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.

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.

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.

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.

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.

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.

Two distinct functions of Lim1 in the Drosophila antenna.

The Lim1 transcription factor is required in Drosophila for patterning the eye-antennal disk. At the adult stage, Lim1 is strongly expressed in Johnston's Organ (JO) neurons, the antennal auditory organ. Using RNAi-mediated knockdown of Lim1 using a strong neuronal driver, we find a significant reduction in electrophysiological responses to auditory stimuli, recorded from the antennal nerve. This reduction can be accounted for by Lim1 knockdown in the auditory subset of JO neurons, with no effect of knockdown in JO neuron subsets associated with wind or gravity detection. Conversely, Lim1 knockdown in JO sense organ precursors had no effect on hearing. Mosaic animals with antennal clones of the Lim1 E9 null mutation showed morphological defects in the antenna, and significant auditory electrophysiological defects. Our results are consistent with two distinct functions for Lim1 in the antenna, including an early patterning function in the eye-antennal disk, and a later neural differentiation function in the JO neurons.

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.

Reflective environment heightens crayfish aggressive and fearful behaviors.

Animals typically respond to their reflection as a conspecific and will respond as if the reflection were another animal that they could interact with, either fearfully or aggressively. We investigated how a modified reflective environment of a standard glass aquarium affects the aggressive and fearful behaviors of the crayfish Orconectes virilis , based on pre-determined behavior criteria. We found that the crayfish were both increasingly aggressive and slightly fearful in the reflective environment compared to minimal behavioral changes in the control non-reflective environment. Thus, our findings support that crayfish recognize their mirror image as a conspecific.

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.

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.

TRPA channels distinguish gravity sensing from hearing in Johnston's organ.

Although many animal species sense gravity for spatial orientation, the molecular bases remain uncertain. Therefore, we studied Drosophila melanogaster, which possess an inherent upward movement against gravity-negative geotaxis. Negative geotaxis requires Johnston's organ, a mechanosensory structure located in the antenna that also detects near-field sound. Because channels of the transient receptor potential (TRP) superfamily can contribute to mechanosensory signaling, we asked whether they are important for negative geotaxis. We identified distinct expression patterns for 5 TRP genes; the TRPV genes nanchung and inactive were present in most Johnston's organ neurons, the TRPN gene nompC and the TRPA gene painless were localized to 2 subpopulations of neurons, and the TRPA gene pyrexia was expressed in cap cells that may interact with the neurons. Likewise, mutating specific TRP genes produced distinct phenotypes, disrupting negative geotaxis (painless and pyrexia), hearing (nompC), or both (nanchung and inactive). Our genetic, physiological and behavioral data indicate that the sensory component of negative geotaxis involves multiple TRP genes. The results also distinguish between different mechanosensory modalities and set the stage for understanding how TRP channels contribute to mechanosensation.

The role of bHLH genes in ear development and evolution: revisiting a 10-year-old hypothesis.

In mouse ear development, two bHLH genes, Atoh1 and Neurog1, are essential for hair cell and sensory neuron differentiation. Evolution converted the original simple atonal-dependent neurosensory cell formation program of diploblasts into the derived developmental program of vertebrates that generates two neurosensory cell types, the sensory neuron and the sensory hair cell. This transformation was achieved through gene multiplication in ancestral triploblasts resulting in the expansion of the atonal bHLH gene family. Novel genes of the Neurogenin and NeuroD families are upregulated prior to the expression of Atoh1. Recent data suggest that NeuroD and Neurogenin were lost or their function in neuronal specification reduced in flies, thus changing our perception of the evolution of these genes. This sequence of expression changes was accompanied by modification of the E-box binding sites of these genes to regulate different downstream genes and to form inhibitory loops among each other, thus fine-tuning expression transitions.

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.

Drosophila N-cadherin mediates an attractive interaction between photoreceptor axons and their targets.

Classical cadherins have been proposed to mediate interactions between pre- and postsynaptic cells that are necessary for synapse formation. We provide the first direct, genetic evidence in favor of this model by examining the role of N-cadherin in controlling the pattern of synaptic connections made by photoreceptor axons in Drosophila. N-cadherin is required in both individual photoreceptors and their target neurons for photoreceptor axon extension. Cell-by-cell reconstruction of wild-type photoreceptor axons extending within mosaic patches of mutant target cells shows that N-cadherin mediates attractive interactions between photoreceptors and their targets. This interaction is not limited to those cells that will become the synaptic partners of photoreceptors. Multiple N-cadherin isoforms are produced, but single isoforms can substitute for endogenous N-cadherin activity. We propose that N-cadherin mediates a homophilic, attractive interaction between photoreceptor growth cones and their targets that precedes synaptic partner choice.

Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila.

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy (and references therein). Here, we show that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals. We demonstrate that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity.

Unique transgenic animal model for hereditary hearing loss.

OBJECTIVES: This study capitalizes on the unique molecular and developmental similarities between the auditory organs of Drosophila and mammals, to investigate genes implicated in human syndromic and nonsyndromic hearing loss in a genetically tractable experimental animal model, the fruit fly Drosophila. METHODS: The Drosophila counterparts of 3 human deafness genes (DIAPH1/DFNA1, ESPN/DFNB36, and TMHS/DF-NB67) were identified by sequence similarity. An electrophysiological assay was used to record sound-evoked potentials in response to an acoustic stimulus, the Drosophila courtship song. RESULTS: Flies with mutations affecting the diaphanous,forked, and CG12026/TMHS genes displayed significant reductions in the amplitude of sound-evoked potentials compared to wild-type flies (p < 0.05 to p < 0.005). The mean responses were reduced from approximately 500 to 600 microV in wild-type flies to approximately 100 to 300 microV in most mutant flies. CONCLUSIONS: The identification of significant auditory dysfunction in Drosophila orthologs of human deafness genes will facilitate exploration of the molecular biochemistry of auditory mechanosensation. This may eventually allow for novel diagnostic and therapeutic approaches to human hereditary hearing loss.

Development of Johnston's organ in Drosophila.

Hearing is a specialized mechanosensory modality that is refined during evolution to meet the particular requirements of different organisms. In the fruitfly, Drosophila, hearing is mediated by Johnston's organ, a large chordotonal organ in the antenna that is exquisitely sensitive to the near-field acoustic signal of courtship songs generated by male wing vibration. We summarize recent progress in understanding the molecular genetic determinants of Johnston's organ development and discuss surprising differences from other chordotonal organs that likely facilitate hearing. We outline novel discoveries of active processes that generate motion of the antenna for acute sensitivity to the stimulus. Finally, we discuss further research directions that would probe remaining questions in understanding Johnston's organ development, function and evolution.