Comparative exploration of mammalian deafness gene homologues in the Drosophila auditory organ shows genetic correlation between insect and vertebrate hearing.

Johnston's organ, the Drosophila auditory organ, is anatomically very different from the mammalian organ of Corti. However, recent evidence indicates significant cellular and molecular similarities exist between vertebrate and invertebrate hearing, suggesting that Drosophila may be a useful platform to determine the function of the many mammalian deafness genes whose underlying biological mechanisms are poorly characterized. Our goal was a comprehensive screen of all known orthologues of mammalian deafness genes in the fruit fly to better understand conservation of hearing mechanisms between the insect and the fly and ultimately gain insight into human hereditary deafness. We used bioinformatic comparisons to screen previously reported human and mouse deafness genes and found that 156 of them have orthologues in Drosophila melanogaster. We used fluorescent imaging of T2A-GAL4 gene trap and GFP or YFP fluorescent protein trap lines for 54 of the Drosophila genes and found 38 to be expressed in different cell types in Johnston's organ. We phenotypically characterized the function of strong loss-of-function mutants in three genes expressed in Johnston's organ (Cad99C, Msp-300, and Koi) using a courtship assay and electrophysiological recordings of sound-evoked potentials. Cad99C and Koi were found to have significant courtship defects. However, when we tested these genes for electrophysiological defects in hearing response, we did not see a significant difference suggesting the courtship defects were not caused by hearing deficiencies. Furthermore, we used a UAS/RNAi approach to test the function of seven genes and found two additional genes, CG5921 and Myo10a, that gave a statistically significant delay in courtship but not in sound-evoked potentials. Our results suggest that many mammalian deafness genes have Drosophila homologues expressed in the Johnston's organ, but that their requirement for hearing may not necessarily be the same as in mammals.

A nonneural miRNA cluster mediates hearing via repression of two neural targets.

We show here that mir-279/996 are absolutely essential for development and function of Johnston's organ (JO), the primary proprioceptive and auditory organ in Drosophila Their deletion results in highly aberrant cell fate determination, including loss of scolopale cells and ectopic neurons, and mutants are electrophysiologically deaf. In vivo activity sensors and mosaic analyses indicate that these seed-related miRNAs function autonomously to suppress neural fate in nonneuronal cells. Finally, genetic interactions pinpoint two neural targets (elav and insensible) that underlie miRNA mutant JO phenotypes. This work uncovers how critical post-transcriptional regulation of specific miRNA targets governs cell specification and function of the auditory system.

The retrograde IFT dynein is required for normal function of diverse mechanosensory cilia in Drosophila.

Introduction: Cilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In Drosophila, anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. We previously isolated the beethoven (btv) mutant in a mutagenesis screen for auditory mutants. Although many btv mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology. Results: Here we molecularly characterize the btv gene and demonstrate that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. We also describe morphological changes in Johnston's organ as flies age to 30 days, and we find that morphological and electrophysiological phenotypes in this ch organ of btv mutants become more severe with age. We show that NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in btv mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, btv mutants show a 50% reduction in mechanoreceptor potentials. Discussion: Thus, the btv-encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs.

The Voltage-Gated Sodium Channel in Drosophila, Para, Localizes to Dendrites As Well As Axons in Mechanosensitive Chordotonal Neurons.

The fruit fly Drosophila melanogaster has provided important insights into how sensory information is transduced by transient receptor potential (TRP) channels in the peripheral nervous system (PNS). However, TRP channels alone have not been able to completely model mechanosensitive transduction in mechanoreceptive chordotonal neurons (CNs). Here, we show that, in addition to TRP channels, the sole voltage-gated sodium channel (NaV) in Drosophila, Para, is localized to the dendrites of CNs. Para is localized to the distal tip of the dendrites in all CNs, from embryos to adults, and is colocalized with the mechanosensitive TRP channels No mechanoreceptor potential C (NompC) and Inactive/Nanchung (Iav/Nan). Para localization also demarcates spike initiation zones (SIZs) in axons and the dendritic localization of Para is indicative of a likely dendritic SIZ in fly CNs. Para is not present in the dendrites of other peripheral sensory neurons. In both multipolar and bipolar neurons in the PNS, Para is present in a proximal region of the axon, comparable to the axonal initial segment (AIS) in vertebrates, 40-60 µm from the soma in multipolar neurons and 20-40 µm in bipolar neurons. Whole-cell reduction of para expression using RNAi in CNs of the adult Johnston's organ (JO) severely affects sound-evoked potentials (SEPs). However, the duality of Para localization in the CN dendrites and axons identifies a need to develop resources to study compartment-specific roles of proteins that will enable us to better understand Para's role in mechanosensitive transduction.

A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Myogenic contraction of a somatic muscle powers rhythmic flow of hemolymph through Drosophila antennae and generates brain pulsations.

Hemolymph is driven through the antennae of Drosophila melanogaster by the rhythmic contraction of muscle 16 (m16), which runs through the brain. Contraction of m16 results in the expansion of an elastic ampulla, opening ostia and filling the ampulla. Relaxation of the ampullary membrane forces hemolymph through vessels into the antennae. We show that m16 is an auto-active rhythmic somatic muscle. The activity of m16 leads to the rapid perfusion of the antenna by hemolymph. In addition, it leads to the rhythmic agitation of the brain, which could be important for clearing the interstitial space.

Using Sox2 to alleviate the hallmarks of age-related hearing loss.

Age-related hearing loss (ARHL) is the most prevalent sensory deficit. ARHL reduces the quality of life of the growing population, setting seniors up for the enhanced mental decline. The size of the needy population, the structural deficit, and a likely research strategy for effective treatment of chronic neurosensory hearing in the elderly are needed. Although there has been profound advancement in auditory regenerative research, there remain multiple challenges to restore hearing loss. Thus, additional investigations are required, using novel tools. We propose how the (1) flat epithelium, remaining after the organ of Corti has deteriorated, can be converted to the repaired-sensory epithelium, using Sox2. This will include (2) developing an artificial gene regulatory network transmitted by (3) large viral vectors to the flat epithelium to stimulate remnants of the organ of Corti to restore hair cells. We hope to unite with our proposal toward the common goal, eventually restoring a functional human hearing organ by transforming the flat epithelial cells left after the organ of Corti loss.

Goggatomy: A Method for Opening Small Cuticular Compartments in Arthropods for Physiological Experiments.

Most sense organs of arthropods are ensconced in small exoskeletal compartments that hinder direct access to plasma membranes. We have developed a method for exposing live sensory and supporting cells in such structures. The technique uses a viscous light cured resin to embed and support the structure, which is then sliced with a sharp blade. We term the procedure a "goggatomy," from the Khoisan word for a bug, gogga. To demonstrate the utility of the method we show that it can be used to expose the auditory chordotonal organs in the second antennal segment and the olfactory receptor neurons in the third antennal segment of Drosophila melanogaster, preserving the transduction machinery. The procedure can also be used on other small arthropods, like mosquitoes and mites to expose a variety of cells.

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals.

Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs.

Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion.

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs.

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila.

Cilia are essential for cell signaling and sensory perception. In many cell types, a cytoskeletal structure called the ciliary rootlet links the cilium to the cell body. Previous studies indicated that rootlets support the long-term stability of some cilia. Here we report that Drosophila melanogaster Rootletin (Root), the sole orthologue of the mammalian paralogs Rootletin and C-Nap1, assembles into rootlets of diverse lengths among sensory neuron subtypes. Root mutant neurons lack rootlets and have dramatically impaired sensory function, resulting in behavior defects associated with mechanosensation and chemosensation. Root is required for cohesion of basal bodies, but the cilium structure appears normal in Root mutant neurons. We show, however, that normal rootlet assembly requires centrioles. The N terminus of Root contains a conserved domain and is essential for Root function in vivo. Ectopically expressed Root resides at the base of mother centrioles in spermatocytes and localizes asymmetrically to mother centrosomes in neuroblasts, both requiring Bld10, a basal body protein with varied functions.

Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing.

In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, we generated and analyzed dpres mutant flies. We found that dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus we conclude that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, we show that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification.

Noise-induced hearing loss: new animal models.

PURPOSE OF THE REVIEW: This article presents research findings from two invertebrate model systems with potential to advance both the understanding of noise-induced hearing loss mechanisms and the development of putative therapies to reduce human noise damage. RECENT FINDINGS: Work on sea anemone hair bundles, which resemble auditory hair cells, has revealed secretions that exhibit astonishing healing properties not only for damaged hair bundles, but also for vertebrate lateral line neuromasts. We present progress on identifying functional components of the secretions, and their mechanisms of repair. The second model, the Johnston's organ in Drosophila, is also genetically homologous to hair cells and shows noise-induced hearing loss similar to vertebrates. Drosophila offers genetic and molecular insight into noise sensitivity and pathways that can be manipulated to reduce stress and damage from noise. SUMMARY: Using the comparative approach is a productive avenue to understanding basic mechanisms, in this case cellular responses to noise trauma. Expanding study of these systems may accelerate identification of strategies to reduce or prevent noise damage in the human ear.

The Drosophila auditory system.

Development of a functional auditory system in Drosophila requires specification and differentiation of the chordotonal sensilla of Johnston's organ (JO) in the antenna, correct axonal targeting to the antennal mechanosensory and motor center in the brain, and synaptic connections to neurons in the downstream circuit. Chordotonal development in JO is functionally complicated by structural, molecular, and functional diversity that is not yet fully understood, and construction of the auditory neural circuitry is only beginning to unfold. Here, we describe our current understanding of developmental and molecular mechanisms that generate the exquisite functions of the Drosophila auditory system, emphasizing recent progress and highlighting important new questions arising from research on this remarkable sensory system.

Physiological, anatomical, and behavioral changes after acoustic trauma in Drosophila melanogaster.

Noise-induced hearing loss (NIHL) is a growing health issue, with costly treatment and lost quality of life. Here we establish Drosophila melanogaster as an inexpensive, flexible, and powerful genetic model system for NIHL. We exposed flies to acoustic trauma and quantified physiological and anatomical effects. Trauma significantly reduced sound-evoked potential (SEP) amplitudes and increased SEP latencies in control genotypes. SEP amplitude but not latency effects recovered after 7 d. Although trauma produced no gross morphological changes in the auditory organ (Johnston's organ), mitochondrial cross-sectional area was reduced 7 d after exposure. In nervana 3 heterozygous flies, which slightly compromise ion homeostasis, trauma had exaggerated effects on SEP amplitude and mitochondrial morphology, suggesting a key role for ion homeostasis in resistance to acoustic trauma. Thus, Drosophila exhibit acoustic trauma effects resembling those found in vertebrates, including inducing metabolic stress in sensory cells. This report of noise trauma in Drosophila is a foundation for studying molecular and genetic sequelae of NIHL.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Cell-type-specific roles of Na+/K+ ATPase subunits in Drosophila auditory mechanosensation.

Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na(+)/K(+) ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a ß subunit required for its transport to the plasma membrane and for regulating its activity. We show that α and ß subunits are expressed in Johnston's organ (JO), the Drosophila auditory organ. We knocked down expression of α subunits (ATPα and α-like) and ß subunits (nrv1, nrv2, and nrv3) individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among ß subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell-specific support of the catalytic ATPα. Our studies provide an amenable model to investigate generation of endolymph-like extracellular compartments.