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Models of congenital deafness: Mouse and zebrafish
Vol. 2, No. 2 2005
Drug Discovery Today: Disease Models Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Developmental defects
Models of congenital deafness: Mouse and zebrafish Tanya T. Whitfield1, Philomena Mburu2, Rachel E. Hardisty-Hughes2, Steve D.M. Brown2,* 1 2
Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK MRC Mammalian Genetics Unit, Harwell OX11 ORD, UK
Much of our understanding of the molecular mechanisms underlying hearing and deafness has come through the study of vertebrate model organisms.
Section Editor: Per Lindahl – Wallenberg Laboratory for Cardiovascular Research, Go¨teborg University, Sweden
We review here the advantages and disadvantages of the mouse and the zebrafish as genetic model systems for hearing research. Despite anatomical differences, many of the key molecules required for hair cell development and function are highly conserved between the two
species
and
both
provide
complementary
approaches for the development of drugs to treat diseases of the ear.
Introduction Hearing impairment affects around 250 million individuals worldwide (World Health Organization: http://www.who.it/ pbd/deafness/en) and around half of all cases are as a result of genetic causes. Given the sophistication of the auditory machinery it would be expected that a large number of genes would be required for hearing. Indeed, a large number of genetic loci (over 120) that cause nonsyndromic deafness have been mapped in human (Hereditary Hearing Loss Homepage: http://www.uia.ac.be/dnalab/hhh) and many more loci underlie the syndromes in which deafness is one of the symptoms. Though in many cases, the underlying genes remain to be identified, there has been considerable recent progress in identifying key genes and proteins that are *Corresponding author: Steve D.M. Brown ([email protected]) 1740-6757/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2005.05.018
involved in the development and maintenance of the auditory system and both the mouse and the zebrafish have been at the forefront of this research. There is not space here to do justice to the many elegant developmental genetic studies in both species that have unravelled the pathways underlying otic induction, those that assign cell fates in the otic vesicle or that reveal how the exquisite alternating arrangement of hair cells and supporting cells is laid down in the sensory epithelium of the inner ear. Instead, we concentrate on the advantages and disadvantages of the mouse and zebrafish as model systems for hearing research (Table 1) and on the key genes required for the differentiation and maintenance of the stereociliary bundle, hair cell regeneration and diseases of the mammalian middle ear. Genetic mutations that affect these processes underlie some of the most prevalent clinical problems of the auditory system, and their study provides ample opportunity for the investigation of deafness genes as potential therapeutic targets.
Genetic identification of deafness genes Each human cochlea contains just 16,000 hair cells – a tiny number compared with the number of photoreceptors in each eye, for example, which totals over 100 million. This paucity of material means that a biochemical approach to the identification of hair cell components and in particular, the transduction apparatus, is very difficult. A genetic approach, www.drugdiscoverytoday.com
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Table 1. Advantages and disadvantages of the mouse and zebrafish as models for hearing and deafness Technique
Mouse
Zebrafish
Forward mutagenesis screens
Recent large- and small-scale screens have isolated many recessive and dominant mutations affecting the ear
Cheaper than in the mouse, requiring less space. Several large- and small-scale screens have isolated mutants (mostly recessive) with defects in the ear and lateral line
Reverse genetic methods
Targeted gene knockout by homologous recombination possible owing to the availability of embryonic stem cell lines
Morpholino knockdown is a quick and rapid test of gene function in the embryo but only effective for the first few days of development TILLING (targeting induced local lesions in genomes) is now being performed in several labs to isolate mutations in genes of interest
Analysis of the embryo
Poor accessibility (embryo develops in utero)
Good accessibility: external fertilization means that all stages are accessible; embryo is transparent
Anatomy of the ear
Many similarities to the human ear
No outer or middle ear; no cochlea.
Measurements of hair cell function
Adult: Preyer reflex, circling behaviour, several electrophysiological assays for cochlear function
Acoustic/vibrational startle response, FM1–43 dye uptake, microphonic potentials, circling behaviour
Genomic organization
Fully sequenced genome
Genome sequencing project underway; orthologues of many mammalian genes are present as duplicate pairs, sometimes showing subfunctionalization
however, where genes are identified on the basis of function, is a much more powerful method for gene identification. Both the mouse and zebrafish are well suited to forward genetic analysis and their use as models for human deafness has concentrated on the identification and cloning of mutants characterized by auditory or vestibular defects. Both organisms can be screened for hearing and balance defects, indicative of malfunction of the auditory or vestibular components of the inner ear, respectively. Such screening depends on the choice of a robust and reproducible assay. A mouse will flinch in response to a loud noise, a behaviour known as the Preyer reflex; zebrafish embryos respond to sound and vibration (such as a tap on the dish) with a characteristic startle response (a rapid contraction of the body musculature, enabling the fish to swim away from danger). These behavioural assays can be used to screen for auditory defects. In addition, both organisms will display characteristic circling behaviour if the vestibular system is impaired. Many mutants in the zebrafish, however, have also been identified on the basis of visible morphological defects of the inner ear. Whereas both the mouse and zebrafish have been exploited for forward mutagenesis screens, both are also suitable for reverse genetic approaches. In the mouse, embryonic stem (ES) cell lines allow the generation of targeted knockouts, where specific human gene defects can be precisely reproduced at the sequence level. No such targeted knockout has yet been achieved in the zebrafish, but rapid knockdown of gene function in the early embryo through the injection of antisense morpholinos, and the possibility of identifying lesions in a gene of choice through targeting induced local lesions in genomes (TILLING) technology means that reverse 86
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genetic approaches in the fish offer a quick alternative approach to targeted gene knockout.
Comparative anatomy of the inner ear in the mouse and zebrafish Anatomically, the ear of the mouse, compared with that of the zebrafish, is a much closer model of the human ear (Fig. 1). In mammals, sound is collected by the pinnae and funnelled down the external ear canal to the tympanic membrane that separates the middle and external ear cavities. The middle ear is responsible for equalizing pressure across the tympanic membrane and transmitting sound via the bony ossicles to the inner ear. The inner ear comprises the vestibular system and the cochlea, the site at which auditory transduction takes place and sound impulses are converted into neural impulses to the brain [1]. The cochlea comprises a wide variety of tissues and cell types but pivotal is the organ of Corti, a neuroepithelial structure that runs the length of the cochlea (Fig. 2). Auditory transduction in the organ of Corti is mediated by hair cells, so called because they project from their apical surface a remarkable array of actinfilled projections called stereocilia. The organ of Corti is composed of single row of inner hair cells and three rows of outer hair cells that run the length of cochlear duct (Fig. 2b). The apical surfaces of the hair cells are bathed by the potassium-rich endolymph of the scala media. Sound impulses travelling down the cochlear duct cause the deflection of the stereocilia bundle resulting in the opening of ion channels at the stereocilia tips, cation influx, hair cell depolarization, neurotransmitter release, signalling to the spiral ganglion and ultimately the auditory centres in the brain. The complexity of the auditory process is reflected not only
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Drug Discovery Today: Disease Models | Developmental defects
Figure 1. Organs involved in hearing, balance and motion detection in the mouse and zebrafish. (a) Diagram of an embryonic mouse head at 16 days gestation (E16) showing the relative position of the developing inner ear (black), pinna (light blue), tympanum (blue) and middle ear ossicles (red). (b) Diagram of an adult zebrafish head showing the relative position of the inner ear (black), Weberian ossicles (red) and anterior chamber of the swimbladder (blue). There is no middle or external ear. (c) Diagram of the main features of the mouse inner ear at E16. The coiled cochlea is specialized for audition; vestibular regions (semicircular canals, utricle and saccule) function to maintain equilibrium and the endolymphatic system plays a role in the regulation of fluid balance within the inner ear. (d) The adult zebrafish inner ear has some anatomical similarities with that of the mammal but lacks a specialized auditory organ (cochlea). (e) Diagram of the distribution of lateral line organs (neuromasts) in a 5-day zebrafish larva. At this stage, all neuromasts are superficial. Both the inner ear and lateral line contain sensory hair cells. All diagrams are lateral views with anterior to the left. Colour coding implies functional, rather than anatomical homology. Scale bars: (a and b) 1 mm; (c–e) 500 mm.
in the elaborate structures of the organ of Corti but also in a variety of other cell types that are crucial for auditory transduction. Comparison of the fish ear with the mammalian ear reveals several striking differences, which at first sight appear to indicate that the zebrafish makes a rather poor model for human hearing (Fig. 1). Most importantly, there is no cochlea. Nevertheless, many fish can hear well; the adult zebrafish ear is sensitive to sound over a frequency range of 100–4000 Hz [2], compared with 20–20,000 Hz for a human. There is also no middle or external ear. However, in fish that are ‘‘hearing specialists’’ (of which the zebrafish is one), a chain of bones known as the Weberian ossicles links the gas-filled swimbladder to the saccule of the inner ear, which is thought to be the primary endorgan for audition (Fig. 1b). Although of completely distinct embryonic origin to the ossicles of the mammalian middle ear, Weberian ossicles provide a similar function in the
fish, transmitting higher frequency sound to the inner ear and increasing sensitivity to sound pressure [3,4]. Despite the obvious differences in the gross anatomy of the ear, hair cell structure and function appear to be highly conserved between the fish and mammal. For accessibility and visibility of the inner ear and hair cells in the live organism, the zebrafish is unsurpassed as a model system. As it is fertilized externally, all embryonic stages are available for observation; moreover, the embryo and larva are optically transparent, allowing observation of inner ear development (including hair cell formation) in the live organism. The mouse embryo, by contrast, is much less accessible, as it develops in utero. The zebrafish has an additional key advantage for hair cell study: the lateral line system. This consists of a series of sensory organs known as neuromasts, each containing hair cells similar to those found in the inner ear but which are uniquely accessible on the www.drugdiscoverytoday.com
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Figure 2. The mammalian organ of Corti, a complex organ for auditory transduction. (a) Diagram illustrating a cross-section through a mammalian cochlea showing the fluid-filled scalae that transmit sound impulses to the organ of Corti where the process of mechanotransduction takes place (see text). The organ of Corti comprises one row of inner hair cells and three rows of outer hair cells (illustrated in red). (b) Scanning electron micrograph illustrating the structure of the organ of Corti comprising three rows of outer hair cells (OHCs) and a single row of inner hair cells (IHCs). Stereocilia bundles project from the surface of outer and inner hair cells. (c) Stereocilia bundles consist of several rows of stereocilia of increasing height that form a staircase pattern. Stereocilia in adjacent rows are connected by tip-links. (d) As sound impulses travel down the cochlea, movement of the organ of Corti causes deflection of the stereocilia. Tip-link attachment sites harbour the mechanotransduction channels such that movement of stereocilia results in an increase in tension on the tip-links and the gating of the ion channels, leading to cation influx, hair cell depolarization and the transmission of a neural impulse to the brain via the spiral ganglion – the process of mechanotransduction.
surface of the skin in the larva for direct observation and manipulation (Fig. 1e). Note that many measurements of hair cell function in zebrafish mutant lines have been performed on lateral line hair cells, rather than those of the inner ear, for this reason. Development of the zebrafish is rapid; the major structures of the inner ear are present in the 3-day-old larva. Hair cell differentiation begins a mere 24 h after fertilization (hpf) and hair cell activity can be measured from 80 hpf through the uptake of the lipophilic dye FM1–43 (which enters zebrafish hair cells via transduction-dependent endocytosis) or by measurement of microphonic potentials (see for example [5]). The acoustic/vibrational startle reflex is present from 3–5 dpf [4,6]. In the mouse, hair cell differentiation begins at E15 and a number of different assays for cochlear function indicate that hearing in rodents matures after birth [7]. 88
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Development, maintenance and function of stereocilia The development and maintenance of stereocilia at the apical hair cell surface is critical for auditory transduction. Stereocilia are organized into bundles that display a precisely ordered staircase pattern [8]. Bundles comprise several rows of stereocilia of increasing height, with stereocilia in adjacent rows physically connected to each other by tip-links that attach the tips of shorter stereocilia to the sides of neighbouring stereocilia (Fig. 2c). It is thought that tip-link attachment sites harbour mechanotransduction channels such that stereocilia deflection increases tip-link tension and physically gates the channel leading to cation influx (Fig. 2d). The development of stereocilia from microvilli on the surface of hair cells has until recently been a molecular mystery. However, the mouse mutants shaker2 and whirler,
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both of which display shortened stereocilia, have played an important role in uncovering one of the molecular complexes involved. Mutations at the whirler locus disrupt the whirlin gene, which codes for a novel PDZ protein that is located at the stereocilia tips and shows an unusual pattern of expression during stereocilia development [9,10]. Mutations at the shaker2 locus disrupt myosin XVa [11]. Myosin XVa has been shown to interact with whirlin [12,13] and appears to play a critical role in transporting whirlin to the stereocilia tips
Drug Discovery Today: Disease Models | Developmental defects
where whirlin may act as an organizer for stereocilia elongation and actin polymerization (Fig. 3). The conservation of gene function involved in hearing in both fish and mammals is beautifully exemplified by the identification of the genes required for the maintenance and function of the hair cell stereociliary bundle once it has formed. At least five proteins – myosin VIIa, cadherin 23, protocadherin 15, harmonin b and sans – form complexes that play an important function in hair bundle maintenance,
Figure 3. Molecular complexes that mediate stereocilia growth, stereocilia bundle cohesion and mechanotransduction. The figure illustrates the key molecular complexes identified to date associated with the processes of stereocilia growth, stereocilia bundle cohesion and mechanotransduction. The whirlin–myosin XVa complex involved with stereocilia development and actin polymerization is localized to the stereocilia tip. Myosin VIIa, harmonin b, cadherin 23 and protocadherin 15 interact to ensure bundle cohesiveness, whereas sans may play a role in regulating trafficking of these proteins to the stereocilia. Cadherin 23 is also a component of the tip-link, whereas TRPA1 appears to be a component of the transduction channel.
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Table 2. Genes required for the development, maintenance and function of stereocilia Gene
Proposed function of gene product
Genes required for the development of stereocilia Myosin XVa Whirlin transport Whirlin Actin polymerization and stereocilia elongation Genes required for the maintenance of stereocilia Myosin VIIa Maintenance of bundle cohesion Myosin Ic Myosin VI Protocadherin 15 Cadherin 23 Sans Harmonin b
Adaptation motor Maintenance of integrity of hair bundle; vesicle trafficking Maintenance of bundle cohesion Component of the tip-link; maintenance of bundle cohesion Protein trafficking to stereocilia Maintenance of bundle cohesion
Genes coding for components of the transduction channel TRPA1 Component of the transduction channel and gating spring TRPN1 Component of the transduction channel and gating spring (zebrafish)
Human disease
Mouse mutants
Zebrafish mutants/morphants
DFNB3 DFNB31
shaker2 whirler
n.d. n.d.
Usher syndrome Type IB, DFNA11, DFNB2 – DFNA22, DFNB37
shaker1
mariner
– Snell’s waltzer
n.d. satellite, ru920 (myo6b)
Usher syndrome Usher syndrome DFNB12 Usher syndrome Usher syndrome DFNB18
Type IF Type ID,
Ames waltzer waltzer
orbiter (pdch15a) sputnik
Type IG Type IC,
Jackson shaker Deaf circler
n.d. n.d.
siRNA knockdown
Morphant
– Not found in the mammalian genome
Morphant
n.d. = not described.
determining the cohesiveness of the bundle structure [14–16] (Fig. 3). Mouse mutants are known for all five genes (shaker1 – myosin VIIa, waltzer – cadherin 23, Ames waltzer – protocadherin 15, deaf circler – harmonin b, Jackson shaker – sans) and all of them display disorganized stereocilia bundles. In zebrafish, mutants in at least three of these have been identified (mariner – myosinVIIa, sputnik – cadherin 23 and orbiter – protocadherin15a [17–19]; Table 2). Mutations in the corresponding human genes give rise to several forms of Usher syndrome, whose principal characteristics are deafness and retinitis pigmentosa, further reinforcing the interlinked functionality of these proteins. Characterization of the interactions of these five proteins and their localization within stereocilia has revealed a complex interplay among these molecules that is important for maintaining bundle cohesiveness. Harmonin b, a PDZ protein, appears central to the function of these proteins, interacting with all of the other four proteins. Myosin VIIa is required to transport harmonin b to appropriate stereocilia locations, where it anchors cadherin 23 and protocadherin 15, potential components of interstereociliary links, to the actin core of the stereocilia. In contrast, sans is found outwith the stereocilia in the apical region of hair cells beneath the cuticular plate, where it may play a role in regulating trafficking of the other proteins. An additional protein, myosin VI, is required for the integrity of the hair cell bundle in both mammals and zebrafish; without it, stereocilia become disorganized and eventually fuse [20–22]. Interestingly, despite the combination of visual defects with deafness in Usher syndrome, none of the mutants 90
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described here show the devastating retinal degeneration that is seen in the human patients. In the mouse, this might reflect the different retinal organization between the two mammals, the differing effects of secondary modifier genes, or the late age of onset of retinal degeneration in the human. In the fish, however, which has two protocadherin15 genes, knockdown of the second orthologue, pcdh15b, does indeed reveal an early requirement in the retina, illustrating the value of the fish model for studying this aspect of Usher syndrome pathology [17]. This is a classic example of subfunctionalization following gene duplication, which has now been demonstrated for several zebrafish gene pairs. Finally, there have been exciting developments in identifying candidates for the mechanotransduction machinery, including the tip-link and the mechanotransduction channel itself. Evidence from both mouse and zebrafish implicates cadherin 23 as a key component of the tip-link [19,23]. The tip-link is believed to gate the mechanotransduction channel either directly or indirectly. So a recent report that the elusive mechanotransduction channel might finally have been elucidated augurs well for establishing a detailed picture of the mechanotransduction machinery and its constituents. It appears that TRPA1, a member of the transient receptor potential (TRP) ion channel family, is a strong candidate, though it seems probable that this is not the only component of the channel [24]. In zebrafish, a second TRP family protein, TRPN1, also appears to be a component of the channel; here, TRPA1 and TRPN1 may function together as a heteromultimer [5]. The involvement of TRP family members is not a total
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surprise given the involvement of TRP proteins in specialized mechanosensory cells of Drosophila.
death, survival, regeneration and sensitivity to ototoxic agents.
Stem cells and hair cell regeneration
The middle ear and otitis media
In mammals, the organ of Corti is unable to replace hair cells that have been damaged or lost through genetic or environmental causes. This inability of mammals to regenerate lost cochlear hair cells has thrown the spotlight on the identification and manipulation of potential stem cell populations within the cochlea that might be used to repopulate a damaged organ of Corti. Two recent studies have yielded promising results [25,26]. In contrast to the organ of Corti, mammalian vestibular sensory epithelia show limited hair cell regeneration, possibly owing to the presence of stem cells. Indeed, Stefan Heller and co-workers have shown that the mouse adult utricular sensory epithelium contains stem cells that demonstrate self-renewal and form spheres that express markers that are typical of early inner ear development [25]. They are also able to differentiate into cells that express hair cell-specific markers and stereocilia-like structures. These stem cells offer a potential resource for studying drugs that can modulate the pathways controlling their proliferation. Moreover, they present a possible route to cell replacement therapy for hearing loss. In addition, the same group has also developed protocols to create inner ear progenitors from murine ES cells in vitro [26]. These progenitor cells display markers typical of early inner ear development and the developing sensory primordia. Interestingly, the progenitors could be differentiated into cells that expressed markers characteristic of hair cells. Progenitor cells are also able to integrate into damaged sensory epithelia, where they expressed hair cell markers and displayed morphological specializations reminiscent of hair bundles. Recently, mouse mutants in the retinoblastoma (pRb) gene have thrown light on how the process of regeneration in mammalian vestibular sensory epithelia might be initiated [27]. pRb is a mediator of mitotic arrest in sensory hair cells and its suppression leads to hair cells re-entering the cell cycle, although surprisingly, the hair cells divide while appearing to maintain their differentiated state. These observations suggest a therapeutic avenue to hair cell loss involving suppression of pRb activity and restoration of hair cell division. However, this approach is likely to be fraught with difficulties, not least the need to restore pRb activity at an appropriate juncture to prevent runaway proliferation. In zebrafish, hair cells continue to be produced in the ear throughout life (reaching an equilibrium at about 10 months of age) [2] and the larva is able to regenerate lateral line hair cells following treatment with ototoxic agents, such as aminoglycoside antibiotics [28,29]. The zebrafish lateral line, therefore, provides an especially attractive model for genetic screens designed to identify genes that influence hair cell
Otitis media (OM), inflammation of the middle ear, is the commonest cause of hearing impairment in children [30]. Treatment for OM is limited. Although antibiotics may ameliorate acute OM, they have little effect on chronic OM, which is often the target of surgical intervention (by gromit insertion to relieve middle ear pressure); OM remains the most common cause of surgery in children in the developed world. As a consequence, it is important to develop a better understanding of the aetiology of OM and to explore novel therapeutic approaches. There is now considerable evidence from studies of the human population as well as mouse models that there is a significant genetic component predisposing to OM [31–33]. Despite some progress in mapping susceptibility loci [34], we know nothing about the genes involved in OM in the human population. However, the availability of mouse models of OM has the potential to identify candidate genes and genetic pathways. A number of large-scale mouse ENU mutagenesis programmes have employed screens for deafness and as well as identifying novel mutants with sensorineural hearing loss [35] have also uncovered two mutants with OM – Jeff [33] and Junbo (N. Parkinson, R. Hardisty-Hughes and S.D.M. Brown, pers. commun.). The gene underlying the Junbo mutant is the transcription factor Evi1, identifying a novel role for this gene in mammalian disease and implicating a new pathway in the genetic predisposition to OM. It will be important to translate these findings into the human population and to examine Evi1 and other members of related pathways as potential candidates in association studies. Although the zebrafish will not be of direct use for the study of OM because it has no anatomical equivalent of the middle ear, it is, nevertheless, an important model of inflammation and immunity [36].
Drug discovery and development in the mouse and zebrafish The detailed understanding of the molecular basis of hearing that is emerging from model systems such as the mouse and zebrafish is of paramount importance for the development of therapeutic applications for deafness. For sensorineural deafness, recent attention has focussed on gene therapies; the report that Math1 can induce hair cell regeneration and restore hearing in a mature deaf mammal (the guinea pig) is particularly exciting [37]. Gene and stem cell therapy remain complex and sometimes ethically contentious areas, however, and approaches to develop pharmacological agents are also welcome. For OM especially, the accessibility of the middle ear cavity provides a straightforward route for the trial and administration of therapeutic molecules. www.drugdiscoverytoday.com
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The zebrafish is rapidly gaining ground as a high-throughput in vivo system for both drug discovery and drug testing. The embryo is readily accessible to small molecules and its small size makes it feasible for systematic, large-scale wholeorganism screening of small molecules. Screens have already yielded compounds that can generate specific phenotypes in wild type fish and a few that can effect rescue of a mutant defect (reviewed in [38]). The zebrafish is, therefore, set to become a key player in the identification and testing of new drugs but the mouse and other mammalian deafness models – such as the guinea pig – will undoubtedly remain crucial in translating novel therapeutic approaches into the clinic.
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15 16 17
18 19 20 21
Acknowledgements TW thanks M. Holley and D. Wu for help in the preparation of Fig. 1 and N. Monk for critical reading of the manuscript.
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References 1 Hudspeth, A.J. (1997) How hearing happens. Neuron 19, 947–950 2 Higgs, D.M. et al. (2001) Age- and size-related changes in the inner ear and hearing ability of the adult zebrafish (Danio rerio). JARO 3, 174–184 3 Higgs, D.M. et al. (2003) Development of form and function in peripheral auditory structures of the zebrafish (Danio rerio). J. Acoust. Soc. Am. 113, 1145–1154 4 Zeddies, R.G. and Fay, R.R. (2005) Development of acoustically evoked behavioral response in zebrafish to pure tones. J. Exp. Biol. 208, 1363– 1372 5 Sidi, S. et al. (2003) NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science 301, 96–99 6 Nicolson, T. et al. (1998) Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants. Neuron 20, 271–283 7 Rubel, E.W. et al., eds (1998) Development of the Auditory System, SpringerVerlag 8 Forge, A. et al. (1997) Structural development of sensory cells in the ear. Semin. Cell Dev. Biol. 8, 225–237 9 Mburu, P. et al. (2003) Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, causes deafness in the whirler mouse and families with DFNB31. Nat. Genet. 34, 421–428 10 Kikkawa, Y. et al. (2005) Mutant analysis reveals whirlin as a dynamic organizer in the growing hair cell stereocilium. Hum. Mol. Genet. 14, 391–400 11 Probst, F.J. et al. (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science 280, 1444–1447 12 Belyantseva, I.A. et al. (2005) Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia. Nat. Cell Biol. 7, 148–156 13 Delprat, B. et al. (2005) Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly. Hum. Mol. Genet. 14, 401–410
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Drug Discovery Today: Disease Models Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Developmental defects
Models of congenital deafness: Mouse and zebrafish Tanya T. Whitfield1, Philomena Mburu2, Rachel E. Hardisty-Hughes2, Steve D.M. Brown2,* 1 2
Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK MRC Mammalian Genetics Unit, Harwell OX11 ORD, UK
Much of our understanding of the molecular mechanisms underlying hearing and deafness has come through the study of vertebrate model organisms.
Section Editor: Per Lindahl – Wallenberg Laboratory for Cardiovascular Research, Go¨teborg University, Sweden
We review here the advantages and disadvantages of the mouse and the zebrafish as genetic model systems for hearing research. Despite anatomical differences, many of the key molecules required for hair cell development and function are highly conserved between the two
species
and
both
provide
complementary
approaches for the development of drugs to treat diseases of the ear.
Introduction Hearing impairment affects around 250 million individuals worldwide (World Health Organization: http://www.who.it/ pbd/deafness/en) and around half of all cases are as a result of genetic causes. Given the sophistication of the auditory machinery it would be expected that a large number of genes would be required for hearing. Indeed, a large number of genetic loci (over 120) that cause nonsyndromic deafness have been mapped in human (Hereditary Hearing Loss Homepage: http://www.uia.ac.be/dnalab/hhh) and many more loci underlie the syndromes in which deafness is one of the symptoms. Though in many cases, the underlying genes remain to be identified, there has been considerable recent progress in identifying key genes and proteins that are *Corresponding author: Steve D.M. Brown ([email protected]) 1740-6757/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2005.05.018
involved in the development and maintenance of the auditory system and both the mouse and the zebrafish have been at the forefront of this research. There is not space here to do justice to the many elegant developmental genetic studies in both species that have unravelled the pathways underlying otic induction, those that assign cell fates in the otic vesicle or that reveal how the exquisite alternating arrangement of hair cells and supporting cells is laid down in the sensory epithelium of the inner ear. Instead, we concentrate on the advantages and disadvantages of the mouse and zebrafish as model systems for hearing research (Table 1) and on the key genes required for the differentiation and maintenance of the stereociliary bundle, hair cell regeneration and diseases of the mammalian middle ear. Genetic mutations that affect these processes underlie some of the most prevalent clinical problems of the auditory system, and their study provides ample opportunity for the investigation of deafness genes as potential therapeutic targets.
Genetic identification of deafness genes Each human cochlea contains just 16,000 hair cells – a tiny number compared with the number of photoreceptors in each eye, for example, which totals over 100 million. This paucity of material means that a biochemical approach to the identification of hair cell components and in particular, the transduction apparatus, is very difficult. A genetic approach, www.drugdiscoverytoday.com
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Table 1. Advantages and disadvantages of the mouse and zebrafish as models for hearing and deafness Technique
Mouse
Zebrafish
Forward mutagenesis screens
Recent large- and small-scale screens have isolated many recessive and dominant mutations affecting the ear
Cheaper than in the mouse, requiring less space. Several large- and small-scale screens have isolated mutants (mostly recessive) with defects in the ear and lateral line
Reverse genetic methods
Targeted gene knockout by homologous recombination possible owing to the availability of embryonic stem cell lines
Morpholino knockdown is a quick and rapid test of gene function in the embryo but only effective for the first few days of development TILLING (targeting induced local lesions in genomes) is now being performed in several labs to isolate mutations in genes of interest
Analysis of the embryo
Poor accessibility (embryo develops in utero)
Good accessibility: external fertilization means that all stages are accessible; embryo is transparent
Anatomy of the ear
Many similarities to the human ear
No outer or middle ear; no cochlea.
Measurements of hair cell function
Adult: Preyer reflex, circling behaviour, several electrophysiological assays for cochlear function
Acoustic/vibrational startle response, FM1–43 dye uptake, microphonic potentials, circling behaviour
Genomic organization
Fully sequenced genome
Genome sequencing project underway; orthologues of many mammalian genes are present as duplicate pairs, sometimes showing subfunctionalization
however, where genes are identified on the basis of function, is a much more powerful method for gene identification. Both the mouse and zebrafish are well suited to forward genetic analysis and their use as models for human deafness has concentrated on the identification and cloning of mutants characterized by auditory or vestibular defects. Both organisms can be screened for hearing and balance defects, indicative of malfunction of the auditory or vestibular components of the inner ear, respectively. Such screening depends on the choice of a robust and reproducible assay. A mouse will flinch in response to a loud noise, a behaviour known as the Preyer reflex; zebrafish embryos respond to sound and vibration (such as a tap on the dish) with a characteristic startle response (a rapid contraction of the body musculature, enabling the fish to swim away from danger). These behavioural assays can be used to screen for auditory defects. In addition, both organisms will display characteristic circling behaviour if the vestibular system is impaired. Many mutants in the zebrafish, however, have also been identified on the basis of visible morphological defects of the inner ear. Whereas both the mouse and zebrafish have been exploited for forward mutagenesis screens, both are also suitable for reverse genetic approaches. In the mouse, embryonic stem (ES) cell lines allow the generation of targeted knockouts, where specific human gene defects can be precisely reproduced at the sequence level. No such targeted knockout has yet been achieved in the zebrafish, but rapid knockdown of gene function in the early embryo through the injection of antisense morpholinos, and the possibility of identifying lesions in a gene of choice through targeting induced local lesions in genomes (TILLING) technology means that reverse 86
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genetic approaches in the fish offer a quick alternative approach to targeted gene knockout.
Comparative anatomy of the inner ear in the mouse and zebrafish Anatomically, the ear of the mouse, compared with that of the zebrafish, is a much closer model of the human ear (Fig. 1). In mammals, sound is collected by the pinnae and funnelled down the external ear canal to the tympanic membrane that separates the middle and external ear cavities. The middle ear is responsible for equalizing pressure across the tympanic membrane and transmitting sound via the bony ossicles to the inner ear. The inner ear comprises the vestibular system and the cochlea, the site at which auditory transduction takes place and sound impulses are converted into neural impulses to the brain [1]. The cochlea comprises a wide variety of tissues and cell types but pivotal is the organ of Corti, a neuroepithelial structure that runs the length of the cochlea (Fig. 2). Auditory transduction in the organ of Corti is mediated by hair cells, so called because they project from their apical surface a remarkable array of actinfilled projections called stereocilia. The organ of Corti is composed of single row of inner hair cells and three rows of outer hair cells that run the length of cochlear duct (Fig. 2b). The apical surfaces of the hair cells are bathed by the potassium-rich endolymph of the scala media. Sound impulses travelling down the cochlear duct cause the deflection of the stereocilia bundle resulting in the opening of ion channels at the stereocilia tips, cation influx, hair cell depolarization, neurotransmitter release, signalling to the spiral ganglion and ultimately the auditory centres in the brain. The complexity of the auditory process is reflected not only
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Figure 1. Organs involved in hearing, balance and motion detection in the mouse and zebrafish. (a) Diagram of an embryonic mouse head at 16 days gestation (E16) showing the relative position of the developing inner ear (black), pinna (light blue), tympanum (blue) and middle ear ossicles (red). (b) Diagram of an adult zebrafish head showing the relative position of the inner ear (black), Weberian ossicles (red) and anterior chamber of the swimbladder (blue). There is no middle or external ear. (c) Diagram of the main features of the mouse inner ear at E16. The coiled cochlea is specialized for audition; vestibular regions (semicircular canals, utricle and saccule) function to maintain equilibrium and the endolymphatic system plays a role in the regulation of fluid balance within the inner ear. (d) The adult zebrafish inner ear has some anatomical similarities with that of the mammal but lacks a specialized auditory organ (cochlea). (e) Diagram of the distribution of lateral line organs (neuromasts) in a 5-day zebrafish larva. At this stage, all neuromasts are superficial. Both the inner ear and lateral line contain sensory hair cells. All diagrams are lateral views with anterior to the left. Colour coding implies functional, rather than anatomical homology. Scale bars: (a and b) 1 mm; (c–e) 500 mm.
in the elaborate structures of the organ of Corti but also in a variety of other cell types that are crucial for auditory transduction. Comparison of the fish ear with the mammalian ear reveals several striking differences, which at first sight appear to indicate that the zebrafish makes a rather poor model for human hearing (Fig. 1). Most importantly, there is no cochlea. Nevertheless, many fish can hear well; the adult zebrafish ear is sensitive to sound over a frequency range of 100–4000 Hz [2], compared with 20–20,000 Hz for a human. There is also no middle or external ear. However, in fish that are ‘‘hearing specialists’’ (of which the zebrafish is one), a chain of bones known as the Weberian ossicles links the gas-filled swimbladder to the saccule of the inner ear, which is thought to be the primary endorgan for audition (Fig. 1b). Although of completely distinct embryonic origin to the ossicles of the mammalian middle ear, Weberian ossicles provide a similar function in the
fish, transmitting higher frequency sound to the inner ear and increasing sensitivity to sound pressure [3,4]. Despite the obvious differences in the gross anatomy of the ear, hair cell structure and function appear to be highly conserved between the fish and mammal. For accessibility and visibility of the inner ear and hair cells in the live organism, the zebrafish is unsurpassed as a model system. As it is fertilized externally, all embryonic stages are available for observation; moreover, the embryo and larva are optically transparent, allowing observation of inner ear development (including hair cell formation) in the live organism. The mouse embryo, by contrast, is much less accessible, as it develops in utero. The zebrafish has an additional key advantage for hair cell study: the lateral line system. This consists of a series of sensory organs known as neuromasts, each containing hair cells similar to those found in the inner ear but which are uniquely accessible on the www.drugdiscoverytoday.com
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Figure 2. The mammalian organ of Corti, a complex organ for auditory transduction. (a) Diagram illustrating a cross-section through a mammalian cochlea showing the fluid-filled scalae that transmit sound impulses to the organ of Corti where the process of mechanotransduction takes place (see text). The organ of Corti comprises one row of inner hair cells and three rows of outer hair cells (illustrated in red). (b) Scanning electron micrograph illustrating the structure of the organ of Corti comprising three rows of outer hair cells (OHCs) and a single row of inner hair cells (IHCs). Stereocilia bundles project from the surface of outer and inner hair cells. (c) Stereocilia bundles consist of several rows of stereocilia of increasing height that form a staircase pattern. Stereocilia in adjacent rows are connected by tip-links. (d) As sound impulses travel down the cochlea, movement of the organ of Corti causes deflection of the stereocilia. Tip-link attachment sites harbour the mechanotransduction channels such that movement of stereocilia results in an increase in tension on the tip-links and the gating of the ion channels, leading to cation influx, hair cell depolarization and the transmission of a neural impulse to the brain via the spiral ganglion – the process of mechanotransduction.
surface of the skin in the larva for direct observation and manipulation (Fig. 1e). Note that many measurements of hair cell function in zebrafish mutant lines have been performed on lateral line hair cells, rather than those of the inner ear, for this reason. Development of the zebrafish is rapid; the major structures of the inner ear are present in the 3-day-old larva. Hair cell differentiation begins a mere 24 h after fertilization (hpf) and hair cell activity can be measured from 80 hpf through the uptake of the lipophilic dye FM1–43 (which enters zebrafish hair cells via transduction-dependent endocytosis) or by measurement of microphonic potentials (see for example [5]). The acoustic/vibrational startle reflex is present from 3–5 dpf [4,6]. In the mouse, hair cell differentiation begins at E15 and a number of different assays for cochlear function indicate that hearing in rodents matures after birth [7]. 88
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Development, maintenance and function of stereocilia The development and maintenance of stereocilia at the apical hair cell surface is critical for auditory transduction. Stereocilia are organized into bundles that display a precisely ordered staircase pattern [8]. Bundles comprise several rows of stereocilia of increasing height, with stereocilia in adjacent rows physically connected to each other by tip-links that attach the tips of shorter stereocilia to the sides of neighbouring stereocilia (Fig. 2c). It is thought that tip-link attachment sites harbour mechanotransduction channels such that stereocilia deflection increases tip-link tension and physically gates the channel leading to cation influx (Fig. 2d). The development of stereocilia from microvilli on the surface of hair cells has until recently been a molecular mystery. However, the mouse mutants shaker2 and whirler,
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both of which display shortened stereocilia, have played an important role in uncovering one of the molecular complexes involved. Mutations at the whirler locus disrupt the whirlin gene, which codes for a novel PDZ protein that is located at the stereocilia tips and shows an unusual pattern of expression during stereocilia development [9,10]. Mutations at the shaker2 locus disrupt myosin XVa [11]. Myosin XVa has been shown to interact with whirlin [12,13] and appears to play a critical role in transporting whirlin to the stereocilia tips
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where whirlin may act as an organizer for stereocilia elongation and actin polymerization (Fig. 3). The conservation of gene function involved in hearing in both fish and mammals is beautifully exemplified by the identification of the genes required for the maintenance and function of the hair cell stereociliary bundle once it has formed. At least five proteins – myosin VIIa, cadherin 23, protocadherin 15, harmonin b and sans – form complexes that play an important function in hair bundle maintenance,
Figure 3. Molecular complexes that mediate stereocilia growth, stereocilia bundle cohesion and mechanotransduction. The figure illustrates the key molecular complexes identified to date associated with the processes of stereocilia growth, stereocilia bundle cohesion and mechanotransduction. The whirlin–myosin XVa complex involved with stereocilia development and actin polymerization is localized to the stereocilia tip. Myosin VIIa, harmonin b, cadherin 23 and protocadherin 15 interact to ensure bundle cohesiveness, whereas sans may play a role in regulating trafficking of these proteins to the stereocilia. Cadherin 23 is also a component of the tip-link, whereas TRPA1 appears to be a component of the transduction channel.
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Table 2. Genes required for the development, maintenance and function of stereocilia Gene
Proposed function of gene product
Genes required for the development of stereocilia Myosin XVa Whirlin transport Whirlin Actin polymerization and stereocilia elongation Genes required for the maintenance of stereocilia Myosin VIIa Maintenance of bundle cohesion Myosin Ic Myosin VI Protocadherin 15 Cadherin 23 Sans Harmonin b
Adaptation motor Maintenance of integrity of hair bundle; vesicle trafficking Maintenance of bundle cohesion Component of the tip-link; maintenance of bundle cohesion Protein trafficking to stereocilia Maintenance of bundle cohesion
Genes coding for components of the transduction channel TRPA1 Component of the transduction channel and gating spring TRPN1 Component of the transduction channel and gating spring (zebrafish)
Human disease
Mouse mutants
Zebrafish mutants/morphants
DFNB3 DFNB31
shaker2 whirler
n.d. n.d.
Usher syndrome Type IB, DFNA11, DFNB2 – DFNA22, DFNB37
shaker1
mariner
– Snell’s waltzer
n.d. satellite, ru920 (myo6b)
Usher syndrome Usher syndrome DFNB12 Usher syndrome Usher syndrome DFNB18
Type IF Type ID,
Ames waltzer waltzer
orbiter (pdch15a) sputnik
Type IG Type IC,
Jackson shaker Deaf circler
n.d. n.d.
siRNA knockdown
Morphant
– Not found in the mammalian genome
Morphant
n.d. = not described.
determining the cohesiveness of the bundle structure [14–16] (Fig. 3). Mouse mutants are known for all five genes (shaker1 – myosin VIIa, waltzer – cadherin 23, Ames waltzer – protocadherin 15, deaf circler – harmonin b, Jackson shaker – sans) and all of them display disorganized stereocilia bundles. In zebrafish, mutants in at least three of these have been identified (mariner – myosinVIIa, sputnik – cadherin 23 and orbiter – protocadherin15a [17–19]; Table 2). Mutations in the corresponding human genes give rise to several forms of Usher syndrome, whose principal characteristics are deafness and retinitis pigmentosa, further reinforcing the interlinked functionality of these proteins. Characterization of the interactions of these five proteins and their localization within stereocilia has revealed a complex interplay among these molecules that is important for maintaining bundle cohesiveness. Harmonin b, a PDZ protein, appears central to the function of these proteins, interacting with all of the other four proteins. Myosin VIIa is required to transport harmonin b to appropriate stereocilia locations, where it anchors cadherin 23 and protocadherin 15, potential components of interstereociliary links, to the actin core of the stereocilia. In contrast, sans is found outwith the stereocilia in the apical region of hair cells beneath the cuticular plate, where it may play a role in regulating trafficking of the other proteins. An additional protein, myosin VI, is required for the integrity of the hair cell bundle in both mammals and zebrafish; without it, stereocilia become disorganized and eventually fuse [20–22]. Interestingly, despite the combination of visual defects with deafness in Usher syndrome, none of the mutants 90
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described here show the devastating retinal degeneration that is seen in the human patients. In the mouse, this might reflect the different retinal organization between the two mammals, the differing effects of secondary modifier genes, or the late age of onset of retinal degeneration in the human. In the fish, however, which has two protocadherin15 genes, knockdown of the second orthologue, pcdh15b, does indeed reveal an early requirement in the retina, illustrating the value of the fish model for studying this aspect of Usher syndrome pathology [17]. This is a classic example of subfunctionalization following gene duplication, which has now been demonstrated for several zebrafish gene pairs. Finally, there have been exciting developments in identifying candidates for the mechanotransduction machinery, including the tip-link and the mechanotransduction channel itself. Evidence from both mouse and zebrafish implicates cadherin 23 as a key component of the tip-link [19,23]. The tip-link is believed to gate the mechanotransduction channel either directly or indirectly. So a recent report that the elusive mechanotransduction channel might finally have been elucidated augurs well for establishing a detailed picture of the mechanotransduction machinery and its constituents. It appears that TRPA1, a member of the transient receptor potential (TRP) ion channel family, is a strong candidate, though it seems probable that this is not the only component of the channel [24]. In zebrafish, a second TRP family protein, TRPN1, also appears to be a component of the channel; here, TRPA1 and TRPN1 may function together as a heteromultimer [5]. The involvement of TRP family members is not a total
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surprise given the involvement of TRP proteins in specialized mechanosensory cells of Drosophila.
death, survival, regeneration and sensitivity to ototoxic agents.
Stem cells and hair cell regeneration
The middle ear and otitis media
In mammals, the organ of Corti is unable to replace hair cells that have been damaged or lost through genetic or environmental causes. This inability of mammals to regenerate lost cochlear hair cells has thrown the spotlight on the identification and manipulation of potential stem cell populations within the cochlea that might be used to repopulate a damaged organ of Corti. Two recent studies have yielded promising results [25,26]. In contrast to the organ of Corti, mammalian vestibular sensory epithelia show limited hair cell regeneration, possibly owing to the presence of stem cells. Indeed, Stefan Heller and co-workers have shown that the mouse adult utricular sensory epithelium contains stem cells that demonstrate self-renewal and form spheres that express markers that are typical of early inner ear development [25]. They are also able to differentiate into cells that express hair cell-specific markers and stereocilia-like structures. These stem cells offer a potential resource for studying drugs that can modulate the pathways controlling their proliferation. Moreover, they present a possible route to cell replacement therapy for hearing loss. In addition, the same group has also developed protocols to create inner ear progenitors from murine ES cells in vitro [26]. These progenitor cells display markers typical of early inner ear development and the developing sensory primordia. Interestingly, the progenitors could be differentiated into cells that expressed markers characteristic of hair cells. Progenitor cells are also able to integrate into damaged sensory epithelia, where they expressed hair cell markers and displayed morphological specializations reminiscent of hair bundles. Recently, mouse mutants in the retinoblastoma (pRb) gene have thrown light on how the process of regeneration in mammalian vestibular sensory epithelia might be initiated [27]. pRb is a mediator of mitotic arrest in sensory hair cells and its suppression leads to hair cells re-entering the cell cycle, although surprisingly, the hair cells divide while appearing to maintain their differentiated state. These observations suggest a therapeutic avenue to hair cell loss involving suppression of pRb activity and restoration of hair cell division. However, this approach is likely to be fraught with difficulties, not least the need to restore pRb activity at an appropriate juncture to prevent runaway proliferation. In zebrafish, hair cells continue to be produced in the ear throughout life (reaching an equilibrium at about 10 months of age) [2] and the larva is able to regenerate lateral line hair cells following treatment with ototoxic agents, such as aminoglycoside antibiotics [28,29]. The zebrafish lateral line, therefore, provides an especially attractive model for genetic screens designed to identify genes that influence hair cell
Otitis media (OM), inflammation of the middle ear, is the commonest cause of hearing impairment in children [30]. Treatment for OM is limited. Although antibiotics may ameliorate acute OM, they have little effect on chronic OM, which is often the target of surgical intervention (by gromit insertion to relieve middle ear pressure); OM remains the most common cause of surgery in children in the developed world. As a consequence, it is important to develop a better understanding of the aetiology of OM and to explore novel therapeutic approaches. There is now considerable evidence from studies of the human population as well as mouse models that there is a significant genetic component predisposing to OM [31–33]. Despite some progress in mapping susceptibility loci [34], we know nothing about the genes involved in OM in the human population. However, the availability of mouse models of OM has the potential to identify candidate genes and genetic pathways. A number of large-scale mouse ENU mutagenesis programmes have employed screens for deafness and as well as identifying novel mutants with sensorineural hearing loss [35] have also uncovered two mutants with OM – Jeff [33] and Junbo (N. Parkinson, R. Hardisty-Hughes and S.D.M. Brown, pers. commun.). The gene underlying the Junbo mutant is the transcription factor Evi1, identifying a novel role for this gene in mammalian disease and implicating a new pathway in the genetic predisposition to OM. It will be important to translate these findings into the human population and to examine Evi1 and other members of related pathways as potential candidates in association studies. Although the zebrafish will not be of direct use for the study of OM because it has no anatomical equivalent of the middle ear, it is, nevertheless, an important model of inflammation and immunity [36].
Drug discovery and development in the mouse and zebrafish The detailed understanding of the molecular basis of hearing that is emerging from model systems such as the mouse and zebrafish is of paramount importance for the development of therapeutic applications for deafness. For sensorineural deafness, recent attention has focussed on gene therapies; the report that Math1 can induce hair cell regeneration and restore hearing in a mature deaf mammal (the guinea pig) is particularly exciting [37]. Gene and stem cell therapy remain complex and sometimes ethically contentious areas, however, and approaches to develop pharmacological agents are also welcome. For OM especially, the accessibility of the middle ear cavity provides a straightforward route for the trial and administration of therapeutic molecules. www.drugdiscoverytoday.com
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The zebrafish is rapidly gaining ground as a high-throughput in vivo system for both drug discovery and drug testing. The embryo is readily accessible to small molecules and its small size makes it feasible for systematic, large-scale wholeorganism screening of small molecules. Screens have already yielded compounds that can generate specific phenotypes in wild type fish and a few that can effect rescue of a mutant defect (reviewed in [38]). The zebrafish is, therefore, set to become a key player in the identification and testing of new drugs but the mouse and other mammalian deafness models – such as the guinea pig – will undoubtedly remain crucial in translating novel therapeutic approaches into the clinic.
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Acknowledgements TW thanks M. Holley and D. Wu for help in the preparation of Fig. 1 and N. Monk for critical reading of the manuscript.
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