays and surrounded by glial
cells, the Mu ¨ller glia, that resemble the support cells and susten-
tacular cells of the inner ear and olfactory system, respectively.
Phototransduction in the sensory receptors is mediated by G
protein-coupled receptors, the opsins, which are concentrated
in specialized cilia, the so-called outer segments. In addition to
the sensory receptors and glia, the retina contains a group of
projection neurons, called retinal ganglion cells, somewhat
Figure 1. Simplified Schematic Diagrams of the Specialized Sensory Organs that Have Been Most Studied for Their Regeneration Potential
(A) The main olfactory epithelium (MOE) and the vomeronasal organ (VNO) contain specialized sensory receptor neurons (ORNs) and supporting cells, called
sustentacular cells. The axons of the ORNs project directly to the olfactory bulb (OB) in the brain.
Corti in mammals, and the vestibular epithelia—the three cristae, the utricle, and the saccule. The vestibular epithelia (middle) are organized with alternating hair
cells (red) and support cells (blue). In the organ of Corti (bottom), one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) alternate with various
types of supporting cells, the pillar cells (PCs) and the Deiters’ cells (DCs). The hair cells are innervated by afferent fibers from associated ganglia (spiral ganglia)
and from efferent fibers from the CNS.
(C) The neurosensory retina (red) lines the back of the eye; it contains the sensory receptors, the rods and cones (red), supporting Mu ¨ller glia (blue), and other
neurons (light red) that process and relay the light responses of the photoreceptors to the brain via the optic nerve.
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
analogous to the spiral ganglion neurons in the auditory system
as well as a diverse array of interneurons, more reminiscent of
other CNS regions than the other sensory epithelia.
Ongoing Sensory Receptor Cell Production
Although most of the neurons in the nervous system of verte-
brates are generated during a developmental period, some
regions of the nervous system continue to add new neurons
throughout life. For example, in mammals, neurons are gener-
ated in the hippocampus into adulthood (Hodge et al., 2008).
In many vertebrates, new receptor cells are also added to the
sensory organs. The cellular and molecular mechanisms that
enable ongoing genesis of receptor cells in different specialized
sensory epithelia in various species have some features in
common that provide insights into what factors might be critical
for regeneration (Figure 2).
The ongoing genesis of olfactory receptor cells is common to
all vertebrates (see Graziadei and Monti Graziadei, 1978 for
review) and the rate of production is quite high. The production
of new olfactory receptor cells is critical to the maintenance of
this system, as the olfactory receptor cells only last a few
months. The rate of production of new olfactory receptor cells
is balanced by their loss so that a relatively stable population
of these receptors is maintained. In the vestibular epithelium of
fish (Corwin, 1981), amphibians (Corwin, 1985), and birds
(Jørgensen and Mathiesen, 1988; Roberson et al., 1992), there
is also ongoing production of the hair cells. However, in fish
and amphibia, rather than the sensory receptor cell turnover
that occurs in the olfactory epithelium, the ongoing production
of new hair cells in vestibular epithelia results in an increase in
the overall number of these cells as the animal grows (Corwin,
1985). The macula neglecta of skates, for example, adds hair
cells continuously through at least six years increasing more
than 10-fold the number of hair cells with a 500-fold increase in
sensitivity. The number of hair cells appears to scale with overall
body size. In the toad sacculus, new hair cell addition occurs
primarily at the peripheral edges; as a result, the epithelium is
composed of concentric rings of progressively younger cells.
The situation is somewhat different in the vestibular epithelia of
birds. Although there is also good evidence for new hair cell
production throughout life, the newly generated hair cells are
frequently near apoptotic cells, and the number of hair cells
does not increase over the life of the animal as it does in fish.
Therefore, it is likely that the ongoing genesis of hair cells in birds
may serve a maintenance role to replace dying hair cells, much
like that in the olfactory epithelia (Jørgensen and Mathiesen,
1988; Roberson et al., 1992). In the retina of fish, there is also
ongoing production of one type of sensory receptor, the rod
photoreceptors (Johns and Easter, 1977; Raymond and Rivlin,
1987). Rod photoreceptor cells are not generated to replace
dying cells, but rather they are generated as the retina grows,
to keep the density of rod photoreceptors relatively constant
(Fernald, 1990). In fish and amphibians, the retina also grows
throughout life at its peripheral edge, adding new retinal cells
of all types that seamlessly integrate with the existing retina
(for review, see Lamba et al., 2008). This process also occurs
in birds to a limited extent (Fischer and Reh, 2000).
The continued production of sensory receptor cells in these
epithelia requires a mitotic cell population that can act like the
stem cells in nonneural epithelia. In the case of the olfactory
epithelium, there are at least two types of mitotic cells: the
globose basal cells (GBCs) and the horizontal basal cells
(HBCs). The GBCs are mitotically active in the normal, undam-
aged epithelium and act as a multipotent progenitor to generate
all of the other types of olfactory cells, including the sensory
receptors (Caggiano et al., 1994; Chen et al., 2004; Huard
et al., 1998). The more slowly cycling (or even quiescent) HBCs
are more like ‘‘stem cells’’ serving both to replenish the more
actively proliferating GBCs (Iwai et al., 2008) and as a reserve
pool after more extensive damage to the receptors (Leung
et al., 2007). The model of a slow-cycling stem cell (HBC) with
a more rapidly cycling, transit-amplifying progenitor cell (GBC)
has similarities with nonneural epithelia, like the epidermis
(Watt et al., 2006).The situation in the vestibular system of
nonmammals is somewhat different, in that there does not
appear to be a committed hair cell progenitor. Rather, it appears
that some or all of the support cells remain capable of mitotic
division and divide at a low rate to produce both additional hair
cells and support cells as the epithelium grows. In the retina,
the source of the new rods in the fish is a group of cells called
the rod precursors (Johns and Fernald, 1981), which typically
generate only rod photoreceptors under normal conditions and
are likely derived from the Mu ¨ller glia (more on this later).
The different progenitors/precursors in these systems also
share some common molecular expression patterns that are
similar to those expressed during initial development (see
Figure 3 and further discussion below). In the olfactory epithe-
lium, for example, at least some of the GBCs express Ascl1,
Neurog1, Sox2, and Pax6, genes critical during olfactory epithe-
lial development (Guo et al., 2010; Manglapus et al., 2004).
NeuroD1 is expressed at a slightly later stage, in the cells that
will differentiate into the olfactory receptor neurons. In the inner
ear, the support cells also express Sox2 (Oesterle et al., 2008),
and manyof thesupport cellsthat arein theS-phase or M-phase
of the cell cycle, as well as the newly generated postmitotic
daughters, express Atoh1 (Cafaro et al., 2007). In the retina,
the rod precursor expresses NeuroD1 (Hitchcock and Kakuk-
Atkins, 2004; Nelson and Reh, 2008), suggesting that these cells
are at a slightly ‘‘later’’ stage in their development, consistent
with their commitment to develop as rod photoreceptors and
their expression of other photoreceptor specific transcription
factors. The Mu ¨ller glia, which act as the ‘‘stem’’ cell that gives
rise to the rod precursors (Bernardos et al., 2007), express
Sox2 and Pax6 (and Ascl1 after damage, see below), similar to
From this overview, several common features of ongoing
sensory cell production emerge. First, the sensory receptor cells
are derived from what might be called a ‘‘persistent progenitor’’
lium and the retina of fish, the immediate precursor to the
receptor neurons/rods is a cell that seems to have a more limited
capacity for cell division than a true ‘‘stem cell.’’ The rod
precursor of fish is particularly committed to generating rod
photoreceptors, and the GBC of the olfactory epithelium can
generate most, though not all, of the cell types in the sensory
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
Figure 2. There Is a Very Good Correlation between the Process of Ongoing Sensory Receptor Cell Generation throughout Life and the
Capacity for Regeneration in the Epithelium
(A)Inthenormalolfactory epithelium, vestibularsystem,andretinainsomevertebrates,thereisacontinualgenerationofnewsensoryreceptorcells.Theglobose
basal cells (GBCs; yellow) and horizontal basal cells (HBCs; yellow) act as progenitors and stem cells for the other cells in the epithelium, the olfactory receptor
neurons (red) and the sustentacular cells (blue). Similarly, in the vestibular epithelium of some vertebrates, the support cells (SC; blue) can enter the mitotic cell
of the rod precursors (yellow), while the Mu ¨ller glia (MG; blue) are quiescent.
(B) The correlation between robust regeneration and ongoing sensory cell genesis is diagrammed as red (regeneration) and green (ongoing sensory cell
production) bars. Exceptions to the general rule occur in the bird auditory epithelium (a) where there is no ongoing production of sensory receptor cells, but there
is robust regeneration,whereas in the bird vestibular organs (v), both processes occur.The lighter red bar for the bird retinaindicates that whereas there is robust
proliferation of Mu ¨ller glia after damage, much like that observed in fish, the number of new neurons generated is small.
(C) The changes in these epithelia during regeneration are diagrammed. In the olfactory epithelium, the GBCs increase in their proliferation after damage, and the
HBCs are recruited when the damage is extensive. In the auditory system of birds, the support cells express Atoh1 and other developmental genes, and in some
cases directlytransdifferentiateintohair cells. Iftheretina is damaged infish,the Mu ¨ller gliare-enterthemitotic cell cycle andproduce all typesof retinal neurons,
including both rods and cones.
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
epithelium. These cells have some similarity to the immediate
neuronal precursors found in the cerebral cortex or the progen-
itor/stem cells in the hippocampal and subventricular zone in
that (1) they are restricted to generate specific subtypes of
neurons and (2) their mitotic divisions do not occur atthe ventric-
ular surface (Hodge et al., 2008; Pontious et al., 2008). Second,
many of the genes expressed in normal development in the
lineages leading to the differentiated sensory receptor cells are
also expressed in the progenitors responsible for the genesis
of these cells in mature sensory epithelia. Third, the progeni-
tors/precursors in the mature epithelia coexist with differenti-
ated, functioning sensory receptors, underscoring the fact that
the maintenance of a ‘‘neurogenic’’ niche is not inconsistent
with the environment of a mature neural tissue. Fourth, although
the different systems have very different requirements for the
in each system. Lastly, although the rate of new cell addition in
the different systems varies considerably, where the olfactory
epithelium generates new sensory receptors at a much higher
rate than the other epithelia, the production of new cells appears
to be under tight regulation, producing precisely the cell types
necessary for maintenance and growth or regeneration of these
Common Pathways in the Development of the Sensory
Before delving into regeneration in the different sensory
epithelia, it would be worthwhile to provide a development
framework in which to understand the molecular underpinnings
of and constraints on regeneration. The development of the
specialized sensory organs share many mechanisms with one
another and other regions of the nervous system (Figure 3).
Paired-homeodomain (Pax), bHLH proneural/neural differentia-
tion, SRY-related HMG-box (Sox), and homeodomain transcrip-
tion factors are all necessary for these sensory organs. Signaling
factors and their receptors, including BMP, FGF, Shh, Wnt, and
Dll/Notch, are also important in the development of these
systems. In the following paragraphs, we will highlight a few
key features of these molecules in the development of special-
ized sensory receptor cells that are particularly relevant to the
studies on regeneration in these systems, but the reader is
referred to several excellent reviews for a more comprehensive
view (Chatterjee et al., 2010; DeMaria and Ngai, 2010; Driver
and Kelley, 2009; Wallace, 2011; Swaroop et al., 2010).
In the development of all these sensory epithelia, like the other
regions of the nervous system, Sox2 is one of the earliest
required factors. Sox2 is required at a very early stage in the
nasal placode for the initial formation of the olfactory sensory
epithelium (Donner et al., 2007). In the inner ear, loss of Sox2
leads to the failure of production of hair cells and support cells
in all inner ear sensory epithelia, including the auditory and
vestibular sensory organs (Kiernan et al., 2005). Sox2 is thus
thought to specify the ‘‘sensory’’ identity in the otic vesicle,
singling out those regions from the surrounding nonsensory
epithelium. In the retina, a very similar phenotype occurs
following conditional deletion of Sox2: no neurons of any type
are produced and the proneural genes and neural differentiation
genes are not expressed (Taranova et al., 2006). Another key
regulator of sensory development is Pax6. Pax6, a member of
the paired-homeodomain family of transcription factors, plays
a critical role in eye development in animals as diverse as
Drosophila to humans (Callaerts et al., 1997). In the retina, loss
of Pax6 causes the progenitor cells to generate only retinal inter-
neurons; photoreceptors are no longer produced (Marquardt
and Gruss, 2002). Pax6 is thought to directly activate expression
of the proneural genes Ascl1 and Neurog2 in the retina,
thereby providing a link to the process of neurogenesis
(Marquardt et al., 2001). Pax6 may play a similar role in the olfac-
tory epithelium, since it is expressed throughout development
and even in the mature epithelium, but there is an early require-
ment in olfactory placode that precludes the analysis of its
Figure 3. Similarities in Developmental Mechanisms in the Different Specialized Sensory Epithelia Relevant to Studies on Regeneration in
The sensory receptor cells arise from specialized Sox2 expressing regions of the nasal and inner ear epithelia. The progenitor cells in these regions go on to
the neural tube. Notch signaling, and its downstream effectors Hes1 and Hes5, promote support cells and Mu ¨ller glial fates in the inner ear and retina.
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
functions in thelaterdevelopmental stages. Pax2isexpressed in
the inner ear sensory epithelia, and loss of Pax2 leads to defects
in their development; however, few direct targets of Pax6 or
Pax2 are known in the sensory epithelia, so it is difficult to
know at this time whether they have similar functions in the eye
and ear, respectively. In addition, it is important to note that
Pax genes interact with many other transcription factors in
combinatorial ways to regulate their targets. In the retina, for
example, Pax6 is one of a group of ‘‘eye-field’’ transcription
factors, which coordinately regulate one another in a concerted
manner to specify the retinal fate (Zuber et al., 2003). An analo-
gous set of organizing transcription factors may exist for the
tory/vestibular sensory cells emerge from placodes, instead of
the neural tube, the specification mechanisms for these early
stages may differ.
Once specific regions of the epithelia are specified as
‘‘sensory’’ by Sox2 and/or Pax genes, the process of neurogen-
esis begins in these domains, and several different bHLH
transcription factors become important in the production and
differentiation of the sensory receptor cells in these regions.
The proneural gene, Ascl1, is expressed in the developing retina
and olfactory epithelium and is necessary for providing a neural
competence in the progenitor cells (Cau et al., 2002, 1997;
Jasoni et al., 1994; Nelson et al., 2009). The proneural neuroge-
nins are also expressed in the olfactory, retinal, and inner ear
epithelia and play important roles in the production of specific
types of neurons in each region. Loss of Neurogenins in the inner
ear, for example, causes the failure of spiral ganglion neurons to
develop (Ma et al., 2000). In addition to these proneural factors,
other bHLH transcription factors are required for differentiation
of the sensory receptor cells or their associated neurons.
NeuroD1 is expressed in the photoreceptors in the retina, and
targeted deletion of this gene in mice leads to a failure of normal
cone photoreceptor differentiation and the degeneration of the
rod photoreceptors (Liu et al., 2008). In the inner ear, NeuroD1
is required in the ganglion neurons that synapse with the hair
cells (Jahan et al., 2010; Liu et al., 2000). One of the most impor-
tant genes for hair cell development, Atoh1 (Bermingham et al.,
1999), is another member of the bHLH family of transcription
factors and is required for hair cell development. Targeted
deletion of this gene results in the absence of hair cells in all
the inner ear sensory epithelia, and overexpression of Atoh1
during development induces hair cells in nonsensory regions of
the inner ear epithelium. Although not required for the sensory
receptors in the retina, the related Atoh7/Math5 is necessary
for the development of the retinal ganglion neurons (Brown
et al., 1998). The similarity in the expression of the proneural
and neural differentiation bHLH genes during development of
the specialized sensory organs is quite striking and supports
the idea that these systems have well conserved developmental
In addition to the transcription factors discussed above, the
development of the specialized sensory structures is regulated
by many different signaling factors. One of the most important
is Notch signaling. Notch is required in all these systems and
functions at several different stages of their development. For
example, in the inner ear, Notch is initially required in the early
specification of the Sox2 expressing presumptive sensory
domain of the epithelium (Brooker et al., 2006; Daudet and
Lewis, 2005; Kiernan et al., 2001, 2006). This requirement for
Notch is called the prosensory function, and recent studies
have shown that ectopic expression of active Notch is sufficient
to produce sensory patches from the nonsensory epithelium
(Hartman et al., 2010; Pan et al., 2010). A later role for Notch
role: lateral inhibition. When hair cells begin to develop from the
sensory epithelium, they signal via Dll1/Jag2/Notch1 interac-
tions to suppress hair cell differentiation in the adjacent cells
and instead direct them to develop as support cells (Haddon
et al., 1998). The Notch signal induces expression of Hes5,
a downstream effector in the Notch pathway (Kageyama and
Ohtsuka, 1999), and the Hes5 likely represses Atoh1, the bHLH
class transcription factor necessary for hair cell development.
In this way the alternating rows of hair cells and support cells
are set up during development. Notch is also necessary for
appropriate development of the retina. Maintained expression
of Notch causes the progenitor cells to develop as Mu ¨ller glia
(Vetter and Moore, 2001), like the support cells in the ear, and
lossNotcheffectors, Hes5,Hes1, and Hesr2 leadsto areduction
in Mu ¨ller glial production (Hojo et al., 2000). Inhibition of the
Notch pathway in the developing retina causes premature neural
differentiation of the progenitor cells and the loss of Mu ¨ller glia
(Nelson et al., 2007).
Although Notch is perhaps the best-studied signaling system
in the sensory epithelia, several members of the FGF family of
receptor tyrosine kinase ligands also are of critical importance.
In the auditory epithelium of the inner ear, FGF20 and Fgfr1 are
critical for the early stages of cochlear development, including
the initiation of Atoh1 expression (Hayashi et al., 2008b; Pirvola
et al., 2002). Later in cochlear development, FGF8 and Fgfr3
are necessary for the proper differentiation of one type of sup-
et al., 2009; Hayashi et al., 2007; Jacques et al., 2007; Puligilla
et al., 2007). In the retina and olfactory system, FGF8 is also
important for the early specification of the sensory domains,
and several other FGFs and FGF receptors are expressed in
these organs. Other signaling molecules, including members of
the Wnt, BMP, EGF, and IGF families, have been shown to be
involved in the normal development in these systems, and
although the details may be different, there are many conserved
Regeneration in Olfactory Epithelium
Of the specialized sensory epithelia, the olfactory epithelium
shows the most robust regeneration in response to injury
(Graziadei and Monti Graziadei, 1985). All cell types, including
the sensory receptor neurons, can be regenerated in all species
that have been examined. Severing the axons at the lamina
cribosa in rats and mice causes extensive apoptosis in the olfac-
tory receptor neurons within a few days (Cowan and Roskams,
2002). The epithelium at this point contains only the sustentacu-
lar cells and the globose and horizontal basal cells. The number
of mitotic figures increases dramatically within the basal cell
populations and within a few days new receptor neurons are
evident. Labeling the epithelium with traceable thymidine
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analogs demonstrated that the proliferating cells generated new
receptor neurons and sustentacular cells such that by 4 weeks
after the injury, the epithelium was completely restored. The
new receptor neurons extend their axons back to the olfactory
bulb and they function normally. Other types of damage also
trigger a regenerative response. Damage from the toxin methyl
bromide (MeBr) causes an even more massive degeneration of
the sensory epithelium, including the receptor neurons, the sus-
tentacular supporting cells, and many of the GBCs; however,
regeneration of the epithelium to the prelesion state occurs
within 4 weeks of the insult.
Several in vitro and in vivo studies have attempted to identify
the cells involved in the regeneration in this system (Beites
et al., 2005; Calof et al., 2002; Carter et al., 2004; Huard et al.,
1998; Kawauchi et al., 2004; Sicard et al., 1998). The two main
candidates are the GBCs and the HBCs. The cells responsible
for the regeneration of the epithelium under conditions of olfac-
tory nerve transection, where the damage is largely confined to
the olfactory receptor neurons, are likely the GBCs. Olfactory
bulbectomy (essentially the same as olfactory nerve section)
repopulate the missing cell types (Carr and Farbman, 1992).
Under normal conditions, the HBCs are relatively quiescent,
and even after bulbectomy, they are only occasionally found in
the mitotic cycle. After the more extensive damage caused by
MeBr, though, the HBCs also proliferate (Leung et al., 2007).
Utilizing mice expressing Cre-recombinase under the keratin 5
etal.,2007), thesegroupsfoundthatthe lineageof theHBCscan
include all the of different cell types of the epithelium, including
the GBCs (even in normal mice, Iwai et al., 2008). However, after
MeBr lesions, the proliferation of the HBCs is greatly increased,
as is the production of GBCs (Leung et al., 2007).
Thus the current model is that the HBCs normally have a very
low level of proliferation, sufficient to self-renew and replenish
the GBC population, while the GBCs act more like transit ampli-
fying cells or immediate precursors to the cells of the sensory
epithelium. A relatively small amount of damage activates the
GBCs to produce receptor neurons at a higher rate, and these
cells are certainly capable of generating the sustentacular cells
as well. A large amount of damage to the epithelium recruits the
HBCs to replace lost GBCs, which go on to generate receptor
neurons and sustentacular cells. On a molecular level, many of
the features of developmental neurogenesis are recapitulated.
that mimics their expression during the embryonic development
of the olfactory epithelium (Cau et al., 2002; Manglapus et al.,
2004): Mash1+ GBCs are destroyed by the MeBr, but they
reappear in increased numbers two days after the MeBr. Ngn1/
NeuroD+ cells are also lost with MeBr damage, but are present
3 days after the damage (Guo et al., 2010) and precede
production of new receptor neurons, which appear by 4 days
postlesion. Hes1 is expressed by the sustentacular cells in the
normal epithelium, but after MeBr, GBCs also express Hes1,
and some of these go on to differentiate into sustentacular cells.
Since the olfactory epithelium displays such robust regenera-
tion it begs the question as to why we lose olfactory sensation as
we age. The loss of sensory perception can result from changes
in the sensory epithelia or, alternatively, from changes in the
brain critical for processing the sensory information. There is
evidence, however, that the number of receptor cells declines
with age in humans. Moreover, in rats and mice, the density of
proliferating (BrdU+) cells in the epithelium declines as the size
of the epithelium grows (Weiler and Farbman, 1997); thus, while
the overall number of proliferating cells does not decline by very
much, the turnover of the receptor neurons, as indicated by the
number of BrdU/OMP+ cells, declines with age (Kondo et al.,
2010). This decline is also seen in the vomeronasal organ of
mice, where Brann and Firestein (2010) reported thatthe number
of proliferating cells declines with age. Taken together, these
studies suggest that the production of new receptor cells may
not be able to keep pace with the increased loss of these cells
that accompanies increasing age.
Regeneration in the Inner Ear
Some of the first evidence for regeneration of hair cells came
from studies of the lateral line organs in fish and amphibia. The
lateral line organs of fish and amphibia consist of mechanosen-
sory neuromasts distributed along the body surface. In urodeles,
after amputation of the tip of the tail, new neuromasts are gener-
ated in the lateral line organ at the stump and migrate to form
new organs as the tail regenerates (Stone, 1937). Studies by
Jones and Corwin demonstrated that a low level of ongoing
hair cell production is dramatically upregulated after hair cells
in the lateral line are destroyed with a laser (Jones and Corwin,
1993, 1996). Direct time-lapse recordings demonstrated that
the regenerated hair cells arose from support cells (Jones and
Corwin, 1993). A similar increase in mitotic proliferation in the
support cells occurs in zebrafish after various types of ototoxic
damage (Herna ´ndez et al., 2007; Ma et al., 2008; Williams and
the hair cells within 48 hr of the insult.
Hair cell regeneration has also been extensively studied in
both auditory and vestibular sensory organs. In the vestibular
epithelia, regeneration of new hair cells has been reported in
the cristae, and the saccule and macula (Warchol, 2011) of all
nonmammalian vertebrates that have been studied such as
fish (e.g., (Faucher et al., 2009), bullfrogs, newts, and birds. In
the bullfrogsaccule, manyof theregenerated hair cellsarenewly
generated and labeled with BrdU, but at least a fraction of the
new hair cells arise from direct transdifferentiation of the support
cells—i.e., hair cells are regenerated even after inhibition of
proliferation (Baird et al., 2000, 1993). In the newt, hair cell
damage causes many support cells to enter the mitotic cell
cycle, but in this system the proliferating BrdU+ cells do not
contribute to the newhair cells (Taylor andForge, 2005).Instead,
all the new hair cells are thought to be due also to transdifferen-
Birds regenerate hair cells in both their vestibular epithelia and
their auditory epithelia. Since the vestibular organs normally
generate new hair cells throughout life in birds, like the olfactory
epithelium, when the sensory receptor cells are destroyed, the
proliferating cell population increases in the rate of new hair
cell production and the normal number of sensory receptors is
restored (Jørgensen and Mathiesen, 1988; Roberson et al.,
1992; Weisleder and Rubel, 1993). The situation in the auditory
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
the bird shows robust regeneration after hair cells are destroyed
with either ototoxic drugs or from excessive noise (Cotanche
et al., 1987; Cruz et al., 1987). In posthatch chicks, for example,
experimental destruction of thehaircells causesthesurrounding
support cells to re-enter the cell cycle within 16 hr, and new hair
cells appear within 2–3 days (Warchol and Corwin, 1996; Corwin
and Cotanche, 1988; Cotanche et al., 1994; Janas et al., 1995;
Ryals and Rubel, 1988; Weisleder and Rubel, 1993) It is not clear
whether there is a subset of support cells that can re-enter the
cell cycle or whether this is a property of all support cells in the
BP, but it has been estimated that only 10%–15% of the support
cells enter the mitotic cell cycle after damage, and most of these
are concentrated in the neural part of the damaged epithelium
(Bermingham-McDonogh et al., 2001; Cafaro et al., 2007). In
addition to the generation of new hair cells through support
cell divisions, there is also evidence in birds that some of the re-
generated hair cells come from direct transdifferentiation (Adler
et al., 1997; Adler and Raphael, 1996; Roberson et al., 2004; Ru-
bel et al., 1995), like that described above in the amphibian. The
initial response occurs prior to even extrusion of the damaged
hair cells and results in an upregulation of a key hair cell marker
(Atoh1) in some cells with support cell morphology (Cafaro et al.,
2007). Moreover, new hair cells appear to be produced even in
the presence of mitotic inhibitors. The regeneration of hair cells
after damage leads to functional recovery (Bermingham-McDo-
nogh and Rubel, 2003).
The story is quite different in the mammalian inner ear. In
mammals, once the hair cells are lost there appears to be little
if any spontaneous recovery. In the organ of Corti, the hair cells
and surrounding support cells are mitotically quiescent, and hair
cell damage does not induce their re-entry into the mitotic cell
cycle. Further, in the adult mammalian cochlea, there appears
to be no direct transdifferention of support cells into hair cells
after damage. The same is true for the mammalian vestibular
organs. Little proliferation is observed after hair cell damage or
in undamaged organs, either in vivo or in vitro (Oesterle et al.,
1993). Occasional H3-thymidine+ or BrdU+ support cells have
been reported, but most investigators would agree that the level
of proliferation in the mammalian inner ear epithelia is extremely
low (Cotanche and Kaiser, 2010; Groves, 2010). So what are the
differences in mammals that may account for this lack in prolifer-
ative potential? One striking difference in the auditory system is
the structure of the organ itself. In birds the auditory epithelium
resembles the vestibular epithelium, with relatively homoge-
neous support cells. By contrast, the support cells of the
mammalian organ of Corti have highly specialized structures
(Figure 1B) adapted for high frequency hearing. The morpholog-
ical specializations in the mammalian organ of Corti may impose
ative response. However, this structural argument does not
really apply to the mammalian vestibular organs, since they are
quite similar to their counterparts in other vertebrates.
Investigations of regeneration of hair cells in the inner ear and
lateral line have focused on two key questions: (1) what factors
control cell proliferation in the support cells and (2) what factors
control hair cell specification and support cell transdifferentia-
tion? Cell proliferation accompanies most examples of hair cell
regeneration in the inner ear sensory epithelia, and consequently
many investigators in this field have focused on developing
a better understanding of the mechanisms that control prolifera-
tion in normal and damaged epithelia. Attempts to stimulate
proliferation of support cells in explant cultures of inner ear
organs have shown some effects with mitogenic factors,
including EGF, TGF-alpha, TNF-alpha, and IGF (Doetzlhofer
et al., 2004; Oesterle and Hume, 1999; Oesterle et al., 1997;
Warchol, 1999; Yamashita and Oesterle, 1995; Zheng et al.,
1997); however, FGF, which is mitogenic in many systems,
appears to have the opposite effect in the inner ear epithelia
(Oesterle et al., 2000), possibly related is the fact that Fgfr3 is
downregulated in the chick basilar papilla after damage
(Bermingham-McDonogh et al., 2001). Many mitogenic factors
act via the upregulation of cyclin expression, and CyclinD is
particularly important in regulating proliferation of hair cell
precursors (Laine et al., 2010). The Cdkis are also important in
the control of proliferation in the inner ear. During development
of the sensory epithelium in the cochlea, the Cdki, p27kip1is an
early marker of the part of the presumptive sensory region that
will generate the hair cells and support cells (Chen and Segil,
1999) and deletion of p27kip1leads to an extension in the normal
developmental limit in proliferation of cells in the cochlea
(Kil et al., 2011; Lee et al., 2006; Lo ¨wenheim et al., 1999).
Although these experiments demonstrated that p27kip1is an
important developmental regulator of support cell proliferation,
recently it was shown that deletion of p27kip1in adult animals
also causes support cells to enter the mitotic cell cycle (Oesterle
of the factors required in mature mice to maintain mitotic quies-
cence in the support cells. Taken together, the results suggest
that methods to stimulate proliferation in the mammalian inner
ear epithelia might be possible through manipulation of a combi-
nation of known pathways. However, even though some support
cells proliferate in the postnatal cochlea in the p27kip1knockout
mice, very few, if any, generate mature new hair cells as they
would during regeneration in nonmammalian vertebrates; rather,
the proliferating cells appear to generate additional support
cells, or else they undergo apoptosis. This leads to the second
main difference between the nonmammalian vertebrates and
mammals: the support cells of the auditory sensory epithelium
of nonmammalian vertebrates have the capacity to transdiffer-
entiate into hair cells, while mammalian cochlear support cells
What factors enable the support cells of nonmammalian verte-
brates to differentiate into hair cells after damage? Studies of the
factors thatcontrolthe fates of hair cellsand support cells during
regeneration have focused on the developmental regulators of
hair cell determination/differentiation: the bHLH transcription
factor, Atoh1, and the Notch pathway (Cafaro et al., 2007;
Daudet et al., 2009; Stone and Rubel, 1999). Atoh1 is a critical
transcription factor for the specification of the hair cells dur-
ing development (Figure 3), while Notch signaling has both
a ‘‘prosensory’’ role and acts in a more conventional lateral
inhibitory manner to regulate the ratios of hair and support cells.
(Brooker et al., 2006; Kiernan et al., 2001, 2006; Adam et al.,
1998; Brooker et al., 2006; Haddon et al., 1998; Kiernan et al.,
2005; Zine and de Ribaupierre, 2002). In the normal adult
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
vestibular organs in birds, Atoh1 is expressed in scattered cells
throughout the epithelium, suggesting a continued requirement
for specification during the ongoing hair cell production in these
organs (Cafaro et al., 2007). By contrast, in the normal avian
adult BP, there is no Atoh1 expression; however, after hair cell
damage a number of cells express Atoh1 and Notch pathway
genes (Cafaro et al., 2007; Daudet et al., 2009), reflecting
the hair cell production during regeneration. The expression of
Atoh1 in support cells occurs rapidly after hair cell damage in
Studies in zebrafish lateral line have shown that Notch pathway
components are also upregulated very soon after hair cell
damage in this system (Ma et al., 2008) and that blocking the
Notch pathway using a gamma-secretase inhibitor leads to an
excess of regenerated hair cells. Similar results were obtained
in chick BP (Daudet et al., 2009). These studies indicate that
the mechanisms of lateral inhibition function during regeneration
much like they would during normal development; however,
there are two additional interesting findings. First, in the chick,
Atoh1 is upregulated in support cells of the chick basilar papilla
very soon after damage, possibly before the cells enter the
cell cycle. Second, the experimental inhibition of Notch in the
undamaged basilar papilla or lateral line does not activate
Atoh1 expression in the support cells and does not induce trans-
differentiation of support cellsto haircells. Theseresultsindicate
that it is only after damage, when the Notch pathway is upregu-
lated in the BP, that Notch-mediated lateral inhibition is impor-
tant for defining the fates of the hair and support cells. Notch
does not appear to be necessary for the maintenance of these
fates in the undamaged epithelium. Since the loss of hair cells
induces the rapid upregulation of Atoh1 in the support cells, it
also suggests that a different type of signal produced by the
hair cells normally inhibits the expression of the proneural
Atoh1 transcription factor in the neighboring support cells.
Although cell proliferation and transdifferentiation are virtually
absent in the normal mature inner ear epithelia of mammals,
these processes can occur in neonatal mammals. Dissociation
of the support cells from the cochlea and vestibular epithelia
from neonatal mice allows them to proliferate in vitro and turn
on markers of hair cells, Myosin VIIa and Atoh1 (Diensthuber
et al., 2009; Martinez-Monedero et al., 2007; Oshima et al.,
2009; White et al., 2006). However, this proliferative ability is
limited to the first 2 weeks of postnatal development in the
cochlea of mice, though the vestibular organs may harbor these
putative stem cells into adulthood. In addition to these examples
of proliferation, the decline in potential for transdifferentiation
with maturation has also been examined in the cochlea. The
Notch pathway remains active for a brief period of postnatal
development (Hartman et al., 2009); however, after postnatal
day 3 in the mouse, inhibition of this pathway no longer leads
to Deiters’ cell transdifferentiation (Hayashi et al., 2008a; Yama-
moto et al., 2006). Analysis of expression of Notch pathway
components has shown that these decline in all of the epithelia
of the inner ear during the first postnatal week, and even after
damage ofthehaircellsin thecochlea, Notchsignaling isnotup-
regulated in mice as it is in nonmammalian vertebrates (Hartman
et al., 2009). A few studies have attempted to identify the genes
responsible for this loss in the competence for hair cell transdif-
ferentiation by cochlear support cells. One candidate is Sox2,
since it is expressed in sensory epithelial precursors in the
inner ear and is required for their formation. However, Sox2 is
does not correlate with the loss of hair cell competence in
Deiters’ cells (Oesterle et al., 2008). Signaling molecules may
also be critical for limiting the process of transdifferentiation in
the organ of Corti: FGF signaling may also play a role in limiting
the competence of pillar cells to transdifferentiate into hair
cells, though Deiters’ cells may use a different mechanism
(Doetzlhofer et al., 2009).
In sum, successful regeneration of hair cells in nonmammalian
vertebrates requires a coordinated induction of Atoh1 and the
Notch pathway in the support cells. Neonatal mammals still
display some aspects of these phenomena in the cochlea, and
they may extend into adulthood in the vestibular epithelia to
a limited extent. In light of these results, several groups have
asked whether expression of Atoh1 is sufficient to generate
new hair cells from nonsensory cells in the inner ear (Gubbels
et al., 2008; Zheng and Gao, 2000). Studies in the adult guinea
pig have shown that overexpression of Atoh1 can promote
new hair cell formation in the normal and damaged organ of
Corti, by reprogramming of the remaining support cells (Izumi-
kawa et al., 2005; Kawamoto et al., 2003), though most of the
new hair cells appeared in nonsensory regions of the inner ear
epithelium. The potential of support cells to generate hair cells
using Atoh1 appears to be limited to a critical window, since
infection 6 days after the damage no longer induces new hair
cells (Izumikawa et al., 2008). Nevertheless, taken together
with the chick and fish studies, it would appear that the expres-
sion of Atoh1 after damage might be sufficient for direct transdif-
ferentiation of support/nonsensory cells to hair cells and clearly
represents a key step in the regeneration process.
Regeneration in the Retina
In amphibians, particularly urodeles (e.g., salamanders), new
retina can be generated from the nonneuronal cells of the retinal
tation and acquiring gene expression patterns similar to the
retinal progenitors found in embryonic development (for review,
see Lamba et al., 2008; Moshiri et al., 2004). These RPE-derived
progenitors proliferate, recapitulate the sequence of normal
histogenesis, and ultimately generate a complete new retina,
sary to process and relay visual sensory information to central
visual nuclei and restore visual function. The process by which
the RPE cells acquire a retinal progenitor phenotype appears
to be a critical one, since the process after this point resembles
that of normal development. This phenomenon has been vari-
ously termed metaplasia, transdifferentiation (Okada, 1980), or
dedifferentiation (since the RPE cells are reverting to a develop-
mentally earlier state). This phenomenon involves shifting the
pattern of gene expression in a highly regulated way and might
be called ‘‘regulated reprogramming’’ to distinguish it from the
‘‘direct transdifferentiation’’ process that occurs in support cells
in mechanosensory receptor regeneration in the inner ear or
lateral line. Pigmented epithelial cells reprogram to a progenitor
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
state in birds as well; however, this phenomenon is restricted to
the earliest stages of eye development, a few days after the
lineages of the retinal progenitors and the RPE progenitors
have diverged (Coulombre and Coulombre, 1970).
Fish also have considerable ability to regenerate sensory
receptor cells and other retinal neurons from sources within the
retina. When the fish retina is damaged (via surgical, neurotoxic,
or genetic lesions or excessive light), there is a burst of prolifer-
ation. As noted above, the fish retina contains a precursor cell
that continues to generate new rod photoreceptors throughout
the lifetime of the animal. For many years it was believed that
the primary source of new retinal neurons was the rod precursor
(Raymond et al., 1988). More recently, it became clear that the
Mu ¨ller glia were another source, if not the major source of prolif-
erating cells after retinal damage in the fish (Fausett and Gold-
man, 2006; Wu et al., 2001), although rod precursors contribute
as well, particularly when only rods are damaged. After retinal
damage, the Mu ¨ller glia in the fish retina undergo a dedifferentia-
tion process (Bernardos et al., 2007; Fausett and Goldman,
2006; Qin et al., 2009; Ramachandran et al., 2010; Raymond
et al., 2006; Thummel et al., 2008), somewhat like that described
for the RPE in the amphibian; they re-express many, if not all, of
the genes normally expressed in retinal progenitors.
causes nonneuronal cells to change their phenotype into retinal
progenitors: in amphibians, the progenitors are derived from the
RPE cells, while in fish these progenitors are derived from Mu ¨ller
glia. However, damage to cells in the peripheral retina can be re-
paired by a very different mechanism in these species. In both
fish and amphibians, the retina contains a specialized zone of
progenitor cells at the periphery, called the ciliary marginal
zone (or CMZ), which adds new neurons of all types throughout
the lifetime of the animal (for review, see Lamba et al., 2008). The
CMZ progenitors and stem cells generate new neurons at a low
rate, and the progeny are integrated with the existing retina cells
extending the retina as the overall eye grows. This process
matches the eye size with the overall size of the animal. Damage
to cells in the peripheral retina causes an increase in the prolifer-
ation of the progenitor cells in the CMZ and replacement of the
cells that were destroyed by the insult. However, the new cells
regenerated by the CMZ do not migrate to central regions of
retina and only repair the peripheral damage. Nevertheless, the
fact that the retina in fish and amphibians grows throughout their
life may require that developmental mechanisms be preserved
and provide a partial explanation for their regenerative potential.
Because of its ability to regenerate and due to the excellent
molecular tools developed in zebrafish, recent studies have
begun to identify the molecular requirements for regeneration
in this species. Neural progenitor genes are upregulated in
Mu ¨ller glia after damage consistent with their shift to the
phenotype of a retinal progenitor, while some Mu ¨ller glial-
specific genes are downregulated as the regenerative process
proceeds. Although it is not yet known whether the Mu ¨ller glia
are fully reprogrammed to retinal progenitors in fish, several
developmentally important genes have been shown to be
necessary for successful regeneration; for example, knockdown
of the proneural bHLH transcription factor Ascl1a blocks regen-
eration (Fausett et al., 2008), as does knockdown of proliferating
cell nuclear antigen (PCNA) (Thummel et al., 2008). Signaling
factors such as Midkine-a and -b, galectin, and ciliary neurotro-
phic factor (CNTF) are upregulated after injury and potentially
important in the proliferation of the Mu ¨ller cells that underlies
regeneration (Calinescu et al., 2009; Kassen et al., 2009).
Mu ¨ller glia of posthatch chicks also respond to neurotoxin
damage to the retina byre-entering the mitotic cell cycle (Fischer
and Reh, 2001). Unlike the fish, however, the Mu ¨ller glia in the
posthatch chick progress through one or at most two cell cycles
but do not undergo multiple rounds of cell division. Attempts to
stimulate the proliferation with injections of growth factors can
prolong this process somewhat and possibly recruit additional
Mu ¨ller glia into the cell cycle. In addition to a tempered prolifer-
amount of neuronal regeneration. Damage to the retina causes
some of the proliferating Mu ¨ller cells to express most of the
progenitor genes that are upregulated in fish Mu ¨ller glia after
damage (Fischer et al., 2002; Fischer and Reh, 2001, 2003;
Hayes et al., 2007). When the progeny of the proliferating Mu ¨ller
glia are tracked over the weeks after damage, BrdU+ cells are
found that express markers of amacrine cells (calretinin+, HuC/
D+), bipolar cells (Islet1), and occasional ganglion cells (Brn3;
neurofilament). Although it is not known whether the expression
of progenitor genes is required for successful regeneration in
the chick, two studies have tested whether reactivation of
Notch signaling is necessary. Blocking Notch signaling with
the gamma-secretase inhibitor DAPT prior to the regenerative
process blocks the Mu ¨ller cells re-entry into the cell cycle
(Ghai et al., 2010; Hayes et al., 2007). However, inhibition of
Notch signaling after the proliferation has begun causes a higher
percentage of cells to differentiate into amacrine cells than in the
control retinas, a result much like that described in the previous
section on hair cell regeneration in fish and chicks. Thus, Notch
activity might also limit the effectiveness of the process.
The above analysis indicates that retinal damage in both chick
and fish causes a somewhat similar response in the Mu ¨ller cells:
they proliferate and upregulate expression of neural progenitor
genes and Notch signaling. However, a key difference is that in
the fish most of the progeny of the Mu ¨ller glia differentiate into
retinal neurons and sensory receptor cells, whereas in the bird
only a smallpercentage ofthe progeny of theMu ¨llerglia differen-
tiate as neurons, and few, if any, develop into rods or cone
photoreceptor cells. Thus, for functional replacement of neurons
after damage, the proliferative response of the Mu ¨ller glia in
birds is not very effective. Nevertheless, comparisons between
the bird and fish are instructive as we discuss the regenerative
response in mammals below.
Mammalian Mu ¨ller glia show an even more limited regen-
erative response to injury than birds (Karl and Reh, 2010). In
‘‘reactive’’ like the astrocytic response to neuronal damage in
other regions of the CNS, increasing their expression of GFAP;
however, very few of them re-enter the mitotic cell cycle (Dyer
and Cepko, 2000; Levine et al., 2000; Ooto et al., 2004), Never-
theless, when the retinal damage is followed by treatment with
specific mitogenic proteins (e.g., EGF, FGF, IGF, Wnt3a), some
Mu ¨ller glial cells are stimulated to proliferate (Close et al., 2005,
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
2006; Karl et al., 2008). It is also possible to stimulate Mu ¨ller
glial proliferation in the absence of overt neuronal death with
subtoxic doses of alpha-aminoadipic acid (Takeda et al., 2008).
In all of these studies, however, only a relatively small number
of Mu ¨ller glia enter the mitotic cell cycle after damage, when
compared with the chick or the fish. Like the mammalian inner
ear, one of the restrictions on the proliferation of Mu ¨ller glia is
theCdki, p27kip1,andin theretina,the expressionof thisinhibitor
is known to be driven by TGF-beta (Close et al., 2005, 2006;
Levine et al., 2000).
Damage to the retina in fish and birds causes Mu ¨ller glia to
undergo a process of regulated reprogramming, allowing them
to adopt a retinal progenitor pattern of gene expression that
correlates with their ability to regenerate neurons after damage.
Does this same type of reprogramming occur in the mammalian
retina? Some aspects of the developmental program present in
the progenitors are reinitiated in rodent Mu ¨ller cells in the injured
retina: Pax6, Dll1, Notch1, and Nestin are upregulated to some
extent after NMDA damage (e.g., Karl et al., 2008). However,
many key progenitor genes do not appear to be re-expressed.
Two key neurogenic transcription factors, Ascl1 and Neuroge-
nin2, for example, are not upregulated in mammalian Mu ¨ller
glia after damage, even under conditions when these cells are
induced to proliferate with growth factors (Karl et al., 2008;
Karl and Reh, 2010). Thus, mammalian Mu ¨ller glia appear to
undergo only a partial reprogramming in contrast to the more
complete reprogramming to the progenitor phenotype that is
observed in fish and birds.
Although there is evidence for at least a partial reprogramming
cells is not nearly as clear. Following the BrdU+ cells for 2 to
4 weeks after NMDA damage, Ooto et al. (2004) reported that
some expressed markers of bipolar cells and photoreceptors.
Karl et al. (2008) reported that a combination of NMDA and
crine cells from the Mu ¨ller glia. Other studies have reported
regeneration of photoreceptors in the mouse or rat retina after
particular experimental manipulations. Wnt3a, MNU damage,
sonic hedgehog (Shh), and alpha-AA all increase Mu ¨ller glial
proliferation, and after survival periods of several days to weeks,
kada et al., 2007; Takeda et al., 2008; Wan et al., 2008, 2007).
However, in all these studies, the numbers of Mu ¨ller glia that
re-enter the cell cycle is very low and the number that go on to
differentiate into cells expressing neuronal markers of any type
are lower still, overall leading to the conclusion that the regener-
ative response in the mammalian retina is very limited compared
with what is observed in nonmammalian vertebrates.
General Principles of Specialized Sensory Regeneration
The specialized sensory epithelia show a range of regenerative
and the sense organ. Regeneration in the olfactory epithelium is
very good in all species that have been studied. The auditory
and vestibular hair cells regenerate in fish and amphibia and
birds; in mammals, regeneration of new hair cells is very limited
or nonexistent. Retinas regenerate in fish, amphibians, and to
some extent in birds; the regenerative capacity in mammals is
very limited. Why is there such variety in their potential for
intrinsic repair? In the following paragraphs we will attempt to
synthesize the common aspects of the response to injury in
these three systems across species with the aim of developing
general principles for sensory receptor cell regeneration.
One of the more clear conclusions that can be made concern-
ing the regeneration in these organs is that those sensory
epithelia with a continual ongoing replacement or addition of
new sensory receptors have excellent regenerative potential.
The olfactory epithelia of all vertebrates, the vestibular sensory
structures of fish and birds, and the retinas of fish all maintain
active processes for adding new sensory receptor cells through-
out life, and coincidentally, they all regenerate very well after
of the proliferating cells and an increase in their overall output to
produce the increased number of new cells required to restore
the epithelium. An appropriate niche has been maintained to
preserve a part of the embryonic environment in a functioning
organ, and in some ways regeneration in these organs is similar
to the phenomena known as ‘‘regulation’’ that occurs in embry-
onic development—i.e., when parts of a developing organ are
removed, the tissue ‘‘regulates’’ to replace the missing parts.
The increases in cell proliferation and shifts in cell fate determi-
nation that occur during regeneration presumably reflect the
complex developmental interactions among these cells that
control their initial patterning and ratios.
Another conclusion that can be drawn concerning regenera-
tion in sensory epithelia is that regeneration generally follows
In the olfactory epithelium, for example, once the process of
regeneration has begun, the progenitor cells go through the
same sequence that was followed during development, first
expressing Ascl1, then Neurog1, then NeuroD1, etc. This makes
sensory receptors, like the olfactory epithelium. However, even
in cases where a functioning, differentiated cell, like the RPE of
the frog or the Mu ¨ller glia in the fish retina, undergo a process
of reprogramming to generate a progenitor, the sequence of
regeneration from the progenitors closely follows the embryonic
developmental sequence. The Mu ¨ller glial-derived progenitors
in the fish retina dedifferentiate into progenitors that go on to
generate neurons after surgical lesions, in spite of the fact
that the regenerating progenitor cells are producing neurons
and glia in a very different microenvironment than that which
was present during embryonic development. Neurons in the
adjacent, undamaged parts of the retina seem to have little
impact on the progression of regeneration. For the inner ear,
the same signaling regulators (e.g., Notch) and transcription
factors (e.g., Atoh1) are employed during regeneration as were
used to regulate the production of hair and support cells during
embryonic development, and the process of lateral inhibition
appears to function in much the same way as it did during
development to generate the correct ratios of hair and support
cells during regeneration.
A third general conclusion that can be made is that those
specialized sensory epithelia that regenerate well ultimately
connect with the undamaged circuitry and restore function.
The olfactory epithelium in all animals tested can regenerate
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
functional sensory cells that grow axons through the lamina cri-
bosa to reconnect with the olfactory bulb, providing there is not
much scar tissue from the surgery. The lateral line of fish and
amphibians and the inner ear of all nonmammalian vertebrates
can regenerate new hair cells that will function nearly as well
damage have thresholds near normal (for review see Berming-
ham-McDonoghandRubel, 2003).The sameistrue fortheretina
of thefish;visualacuityisnearlyrestoredto normal,thoughthere
is some disruption in the details of cone patterning. The fact that
function can be restored in these systems is a rather amazing
feat, and so it gives us a bit of hope that stimulation of regener-
ation in the mammalian retina or inner ear sensory receptor cells
might be sufficient to trigger the associated processes that must
take placeforeffectiverestoration offunction.Eveninmammals,
there is evidence from both the inner ear and the retina that
considerable plasticity remains in the cells that synaptically
connect with the sensory receptor cells; ectopic hair cells
induced form Atoh1 overexpression can be innervated by the
ganglion neurons, and rod photoreceptors transplanted into
normal adult mice will reconnect to host bipolar cells and appear
to function (MacLaren et al., 2006).
Lastly, we can conclude that those sensory epithelia that
display little or no capacity for regeneration generally do not
have ongoing proliferation or addition of new neural cells
anywhere in the organ. What is even more striking about most
of the systems where regeneration is absent is that the cells in
these epithelia don’t respond to damage by increasing prolifera-
tion; in both the mammalian inner ear and retina there is very little
proliferation of the support cells or Mu ¨ller glia, respectively, after
injury. In addition, the nonneuronal support/glial cells in these
epithelia do not undergo extensive reprogramming after injury
but for the most part retain most morphological and gene
expression characteristics asthey have in the undamaged tissue
(though they frequently become ‘‘reactive’’). So in the special-
ized sensory epithelia that do not have ongoing new sensory
cell addition, like the mammalian inner ear and retina, the tissue
may no longer retain the ‘‘developmental niche’’ that is charac-
teristic of the olfactory epithelium, or the retina and inner ear of
There are two interesting exceptions to some of the above
conclusions: the retina and the cochlea of birds. The avian retina
responds to damage with robustMu ¨ller glial proliferation, though
the reprogramming of these cells to a progenitor pattern of gene
expression is much more limited than in the fish, and very few of
from studies of regenerating fish retina suggest that proliferation
might berequiredfor reprogramming in thissystem, sinceblock-
ing the proliferation with antisense to PCNA blocks regeneration.
However, most of the Mu ¨ller glia in the chick retina enter the
cell cycle after damage, so why do they not reprogram more
effectively? One possible answer might be that chick Mu ¨ller glial
cells only go through a single round of cell division after damage,
while fish Mu ¨ller cells appear to undergo multiple rounds; it is
possible that full reprogramming requires multiple rounds of
division. In vitro studies of reprogramming also suggest that
cell division is important for the more complete reprogramming
required to generate iPS cells (e.g., Takahashi and Yamanaka,
2006), though fibroblasts can be directly converted to neurons
by misexpressing neurogenic transcription factors without
multiple rounds of cell division (Vierbuchen et al., 2010). Exam-
ples from the other sensory systems also suggest that cell
division is not absolutely required for reprogramming; the lateral
line of the amphibian and the chick basilar papilla support cells
can directly transdifferentiate to hair cells.
Another related puzzle concerns the chick inner ear. The avian
vestibularsystem has ongoingproliferation yetthe aviancochlea
does not, but they both regenerate very well. How has the chick
cochlea retained a ‘‘developmentally immature’’ state equivalent
to that of the best regenerating epithelia, without apparently
adding new cells? An analogous situation can be also seen in
the regeneration of the newt retina from RPE cells, which are
not actively dividing in the mature organism. Despite this lack
of continual renewal, both the support cells of the chick basilar
and reprogramming after injury to replace the lost cells. An inter-
esting feature of both systems is that while they do not have
ongoing cell replacement within the specific cells that provide
the source for the regeneration, both of these organs have
ongoing sensory cell replacement ‘‘nearby.’’ For the newt, the
stem cells at the margin of the retina continue to produce new
retinal neurons at its peripheral edge; in the chick inner ear,
vestibular organs with ongoing hair cell genesis (i.e., the lagena)
are immediately adjacent to the basilar papilla in chick. It is
possible that some type of long-distance nonautonomous
property of the organs allows more plasticity in cell phenotype
throughout the epithelia. Alternatively, the genetic program of
development that allowed some part of the retina or inner ear
to retain developmental character into adulthood might also
enable regeneration more broadly across the sensory organ.
For example, perhaps in the inner ear of the chick, the vestibular
system and basilar papilla share a common developmental
program that does not preclude new cell addition, and this is
utilized continuously by the vestibular system and only after
injury in the basilar papilla. Answers to these questions might
provide insight into the reasons why mammalian regeneration
in the retina and inner ear are so limited.
What have we learned from studies of regeneration in the
systems capable of this process to inform our future progress
in promoting regeneration in the mammalian retina and audi-
tory/vestibular epithelia? Despite many years of study, it has
proven to be very difficult to stimulate regeneration in an organ
cells, like the mammalian retina or inner ear. Nevertheless, we
have really only scratched the surface in our understanding of
the molecular mechanisms underlying successful regeneration,
such as that in the olfactory epithelium. The studies of regener-
ation in both the retina and the inner ear have shown that cell
proliferation is quite limited in the species that do not regenerate
their sensory receptors in these organs. There are few, if any,
mitotic cells in the mouse retina or cochlea after photoreceptor
or hair cell damage, respectively. At least some of the regulators
of proliferation have been identified in these structures; prolifer-
ation of support cells and Mu ¨ller glia in both the inner ear and
the retina is regulated in part by the Cdki, p27kip1, and the tumor
suppressor,Rb. Lossofp27kip1leadstoextra celldivisions inthe
Neuron 71, August 11, 2011 ª2011 Elsevier Inc.
of mitotic divisions is still very limited. Studies in other systems
suggest that multiple pathways may need to be targeted to
stimulate proliferation in otherwise quiescent tissues (Pajcini
et al., 2010). More importantly, the new cells that are produced
in the retina and inner ear of mammals, even when the prolifera-
tion is stimulated, for the most part do not generate sensory
receptor cells. Simply getting the cells to divide again is not
sufficient for regeneration; some reprogramming appears to be
necessary for regeneration.
The reprogramming or transdifferentiation that occurs natu-
rally during regeneration in the retinas of fish and newts involves
the silencing of glial/RPE genes and the reactivation of a progen-
itor gene expression program. However, the molecular mecha-
nisms that maintain cell identity are still not very well understood
and further research into the epigenetic response of cells to
injury and during regeneration is warranted. The degree of re-
programming that takes place in the retinas of these animals
does not appear to be required in the inner ear, where the
support cells seem poised to activate Atoh1 expression. Several
rounds of cell division might be needed to effectively reprogram
the RPE cells or the Mu ¨ller glia, whereas no cell division at all is
required in the inner ear of fish and chicks. In both the retina and
the inner ear, Notch signaling also plays a role in regeneration. In
the olfactory epithelium, the Notch pathway is upregulated after
damage. In both the inner ear, and in the retina, Notch and its
ligands are upregulated after damage, and in the retina, the up-
regulation of Notch seems to be required at a very early stage in
the process of reprogramming. By contrast, in the inner ear the
support cells upregulate the Notch pathway, but inhibiting the
pathway does not prevent support cell transdifferentiation to
hair cells. These results suggest that the upregulation of Notch
after damage may not be required in the inner ear, but neverthe-
less the presence (and upregulation) of Notch signaling is a reli-
able indicator of a regenerating system. This is an important
conclusion in light of thefact that the downstreamNotch effector
Hes5 is not expressed in either the normal adult mouse cochlea,
or after damage to the hair cells (Hartman et al., 2009). Unbiased
screening for critical factors in the regeneration process, using
small molecule libraries, microarray studies, and genetics
(Brignull et al., 2009), will certainly lead to a better understanding
of the differences between the successful and non-successful
Outlook: Regenerative Medicine in the Special Senses
A process of ongoing sensory receptor cell replacement
characterizes the sensory epithelia that show robust regenera-
of mammals. Therefore, the main options for therapy will likely
involve reinitiating the process of regulated reprogramming
to a proliferative progenitor state in the glia and support cells.
Although stimulation of regeneration in mammalian inner ear
and retina to the level present nonmammalian vertebrates would
be ideal, considerably less effective regeneration could still be
useful for patients. For example, stimulation of proliferation in
support cells in the cochlea may not be necessary for some
recovery. In many individuals with age-related hearing loss, the
inner hair cells are thought to survive, longer than the outer hair
function (e.g., Chen et al., 2009). Therefore, the restoration of
only 30%–40% of the outer hair cells, by stimulation of transdif-
icant hearing improvement. The same may be true for the retina.
The degeneration of foveal cones in late stages of macular
degeneration leads to significant vision loss, though these cells
make up less than 1% of the total retinal cell population. As
the molecular pathways are further elucidated, one can imagine
a scheme in which these pathways could be targeted by gene
therapy to initiate a process of regulated reprogramming; in the
mammalian inner ear, viral expression of Atoh1 has already
been shown to restore some hair cells to damaged cochlea.
ulate this process, since these have proven successful in the
more drastic reprogramming that is required to generate iPS
cells (Li et al., 2009). The types of phenomena that we currently
lump together under the terms ‘‘transdifferentiation,’’ ‘‘dediffer-
entiation,’’ and ‘‘reprogramming’’ are complex and highly regu-
lated processes that are key to regeneration in the specialized
sensory epithelia. Our understanding of the mechanisms that
underlie these phenomena are in their infancy, but there is
a newsenseof optimism thatregenerativemedicine approaches
will someday provide treatments for sensory disorders.
The authors acknowledge the many discussions on regeneration in sensory
epithelia that they have had over the years with the members of the current
Reh/Bermingham-McDonogh labs, past members of our labs, especially
Drs. M.O. Karl, B. Nelson, T. Hayashi, and B. Hartman, and colleagues at
the University of Washington, including Drs. E. Rubel, D. Raible, J. Stone, E.
Oesterle, and C. Hume. Research in the authors’ labs are supported by the
grants DC005953, DC009991, DC010862, EY021482, EY021374, and PO1
GM081619-01 from the National Institutes of Health and TA-CBT-0608-
0464-UWA-WG from the Foundation Fighting Blindness.
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