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Using Drosophila to study mechanisms of hereditary hearing lossTongchao Li1,*, Hugo J. Bellen1,2,3,4,5 and Andrew K. Groves1,3,5,‡

ABSTRACTJohnston’s organ – the hearing organ of Drosophila – has a verydifferent structure and morphology to that of the hearing organs ofvertebrates. Nevertheless, it is becoming clear that vertebrate andinvertebrate auditory organs share many physiological, molecularand genetic similarities. Here, we compare the molecular and cellularfeatures of hearing organs in Drosophila with those of vertebrates,and discuss recent evidence concerning the functional conservationof Usher proteins between flies and mammals. Mutations in Ushergenes cause Usher syndrome, the leading cause of human deafnessand blindness. In Drosophila, some Usher syndrome proteins appearto physically interact in protein complexes that are similar to thosedescribed in mammals. This functional conservation highlights arational role for Drosophila as a model for studying hearing, and forinvestigating the evolution of auditory organs, with the aim ofadvancing our understanding of the genes that regulate humanhearing and the pathogenic mechanisms that lead to deafness.

KEY WORDS: Cochlea, Deafness, Drosophila, Hair cells, Hearing,Usher syndrome

IntroductionHearing loss is one of the most common neurological disorders inhumans, affecting 2 to 3 of every 1000 children in the USA (Imtiazet al., 2016; Naz et al., 2017). Approximately 50% of congenitalhearing loss is caused by genetic mutations, with a variety ofother factors, particularly infectious disease, contributing to theremaining forms of congenital deafness (Korver et al., 2017).Genetic mutations affect many components of the auditory pathway,and can affect only hearing (nonsyndromic deafness), or otherorgans and tissues as well (syndromic deafness). Of the geneticmutations that cause nonsyndromic deafness, ∼30% are autosomaldominant, ∼70% are autosomal recessive, and five are X-linked(hereditaryhearingloss.org). Among the many forms of syndromichearing loss are Alport syndrome, Pendred syndrome, Branchio-oto-renal syndrome, Stickler syndrome and Usher syndrome(Korver et al., 2017). Usher syndrome is the leading cause ofhuman deafness and blindness, with a prevalence of 1-4 per 25,000people in the USA (Boughman et al., 1983; Hartong et al., 2006;

Keats and Corey, 1999; Kimberling et al., 2010; Mathur and Yang,2015). It is an autosomal recessive genetic disease, characterized byvarying degrees of deafness and retinitis pigmentosa-induced visionloss. Although our understanding of genetic hearing loss hasadvanced greatly over the past 20 years (Vona et al., 2015), there is apressing need for experimental systems to understand the functionof the proteins encoded by deafness genes. The mouse is wellestablished as a model for studying human genetic deafness (Brownet al., 2008), but other model organisms, such as the fruit flyDrosophila, might also provide convenient and more rapid ways toassay the function of candidate deafness genes.

In mammals, mechanosensitive hair cells reside in a specializedepithelial structure, the organ of Corti (see Glossary, Box 1), thatruns along the length of the cochlear duct (Box 1) of the inner ear(Basch et al., 2016). Vibration of the organ of Corti in response tosound waves that are captured and amplified by the external andmiddle ears causes a deflection of the hair-like stereocilia of haircells (Box 1). This deflection opens ion channels, leading to therelease of neurotransmitters by hair cells and the activation of thefirst neurons in the auditory pathway (Fettiplace and Hackney,2006). Drosophila also have a hearing organ – Johnston’s organ(Box 1) – located in the antenna, the second segment of whichcontains several hundred mechanosensitive units called scolopidia(Eberl and Boekhoff-Falk, 2007). Bulk air displacements caused bynear-field sound stimuli such as courtship song rotate the distalantennal segments, stretching the scolopidia (Boekhoff-Falk andEberl, 2014). Accumulating evidence from Drosophila suggeststhat there are many physiological, molecular and genetic similaritiesbetween vertebrate and invertebrate auditory organs (Albert andGöpfert, 2015; Bokolia and Mishra, 2015; Jarman and Groves,2013; Lu et al., 2009; Senthilan et al., 2012). For example,vertebrates and invertebrates share homologous transcription factorsthat specify the development of their sensory cells, the analogousaccessory structures to capture and transduce sound energy, and theuse of analogous systems to transduce and amplify mechanical forceinto electrical energy.

Here, we review the evidence that the genes, cells and molecularnetworks that specify the Drosophila hearing organ havemammalian counterparts that function in the differentiated cells ofthe cochlea and are required for hearing in mammals. In particular,we focus on recent findings that Usher syndrome proteins inDrosophila appear to physically interact in protein complexes thatare similar to those described in mammals. We also describe recentwork that indicates that some Usher syndrome proteins interactphysically with Myosin II, the variants of which can cause MYH9-related diseases, including deafness. Various approaches, includingforward genetic screens in Drosophila (Box 1) to identify mutantswith behavioral defects associated with mechanosensorytransduction (Eberl et al., 1997; Kernan et al., 1994), andmicroarray screens to identify transcripts enriched in Johnston’sorgan and regulated by atonal, have revealed striking similaritiesbetween the genes that are implicated in human deafness and thosethat affect the function and development of Johnston’s organ in

1Program in Developmental Biology, Baylor College of Medicine, Houston, TX77030, USA. 2Jan and Dan Duncan Neurological Research Institute, TexasChildren’s Hospital, Houston, TX 77030, USA. 3Department of Molecular andHuman Genetics, Baylor College of Medicine, Houston, TX 77030, USA. 4HowardHughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.5Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030,USA.*Present address: Department of Biological Sciences, Stanford University, PaloAlto, CA 94305, USA.

‡Author for correspondence (akgroves@bcm.edu)

A.K.G., 0000-0002-0784-7998

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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Drosophila. We also review the recently developed genetic toolkitsin Drosophila that can characterize the function of novel genes inDrosophila hearing. It is estimated that several hundred genesinvolved in hearing still await discovery on the basis of mouse

deafness mutants that have been isolated in mutagenesis andknockout screens (Steel, 2014). We suggest that by improving ourunderstanding of the mechanisms of hearing in Drosophila, wecould discover new genes required for hair cell function invertebrates, better understand the evolution of auditory organs,and gain new insights into the pathogenic mechanisms of somedeafness-related genes.

Functional similarities between Drosophila and mammalianhearing organsJohnston’s organ, the largest chordotonal organ (Box 1) inDrosophila, is located in the second antennal segment (Box 1)and consists of ∼200 functional units or scolopidia (Kernan, 2007).Each scolopidium is suspended between the cuticle of the secondantennal segment and the insertion of the third antennal segment(Fig. 1A,B) (Todi et al., 2004). During courtship, male flies beattheir wings, causing the air to move, which in turn causes thefeather-like arista (Box 1) on the third antennal segment of thefemale to vibrate. The resulting movement of the third antennalsegment applies force to the scolopidia as the a2/a3 joint rotates(Boekhoff-Falk and Eberl, 2014; Eberl and Boekhoff-Falk, 2007).This stretch is transmitted to the mechanosensory cilia of sensoryneurons, opening stretch-gated mechanotransduction channels(Kernan et al., 1994; Kernan, 2007). A transient receptor potential(TRP) channel – TRPN1 (also known as NompC) – was firstidentified as a candidate component of a mechanosensitive channelin a genetic screen in Drosophila (Walker et al., 2000), and wassubsequently shown to be expressed in the distal region of the ciliarydendrite of Johnston’s organ neurons (Lee et al., 2010). Mutation ofDrosophila nompC attenuates, but does not completely abolish,sound-evoked potentials (Eberl et al., 2000; Effertz et al., 2012,2011; Göpfert et al., 2006; Lee et al., 2010; Liang et al., 2011). TwoTRPV family members, Nanchung (Nan) and Inactive (Iav) are alsoexpressed in Johnston’s organ neurons, although in a more proximalregion of the ciliated dendrite than NompC (Cheng et al., 2010;Gong et al., 2004; Lee et al., 2010; Liang et al., 2011). Nan and Iavare also required for the generation of sound-evoked potentials inthe antennal nerve (Gong et al., 2004; Kim, 2007; Kim et al., 2003).It is less clear whether Nan and Iav can be directly gated bymechanical force, as studies in which either or both channels wereexpressed in heterologous cells reached different conclusions overthe degree to which the channels could be opened by osmotic stress[compare Gong et al. (2004) and Kim et al., (2003) with Nesterovet al. (2015)]. The physical separation of the TRPN and TRPVchannels in the dendrite of Johnston’s organ neurons, together withthe effects of these mutations on sound-evoked potentials and onactive amplification, has led to the idea that NompC plays a directrole in mechanotransduction (Effertz et al., 2012; Göpfert et al.,2006), whereas Nan and Iav, which can form heteromers (Gonget al., 2004), are thought to amplify weak signals transmitted fromthe NompC complex (Gong et al., 2004; Göpfert et al., 2006;Kamikouchi et al., 2009; Kim et al., 2003).

Despite the progress made in characterizing the properties ofthese channels, it has been hard to resolve the relative contributionsof these three channels to mechanotransduction, in part because ofthe technical challenges involved in recording from single auditoryneurons in Johnston’s organ. A recent innovation by Wilson andcolleagues (Lehnert et al., 2013) exploited the fact that auditoryneurons terminate on, and are dye-coupled to, a single giant fiberneuron in the brain (Kamikouchi et al., 2009; Tootoonian et al.,2012). Recording the activity of this neuron allows currents fromJohnston’s organ neurons to be indirectly measured. Results from

Box 1. GlossaryAntennal segments: the Drosophila antenna is made up of threesegments: a1 (scape), a2 (or pedicelus), which contains Johnston’sorgan and responds to sound, gravity and wind flow, and a3 (orfuniculus), the most distal segment, which is an olfactory organ.Arista: a delicate, feather-like structure projecting from the third (mostdistal) antennal segment of Drosophila that is involved in sounddetection.Chordotonal organs: these organs are ciliated stretch receptors thatdetect motion in joints of the insect body. They are made up of repeatingmulticellular units called scolopidia (see below).Cochlea: the coiled, snail shell-shaped part of the mammalian inner ear,the innermost portion of which, the cochlear duct, contains the organ ofCorti (see below), which detects sound.CRISPR-mediated homology-directed repair: the CRISPR-Cas9system can be used to generate targeted double-stranded DNA breaks(DSBs) when coupled with a sequence-specific guide RNA (gRNA).When Cas9 and a gRNA are introduced into a cell with a piece of DNAflanked by homology arms, the DSB can be repaired by homologousrecombination, inserting the new piece of DNA into the locus of interest.Forward genetic screens: a genetic screen, typically of inducedmutations, which identifies genes responsible for a particular phenotype,for example, hearing defects.GAL4-UAS system: a two-component genetic system that activatesgene expression in Drosophila. It uses the yeast GAL4 transcriptionfactor to activate transcription of a transgene adjacent to an upstreamactivation sequence (UAS).Johnston’s organ: the largest chordotonal organ in Drosophila,containing ∼200 scolopidia. It is located in the second antennalsegment and detects auditory signals (courtship songs), as well aswind flow and gravity.Lethal mosaic screen: lethal homozygous mutations cannot be studiedbeyond the age they cause lethality. To circumvent this, clones of cellscarrying two lethal mutant alleles can be generated in an otherwiseheterozygous organism using a variety of recombination techniques.This approach allows researchers to assess the phenotypes caused bylethal mutations.Mechanotransduction channels: ion channels that are gated bymechanical force.Organ of Corti: an epithelial specialization of the cochlear duct, whichdetects sound through its mechanosensitive hair cells. The organ of Cortisits on a thin, flexible basilar membrane, which oscillates at differentfrequencies corresponding to its position along the cochlea.Recombination-mediated cassette exchange (RMCE): regions ofgenomic DNA can be flanked by DNA sequences that act as targets forsite-specific integrases. For example, the attB and attP DNA motifs aretargets for the phiC31 integrase. If a piece of DNA bearing attB sites (arecombination cassette) is introduced into a cell that carries a DNAsequence flanked by attP sites in the presence of the phiC31 integrase,the intervening sequencewill be replaced by the recombination cassette.Scolopidium: a sensory unit of chordotonal organs, each containing 1-3mechanosensitive sensory neurons, the cilia of which are surrounded bya scolopale cell. The scolopidium is anchored to the insect cuticle at itsproximal and distal ends by a ligament cell and a cap cell, respectively.When flexional or rotational force is applied to the insect’s cuticle, it istransferred to the sensory neurons in each scolopidium.Stereocilia: elongated, actin-rich microvilli that protrude from the apicalsurface of sensory hair cells in vertebrates. Their displacement by forceopens mechanosensitive ion channels, with their distal ends acting aslikely sites of active mechanotransduction in hair cells. Rows ofstereocilia of increasing length form a staircase-like bundle structure inhair cells, and the coupling of adjacent stereocilia by tip links transfersforce to the entire bundle.

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this approach suggest that Nan and Iav are required to respond tosound, whereas NompC is required for the modulation andamplification of force generated during sound reception (Lehnertet al., 2013). It is notable that NompC contains numerous ankyrinrepeats (Cheng et al., 2010; Liang et al., 2013; Zhang et al., 2015)that might interact with motile components of the ciliated dendrite tomodulate force, an idea that is supported circumstantially by thepresence of NompC in the distal cilium, between the site of ciliainsertion, and by more proximal location of the Nan/Iav complex inthe cilium.Unlike the Drosophila hearing organ, which responds optimally

to a narrow frequency range of the beating male wing, themammalian inner ear has evolved to respond to a wide range ofsound frequencies. The hearing organ of the cochlea, the organ ofCorti, sits on a thin basilar membrane that runs the length of thecochlear duct (Fig. 1B). Both the thickness and width of the basilarmembrane change along the length of the cochlear duct (Dallos,1996); therefore, it can resonate in response to many differentfrequencies, allowing the basilar membrane to transform complexsounds into their component sound frequencies (Reichenbach andHudspeth, 2014). The rising and falling oscillation of the basilarmembrane in response to mechanical waves traveling along thecochlea is transmitted to the mechanosensory hair cells of the organof Corti (Fettiplace and Hackney, 2006). One row of inner hair cells

runs the length of the organ of Corti, and these cells form synapseswith sensory afferent neurons of the acoustic (spiral) ganglion(Goodrich, 2016), while three rows of outer hair cells receive mostlyefferent input from the brainstem (Basch et al., 2016; Yu andGoodrich, 2014). Hair cells bear elongated microvilli or stereociliaon their apical surface that insert into an acellular tectorialmembrane above them (Fig. 2). As the basilar membrane vibrates,the motion of the organ of Corti causes the stereociliary bundles ofthe hair cells to be displaced by contact with the tectorial membrane(outer hair cells) or fluid flow (inner hair cells). This opensmechanically gated ion channels in the tips of the stereocilia (Coreyand Holt, 2016; Fettiplace, 2016; Wu and Muller, 2016). Adjacentstereocilia connect to each other close to their tips by a tip linkprotein complex consisting of protocadherin 15 and cadherin 23(Pepermans and Petit, 2015). This complex allows the opening ofmechanotransduction channels to be coordinated as the hair bundleis displaced (Hudspeth, 1997, 2008). As in Drosophila, the identityof the mechanosensitive ion channels in these hair cells is still beinginvestigated and debated (Corey and Holt, 2016; Fettiplace, 2016;Wu and Muller, 2016). Current work is focused on fourtransmembrane proteins, transmembrane inner ear (TMIE),tetraspan membrane protein of hair cell stereocilia/lipomaHMGIC fusion partner-like 5 (TMHS; Lhfpl5) andtransmembrane channel-like 1 and 2 (TMC1/2), all of which are

Cap cell

Scolopale cell

Neuron

Ligament cell

Cillium (NompC,Nan/Iav)

Ciliary dilation

Dendritic cap (NompA)

Scolopale rods(actin bundles)

Cap rods(actin bundles)

Ciliary rootlets

Distal basal bodies

CA

Arista Antennae

BSecond antennalsegment

Third antennal segment

Arista

Fig. 1. Structure of theDrosophila auditory organ. (A) A frontal view of an adultDrosophila head, with antennaemarked in gray (eyes are in red). (B) Magnifiedimage of an antenna (area highlighted in light green in A). The auditory organ (Johnston’s organ) is located in the second segment of the antenna (A2) andconsists of functional units called scolopidia. These are attached to the cuticle of the second antennal segment and to the A2/A3 joint. Sound particles cause thedisplacement of the antennal arista, which induces the third antennal segment to rotate (arrow). This acoustic, stimulus-induced rotation applies force to the cilia ofmechanosensory neurons in the scolopidia, resulting in the firing of action potentials along the antennal nerve. A single scolopidium is highlighted in the light greenbox. Each scolopidium is attached to the antenna cuticle (arrows) by a cap cell (red) and a ligament cell (gray), as detailed in C. (C) Cellular components of anindividual scolopium. Two to three mechanosensory neurons (two neurons are shown) have their ciliary dendrites enclosed by an actin-rich scolopale cell.Mechanosensory neurons express several ion channels that regulate mechanosensation, including Nan/Iav (green) and NompC (yellow). Scolopale cells formseptate junctions with the ligament and cap cells at the basal and apical ends of the scolopidium, respectively, ensuring the formation of an enclosed scolopalespace between the scolopale cell and neuronal cilia. The ciliary dendrites of each neuron insert into an acellular cap that contains the NompA glycoprotein (red),which is anchored to the A2/A3 joint by a cap cell. The scolopidium is attached to the A2 cuticle at its proximal end by a ligament cell. Approximately 200 scolopidiaare located in Johnston’s organ.

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necessary for hearing in mice (Kawashima et al., 2011; Kurimaet al., 2015; Xiong et al., 2012; Zhao et al., 2014). It is possible thatother proteins, such as the large transmembrane protein Piezo2,might also contribute to mechanotransduction in other parts of thehair cell (Wu et al., 2017).Although the appearance and organization of mammalian and

Drosophila hearing organs are clearly very different, they share anumber of genes that regulate their formation and cellular functions.We describe some of these in the following section.

Cellular and molecular similarities between Drosophila andmammalian hearing organsDespite being separated by several hundred million years ofevolutionary time, the mechanosensitive cells and supporting cellsof mammalian and Drosophila hearing organs are specified bysimilar sets of transcription factors, and have similarities in theaccessory cells and proteins that allow these receptor cells to bemechanically gated. We first compare the axonless hair cells ofvertebrates with the mechanosensitive neurons of Drosophila andthen describe similarities in how these cells are formed and howthey function.Vertebrate hair cells are secondary receptor cells: they elaborate

neither axons nor dendrites but are innervated by axons of bipolarsensory neurons that also send processes to brainstem nuclei. Inaddition to their apical stereociliary bundle, they also have a truecilium, the kinocilium, which disappears in some hair cell types asthey mature, including inner and outer hair cells of the mammaliancochlea (Nayak et al., 2007). Among invertebrates, only insects and

crustaceans have true hearing; however, other invertebrate groupspossess mechanoreceptor cells with similar apical specializationsthat can detect gravity and acceleration. These receptor cells are alsosometimes referred to as hair cells, although their homology to truevertebrate hair cells is still under debate (Burighel et al., 2011, 2008,2003; Fritzsch and Straka, 2014; Manley and Ladher, 2007). Theseinvertebrate ‘hair cells’ can be either secondary receptors or primarysensory neurons that have intrinsic mechanosensitivespecializations (Burighel et al., 2011). These specializations canbe actin-rich structures similar to vertebrate stereocilia ormechanosensitive organelles made up of one or more true cilia,reminiscent of vertebrate photoreceptors. Some nonvertebrategroups, such as urochordates and cephalopod mollusks, use bothkinds of receptor cells in their sensory organs (Budelmann andThies, 1977; Burighel et al., 2011). In contrast, secondary receptorcells have not been observed in insects and crustaceans (Fritzsch andStraka, 2014), and these groups instead have a wide range ofmechanoreceptive sensory neurons arranged in clusters all over theirbodies (Kernan, 2007), including the ciliated sensory neurons inscolopidia of Johnston’s organ. It is not clear whether amechanosensitive sensory neuron represents the ancestral cell typefrom which hair cells and sensory neurons emerged as sister celltypes in multiple taxa, or whether secondary mechanosensitive cellswere lost in the arthropod lineage (Arendt et al., 2016).

Despite the structural differences between insect and vertebratesound-detecting cells, molecular evidence suggests that certainaspects of the development of the various cell types found inauditory organs are conserved between invertebrates and vertebrates

Tectorial membrane

Basilar membrane

Inner hair cell Outer hair cell

Deiters’ cellsInner phalangeal cell

Border cell

Stri

a va

scul

aris

A B

C

Oval window

Round window

Organ ofCorti

Vestibular canal(perilymph)

Tympanic canal(perilymph)

Cochlear duct(endolymph)

Fig. 2. Structure of the mammalian auditory organ.(A) In the mouse inner ear, the organ of Corti is locatedon the basilar membrane, which runs the length of thecochlear duct. Mechanical waves conducted by themiddle ear bones are transmitted to the oval windowand travel through the perilymph surrounding thecochlear duct (arrows). Compression of the ovalwindow is matched by a corresponding outwardmovement at the round window. (B) A cross-section ofthe cochlear duct, showing three rows of outer hair cells(dark blue) and one row of inner hair cells (dark green).(C) A magnified view of the arrangement of hair cells inthe organ of Corti. Inner hair cells are surrounded byinner phalangeal and border cells and are separatedfrom the outer hair cells by two pillar cells that togetherform the tunnel of Corti. Outer hair cells are surroundedby Deiters’ cells. Inner and outer hair cells receiveafferent and efferent innervation, respectively.Stereocilia located on the apical surface of hair cells areembedded into the tectorial membrane (outer hair cells)or lie just beneath the membrane (inner hair cells).During sound reception, the sound-induced vibration ofthe basilar membrane applies mechanical force to thetip links of the stereocilia, causing an influx of potassiumand calcium and the depolarization of hair cells. Thepotassium gradient in the cochlear duct endolymph ismaintained by the stria vascularis in the lateral wall ofthe cochlear duct.

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(Bechstedt and Howard, 2008; Bokolia and Mishra, 2015; Caldwelland Eberl, 2002; Lewis and Steel, 2012) (Table 1). The atonal (ato)basic helix-loop-helix (bHLH) transcription factor was discovered

in Drosophila as a proneural gene required for the formation ofphotoreceptors and of some mechanoreceptors (Jarman et al., 1993,1994). In Drosophila, the loss of ato leads to loss of all chordotonal

Table 1. Molecular and cellular conservation between Drosophila and mammalian auditory organs

Drosophila Mammals References

Transcription factors specifying hearing organ cellsAto Loss of function leads to loss of all

chordotonal organs. Overexpressioncauses ectopic chordotonal organs.Mouse Atoh1 rescues fly Ato mutantphenotype.

Atoh1 Necessary and sufficient for differentiationand survival of hair cells. Fly Ato rescuesmouse Atoh1 mutant phenotype.

Ben-Arie et al., 2000; Berminghamet al., 1999; Cai et al., 2013;Jarman et al., 1993, 1994; Wanget al., 2002

Sens Required for the development ofchordotonal organ in Drosophilaembryos.

Gfi1 Required for the survival of hair cells inmouse.

Acar et al., 2006; Jafar-Nejad et al.,2003, 2006; Nolo et al., 2000;Wallis et al., 2003; Wang et al.,2002

Pros Specifically expressed by scolopale cells inJohnston’s organ.

Prox1 Initially expressed in the progenitors of haircells and supporting cells, but becomesrestricted to supporting cells as thesensory epithelium differentiates.

Bermingham-McDonogh et al.,2006; Boekhoff-Falk and Eberl,2014

Pax2 (Sv) Specifically expressed by scolopale cells inJohnston’s organ.

Pax2 Initially expressed in the progenitors of theentire inner ear but becomes restricted tothe stria vascularis of the cochlear duct.

Burton et al., 2004; Kavaler et al.,1999

Rfx Rfx regulates many ciliogenic genes in avariety of organisms, including inDrosophila sensory neurons.

Rfx1-3 Loss of Rfx genes in mice leads to a rapidloss of initially well-formed outer haircells in the mouse cochlea and causesdeafness.

Dubruille et al., 2002; Elkon et al.,2015; Jarman and Groves, 2013;Laurencon et al., 2007; Vandaeleet al., 2001

Structural proteins required for mechanosensationNompA Produced by scolopale cells and forms

acellular filaments connecting themechanosensory neuronal dendrites tothe third antennal cuticle.

α- and β-tectorin

Produced and secreted by supportingcells, they constitute the major structuralproteins in the acellular tectorialmembrane that lies above the hair cells.

Chung et al., 2001; Legan et al.,2000, 1997; Xia et al., 2010

Myosin VIIA Expressed in scolopale cells and enrichedin apical tips; required for apicalattachment of scolopidia and auditorysensation; physically interacts withhomologs of other Usher proteins,Cad99C and Sans.

MYO7A(USH1B)

Recessive mutations cause Ushersyndrome and nonsyndromic deafnessin humans; physically interacts with otherUsher proteins in a protein complex toform stereocilia tip links.

Ahmed et al., 2013; Cosgrove andZallocchi, 2014; Glowinski et al.,2014; Li et al., 2016; Pan andZhang, 2012; Todi et al., 2005,2008

Cad99C Expressed in Johnston’s organ andlocalizes to apical tips of scolopidia;regulates apical attachment ofscolopidia; physically interacts withhomologs of other Usher proteins,Myosin VIIA and Sans.

PCDH19(USH1F)

Recessive mutations cause Ushersyndrome in humans; complexes withother Usher proteins to form stereociliatip links.

Ahmed et al., 2013; Cosgrove andZallocchi, 2014; D’Alterio et al.,2005; Glowinski et al., 2014; Liet al., 2016; Pan and Zhang,2012

Sans Regulates apical attachment of scolopidia;physically interacts with homologs ofother Usher proteins, Myosin VIIA andCad99C.

SANS Recessive mutations cause Ushersyndrome in humans; physicallyinteracts with other Usher proteins in aprotein complex to form stereocilia tiplinks.

Ahmed et al., 2013; Cosgrove andZallocchi, 2014; Demontis andDahmann, 2009; Glowinski et al.,2014; Li et al., 2016; Pan andZhang, 2012

Myosin II Expressed in Johnston’s organ andlocalizes to apical tips of scolopidia, alsoregulates apical attachment ofscolopidia.

MYH9 Dominant mutations cause MYH9-relateddiseases and nonsyndromic deafnessdisorder, DFNA17.

Heath et al., 2001; Lalwani et al.,2000; Li et al., 2016; Savoia andPecci, 2008

TMC Regulates mechanosensation in larvallocomotion.

TMC1/2 Required for auditory mechanosensation. Guo et al., 2016; Kawashima et al.,2015; Kurima et al., 2015, 2002

Conserved cells and accessory structuresAccessorycells

The scolopale, cap and ligament cells ofJohnston’s organ scolopidia.

Supportingcells

Deiters’, pillar, inner phalangeal, borderand Hensen’s cells of the organ of Corti.

Boekhoff-Falk, 2005; Carlson et al.,1997; Eberl and Boekhoff-Falk,2007

Scolopalefluid

Potassium-rich fluid secreted by scolopalecells, bathing the ciliary dendrites ofmechanosensitive neurons.

Endolymph Potassium-rich fluid secreted by the striavascularis of the cochlear duct wall,bathing the apical surface of sensory haircells.

Fettiplace and Hackney, 2006;Kikuchi et al., 2000; Küppers,1974; Roy et al., 2013

Dendriticcap

Secreted extracellular structure thatanchors neuronal cilia and transfersforce to them. Composed of the zonapellucida (ZP) domain glycoproteinNompA.

Tectorialmembrane

Secreted extracellular structure that liesabove hair cell stereocilia and appliesforce to the hair bundles of outer haircells. Major components include the ZPdomain glycoproteins α- and β-tectorin.

Chung et al., 2001; Legan et al.,2000, 1997; Xia et al., 2010

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organs, including Johnston’s organ, while its misexpression resultsin the formation of ectopic chordotonal organs (Jarman et al., 1993).Similarly, the vertebrate homolog of ato, Atoh1, is both necessaryand sufficient for the differentiation and survival of hair cells(Bermingham et al., 1999; Cai et al., 2013). Remarkably,Drosophila ato and mouse Atoh1 can functionally replace eachother in loss-of-function mutants (Ben-Arie et al., 2000;Wang et al.,2002), and are both regulated through conserved post-translationalregulation (Quan et al., 2016), even though they share littlehomology outside the bHLH domain. A conserved post-translational control of the temporal dynamics of ato/Atoh1expression during neurogenesis further supports the functionalsimilarities between ato and Atoh1 (Quan et al., 2016). Theconservation betweenDrosophila ato and the vertebrate Atoh1 generegulatory networks is reflected by conservation of some of theirdownstream target genes. The zinc finger transcription factorsenseless (sens)/Gfi1, which can be regulated by ato/Atoh1, isrequired for the development of the chordotonal organ inDrosophila embryos and for the survival of hair cells in themouse (Acar et al., 2006; Jafar-Nejad et al., 2003, 2006; Nolo et al.,2000; Wallis et al., 2003; Wang et al., 2002). Regulatory factor X(Rfx) is another downstream transcription factor activated by Atonal(Jarman and Groves, 2013; Laurençon et al., 2007) that regulatesmany ciliogenic genes in a variety of organisms, including inDrosophila sensory neurons (Dubruille et al., 2002; Vandaele et al.,2001). A recent study showed that combined loss of Rfx1 and Rfx3in mice leads to a rapid loss of initially well-formed outer hair cellsin the mouse cochlea and causes deafness (Elkon et al., 2015).The division of labor between mechanosensitive secondary

receptor cells and their afferent sensory neurons in mammals andsome invertebrate taxa is reflected by an analogous division of laborbetween the transcription factors that specify the neuronal andsensory lineages. Neurog1 is a bHLH factor closely related to Atoh1and is expressed in the progenitor cells of the inner ear primordiumshortly before they delaminate as neuroblasts (Zheng and Gao,2000). Its expression is then extinguished and replaced by anotherclosely related bHLH gene, Neurod1; both genes are necessary forthe generation of inner ear neurons (Kim et al., 2001; Liu et al.,2000). The Atoh, Neurogenin and NeuroD bHLH transcriptionfactor families are ancient, with members of each family observed inbasal taxa, such as sponges (Simionato et al., 2008, 2007).Arthropods appear to have dispensed with the need for theNeurog genes in sensory development; the one identified memberof this family in Drosophila, target of pox neuro (tap), appears tofunction in neurite outgrowth rather than in neuronal specification(Gautier et al., 1997; Yuan et al., 2016). In contrast, Drosophila atohas undergone duplication to give rise to two additional orthologs:absent multidendritic and olfactory sensilla (amos) and cousin ofatonal (cato) (Jarman and Groves, 2013). Although the Atoh,Neurogenin and NeuroD bHLH families are more closely related toeach other than to other bHLH genes, they nevertheless haveextremely specific functions. For example, while Drosophila atocan functionally replace Atoh1 in mice, and vice versa (Ben-Arieet al., 2000; Wang et al., 2002), Neurog1 can only rescue loss ofAtoh1 in mice to a very modest degree (Jahan et al., 2012, 2015),suggesting that Neurog1 is only able to activate a subset of Atoh1target genes. Thus, these different bHLH factors can specifydifferent cell fates – neuron or hair cell – depending on the bindingaffinities and range of their targets.A common feature of sensory systems is the presence of

accessory cells that surround, support, nourish or modulate theoutput of sensory cells. In each scolopidium of Johnston’s organ,

neuronal cilia are enclosed by a tube-like scolopale cell (Fig. 1). Theshape of the scolopale cell is thought to be maintained by a cage-likescolopale consisting of actin rods, microtubules and somemicrotubule-associated proteins (Todi et al., 2004; Wolfrum,1997). Scolopale cells form septate junctions with the ligamentand cap cells at the basal and apical ends of the scolopidium,respectively, ensuring the formation of an enclosed scolopale spacebetween the scolopale cell and neuronal cilia (Boekhoff-Falk, 2005;Carlson et al., 1997; Eberl and Boekhoff-Falk, 2007; Todi et al.,2004). Although the ionic composition of the scolopale space hasnot been measured directly in Drosophila, studies of the fluid thatbathes a variety of insect and crustacean mechanoreceptors havereported that this fluid has a positive potential of about +70 mVrelative to the hemolymph (Thurm and Wessel, 1979). In addition,studies of blowfly mechanoreceptor organs have found such fluid tobe high in potassium (Küppers, 1974), the concentration of which isactively maintained by transport ATPases (French, 1988; Thurm,1974; Wieczorek, 1982). Scolopale cells express Na+/K+ ATPasesthat localize to their ablumenal plasma membrane and are requiredfor hearing in Drosophila. These Na+ pumps function to activelytransport K+ into the scolopale space to maintain ion homeostasis(Roy et al., 2013). In the mammalian organ of Corti, inner hair cellsand outer hair cells are surrounded by different types of supportingcells: inner phalangeal and border cells surround the inner hair cells,while Deiters’ cells surround the outer hair cells (Fig. 2), and innerand outer pillar cells located between these two domains form thetunnel of Corti. During sound perception, sound-induced vibrationof the basilar membrane applies force to the tip links of thestereocilia. This results in their deflection, in the opening of cationchannels in the stereocilia and in the influx of K+ and Ca2+ from thesurrounding potassium-enriched endolymph (Fettiplace andHackney, 2006). Potassium homeostasis in the endolymph iscrucial for the maintenance of the endocochlear potential and forhair cell function. The K+ that enters hair cells is recycled throughthe supporting cells and is ultimately transported back into theendolymph by cells in the stria vascularis of the lateral wall of thecochlear duct (Kikuchi et al., 2000). It is not clear whether the K+-rich extracellular fluid of the endolymph and scolopale space reflecta common or convergent evolutionary origin. For example, sensoryhair cells of the lateral line organs of vertebrates are not enclosed,but open to the external fluid environment, and a number of studiesmeasuring the endolymph-like fluid bathing cephalopod statocystsensory organs have found it to have low levels of K+ compared withNa+ (reviewed in Vinnikov, 2011).

Some molecular and cellular similarities have been identifiedbetween scolopale cells and cochlear-supporting cells. prospero( pros), a homeodomain transcription factor, is specificallyexpressed by scolopale cells in Johnston’s organ (Boekhoff-Falkand Eberl, 2014). The mammalian homolog of pros, Prox1, isinitially expressed in the progenitors of hair cells and supportingcells, but becomes restricted to supporting cells as the sensoryepithelium differentiates (Bermingham-McDonogh et al., 2006).On the apical side of Johnston’s organ, cap cells participate in thecoupling of neuronal cilia to the cuticle of the third antennalsegment, and help impart mechanical force to the cilia during soundperception. nompA encodes a zona pellucida (ZP) domainglycoprotein that is produced and secreted by scolopale cells. Itforms acellular filaments that connect the mechanosensory neuronaldendrites to the third antennal cuticle (Chung et al., 2001). Loss ofNompA leads to the detachment of both the scolopidia from thethird antennal segment cuticle and of the ciliary dendrites from thecuticular structures in other sensory organs, such as the external

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sensory organs and other campaniform organs (Chung et al., 2001).Mammals also have ZP domain-containing proteins – α- and β-tectorin (Legan et al., 1997) – which are produced and secreted bysupporting cells, and are the major structural proteins in the acellulartectorial membrane that lies above the hair cells, and that comes intocontact with the hair bundles of the outer hair cells. Loss of α-tectorin in mice leads to the detachment of hair cells from thetectorial membranes (Legan et al., 2000; Xia et al., 2010).Intriguingly, a Caenorhabditis elegans ZP domain protein, DYF-7, resembles tectorins and anchors the tips of sensory dendrites tothe body wall as the neuronal cell bodies in the developing amphidmigrate away (Heiman and Shaham, 2009). In addition, thetransmembrane channel-like (TMC) proteins are required forauditory mechanosensation in mice and humans (Kawashimaet al., 2015). Although it has yet to be shown that the Drosophilahomolog of the TMC proteins functions directly in auditorymechanosensation, it has been shown to regulate mechanosensationin larval locomotion (Guo et al., 2016).A striking illustration of the molecular and genetic similarities

between Drosophila and mammalian hearing organs comes fromthe work of Göpfert and colleagues (Senthilan et al., 2012). Here, amicroarray survey of genes expressed in Johnston’s organ of wild-type and ato mutant flies identified 274 genes expressed in thisauditory organ. Approximately 20% of these genes had a humanortholog implicated in hearing loss. This suggests that the fruit flymight be a rich resource for identifying and characterizing genesinvolved in human deafness. In the next section, we describe recentwork in which forward genetic screens identified aDrosophila genethat linked together two myosin genes previously implicated inhuman syndromic hearing loss.

Can Drosophila be used to model hereditary deafness andhearing loss?The molecular and functional similarities between Johnston’s organand the mammalian cochlea summarized above suggest that somegenes implicated in human deafness might have similar functions inJohnston’s organ. Here, we focus on two particular forms ofsyndromic hearing loss in humans, Usher syndrome and MYH9-related disorders, as these well-characterized syndromes provideevidence of such functional similarity and highlight the potentialadvantages of using Drosophila as a model for studying hereditarydeafness.

Conservation of Usher syndrome proteins and theirinteractions in DrosophilaUsher syndrome is an autosomal recessive genetic disease [OnlineMendelian Inheritance in Man (OMIM) #276900, #276904,#601067, #276901, #605472, #611383] that is characterized byvarying degrees of deafness and retinitis pigmentosa-induced visionloss, and is the leading cause of deaf-blindness in humans(Boughman et al., 1983; Hartong et al., 2006; Keats and Corey,1999; Kimberling et al., 2010). Based on the severity of clinicalsymptoms in patients, Usher syndrome is subdivided into threetypes: USH1, 2 and 3. To date, 16 genetic loci have been associatedwith this syndrome: nine for USH1, three for USH2, two for USH3and two that have yet to be identified (Cosgrove and Zallocchi,2014; Mathur and Yang, 2015). Some missense mutations of Usher(USH) genes that retain residual function are also associated withnonsyndromic forms of deafness that do not cause vision loss (see,for example, Schultz et al., 2011).USH proteins regulate a variety of cellular processes, including

actin-based trafficking, cell adhesion, scaffolding, and G-protein- or

Ca2+-mediated signaling (Table 2). Evidence from colocalizationdata, in vitro interaction studies, and mouse genetic studies suggestthat USH proteins interact with each other to form multiproteincomplexes in a variety of cell types (Fig. 3A) (Ahmed et al., 2013;Pan and Zhang, 2012). In mature mammalian hair cells, many Ushersyndrome proteins localize to the tips of stereocilia wheremechanotransduction takes place (Fig. 3A). Adjacent stereociliaare connected by complexes at their tips, called tip links, which arecomposed of cadherin 23 (USH1D; Cdh23) and protocadherin 15(USH1F; Pcdh15). Protocadherin 15 is thought to interact withcomponents of the mechanotransduction channel (TMHS), andboth ends of the tip links are indirectly coupled to the actin core ofthe stereocilia through other Usher proteins, including myosin VIIA(USH1B; Myo7A), Sans (USH1G), harmonin (USH1C), Cib2 andwhirlin (USH2D) (Ahmed et al., 2013; Cosgrove and Zallocchi,2014).

The Drosophila genome harbors homologs of several Ushergenes and some of these have been characterized in Johnston’sorgan (Table 2). The Drosophila homolog of myosin VIIA[myosin VIIA; also known as crinkled (ck)] is required for hearing:loss of ck abolishes a fly’s auditory responses and causes thescolopidia to detach from the cuticle of the a2/a3 antennal joint inJohnston’s organ (Li et al., 2016; Todi et al., 2005, 2008) (Fig. 4).Interestingly, Myosin VIIA is predominantly expressed in theactin-rich scolopale cells and is enriched at their apical tips closeto where a scolopidium inserts into the cuticle (Li et al., 2016), apattern that is superficially similar to the localization of myosinVIIA in the actin-rich stereocilia tips of mammalian hair cells.However, whereas myosin VIIA functions in hair cell stereocilia tomechanically couple mechanotransduction channels to the actincytoskeleton, Myosin VIIA is unlikely to have the same functionin scolopale cells, which ensheath the mechanosensitive cilia ofthe Johnston’s organ sensory neurons and secrete the dendritic capthat anchors them to the cap cell and cuticle. However, it is likelythat myosin VIIA serves an anchoring role in both cell types,because it is a high duty ratio motor – i.e. it predominantly binds toactin filaments rather than processing along them (Haithcock et al.,2011; Watanabe et al., 2006; Yang et al., 2006). Pcdh15 and Sansalso have Drosophila homologs, named Cad99C and Sans,respectively (D’Alterio et al., 2005; Demontis and Dahmann,2009). Recent studies have shown that Cad99C, Sans and MyosinVIIA form a complex in Johnston’s organ and in the ovary ofDrosophila (Glowinski et al., 2014; Li et al., 2016). In Johnston’sorgan, a subtle, low-penetrant phenotype of scolopidialdetachment can be observed in Cad99C mutants, resemblingthat ofmyosin VIIAmutants. Cad99C localizes to the apical tips ofscolopidia, reminiscent of the localization of Pcdh15 to the tip linkregion in mammalian hair cells. In addition, myosin VIIA, Cad99Cand Sans genetically interact with Ubr3, a regulator of Usherproteins in Johnston’s organ (Li et al., 2016).

These data suggest that USH1 proteins are not only conserved inDrosophila, but that their interactions are important for thedevelopment and function of the Drosophila auditory apparatus.USH1 proteins appear to interact in the scolopidia tips to link thescolopale cell and cap cell, which might stabilize the scolopidialunit during mechanotransduction where force is applied to theneuronal cilia through the extracellular dendritic cap (Fig. 3B,C).This function of USH proteins in Johnston’s organ – where theproteins appear to mediate intercellular interaction and attachment –is somewhat different to their functions in the mammalian hair cell,in which the USH complex acts intracellularly to anchor adjacentstereocilia.

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Several other USH genes have Drosophila homologs, such asharmonin (CG5921), Cib2 (cib2), whirlin (dyschronic) and clarin 1(CG1103) (Table 2). Although cib2 is expressed in the eye and isrequired for normal phototransduction inDrosophila, whether thesehomologs are also present in the scolopidia of Johnston’s organ and/or participate in hearing remains unclear. Other vertebrate USHproteins that are present in the ankle links that connect the base ofstereocilia, such as usherin and VLGR1/GPR98 (Adgrv1), do notappear to have any obvious homologs in Drosophila (Table 2).Moreover, the mechanism by which Drosophila USH proteinscause scolopidial attachment – for example, how the scolopidiuminserts into the cuticle, or whether additional cadherins act withCad99C/Pcdh15 – remains unclear. Nevertheless, the clear andquantifiable phenotypes seen in Usher syndrome gene mutants inDrosophila (Fig. 4), and the strong genetic interactions betweenUsher genes, supports the use of Drosophila, and the Johnston’sorgan, as a model in which to study the functions of known andnewly identified Usher genes and to identify their regulators.Finally, one recently reported unexpected finding is that USHhomologs in the Drosophila hearing organ genetically andphysically interact with Myosin II (Li et al., 2016), themammalian homologs of which also play a role in hearing. Wedescribe the function of Myosin II in hearing in flies and mammalsin the next section.

MYH9-related diseaseAutosomal dominant MYH9-related diseases are caused bymutations in MYH9, which encodes nonmuscle myosin IIA.These diseases are characterized by large platelets andthrombocytopenia from birth, and are associated with a variableonset of progressive sensorineural hearing loss, presenile cataracts,elevated liver enzymes and renal disease, which initially manifestsas a glomerular nephropathy (Heath et al., 2001). Prior to theidentification of the underlying molecular cause, individuals withMYH9-related disease were variously diagnosed as having Epsteinsyndrome, Fechtner syndrome, May-Hegglin anomaly or Sebastiansyndrome (Heath et al., 2001; Newell-Litwa et al., 2015). The onsetof hearing loss in patients with a MYH9-related disease can occurover a wide age range, from the first to the sixth decade (Savoia andPecci, 2008), and frequently progresses over time. In addition, adominant mutation,MYH9R705H, causes the nonsyndromic deafnessdisorder called DFNA17 (Lalwani et al., 2000).

Mammals have three isoforms of nonmuscle myosin II encodedby three different genes: NMIIA (encoded by MYH9), NMIIB(encoded by MYH10) and NMIIC (encoded by MYH14). Mice thatare homozygous for aMyh9 null mutation die during embryogenesisby embryonic day (E) 7.5 (Conti et al., 2004; Mhatre et al., 2007).Mice heterozygous for Myh9 do not exhibit any hearing loss orchanges in age-dependent hearing thresholds (Mhatre et al., 2007).

Table 2. Summary of USH loci, their human and Drosophila proteins, and predicted functions

USHtype Locus Human gene

Humanprotein

Drosophilaortholog Function References

USH1 USH1B MYO7A myosinVIIA

(crinkled) ck Cytoskeleton-associated motor protein Li et al., 2016; Todi et al., 2005,2008

USH1C USH1C harmonin CG5921* Scaffold protein Ahmed et al., 2013; Cosgrove andZallocchi, 2014; Pan andZhang, 2012

USH1D CDH23 CDH23 No candidatesidentified

Cell-cell adhesion; tip link protein Alagramam et al., 2011;Kazmierczak et al., 2007;Sollner et al., 2004

USH1E Unknown(21q21)

Unknown No candidatesidentified

Unknown

USH1F PCDH15 PCDH15 Cad99C Cell-cell adhesion; tip link protein D’Alterio et al., 2005; Li et al.,2016

USH1G SANS SANS Sans Scaffold protein Demontis and Dahmann, 2009;Glowinski et al., 2014; Li et al.,2016

USH1H Unknown(15q22-23)

Unknown No candidatesidentified

Unknown

USH1J CIB2 CIB2 Cib2 Calcium and integrin binding protein Riazuddin et al., 2012USH1K Unknown

(10p11.21-q21.1)

Unknown No candidatesidentified

Unknown

USH2 USH2A USH2A usherin No candidatesidentified

Stereocilia links; extracellular matrix Adato et al., 2005; Liu et al., 2007;Weston et al., 2000

USH2B Unknown(3p23-24.2)

Unknown No candidatesidentified

Unknown

USH2C GPR98 VLGR1/GPR98

No candidatesidentified

Stereocilia links; G-protein signaling Schwartz et al., 2005; Westonet al., 2004

USH2D WHRN whirlin (dyschronic)dysc*

Scaffold protein; stereocilia elongation andstaircase formation

Jepson et al., 2012

USH3 USH3A CLRN1 clarin 1 CG1103* Extracellular matrix structure Adato et al., 2002; Fields et al.,2002; Joensuu et al., 2001

USH3B HARS HARS No candidatesidentified

Histidyl transfer RNA synthetase Abbott et al., 2017; Puffenbergeret al., 2012

– PDZD7 PDZD7 No candidatesidentified

Scaffold protein with homology to harmoninand whirlin; interacts genetically withUSH2A and GPR98

Chen et al., 2014; Ebermannet al., 2010; Schneider et al.,2009

For human USH loci for which a causative gene has not been identified, we indicate the cytogenetic band in which the locus is situated. Asterisks indicateDrosophila homologs predicted by protein sequence homology that have yet to be functionally validated.

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This suggests that MYH9-related diseases are not caused by MYH9haplo-insufficiency. However, knock-in mice, in which one Myh9allele has been modified to contain a specific dominant pathogenicMyh9mutation, R702C, develop defects, including hearing loss andkidney disease, that are similar to symptoms associated with severalMYH9-related syndromes such as May-Hegglin anomaly (Suzukiet al., 2013). Although Myh9 is expressed in hair cells and NMIIAlocalizes to the stereocilia and apical surface of the hair cell (Li et al.,2016; Meyer Zum Gottesberge and Hansen, 2014; Mhatre et al.,2006), NMIIA is also broadly expressed in many other parts of thecochlea. It is therefore possible that the deafness that occurs inMYH9-related diseases might not be directly caused by hair celldefects. Thus, the molecular mechanism by whichMYH9mutationscause hearing loss remains unclear.

The single Drosophila homolog of NMIIA is encoded by themyosin II gene, also known as zipper. This gene is expressed inJohnston’s organ, and Myosin II is enriched at the apical tips ofscolopidia, adjacent to Myosin VIIA (Fig. 3B) (Li et al., 2016).Myosin II is also required for the normal apical attachment ofscolopidia. Consistent with this, the gene encoding the regulatorylight chain of Myosin II, spaghetti squash (sqh), geneticallyinteracts with myosin VIIA in Johnston’s organ and affects hearing(Todi et al., 2008). Recent studies in Drosophila show that MyosinII physically interacts with Myosin VIIA, probably through themono-ubiquitination of Myosin II, which is regulated by the E3ligase, Ubr3 (Li et al., 2016). The myosin IIA-myosin VIIAinteraction and the mono-ubiquitination of myosin IIA also occur inthe mammalian cochlea (Li et al., 2016), although an interaction

Hair cell

Stereocilia

Actin bundles

UTLD

LTLD Myosin VIIA

Harmonin

Sans

Cdh23

Pcdh15

Mechanosensory

ion channel

Myosin IIA

Myosin VIIA

Sans

Harmonin

Cad23

Pcdh15 Ion channel

A

B Apical tip of a scolopidium in Drosophila Johnston’s organ

Cad99C

C Stereocilia tip links of a hair cell in mammalian cochlea

Cap cell

Scolopalecell

Scolopale cell

Key

Key

Fig. 3. USH1 proteins and Myosin II function in the apical region of Drosophila scolopidia and mammalian stereocilia. (A) Left: a schematic of theapical surface of a hair bundle in a single mouse hair cell, showing individual stereocilia. The red box outlines the tip link region, which is expanded to highmagnification on the right. Right: USH proteins form a protein complex and link the upper tip link density (UTLD) in the taller stereocilia to the lower tip link density(LTLD) of the neighboring shorter stereocilia in the same hair cell. In the prevailing model of USH protein interactions, the Cdh23-Pchd15 heterodimer islocated directly or in close proximity to other protein components of the mechanotransduction (MET) channel complex, including TMIE and TMHS. The adaptorproteins harmonin and Sans link Cdh23 to myosin VIIA and hence to the actin core of the stereocilium. (B) A schematic of a scolopidium. In Drosophila,Myosin VIIA and Myosin II are present in the apical regions of the scolopidia of Johnston’s organ and are enriched at the tips of scolopale cells, where theycontact the cap cell. Myosin II ubiquitination promotes its interaction with Myosin VIIA, the levels of which are crucial for anchoring the apical junction complexesof the scolopidia. The motor activity of these myosins might also be required to transport the myosin complex to the tips of the scolopale cell. Both MyosinVIIA andMyosin II likely bind to actin bundles in the scolopale cells and regulate the apical attachment of scolopidia. TwoDrosophila homologs of Usher syndrometype I proteins, Cad99C (Pcdh15) and Sans, interact with Myosin VIIA in a protein complex. It is not clear whether Cad99C mediates attachment to neuronalcilia or cap cells as a homodimer or as a heterodimer with another adhesion molecule. (C) In mammalian hair cells, a USH1 protein complex that includesmyosin VIIA, Sans, harmonin and Cdh23 is located close to the stereocilia tips. By analogy withDrosophila, we propose that myosin IIA interacts with myosin VIIA,and that this interaction is promoted by myosin II ubiquitination. The motor activity of either myosin might be required for the transport of the myosinVIIA-myosin II-USH1 protein complex to the stereocilia tips.

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between these two myosins in stereocilia (Fig. 3C) remainsspeculative. The overexpression of different pathogenic variantsof Myosin II in Johnston’s organ leads to the apical detachment ofscolopidia, which also occurs inDrosophilaUSHmutants (Li et al.,2016). These data indicate that Myosin II functions in Drosophilahearing, together with Myosin VIIA and/or other USH1 proteins;this interaction might also underlie the pathogenic effects of somepathogenic variants of myosin IIA in MYH9-related disease. Theclear cut and quantitative scolopidial detachment phenotype (Fig. 4)associated with myosin II and myosin VIIA mutations in Johnston’sorgan provides a quick and easy assay with which to investigate themolecular function of Myosin II in hearing and the mechanisms bywhich pathogenic Myosin II variants cause hearing loss. In the nextsection, we describe new technology that exploits the Drosophila tohuman conservation of genes implicated in hearing loss to rapidlytest the function of human genes and their variants in Drosophila.

Newapproaches formodeling human deafness inDrosophilaMutagenesis-based screens in mice and Drosophila have identifiedcandidate genes responsible for hearing loss and age-related hearingloss (Eberl et al., 1997; Hrabe de Angelis et al., 2000; Kernan et al.,1994; Nolan et al., 2000; Potter et al., 2016; Senthilan et al., 2012).Researchers used a large-scale lethal mosaic screen (Box 1) toisolate mutations that affect the Drosophila peripheral nervoussystem (Yamamoto et al., 2014). A secondary mosaic screen wasthen performed in this mutant collection by creating mutant clonesin the antenna to identify mutants with an abnormal Johnston’sorgan (Li et al., 2016). As discussed in previous sections, genesimplicated in hearing in these Drosophila screens include thoseknown to cause hearing loss in humans or mice. The convenience ofDrosophila as a model organism, including its short life span andarmory of genetic tools, makes it an attractive system to investigatethe function of genes involved in hearing. Historically,overexpression experiments have been used to test gene functionin Drosophila, either using the relevant fly gene or a conservedmammalian homolog. However, it is not always possible tounambiguously identify mammalian homologs of Drosophilagenes based on sequence alone. Moreover, these approaches arenot necessarily able to dissect why particular human gene variantsare pathogenic, especially in the case of missense mutations. Inaddition, overexpressing a gene or gene variant at nonphysiological

levels can lead to experimental artifacts. In recent years, advances inrecombination-mediated cassette exchange (RMCE; Box 1)technology and the ability to perform homologous recombinationinDrosophila using the CRISPR-Cas9 system (Box 1) have allowedresearchers to rapidly test the function of human genes inDrosophila and to rapidly assay the effects of potentiallypathogenic human variants far more quickly than in mice. In thissection, we describe the components of this technology, which issummarized in Fig. 5.

Gene trap cassette integration and exchangeAn identified ortholog of a human or mouse gene is a prerequisitefor functional testing in Drosophila. Although not all candidatedisease genes will have invertebrate orthologs,∼60% of the protein-coding genes in theDrosophila genome are conserved in mammals,and Drosophila orthologs have been identified for about two-thirdsof human disease genes (Chien et al., 2002; Hu et al., 2011; Reiterand Bier, 2002; Spradling et al., 2006). Numerous DrosophilaMinos-mediated integration cassette (MiMIC) lines are available(Venken et al., 2011) that can be further modified using RMCE(Nagarkar-Jaiswal et al., 2015a,b). Using this technique, an artificialexon that serves as a gene trap – such as a GAL4 Trojan exoncassette (Diao et al., 2015) – can be inserted into a MiMIC line tocreate a premature termination signal that disrupts the function of theendogenous gene. If MiMIC lines are not available or suitable for aparticular gene, it is now possible to insert MiMIC-like cassettes inDrosophila genes using CRISPR-mediated homology-directedrepair (Port et al., 2014; Ren et al., 2013).

Green fluorescent protein tagging to characterize expression andfunctionArtificial exons can be created to have flanking sites that facilitateRMCE (Nagarkar-Jaiswal et al., 2015a,b; Venken et al., 2011).These exons allow fluorescent tags to be introduced into a gene ofinterest to reveal its cellular and subcellular expression pattern(Fig. 5). This approach has been used to modify hundreds ofDrosophila genes, providing detailed expression data and with∼75% of the genes retaining their function after incorporation of theinternal exon-encoding green fluorescent protein (GFP), as shownby their ability to rescue null alleles (Nagarkar-Jaiswal et al.,2015b). To date, nearly 10,000 Drosophila genes have been GFP

10 µm

A B

A2 antennal segment

Detachedscolopidia

Fig. 4. Apical detachment of scolopidia in Drosophila Ubr3mutants. (A) A confocal image of the second antennal segmentof a mosaic adult Drosophila in which GFP+ marked cells areUbr3−/− (mutant) andGFP– cells areUbr3+/− (wild-type control).The apical junction protein NompA is shown in red and actin(scolopale cells) in blue. Arrows indicate two detached Ubr3−/−

scolopidia, which also exhibit abnormal puncta pattern ofNompA. The white outline box (upper) and yellow outline box(lower) show high-power images of the NompA pattern in theapical junction from wild-type or Ubr3 mutant scolopidia,respectively. (B) A schematic summary of the apicaldetachment of scolopidia in Ubr3 and other Usher mutants,such as myosin VIIA and Cad99c. Arrows indicate detachedscolopidia. Image in A taken from Li et al. (2016).

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tagged in this way (Kanca et al., 2017). These lines also allow geneand protein function to be tested through the targeting of the GFP-encoding exon with RNA interference (RNAi) approaches(Neumuller et al., 2012; Pastor-Pareja and Xu, 2011). In theadult fly, however, the stability of some proteins in postmitoticadult cells can preclude efficient RNAi knockdown. To addressthis, a genetic system known as the deGradFP system can be usedto directly degrade the GFP-tagged protein by using a modifiedform of the ubiquitin-proteasome pathway to specifically targetand degrade GFP-tagged proteins (Caussinus et al., 2012, 2013)(Fig. 5).

Testing auditory function in DrosophilaDrosophila mutants can be assessed for auditory function inseveral ways. In adult flies, a sound-evoked compound actionpotential can be recorded from the antennal nerve (Eberl et al.,2000; Todi et al., 2008), although this method is less sensitive ifsmall clones of mutant cells are being assayed in an otherwisewild-type background (Li et al., 2016). Behavioral assays can alsobe used to detect hearing in response to stimuli such as matingsongs (Eberl et al., 1997), or the response of flies to the force ofgravity (Kamikouchi et al., 2009), although, once again, thesesuffer from a limited sensitivity. It is also possible to measureactive movement of the antenna by laser vibrometry and to recordthe activity of single giant neurons in the Drosophila brain(Kamikouchi et al., 2009; Tootoonian et al., 2012). Finally, it ispossible to examine the morphology of Johnston’s organ indifferent mutants using an array of antibodies and GAL4 lines(Box 1) that express fluorescent proteins in the cellularcomponents of scolopidia.

Testing mammalian homologs and pathogenic variantsHaving identified and characterized the function of Drosophilaorthologs of mammalian genes in hearing, it is possible to test theirfunctional conservation by rescuing aDrosophilamutation with oneof its human or mouse orthologs. The use of GAL4-Trojan genetraps (Fig. 5) is particularly useful in this regard. In this approach,the GAL4 transcription factor is targeted to an endogenousDrosophila exon. By fusing the GAL4 sequence to a T2Apeptide-coding sequence, the endogenous protein begins to betranslated, but is then truncated and GAL4 protein is expressedinstead. This transcriptional activator then activates the expressionof a mammalian complementary DNA (cDNA) of interest under thecontrol of a UAS promoter from a separate locus in the Drosophilagenome (Fig. 5). Thus, it is possible to inactivate aDrosophila geneand simultaneously assay whether a mammalian homolog canfunctionally rescue the inactivated gene. Moreover, once it has beenestablished that a mammalian cDNA can rescue its Drosophilaortholog, it is then possible to test variants of the mammalian geneto determine whether they are pathogenic, and if so, to evaluate thecellular or biochemical consequences of such pathogenic variants(Bellen and Yamamoto, 2015). The function of potentiallydominant variants can also be assessed in this way byoverexpressing the variant of interest in Johnston’s organ to see ifit elicits a phenotype. For example, pathogenic variants of Myh9known to cause deafness in humans also cause morphologicaldefects in scolopidia when overexpressed in Johnston’s organ (Liet al., 2016).

The GAL4-Trojan system has several advantages overconventional methods to test gene function by overexpression.First, it can test the function of a mammalian gene in the context of a

GFP exonFluorescent proteintagging

Candidate genes

Drosophila gene

SA 2A GAL4 PolyA

SA 2A GAL4 PolyA

SA GFP SD

SA GFP SD

Ortholog prediction

CRISPR

A

B

CForward genetics: identify novel regulators in hearing

Mutagenesis-based screensfollowed by mapping

Reverse genetics: study function of candidate genes

Traditional strategies

Overexpress the human variants or‘humanized’ version of fly genes

New strategies

Characterize expression patternand protein localization usingGFP-tagged protein trap lines

Knockdown of proteins throughiGFPi or deGradFP

(efficient knockdown; bypass mapping)

Assess LOF phenotypes associatedwith T2A-GAL4 gene trap mutants

Rescue with human cDNA; express humanpathogenic variants using T2A-GAL4

(precise temporal and spatial expression)

Expression pattern in auditory organs

Microarray, RNA-seq,antibody staining,

GAL4 reporter lines

RMCE

Insertion of Trojan exonPremature terminationand GAL4 expression

Fig. 5. Approaches to functionally test candidate mammalian deafness genes in Drosophila. (A) Candidate genes can include known deafness genes ormouse genes with deafness phenotypes. Their Drosophila homologs are predicted by protein sequence homology. (B) An artificial exon that acts as a genetrap can be inserted into a MiMIC line by recombination-mediated cassette exchange (RMCE; Venken et al., 2011) or by CRISPR-mediated homology-directedrepair, which prematurely terminates transcription of the endogenous gene through a splice acceptor site (SA) and poly-adenylation signal (PolyA), andexpresses the GAL4 transcriptional activator as part of the endogenous transcript through a picornavirus self-cleaving 2A peptide sequence (2A). (C) Genetrap/GAL4 lines are screened for hearing phenotypes. The GAL4 protein expressed by the endogenous regulatory elements of the gene of interest can also beused to drive the expression of transgenes under the control of the UAS DNA element inserted elsewhere in the fly genome. These transgenes can be wild type,or pathogenic human or mouse orthologs, and the insertion of GAL4 into the endogenous locus allows the transgene to be expressed in a spatiotemporalpattern that is identical to the endogenous Drosophila gene. RMCE through attP and attB sites (yellow triangles) can convert the Trojan exon into an exoncontaining GFP flanked by SA and splice donor (SD) sites, to reveal the cellular and subcellular localization of the trapped gene (Nagarkar-Jaiswal et al., 2015b).The GFP exon can also be used to knockdown the encoded transcript or protein by in vivo GFP interference (iGFPi) or the deGradFP system, respectively.LOF, loss of function; RNA-seq, RNA sequencing.

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null mutation for its Drosophila counterpart. Second, targeting theGAL4 cassette to the Drosophila gene of interest significantlyincreases the likelihood that the GAL4 activator will be expressed inthe same spatial and temporal pattern as the endogenousDrosophilagene. Finally, the incorporation of DNA sequences in the Trojancassette that permit RMCE allows the locus to be modified furtherwith little effort. Although these advances are significant, they stillhave limitations. For example, this approach is only feasible if cleargene homologs can be identified between Drosophila andmammals. Moreover, although expression of the GAL4transactivator might be faithful, the UAS promoter might driveexpression of the mammalian gene at levels greater than those of theendogenous Drosophila gene.

ConclusionThe field of genetic hearing loss research has advanced significantlyin the last several decades, but three challenges and opportunitiesremain. First, it is likely that many new genes that regulate hearingawait discovery. The study of extended families, particularly thosethat feature consanguineous marriages, has revealed new deafnessgene candidates, but following up these new candidates is hamperedby the limited availability of these large families in restricted regionsof the world. The National Institutes of Health (NIH)-fundedUndiagnosed Diseases Network (http://undiagnosed.hms.harvard.edu) seeks to find possible genetic etiologies for rare humandiseases (Wangler et al., 2017), some of which include hearing loss,and it is likely that this initiative will reveal new forms of syndromichearing loss. In addition to the ongoing study of human familieswith hereditary deafness (Smith et al., 2014), there is currently aworldwide effort to create, characterize and curate mutations inevery mouse gene coordinated through the International MousePhenotyping Consortium (IMPC) (www.mousephenotype.org),with the US effort coordinated through an NIH-funded KnockoutMouse Project (www.komp.org). Because auditory testing is part ofthe standard IMPC phenotyping pipeline, it is likely that many newmouse genes will be identified that can cause hearing loss inhomozygous or heterozygous mutants, and this might support theidentification of human variants that also cause hearing loss.Mutagenesis-based screens in mice have also revealed new genesresponsible for hearing loss and age-related hearing loss (Hrabe deAngelis et al., 2000; Nolan et al., 2000; Potter et al., 2016).Although over 100 deafness genes have been identified, a second

challenge is to understand the function of these genes in hearing.While the cellular and biochemical functions of some of these geneshave been extensively characterized, many other known deafnessgenes remain poorly characterized. Moreover, the pathogenic basisof many human deafness gene variants await elucidation. In thisReview, we have suggested that the homologies between insect andmammals, in terms of the genes involved in hearing organdevelopment and function, make Drosophila an attractive systemfor assaying the function of deafness-associated gene variants. Webelieve that the technology described in this Review will make itpossible to perform medium- and high-throughput screens ofcandidate mammalian genes and known deafness-associated genesin Drosophila, in addition to identifying novel genes that areessential for hearing in Drosophila that might have mammalianhomologs.Once the pathogenic mechanisms of human deafness gene

variants have been established, the final challenge is to developtherapies for particular forms of deafness that do not rely on hearingaids or cochlear implants. As with other forms of genetic disease, itis possible to improve function by pharmacological approaches that,

to name but three, stabilize protein complexes, enhance the functionof the affected protein(s) or proteins acting downstream of theaffected proteins, and slow the aggregation of pathogenic proteinvariants. Once again, we suggest that Drosophila might offer apromising platform in which to rapidly screen for compounds orsignaling pathways that improve auditory function in animalsbearing pathogenic hearing gene variants.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported by the National Institute on Deafness and OtherCommunication Disorders (DC014932) to A.K.G. H.J.B. is a member of the HowardHughes Medical Institute.

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