© 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm031492. doi:10.1242/dmm.031492

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

ABSTRACT Keats and Corey, 1999; Kimberling et al., 2010; Mathur and Yang, Johnston’s organ – the hearing organ of Drosophila – has a very 2015). It is an autosomal recessive genetic disease, characterized by different structure and morphology to that of the hearing organs of varying degrees of and retinitis pigmentosa-induced vision vertebrates. Nevertheless, it is becoming clear that vertebrate and loss. Although our understanding of genetic has invertebrate auditory organs share many physiological, molecular advanced greatly over the past 20 years (Vona et al., 2015), there is a and genetic similarities. Here, we compare the molecular and cellular pressing need for experimental systems to understand the function features of hearing organs in Drosophila with those of vertebrates, of the proteins encoded by deafness genes. The mouse is well and discuss recent evidence concerning the functional conservation established as a model for studying human genetic deafness (Brown of Usher proteins between and mammals. Mutations in Usher et al., 2008), but other model organisms, such as the fruit genes cause , the leading cause of human deafness Drosophila, might also provide convenient and more rapid ways to and blindness. In Drosophila, some Usher syndrome proteins appear assay the function of candidate deafness genes. to physically interact in protein complexes that are similar to those In mammals, mechanosensitive hair cells reside in a specialized described in mammals. This functional conservation highlights a epithelial structure, the organ of Corti (see Glossary, Box 1), that rational role for Drosophila as a model for studying hearing, and for runs along the length of the cochlear duct (Box 1) of the inner ear investigating the evolution of auditory organs, with the aim of (Basch et al., 2016). Vibration of the organ of Corti in response to advancing our understanding of the genes that regulate human sound waves that are captured and amplified by the external and hearing and the pathogenic mechanisms that lead to deafness. middle ears causes a deflection of the hair-like stereocilia of hair cells (Box 1). This deflection opens ion channels, leading to the KEY WORDS: , Deafness, Drosophila, Hair cells, Hearing, release of neurotransmitters by hair cells and the activation of the Usher syndrome first neurons in the auditory pathway (Fettiplace and Hackney, 2006). Drosophila also have a hearing organ – Johnston’s organ Introduction (Box 1) – located in the antenna, the second segment of which Hearing loss is one of the most common neurological disorders in contains several hundred mechanosensitive units called scolopidia humans, affecting 2 to 3 of every 1000 children in the USA (Imtiaz (Eberl and Boekhoff-Falk, 2007). Bulk air displacements caused by et al., 2016; Naz et al., 2017). Approximately 50% of congenital near-field sound stimuli such as courtship song rotate the distal hearing loss is caused by genetic mutations, with a variety of antennal segments, stretching the scolopidia (Boekhoff-Falk and other factors, particularly infectious disease, contributing to the Eberl, 2014). Accumulating evidence from Drosophila suggests remaining forms of congenital deafness (Korver et al., 2017). that there are many physiological, molecular and genetic similarities Genetic mutations affect many components of the auditory pathway, between vertebrate and invertebrate auditory organs (Albert and and can affect only hearing (nonsyndromic deafness), or other Göpfert, 2015; Bokolia and Mishra, 2015; Jarman and Groves, organs and tissues as well (syndromic deafness). Of the genetic 2013; Lu et al., 2009; Senthilan et al., 2012). For example, mutations that cause nonsyndromic deafness, ∼30% are autosomal vertebrates and invertebrates share homologous transcription factors dominant, ∼70% are autosomal recessive, and five are X-linked that specify the development of their sensory cells, the analogous (hereditaryhearingloss.org). Among the many forms of syndromic accessory structures to capture and transduce sound energy, and the hearing loss are , , Branchio- use of analogous systems to transduce and amplify mechanical force oto-renal syndrome, and Usher syndrome into electrical energy. (Korver et al., 2017). Usher syndrome is the leading cause of Here, we review the evidence that the genes, cells and molecular human deafness and blindness, with a prevalence of 1-4 per 25,000 networks that specify the Drosophila hearing organ have people in the USA (Boughman et al., 1983; Hartong et al., 2006; mammalian counterparts that function in the differentiated cells of the cochlea and are required for hearing in mammals. In particular, we focus on recent findings that Usher syndrome proteins in 1Program in Developmental , Baylor College of Medicine, Houston, TX 77030, USA. 2Jan and Dan Duncan Neurological Research Institute, Texas Drosophila appear to physically interact in protein complexes that Children’s Hospital, Houston, TX 77030, USA. 3Department of Molecular and are similar to those described in mammals. We also describe recent Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. 4Howard work that indicates that some Usher syndrome proteins interact Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA. 5Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, physically with II, the variants of which can cause MYH9- USA. related diseases, including deafness. Various approaches, including *Present address: Department of Biological Sciences, Stanford University, Palo forward genetic screens in Drosophila (Box 1) to identify mutants Alto, CA 94305, USA. with behavioral defects associated with mechanosensory ‡Author for correspondence ([email protected]) transduction (Eberl et al., 1997; Kernan et al., 1994), and microarray screens to identify transcripts enriched in Johnston’s A.K.G., 0000-0002-0784-7998 organ and regulated by atonal, have revealed striking similarities This is an Open Access article distributed under the terms of the Creative Commons Attribution between the genes that are implicated in human deafness and those License (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. that affect the function and development of Johnston’s organ in Disease Models & Mechanisms

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deafness mutants that have been isolated in mutagenesis and Box 1. Glossary knockout screens (Steel, 2014). We suggest that by improving our Antennal segments: the Drosophila antenna is made up of three understanding of the mechanisms of hearing in Drosophila,we segments: a1 (scape), a2 (or pedicelus), which contains Johnston’s could discover new genes required for hair function in organ and responds to sound, gravity and wind flow, and a3 (or vertebrates, better understand the evolution of auditory organs, funiculus), the most distal segment, which is an olfactory organ. and gain new insights into the pathogenic mechanisms of some Arista: a delicate, feather-like structure projecting from the third (most distal) antennal segment of Drosophila that is involved in sound deafness-related genes. detection. Chordotonal organs: these organs are ciliated stretch receptors that Functional similarities between Drosophila and mammalian detect motion in joints of the body. They are made up of repeating hearing organs multicellular units called scolopidia (see below). Johnston’s organ, the largest chordotonal organ (Box 1) in Cochlea: the coiled, snail shell-shaped part of the mammalian inner ear, Drosophila, is located in the second antennal segment (Box 1) the innermost portion of which, the cochlear duct, contains the organ of ∼ Corti (see below), which detects sound. and consists of 200 functional units or scolopidia (Kernan, 2007). CRISPR-mediated homology-directed repair: the CRISPR-Cas9 Each scolopidium is suspended between the cuticle of the second system can be used to generate targeted double-stranded DNA breaks antennal segment and the insertion of the third antennal segment (DSBs) when coupled with a sequence-specific guide RNA (gRNA). (Fig. 1A,B) (Todi et al., 2004). During courtship, male flies beat When Cas9 and a gRNA are introduced into a cell with a piece of DNA their wings, causing the air to move, which in turn causes the flanked by homology arms, the DSB can be repaired by homologous feather-like arista (Box 1) on the third antennal segment of the recombination, inserting the new piece of DNA into the locus of interest. Forward genetic screens: a genetic screen, typically of induced female to vibrate. The resulting movement of the third antennal mutations, which identifies genes responsible for a particular phenotype, segment applies force to the scolopidia as the a2/a3 joint rotates for example, hearing defects. (Boekhoff-Falk and Eberl, 2014; Eberl and Boekhoff-Falk, 2007). GAL4-UAS system: a two-component genetic system that activates This stretch is transmitted to the mechanosensory cilia of sensory gene expression in Drosophila. It uses the yeast GAL4 transcription neurons, opening stretch-gated channels factor to activate transcription of a transgene adjacent to an upstream (Kernan et al., 1994; Kernan, 2007). A transient receptor potential activation sequence (UAS). (TRP) channel – TRPN1 (also known as NompC) – was first Johnston’s organ: the largest chordotonal organ in Drosophila, containing ∼200 scolopidia. It is located in the second antennal identified as a candidate component of a mechanosensitive channel segment and detects auditory signals (courtship songs), as well as in a genetic screen in Drosophila (Walker et al., 2000), and was wind flow and gravity. subsequently shown to be expressed in the distal region of the ciliary Lethal mosaic screen: lethal homozygous mutations cannot be studied dendrite of Johnston’s organ neurons (Lee et al., 2010). Mutation of beyond the age they cause lethality. To circumvent this, clones of cells Drosophila nompC attenuates, but does not completely abolish, carrying two lethal mutant alleles can be generated in an otherwise sound-evoked potentials (Eberl et al., 2000; Effertz et al., 2012, heterozygous organism using a variety of recombination techniques. This approach allows researchers to assess the phenotypes caused by 2011; Göpfert et al., 2006; Lee et al., 2010; Liang et al., 2011). Two lethal mutations. TRPV family members, Nanchung (Nan) and Inactive (Iav) are also Mechanotransduction channels: ion channels that are gated by expressed in Johnston’s organ neurons, although in a more proximal mechanical force. region of the ciliated dendrite than NompC (Cheng et al., 2010; Organ of Corti: an epithelial specialization of the cochlear duct, which Gong et al., 2004; Lee et al., 2010; Liang et al., 2011). Nan and Iav detects sound through its mechanosensitive hair cells. The organ of Corti are also required for the generation of sound-evoked potentials in sits on a thin, flexible basilar membrane, which oscillates at different frequencies corresponding to its position along the cochlea. the antennal nerve (Gong et al., 2004; Kim, 2007; Kim et al., 2003). Recombination-mediated cassette exchange (RMCE): regions of It is less clear whether Nan and Iav can be directly gated by genomic DNA can be flanked by DNA sequences that act as targets for mechanical force, as studies in which either or both channels were site-specific integrases. For example, the attB and attP DNA motifs are expressed in heterologous cells reached different conclusions over targets for the phiC31 integrase. If a piece of DNA bearing attB sites (a the degree to which the channels could be opened by osmotic stress recombination cassette) is introduced into a cell that carries a DNA [compare Gong et al. (2004) and Kim et al., (2003) with Nesterov sequence flanked by attP sites in the presence of the phiC31 integrase, et al. (2015)]. The physical separation of the TRPN and TRPV the intervening sequence will be replaced by the recombination cassette. ’ Scolopidium: a sensory unit of chordotonal organs, each containing 1-3 channels in the dendrite of Johnston s organ neurons, together with mechanosensitive sensory neurons, the cilia of which are surrounded by the effects of these mutations on sound-evoked potentials and on a scolopale cell. The scolopidium is anchored to the insect cuticle at its active amplification, has led to the idea that NompC plays a direct proximal and distal ends by a ligament cell and a cap cell, respectively. role in mechanotransduction (Effertz et al., 2012; Göpfert et al., When flexional or rotational force is applied to the insect’s cuticle, it is 2006), whereas Nan and Iav, which can form heteromers (Gong transferred to the sensory neurons in each scolopidium. et al., 2004), are thought to amplify weak signals transmitted from Stereocilia: elongated, -rich microvilli that protrude from the apical surface of sensory hair cells in vertebrates. Their displacement by force the NompC complex (Gong et al., 2004; Göpfert et al., 2006; opens mechanosensitive ion channels, with their distal ends acting as Kamikouchi et al., 2009; Kim et al., 2003). likely sites of active mechanotransduction in hair cells. Rows of Despite the progress made in characterizing the properties of stereocilia of increasing length form a staircase-like bundle structure in these channels, it has been hard to resolve the relative contributions hair cells, and the coupling of adjacent stereocilia by tip links transfers of these three channels to mechanotransduction, in part because of force to the entire bundle. the technical challenges involved in recording from single auditory neurons in Johnston’s organ. A recent innovation by Wilson and colleagues (Lehnert et al., 2013) exploited the fact that auditory Drosophila. We also review the recently developed genetic toolkits neurons terminate on, and are dye-coupled to, a single giant fiber in Drosophila that can characterize the function of novel genes in neuron in the brain (Kamikouchi et al., 2009; Tootoonian et al., Drosophila hearing. It is estimated that several hundred genes 2012). Recording the activity of this neuron allows currents from involved in hearing still await discovery on the basis of mouse Johnston’s organ neurons to be indirectly measured. Results from Disease Models & Mechanisms

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A C

Cap rods (actin bundles) Dendritic cap Cap cell (NompA)

Cillium (NompC, Nan/Iav) Arista Antennae Ciliary dilation

Scolopale rods B (actin bundles)

Second antennal Distal basal bodies segment Ciliary rootlets Scolopale cell Arista

Neuron

Third antennal Ligament cell segment

Fig. 1. Structure of the Drosophila auditory organ. (A) A frontal view of an adult Drosophila head, with antennae marked in gray (eyes are in red). (B) Magnified image 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) and consists 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 the displacement of the antennal arista, which induces the third antennal segment to rotate (arrow). This acoustic, stimulus-induced rotation applies force to the cilia of mechanosensory neurons in the scolopidia, resulting in the firing of action potentials along the antennal nerve. A single scolopidium is highlighted in the light green box. 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 an individual 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 form septate junctions with the ligament and cap cells at the basal and apical ends of the scolopidium, respectively, ensuring the formation of an enclosed scolopale space 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 scolopidia are located in Johnston’s organ. this approach suggest that Nan and Iav are required to respond to runs the length of the organ of Corti, and these cells form synapses sound, whereas NompC is required for the modulation and with sensory afferent neurons of the acoustic (spiral) ganglion amplification of force generated during sound reception (Lehnert (Goodrich, 2016), while three rows of outer hair cells receive mostly et al., 2013). It is notable that NompC contains numerous efferent input from the brainstem (Basch et al., 2016; Yu and repeats (Cheng et al., 2010; Liang et al., 2013; Zhang et al., 2015) Goodrich, 2014). Hair cells bear elongated microvilli or stereocilia that might interact with motile components of the ciliated dendrite to on their apical surface that insert into an acellular tectorial modulate force, an idea that is supported circumstantially by the membrane above them (Fig. 2). As the basilar membrane vibrates, presence of NompC in the distal , between the site of cilia the motion of the organ of Corti causes the stereociliary bundles of insertion, and by more proximal location of the Nan/Iav complex in the hair cells to be displaced by contact with the tectorial membrane the cilium. (outer hair cells) or fluid flow (inner hair cells). This opens Unlike the Drosophila hearing organ, which responds optimally mechanically gated ion channels in the tips of the stereocilia (Corey to a narrow frequency range of the beating male wing, the and Holt, 2016; Fettiplace, 2016; Wu and Muller, 2016). Adjacent mammalian inner ear has evolved to respond to a wide range of stereocilia connect to each other close to their tips by a sound frequencies. The hearing organ of the cochlea, the organ of protein complex consisting of protocadherin 15 and 23 Corti, sits on a thin basilar membrane that runs the length of the (Pepermans and Petit, 2015). This complex allows the opening of cochlear duct (Fig. 1B). Both the thickness and width of the basilar mechanotransduction channels to be coordinated as the hair bundle membrane change along the length of the cochlear duct (Dallos, is displaced (Hudspeth, 1997, 2008). As in Drosophila, the identity 1996); therefore, it can resonate in response to many different of the mechanosensitive ion channels in these hair cells is still being frequencies, allowing the basilar membrane to transform complex investigated and debated (Corey and Holt, 2016; Fettiplace, 2016; sounds into their component sound frequencies (Reichenbach and Wu and Muller, 2016). Current work is focused on four Hudspeth, 2014). The rising and falling oscillation of the basilar transmembrane proteins, transmembrane inner ear (TMIE), membrane in response to mechanical waves traveling along the tetraspan membrane protein of stereocilia/lipoma cochlea is transmitted to the mechanosensory hair cells of the organ HMGIC fusion partner-like 5 (TMHS; Lhfpl5) and of Corti (Fettiplace and Hackney, 2006). One row of inner hair cells transmembrane channel-like 1 and 2 (TMC1/2), all of which are Disease Models & Mechanisms

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Fig. 2. Structure of the mammalian auditory organ. A B (A) In the mouse inner ear, the organ of Corti is located on the basilar membrane, which runs the length of the cochlear duct. Mechanical waves conducted by the middle ear are transmitted to the oval window Vestibular canal and travel through the perilymph surrounding the (perilymph) cochlear duct (arrows). Compression of the oval Cochlear duct window is matched by a corresponding outward (endolymph) movement at the round window. (B) A cross-section of the cochlear duct, showing three rows of outer hair cells (dark blue) and one row of inner hair cells (dark green). Oval window (C) A magnified view of the arrangement of hair cells in the organ of Corti. Inner hair cells are surrounded by inner phalangeal and border cells and are separated Tympanic canal from the outer hair cells by two pillar cells that together Organ of (perilymph) form the tunnel of Corti. Outer hair cells are surrounded Corti by Deiters’ cells. Inner and outer hair cells receive Round window afferent and efferent innervation, respectively. Stereocilia located on the apical surface of hair cells are embedded into the tectorial membrane (outer hair cells) or lie just beneath the membrane (inner hair cells). During sound reception, the sound-induced vibration of C the basilar membrane applies mechanical force to the Tectorial membrane tip links of the stereocilia, causing an influx of potassium and calcium and the depolarization of hair cells. The potassium gradient in the cochlear duct endolymph is maintained by the stria vascularis in the lateral wall of the cochlear duct.

Stria vascularis

Basilar membrane

Border cell Inner hair cell Outer hair cell Inner phalangeal cell Deiters’ cells necessary for hearing in mice (Kawashima et al., 2011; Kurima crustaceans have true hearing; however, other invertebrate groups et al., 2015; Xiong et al., 2012; Zhao et al., 2014). It is possible that possess cells with similar apical specializations other proteins, such as the large transmembrane protein Piezo2, that can detect gravity and acceleration. These receptor cells are also might also contribute to mechanotransduction in other parts of the sometimes referred to as hair cells, although their homology to true hair cell (Wu et al., 2017). vertebrate hair cells is still under debate (Burighel et al., 2011, 2008, Although the appearance and organization of mammalian and 2003; Fritzsch and Straka, 2014; Manley and Ladher, 2007). These Drosophila hearing organs are clearly very different, they share a invertebrate ‘hair cells’ can be either secondary receptors or primary number of genes that regulate their formation and cellular functions. sensory neurons that have intrinsic mechanosensitive We describe some of these in the following section. specializations (Burighel et al., 2011). These specializations can be actin-rich structures similar to vertebrate stereocilia or Cellular and molecular similarities between Drosophila and mechanosensitive made up of one or more true cilia, mammalian hearing organs reminiscent of vertebrate photoreceptors. Some nonvertebrate Despite being separated by several hundred million years of groups, such as urochordates and cephalopod mollusks, use both evolutionary time, the mechanosensitive cells and supporting cells kinds of receptor cells in their sensory organs (Budelmann and of mammalian and Drosophila hearing organs are specified by Thies, 1977; Burighel et al., 2011). In contrast, secondary receptor similar sets of transcription factors, and have similarities in the cells have not been observed in and crustaceans (Fritzsch and accessory cells and proteins that allow these receptor cells to be Straka, 2014), and these groups instead have a wide range of mechanically gated. We first compare the axonless hair cells of mechanoreceptive sensory neurons arranged in clusters all over their vertebrates with the mechanosensitive neurons of Drosophila and bodies (Kernan, 2007), including the ciliated sensory neurons in then describe similarities in how these cells are formed and how scolopidia of Johnston’s organ. It is not clear whether a they function. mechanosensitive represents the ancestral cell type Vertebrate hair cells are secondary receptor cells: they elaborate from which hair cells and sensory neurons emerged as sister cell neither nor dendrites but are innervated by axons of bipolar types in multiple taxa, or whether secondary mechanosensitive cells sensory neurons that also send processes to brainstem nuclei. In were lost in the lineage (Arendt et al., 2016). addition to their apical stereociliary bundle, they also have a true Despite the structural differences between insect and vertebrate cilium, the , which disappears in some hair cell types as sound-detecting cells, molecular evidence suggests that certain they mature, including inner and outer hair cells of the mammalian aspects of the development of the various cell types found in cochlea (Nayak et al., 2007). Among invertebrates, only insects and auditory organs are conserved between invertebrates and vertebrates Disease Models & Mechanisms

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(Bechstedt and Howard, 2008; Bokolia and Mishra, 2015; Caldwell in Drosophila as a proneural gene required for the formation of and Eberl, 2002; Lewis and Steel, 2012) (Table 1). The atonal (ato) photoreceptors and of some (Jarman et al., 1993, basic helix-loop-helix (bHLH) was discovered 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 cells Ato Loss of function leads to loss of all Atoh1 Necessary and sufficient for differentiation Ben-Arie et al., 2000; Bermingham chordotonal organs. Overexpression and survival of hair cells. Fly Ato rescues et al., 1999; Cai et al., 2013; causes ectopic chordotonal organs. mouse Atoh1 mutant phenotype. Jarman et al., 1993, 1994; Wang Mouse Atoh1 rescues fly Ato mutant et al., 2002 phenotype. Sens Required for the development of Gfi1 Required for the survival of hair cells in Acar et al., 2006; Jafar-Nejad et al., chordotonal organ in Drosophila mouse. 2003, 2006; Nolo et al., 2000; embryos. Wallis et al., 2003; Wang et al., 2002 Pros Specifically expressed by scolopale cells in Prox1 Initially expressed in the progenitors of hair Bermingham-McDonogh et al., Johnston’s organ. cells and supporting cells, but becomes 2006; Boekhoff-Falk and Eberl, restricted to supporting cells as the 2014 sensory differentiates. Pax2 (Sv) Specifically expressed by scolopale cells in Pax2 Initially expressed in the progenitors of the Burton et al., 2004; Kavaler et al., Johnston’s organ. entire inner ear but becomes restricted to 1999 the stria vascularis of the cochlear duct. Rfx Rfx regulates many ciliogenic genes in a Rfx1-3 Loss of Rfx genes in mice leads to a rapid Dubruille et al., 2002; Elkon et al., variety of organisms, including in loss of initially well-formed outer hair 2015; Jarman and Groves, 2013; Drosophila sensory neurons. cells in the mouse cochlea and causes Laurencon et al., 2007; Vandaele deafness. et al., 2001 Structural proteins required for mechanosensation NompA Produced by scolopale cells and forms α- and β- Produced and secreted by supporting Chung et al., 2001; Legan et al., acellular filaments connecting the tectorin cells, they constitute the major structural 2000, 1997; Xia et al., 2010 mechanosensory neuronal dendrites to proteins in the acellular tectorial the third antennal cuticle. membrane that lies above the hair cells. Myosin VIIA Expressed in scolopale cells and enriched MYO7A Recessive mutations cause Usher Ahmed et al., 2013; Cosgrove and in apical tips; required for apical (USH1B) syndrome and nonsyndromic deafness Zallocchi, 2014; Glowinski et al., attachment of scolopidia and auditory in humans; physically interacts with other 2014; Li et al., 2016; Pan and sensation; physically interacts with Usher proteins in a protein complex to Zhang, 2012; Todi et al., 2005, homologs of other Usher proteins, form stereocilia tip links. 2008 Cad99C and Sans. Cad99C Expressed in Johnston’s organ and PCDH19 Recessive mutations cause Usher Ahmed et al., 2013; Cosgrove and localizes to apical tips of scolopidia; (USH1F) syndrome in humans; complexes with Zallocchi, 2014; D’Alterio et al., regulates apical attachment of other Usher proteins to form stereocilia 2005; Glowinski et al., 2014; Li scolopidia; physically interacts with tip links. et al., 2016; Pan and Zhang, homologs of other Usher proteins, 2012 Myosin VIIA and Sans. Sans Regulates apical attachment of scolopidia; SANS Recessive mutations cause Usher Ahmed et al., 2013; Cosgrove and physically interacts with homologs of syndrome in humans; physically Zallocchi, 2014; Demontis and other Usher proteins, Myosin VIIA and interacts with other Usher proteins in a Dahmann, 2009; Glowinski et al., Cad99C. protein complex to form stereocilia tip 2014; Li et al., 2016; Pan and links. Zhang, 2012 Myosin II Expressed in Johnston’s organ and MYH9 Dominant mutations cause MYH9-related Heath et al., 2001; Lalwani et al., localizes to apical tips of scolopidia, also diseases and nonsyndromic deafness 2000; Li et al., 2016; Savoia and regulates apical attachment of disorder, DFNA17. Pecci, 2008 scolopidia. TMC Regulates mechanosensation in larval TMC1/2 Required for auditory mechanosensation. Guo et al., 2016; Kawashima et al., locomotion. 2015; Kurima et al., 2015, 2002 Conserved cells and accessory structures Accessory The scolopale, cap and ligament cells of Supporting Deiters’, pillar, inner phalangeal, border Boekhoff-Falk, 2005; Carlson et al., cells Johnston’s organ scolopidia. cells and Hensen’s cells of the organ of Corti. 1997; Eberl and Boekhoff-Falk, 2007 Scolopale Potassium-rich fluid secreted by scolopale Endolymph Potassium-rich fluid secreted by the stria Fettiplace and Hackney, 2006; fluid cells, bathing the ciliary dendrites of vascularis of the cochlear duct wall, Kikuchi et al., 2000; Küppers, mechanosensitive neurons. bathing the apical surface of sensory hair 1974; Roy et al., 2013 cells. Dendritic Secreted extracellular structure that Tectorial Secreted extracellular structure that lies Chung et al., 2001; Legan et al., cap anchors neuronal cilia and transfers membrane above hair cell stereocilia and applies 2000, 1997; Xia et al., 2010 force to them. Composed of the zona force to the hair bundles of outer hair pellucida (ZP) domain glycoprotein cells. Major components include the ZP NompA. domain glycoproteins α- and β-tectorin. Disease Models & Mechanisms

5 REVIEW Disease Models & Mechanisms (2018) 11, dmm031492. doi:10.1242/dmm.031492 organs, including Johnston’s organ, while its misexpression results neuronal cilia are enclosed by a tube-like scolopale cell (Fig. 1). The in the formation of ectopic chordotonal organs (Jarman et al., 1993). shape of the scolopale cell is thought to be maintained by a cage-like Similarly, the vertebrate homolog of ato, Atoh1, is both necessary scolopale consisting of actin rods, and some and sufficient for the differentiation and survival of hair cells -associated proteins (Todi et al., 2004; Wolfrum, (Bermingham et al., 1999; Cai et al., 2013). Remarkably, 1997). Scolopale cells form septate junctions with the ligament Drosophila ato and mouse Atoh1 can functionally replace each and cap cells at the basal and apical ends of the scolopidium, other in loss-of-function mutants (Ben-Arie et al., 2000; Wang et al., respectively, ensuring the formation of an enclosed scolopale space 2002), and are both regulated through conserved post-translational between the scolopale cell and neuronal cilia (Boekhoff-Falk, 2005; regulation (Quan et al., 2016), even though they share little Carlson et al., 1997; Eberl and Boekhoff-Falk, 2007; Todi et al., homology outside the bHLH domain. A conserved post- 2004). Although the ionic composition of the scolopale space has translational control of the temporal dynamics of ato/Atoh1 not been measured directly in Drosophila, studies of the fluid that expression during neurogenesis further supports the functional bathes a variety of insect and crustacean mechanoreceptors have similarities between ato and Atoh1 (Quan et al., 2016). The reported that this fluid has a positive potential of about +70 mV conservation between Drosophila ato and the vertebrate Atoh1 gene relative to the hemolymph (Thurm and Wessel, 1979). In addition, regulatory networks is reflected by conservation of some of their studies of blowfly mechanoreceptor organs have found such fluid to downstream target genes. The zinc finger transcription factor be high in potassium (Küppers, 1974), the concentration of which is senseless (sens)/Gfi1, which can be regulated by ato/Atoh1,is actively maintained by transport ATPases (French, 1988; Thurm, required for the development of the chordotonal organ in 1974; Wieczorek, 1982). Scolopale cells express Na+/K+ ATPases Drosophila embryos and for the survival of hair cells in the that localize to their ablumenal plasma membrane and are required mouse (Acar et al., 2006; Jafar-Nejad et al., 2003, 2006; Nolo et al., for hearing in Drosophila. These Na+ pumps function to actively 2000; Wallis et al., 2003; Wang et al., 2002). Regulatory factor X transport K+ into the scolopale space to maintain ion homeostasis (Rfx) is another downstream transcription factor activated by Atonal (Roy et al., 2013). In the mammalian organ of Corti, inner hair cells (Jarman and Groves, 2013; Laurençon et al., 2007) that regulates and outer hair cells are surrounded by different types of supporting many ciliogenic genes in a variety of organisms, including in cells: inner phalangeal and border cells surround the inner hair cells, Drosophila sensory neurons (Dubruille et al., 2002; Vandaele et al., while Deiters’ cells surround the outer hair cells (Fig. 2), and inner 2001). A recent study showed that combined loss of Rfx1 and Rfx3 and outer pillar cells located between these two domains form the in mice leads to a rapid loss of initially well-formed outer hair cells tunnel of Corti. During sound perception, sound-induced vibration in the mouse cochlea and causes deafness (Elkon et al., 2015). of the basilar membrane applies force to the tip links of the The division of labor between mechanosensitive secondary stereocilia. This results in their deflection, in the opening of cation receptor cells and their afferent sensory neurons in mammals and channels in the stereocilia and in the influx of K+ and Ca2+ from the some invertebrate taxa is reflected by an analogous division of labor surrounding potassium-enriched endolymph (Fettiplace and between the transcription factors that specify the neuronal and Hackney, 2006). Potassium homeostasis in the endolymph is sensory lineages. Neurog1 is a bHLH factor closely related to Atoh1 crucial for the maintenance of the endocochlear potential and for and is expressed in the progenitor cells of the inner ear primordium hair cell function. The K+ that enters hair cells is recycled through shortly before they delaminate as neuroblasts (Zheng and Gao, the supporting cells and is ultimately transported back into the 2000). Its expression is then extinguished and replaced by another endolymph by cells in the stria vascularis of the lateral wall of the closely related bHLH gene, Neurod1; both genes are necessary for cochlear duct (Kikuchi et al., 2000). It is not clear whether the K+- the generation of inner ear neurons (Kim et al., 2001; Liu et al., rich extracellular fluid of the endolymph and scolopale space reflect 2000). The Atoh, Neurogenin and NeuroD bHLH transcription a common or convergent evolutionary origin. For example, sensory factor families are ancient, with members of each family observed in hair cells of the organs of vertebrates are not enclosed, basal taxa, such as sponges (Simionato et al., 2008, 2007). but open to the external fluid environment, and a number of studies appear to have dispensed with the need for the measuring the endolymph-like fluid bathing cephalopod statocyst Neurog genes in sensory development; the one identified member sensory organs have found it to have low levels of K+ compared with of this family in Drosophila, target of pox neuro (tap), appears to Na+ (reviewed in Vinnikov, 2011). function in neurite outgrowth rather than in neuronal specification Some molecular and cellular similarities have been identified (Gautier et al., 1997; Yuan et al., 2016). In contrast, Drosophila ato between scolopale cells and cochlear-supporting cells. prospero has undergone duplication to give rise to two additional orthologs: ( pros), a homeodomain transcription factor, is specifically absent multidendritic and olfactory sensilla (amos) and cousin of expressed by scolopale cells in Johnston’s organ (Boekhoff-Falk atonal (cato) (Jarman and Groves, 2013). Although the Atoh, and Eberl, 2014). The mammalian homolog of pros, Prox1,is Neurogenin and NeuroD bHLH families are more closely related to initially expressed in the progenitors of hair cells and supporting each other than to other bHLH genes, they nevertheless have cells, but becomes restricted to supporting cells as the sensory extremely specific functions. For example, while Drosophila ato epithelium differentiates (Bermingham-McDonogh et al., 2006). can functionally replace Atoh1 in mice, and vice versa (Ben-Arie On the apical side of Johnston’s organ, cap cells participate in the et al., 2000; Wang et al., 2002), Neurog1 can only rescue loss of coupling of neuronal cilia to the cuticle of the third antennal Atoh1 in mice to a very modest degree (Jahan et al., 2012, 2015), segment, and help impart mechanical force to the cilia during sound suggesting that Neurog1 is only able to activate a subset of Atoh1 perception. nompA encodes a zona pellucida (ZP) domain target genes. Thus, these different bHLH factors can specify glycoprotein that is produced and secreted by scolopale cells. It different cell fates – neuron or hair cell – depending on the binding forms acellular filaments that connect the mechanosensory neuronal affinities and range of their targets. dendrites to the third antennal cuticle (Chung et al., 2001). Loss of A common feature of sensory systems is the presence of NompA leads to the detachment of both the scolopidia from the accessory cells that surround, support, nourish or modulate the third antennal segment cuticle and of the ciliary dendrites from the output of sensory cells. In each scolopidium of Johnston’s organ, cuticular structures in other sensory organs, such as the external Disease Models & Mechanisms

6 REVIEW Disease Models & Mechanisms (2018) 11, dmm031492. doi:10.1242/dmm.031492 sensory organs and other campaniform organs (Chung et al., 2001). Ca2+-mediated signaling (Table 2). Evidence from colocalization Mammals also have ZP domain-containing proteins – α- and β- data, in vitro interaction studies, and mouse genetic studies suggest tectorin (Legan et al., 1997) – which are produced and secreted by that USH proteins interact with each other to form multiprotein supporting cells, and are the major structural proteins in the acellular complexes in a variety of cell types (Fig. 3A) (Ahmed et al., 2013; tectorial membrane that lies above the hair cells, and that comes into Pan and Zhang, 2012). In mature mammalian hair cells, many Usher contact with the hair bundles of the outer hair cells. Loss of α- syndrome proteins localize to the tips of stereocilia where tectorin in mice leads to the detachment of hair cells from the mechanotransduction takes place (Fig. 3A). Adjacent stereocilia tectorial membranes (Legan et al., 2000; Xia et al., 2010). are connected by complexes at their tips, called tip links, which are Intriguingly, a Caenorhabditis elegans ZP domain protein, DYF- composed of cadherin 23 (USH1D; Cdh23) and protocadherin 15 7, resembles tectorins and anchors the tips of sensory dendrites to (USH1F; Pcdh15). Protocadherin 15 is thought to interact with the body wall as the neuronal cell bodies in the developing amphid components of the mechanotransduction channel (TMHS), and migrate away (Heiman and Shaham, 2009). In addition, the both ends of the tip links are indirectly coupled to the actin core of transmembrane channel-like (TMC) proteins are required for the stereocilia through other Usher proteins, including myosin VIIA auditory mechanosensation in mice and humans (Kawashima (USH1B; Myo7A), Sans (USH1G), harmonin (USH1C), Cib2 and et al., 2015). Although it has yet to be shown that the Drosophila whirlin (USH2D) (Ahmed et al., 2013; Cosgrove and Zallocchi, homolog of the TMC proteins functions directly in auditory 2014). mechanosensation, it has been shown to regulate mechanosensation The Drosophila genome harbors homologs of several Usher in larval locomotion (Guo et al., 2016). genes and some of these have been characterized in Johnston’s A striking illustration of the molecular and genetic similarities organ (Table 2). The Drosophila homolog of myosin VIIA between Drosophila and mammalian hearing organs comes from [myosin VIIA; also known as crinkled (ck)] is required for hearing: the work of Göpfert and colleagues (Senthilan et al., 2012). Here, a loss of ck abolishes a fly’s auditory responses and causes the microarray survey of genes expressed in Johnston’s organ of wild- scolopidia to detach from the cuticle of the a2/a3 antennal joint in type and ato mutant flies identified 274 genes expressed in this Johnston’s organ (Li et al., 2016; Todi et al., 2005, 2008) (Fig. 4). auditory organ. Approximately 20% of these genes had a human Interestingly, Myosin VIIA is predominantly expressed in the ortholog implicated in hearing loss. This suggests that the fruit fly actin-rich scolopale cells and is enriched at their apical tips close might be a rich resource for identifying and characterizing genes to where a scolopidium inserts into the cuticle (Li et al., 2016), a involved in human deafness. In the next section, we describe recent pattern that is superficially similar to the localization of myosin work in which forward genetic screens identified a Drosophila gene VIIA in the actin-rich stereocilia tips of mammalian hair cells. that linked together two myosin genes previously implicated in However, whereas myosin VIIA functions in hair cell stereocilia to human syndromic hearing loss. mechanically couple mechanotransduction channels to the actin , Myosin VIIA is unlikely to have the same function Can Drosophila be used to model hereditary deafness and in scolopale cells, which ensheath the mechanosensitive cilia of hearing loss? the Johnston’s organ sensory neurons and secrete the dendritic cap The molecular and functional similarities between Johnston’s organ that anchors them to the cap cell and cuticle. However, it is likely and the mammalian cochlea summarized above suggest that some that myosin VIIA serves an anchoring role in both cell types, genes implicated in human deafness might have similar functions in because it is a high duty ratio motor – i.e. it predominantly binds to Johnston’s organ. Here, we focus on two particular forms of actin filaments rather than processing along them (Haithcock et al., syndromic hearing loss in humans, Usher syndrome and MYH9- 2011; Watanabe et al., 2006; Yang et al., 2006). Pcdh15 and Sans related disorders, as these well-characterized syndromes provide also have Drosophila homologs, named Cad99C and Sans, evidence of such functional similarity and highlight the potential respectively (D’Alterio et al., 2005; Demontis and Dahmann, advantages of using Drosophila as a model for studying hereditary 2009). Recent studies have shown that Cad99C, Sans and Myosin deafness. VIIA form a complex in Johnston’s organ and in the ovary of Drosophila (Glowinski et al., 2014; Li et al., 2016). In Johnston’s Conservation of Usher syndrome proteins and their organ, a subtle, low-penetrant phenotype of scolopidial interactions in Drosophila detachment can be observed in Cad99C mutants, resembling Usher syndrome is an autosomal recessive genetic disease [Online that of myosin VIIA mutants. Cad99C localizes to the apical tips of Mendelian Inheritance in Man (OMIM) #276900, #276904, scolopidia, reminiscent of the localization of Pcdh15 to the tip link #601067, #276901, #605472, #611383] that is characterized by region in mammalian hair cells. In addition, myosin VIIA, Cad99C varying degrees of deafness and retinitis pigmentosa-induced vision and Sans genetically interact with Ubr3, a regulator of Usher loss, and is the leading cause of deaf-blindness in humans proteins in Johnston’s organ (Li et al., 2016). (Boughman et al., 1983; Hartong et al., 2006; Keats and Corey, These data suggest that USH1 proteins are not only conserved in 1999; Kimberling et al., 2010). Based on the severity of clinical Drosophila, but that their interactions are important for the symptoms in patients, Usher syndrome is subdivided into three development and function of the Drosophila auditory apparatus. types: USH1, 2 and 3. To date, 16 genetic loci have been associated USH1 proteins appear to interact in the scolopidia tips to link the with this syndrome: nine for USH1, three for USH2, two for USH3 scolopale cell and cap cell, which might stabilize the scolopidial and two that have yet to be identified (Cosgrove and Zallocchi, unit during mechanotransduction where force is applied to the 2014; Mathur and Yang, 2015). Some missense mutations of Usher neuronal cilia through the extracellular dendritic cap (Fig. 3B,C). (USH) genes that retain residual function are also associated with This function of USH proteins in Johnston’s organ – where the nonsyndromic forms of deafness that do not cause vision loss (see, proteins appear to mediate intercellular interaction and attachment – for example, Schultz et al., 2011). is somewhat different to their functions in the mammalian hair cell, USH proteins regulate a variety of cellular processes, including in which the USH complex acts intracellularly to anchor adjacent actin-based trafficking, cell adhesion, scaffolding, and G-protein- or stereocilia. Disease Models & Mechanisms

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Table 2. Summary of USH loci, their human and Drosophila proteins, and predicted functions USH Human Drosophila type Locus Human gene protein ortholog Function References USH1 USH1B MYO7A myosin (crinkled) ck Cytoskeleton-associated motor protein Li et al., 2016; Todi et al., 2005, VIIA 2008 USH1C USH1C harmonin CG5921* Scaffold protein Ahmed et al., 2013; Cosgrove and Zallocchi, 2014; Pan and Zhang, 2012 USH1D CDH23 CDH23 No candidates Cell-cell adhesion; tip link protein Alagramam et al., 2011; identified Kazmierczak et al., 2007; Sollner et al., 2004 USH1E Unknown Unknown No candidates Unknown (21q21) identified 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 Unknown No candidates Unknown (15q22-23) identified USH1J CIB2 CIB2 Cib2 Calcium and integrin binding protein Riazuddin et al., 2012 USH1K Unknown Unknown No candidates Unknown (10p11.21- identified q21.1) USH2 USH2A USH2A usherin No candidates Stereocilia links; extracellular matrix Adato et al., 2005; Liu et al., 2007; identified Weston et al., 2000 USH2B Unknown Unknown No candidates Unknown (3p23-24.2) identified USH2C GPR98 VLGR1/ No candidates Stereocilia links; G-protein signaling Schwartz et al., 2005; Weston GPR98 identified et al., 2004 USH2D WHRN whirlin (dyschronic) Scaffold protein; stereocilia elongation and Jepson et al., 2012 dysc* staircase formation 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 candidates Histidyl transfer RNA synthetase Abbott et al., 2017; Puffenberger identified et al., 2012 – PDZD7 PDZD7 No candidates Scaffold protein with homology to harmonin Chen et al., 2014; Ebermann identified and whirlin; interacts genetically with et al., 2010; Schneider et al., USH2A and GPR98 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 indicate Drosophila homologs predicted by protein sequence homology that have yet to be functionally validated.

Several other USH genes have Drosophila homologs, such as MYH9-related disease harmonin (CG5921), Cib2 (cib2), whirlin (dyschronic) and clarin 1 Autosomal dominant MYH9-related diseases are caused by (CG1103) (Table 2). Although cib2 is expressed in the eye and is mutations in MYH9, which encodes nonmuscle myosin IIA. required for normal phototransduction in Drosophila, whether these These diseases are characterized by large platelets and homologs are also present in the scolopidia of Johnston’s organ and/ thrombocytopenia from birth, and are associated with a variable or participate in hearing remains unclear. Other vertebrate USH onset of progressive sensorineural hearing loss, presenile cataracts, proteins that are present in the ankle links that connect the base of elevated liver enzymes and renal disease, which initially manifests stereocilia, such as usherin and VLGR1/GPR98 (Adgrv1), do not as a glomerular nephropathy (Heath et al., 2001). Prior to the appear to have any obvious homologs in Drosophila (Table 2). identification of the underlying molecular cause, individuals with Moreover, the mechanism by which Drosophila USH proteins MYH9-related disease were variously diagnosed as having Epstein cause scolopidial attachment – for example, how the scolopidium syndrome, Fechtner syndrome, May-Hegglin anomaly or Sebastian inserts into the cuticle, or whether additional act with syndrome (Heath et al., 2001; Newell-Litwa et al., 2015). The onset Cad99C/Pcdh15 – remains unclear. Nevertheless, the clear and of hearing loss in patients with a MYH9-related disease can occur quantifiable phenotypes seen in Usher syndrome gene mutants in over a wide age range, from the first to the sixth decade (Savoia and Drosophila (Fig. 4), and the strong genetic interactions between Pecci, 2008), and frequently progresses over time. In addition, a Usher genes, supports the use of Drosophila, and the Johnston’s dominant mutation, MYH9R705H, causes the nonsyndromic deafness organ, as a model in which to study the functions of known and disorder called DFNA17 (Lalwani et al., 2000). newly identified Usher genes and to identify their regulators. Mammals have three isoforms of nonmuscle myosin II encoded Finally, one recently reported unexpected finding is that USH by three different genes: NMIIA (encoded by MYH9), NMIIB homologs in the Drosophila hearing organ genetically and (encoded by MYH10) and NMIIC (encoded by MYH14). Mice that physically interact with Myosin II (Li et al., 2016), the are homozygous for a Myh9 null mutation die during embryogenesis mammalian homologs of which also play a role in hearing. We by embryonic day (E) 7.5 (Conti et al., 2004; Mhatre et al., 2007). describe the function of Myosin II in hearing in flies and mammals Mice heterozygous for Myh9 do not exhibit any hearing loss or in the next section. changes in age-dependent hearing thresholds (Mhatre et al., 2007). Disease Models & Mechanisms

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A Key

LTLD Myosin VIIA

Sans

Harmonin

Stereocilia UTLD Cdh23

Pcdh15

Hair cell Mechanosensory

Scolopale cell Actin bundles

B Apical tip of a scolopidium in C Stereocilia tip links of a hair cell Drosophila Johnston’s organ in mammalian cochlea

Cap cell

Scolopale cell

Key Myosin IIA Sans Cad23 Cad99C

Myosin VIIA Harmonin Pcdh15 Ion channel

Fig. 3. USH1 proteins and Myosin II function in the apical region of Drosophila scolopidia and mammalian stereocilia. (A) Left: a schematic of the apical 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 high magnification 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 is located directly or in close proximity to other protein components of the mechanotransduction (MET) channel complex, including TMIE and TMHS. The adaptor proteins 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 they contact the cap cell. Myosin II ubiquitination promotes its interaction with Myosin VIIA, the levels of which are crucial for anchoring the apical junction complexes of the scolopidia. The motor activity of these might also be required to transport the myosin complex to the tips of the scolopale cell. Both Myosin VIIA and Myosin II likely bind to actin bundles in the scolopale cells and regulate the apical attachment of scolopidia. Two Drosophila homologs of Usher syndrome type I proteins, Cad99C (Pcdh15) and Sans, interact with Myosin VIIA in a protein complex. It is not clear whether Cad99C mediates attachment to neuronal cilia or cap cells as a homodimer or as a heterodimer with another adhesion molecule. (C) In mammalian hair cells, a USH1 protein complex that includes myosin VIIA, Sans, harmonin and Cdh23 is located close to the stereocilia tips. By analogy with Drosophila, 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 myosin VIIA-myosin II-USH1 protein complex to the stereocilia tips.

This suggests that MYH9-related diseases are not caused by MYH9 The single Drosophila homolog of NMIIA is encoded by the haplo-insufficiency. However, knock-in mice, in which one Myh9 myosin II gene, also known as zipper. This gene is expressed in allele has been modified to contain a specific dominant pathogenic Johnston’s organ, and Myosin II is enriched at the apical tips of Myh9 mutation, R702C, develop defects, including hearing loss and scolopidia, adjacent to Myosin VIIA (Fig. 3B) (Li et al., 2016). kidney disease, that are similar to symptoms associated with several Myosin II is also required for the normal apical attachment of MYH9-related syndromes such as May-Hegglin anomaly (Suzuki scolopidia. Consistent with this, the gene encoding the regulatory et al., 2013). Although Myh9 is expressed in hair cells and NMIIA light chain of Myosin II, spaghetti squash (sqh), genetically localizes to the stereocilia and apical surface of the hair cell (Li et al., interacts with myosin VIIA in Johnston’s organ and affects hearing 2016; Meyer Zum Gottesberge and Hansen, 2014; Mhatre et al., (Todi et al., 2008). Recent studies in Drosophila show that Myosin 2006), NMIIA is also broadly expressed in many other parts of the II physically interacts with Myosin VIIA, probably through the cochlea. It is therefore possible that the deafness that occurs in mono-ubiquitination of Myosin II, which is regulated by the E3 MYH9-related diseases might not be directly caused by hair cell ligase, Ubr3 (Li et al., 2016). The myosin IIA-myosin VIIA defects. Thus, the molecular mechanism by which MYH9 mutations interaction and the mono-ubiquitination of myosin IIA also occur in cause hearing loss remains unclear. the mammalian cochlea (Li et al., 2016), although an interaction Disease Models & Mechanisms

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Fig. 4. Apical detachment of scolopidia in Drosophila Ubr3 A B mutants. (A) A confocal image of the second antennal segment of a mosaic adult Drosophila in which GFP+ marked cells are Detached Ubr3−/− (mutant) and GFP– cells are Ubr3+/− (wild-type control). scolopidia 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 of NompA. The white outline box (upper) and yellow outline box (lower) show high-power images of the NompA pattern in the apical junction from wild-type or Ubr3 mutant scolopidia, respectively. (B) A schematic summary of the apical detachment of scolopidia in Ubr3 and other Usher mutants, such as myosin VIIA and Cad99c. Arrows indicate detached scolopidia. Image in A taken from Li et al. (2016).

A2 antennal segment

10 µm

between these two myosins in stereocilia (Fig. 3C) remains levels can lead to experimental artifacts. In recent years, advances in speculative. The overexpression of different pathogenic variants recombination-mediated cassette exchange (RMCE; Box 1) of Myosin II in Johnston’s organ leads to the apical detachment of technology and the ability to perform homologous recombination scolopidia, which also occurs in Drosophila USH mutants (Li et al., in Drosophila using the CRISPR-Cas9 system (Box 1) have allowed 2016). These data indicate that Myosin II functions in Drosophila researchers to rapidly test the function of human genes in hearing, together with Myosin VIIA and/or other USH1 proteins; Drosophila and to rapidly assay the effects of potentially this interaction might also underlie the pathogenic effects of some pathogenic human variants far more quickly than in mice. In this pathogenic variants of myosin IIA in MYH9-related disease. The section, we describe the components of this technology, which is clear cut and quantitative scolopidial detachment phenotype (Fig. 4) summarized in Fig. 5. associated with myosin II and myosin VIIA mutations in Johnston’s organ provides a quick and easy assay with which to investigate the Gene trap cassette integration and exchange molecular function of Myosin II in hearing and the mechanisms by An identified ortholog of a human or mouse gene is a prerequisite which pathogenic Myosin II variants cause hearing loss. In the next for functional testing in Drosophila. Although not all candidate section, we describe new technology that exploits the Drosophila to disease genes will have invertebrate orthologs, ∼60% of the protein- human conservation of genes implicated in hearing loss to rapidly coding genes in the Drosophila genome are conserved in mammals, test the function of human genes and their variants in Drosophila. and Drosophila orthologs have been identified for about two-thirds of human disease genes (Chien et al., 2002; Hu et al., 2011; Reiter New approaches for modeling human deafness in Drosophila and Bier, 2002; Spradling et al., 2006). Numerous Drosophila Mutagenesis-based screens in mice and Drosophila have identified Minos-mediated integration cassette (MiMIC) lines are available candidate genes responsible for hearing loss and age-related hearing (Venken et al., 2011) that can be further modified using RMCE loss (Eberl et al., 1997; Hrabe de Angelis et al., 2000; Kernan et al., (Nagarkar-Jaiswal et al., 2015a,b). Using this technique, an artificial 1994; Nolan et al., 2000; Potter et al., 2016; Senthilan et al., 2012). exon that serves as a gene trap – such as a GAL4 Trojan exon Researchers used a large-scale lethal mosaic screen (Box 1) to cassette (Diao et al., 2015) – can be inserted into a MiMIC line to isolate mutations that affect the Drosophila peripheral nervous create a premature termination signal that disrupts the function of the system (Yamamoto et al., 2014). A secondary mosaic screen was endogenous gene. If MiMIC lines are not available or suitable for a then performed in this mutant collection by creating mutant clones particular gene, it is now possible to insert MiMIC-like cassettes in in the antenna to identify mutants with an abnormal Johnston’s Drosophila genes using CRISPR-mediated homology-directed organ (Li et al., 2016). As discussed in previous sections, genes repair (Port et al., 2014; Ren et al., 2013). implicated in hearing in these Drosophila screens include those known to cause hearing loss in humans or mice. The convenience of Green fluorescent protein tagging to characterize expression and Drosophila as a model organism, including its short life span and function armory of genetic tools, makes it an attractive system to investigate Artificial exons can be created to have flanking sites that facilitate the function of genes involved in hearing. Historically, RMCE (Nagarkar-Jaiswal et al., 2015a,b; Venken et al., 2011). overexpression experiments have been used to test gene function These exons allow fluorescent tags to be introduced into a gene of in Drosophila, either using the relevant fly gene or a conserved interest to reveal its cellular and subcellular expression pattern mammalian homolog. However, it is not always possible to (Fig. 5). This approach has been used to modify hundreds of unambiguously identify mammalian homologs of Drosophila Drosophila genes, providing detailed expression data and with genes based on sequence alone. Moreover, these approaches are ∼75% of the genes retaining their function after incorporation of the not necessarily able to dissect why particular human gene variants internal exon-encoding green fluorescent protein (GFP), as shown are pathogenic, especially in the case of missense mutations. In by their ability to rescue null alleles (Nagarkar-Jaiswal et al., addition, overexpressing a gene or gene variant at nonphysiological 2015b). To date, nearly 10,000 Drosophila genes have been GFP Disease Models & Mechanisms

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Reverse genetics: study function of candidate genes Candidate genes A Assess LOF phenotypes associated Ortholog prediction with T2A-GAL4 gene trap mutants Overexpress the human variants or Drosophila gene ‘humanized’ version of fly genes Rescue with human cDNA; express human pathogenic variants using T2A-GAL4 B CRISPR (precise temporal and spatial expression) SA 2A GAL4 PolyA

Forward genetics: identify novel regulators in hearing Insertion of Trojan exon Premature termination Knockdown of proteins through and GAL4 expression Mutagenesis-based screens C iGFPi or deGradFP SA followed by mapping 2A GAL4 PolyA (efficient knockdown; bypass mapping) RMCE Expression pattern in auditory organs SA GFP SD

Microarray, RNA-seq, Characterize expression pattern antibody staining, and protein localization using GFP exon GAL4 reporter lines GFP-tagged protein trap lines Fluorescent protein tagging

Traditional strategies New strategies SA GFP SD

Fig. 5. Approaches to functionally test candidate mammalian deafness genes in Drosophila. (A) Candidate genes can include known deafness genes or mouse genes with deafness phenotypes. Their Drosophila homologs are predicted by protein sequence homology. (B) An artificial exon that acts as a gene trap can be inserted into a MiMIC line by recombination-mediated cassette exchange (RMCE; Venken et al., 2011) or by CRISPR-mediated homology-directed repair, which prematurely terminates transcription of the endogenous gene through a splice acceptor site (SA) and poly-adenylation signal (PolyA), and expresses the GAL4 transcriptional activator as part of the endogenous transcript through a picornavirus self-cleaving 2A peptide sequence (2A). (C) Gene trap/GAL4 lines are screened for hearing phenotypes. The GAL4 protein expressed by the endogenous regulatory elements of the gene of interest can alsobe used 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 spatiotemporal pattern that is identical to the endogenous Drosophila gene. RMCE through attP and attB sites (yellow triangles) can convert the Trojan exon into an exon containing 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. tagged in this way (Kanca et al., 2017). These lines also allow gene Testing mammalian homologs and pathogenic variants and protein function to be tested through the targeting of the GFP- Having identified and characterized the function of Drosophila encoding exon with RNA interference (RNAi) approaches orthologs of mammalian genes in hearing, it is possible to test their (Neumuller et al., 2012; Pastor-Pareja and Xu, 2011). In the functional conservation by rescuing a Drosophila mutation with one adult fly, however, the stability of some proteins in postmitotic of its human or mouse orthologs. The use of GAL4-Trojan gene adult cells can preclude efficient RNAi knockdown. To address traps (Fig. 5) is particularly useful in this regard. In this approach, this, a genetic system known as the deGradFP system can be used the GAL4 transcription factor is targeted to an endogenous to directly degrade the GFP-tagged protein by using a modified Drosophila exon. By fusing the GAL4 sequence to a T2A form of the ubiquitin-proteasome pathway to specifically target peptide-coding sequence, the endogenous protein begins to be and degrade GFP-tagged proteins (Caussinus et al., 2012, 2013) translated, but is then truncated and GAL4 protein is expressed (Fig. 5). instead. This transcriptional activator then activates the expression of a mammalian complementary DNA (cDNA) of interest under the Testing auditory function in Drosophila control of a UAS promoter from a separate locus in the Drosophila Drosophila mutants can be assessed for auditory function in genome (Fig. 5). Thus, it is possible to inactivate a Drosophila gene several ways. In adult flies, a sound-evoked compound action and simultaneously assay whether a mammalian homolog can potential can be recorded from the antennal nerve (Eberl et al., functionally rescue the inactivated gene. Moreover, once it has been 2000; Todi et al., 2008), although this method is less sensitive if established that a mammalian cDNA can rescue its Drosophila small clones of mutant cells are being assayed in an otherwise ortholog, it is then possible to test variants of the mammalian gene wild-type background (Li et al., 2016). Behavioral assays can also to determine whether they are pathogenic, and if so, to evaluate the be used to detect hearing in response to stimuli such as mating cellular or biochemical consequences of such pathogenic variants songs (Eberl et al., 1997), or the response of flies to the force of (Bellen and Yamamoto, 2015). The function of potentially gravity (Kamikouchi et al., 2009), although, once again, these dominant variants can also be assessed in this way by suffer from a limited sensitivity. It is also possible to measure overexpressing the variant of interest in Johnston’s organ to see if active movement of the antenna by laser vibrometry and to record it elicits a phenotype. For example, pathogenic variants of Myh9 the activity of single giant neurons in the Drosophila brain known to cause deafness in humans also cause morphological (Kamikouchi et al., 2009; Tootoonian et al., 2012). Finally, it is defects in scolopidia when overexpressed in Johnston’s organ (Li possible to examine the morphology of Johnston’sorganin et al., 2016). different mutants using an array of antibodies and GAL4 lines The GAL4-Trojan system has several advantages over (Box 1) that express fluorescent proteins in the cellular conventional methods to test gene function by overexpression. components of scolopidia. First, it can test the function of a mammalian gene in the context of a Disease Models & Mechanisms

11 REVIEW Disease Models & Mechanisms (2018) 11, dmm031492. doi:10.1242/dmm.031492 null mutation for its Drosophila counterpart. Second, targeting the to name but three, stabilize protein complexes, enhance the function GAL4 cassette to the Drosophila gene of interest significantly of the affected protein(s) or proteins acting downstream of the increases the likelihood that the GAL4 activator will be expressed in affected proteins, and slow the aggregation of pathogenic protein the same spatial and temporal pattern as the endogenous Drosophila variants. Once again, we suggest that Drosophila might offer a gene. Finally, the incorporation of DNA sequences in the Trojan promising platform in which to rapidly screen for compounds or cassette that permit RMCE allows the locus to be modified further signaling pathways that improve auditory function in animals with little effort. Although these advances are significant, they still bearing pathogenic hearing gene variants. have limitations. For example, this approach is only feasible if clear gene homologs can be identified between Drosophila and Competing interests mammals. Moreover, although expression of the GAL4 The authors declare no competing or financial interests. transactivator might be faithful, the UAS promoter might drive Funding expression of the mammalian gene at levels greater than those of the This work was supported by the National Institute on Deafness and Other endogenous Drosophila gene. Communication Disorders (DC014932) to A.K.G. H.J.B. is a member of the Howard Hughes Medical Institute. Conclusion References The field of genetic hearing loss research has advanced significantly Abbott, J. A., Guth, E., Kim, C., Regan, C., Siu, V. M., Rupar, C. A., Demeler, B., in the last several decades, but three challenges and opportunities Francklyn, C. S. and Robey-Bond, S. M. (2017). The usher syndrome type IIIB remain. 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