Mechanisms and Genes in Drosophila Hearing

Review article

e-Neuroforum 2014 · 5:72–76 M. Kittelmann · M.C. Göpfert DOI 10.1007/s13295-014-0063-7 Abteilung Zelluläre Neurobiologie, Universität Göttingen, Göttingen © Springer-Verlag 2014 Mechanisms and genes in Drosophila hearing

Introduction Another important aspect was the ability ceivers, functionally corresponding to our to analyze molecular hearing mechanisms tympanic membrane. As the arista is stiff- The genome sequence of the fruit fly noninvasively, due to the direct coupling ly coupled to the funiculus, both compart- Drosophila melanogaster was published between molecular processes and the sys- ments swing around the longitudinal axis in 2000. One year later, a study screening tematic performance of the fly ear. of the latter upon exposure to sound. The the fly genome for disease-related genes funiculus rotation is directly detected by followed: 548 genes implicated in 714 hu- The Drosophila ear the mechanosensory cells of the Johnston’s man diseases were found. The first au- organ—the fly equivalent of the organ of thor of the study, Laurence Reiter, com- Drosophila males use courtship songs to Corti in the human ear. The Johnston’s or- mented “this came as a bit of a surprise as attract females. When females are in the gan is located in the pedicellus of the an- most people don’t think to study hearing proximity, males extend one of their wings tenna and consists of about 200 chordo- or cancer in Drosophila”. In terms of hear- and vibrate it in a regular pattern, result- tonal stretch receptors. Each of these re- ing, this is entirely true: we have known ing in a courtship song dominated by fre- ceptors contains two or three sensory that Drosophila can hear since the 1960s; quencies of around 200 Hz. These songs cells and several supporting cells, which however, for a long time, only few sci- enhance the receptivity of the females to form via mitotic division of a precursor entists investigated hearing in flies. The copulation and animate other males to cell. This precursor cell is specified via findings of these studies were astonishing sing along. the transcription factor atonal, which al- and hearing in fruit flies has thus become Males as well as females detect the so regulates the development of hair cells a very competitive field of research over courtship songs with their antennae. in the vertebrate ear. Despite this simi- the past years. Not only was the availabili- These are located at the front of the fly larity, fly auditory sensory cells and ver- ty of new genetic methods to analyze and head (. Fig. 1). Every antenna is com- tebrate hair cells are anatomically differ- manipulate the function of sensory cells posed of three main compartments—the ent: in flies these cells are bipolar mecha- in Drosophila essential, but the identifica- proximal scapus, the pedicellus and the nosensory neurons with a proximal axon tion of many functional parallels between distal funiculus. The club-shaped funicu- and a distal ciliated dendrite that is direct- fly and vertebrate hearing was also crucial. lus and its feathery arista are the sound rely attached to the funiculus via an extra-

Fig. 1 8 Fly hearing organ a lateral view of the fly head. The antenna comprises the scapus (s), the pedicellus (p) and a funiculus (f) with a laterally attached arista (a). b Schemata of the antennal ear. The arista (a) and the funiculus (f) together form the sound receiver and rotate around the longitudinal axis of the latter when stimulated acoustically (dotted line and black arrows). Vibration of the funiculus is directly detected by the mechanosensory neurons of the Johnston’s organ in the pedicellus (p), which thus act as gravity and wind receptors or auditory sound receptors. c Schemata of a single mechanosensory neuron of the Johnston’s organ with its proximal dendritic cilium, soma and axon. The Nan-Iav TRP ion channels localize to the proximal part of the cilium, the NOMPC TRP channels to the distal ciliary tip

72 | e-Neuroforum 3 · 2014 the loss of the other neuron classes does not affect this process. In addition to active amplification characteristics, mechanical signatures of transduction can be identified by analyzing the performance of the fly antennal receiver and these are similar to the mechanisms detectable in the mechanosensory hair bundles of hair cells (. Fig. 2). Quantitatively, these transduction signatures in the fly ear can be well explained by the “gating spring model” of hair cell transduction. This model assumes a serial arrangement of mechanically activated ion channels, “gating springs” and motors. The gating springs are elastic elements that transfer force to the ion channel, whereby the deformation of the spring determines the opening probability of the channel. Upon opening of the channel, the gating springs are relieved, which reduces the stiffness of the antennal receiver. After prolonged deflection, channels are closed by motors and the original stiffness is restored. Physical simulations have revealed that Fig. 2 8 a Active amplification and transduction. Sound-induced vibrations of the antennal receiv- the interplay between opening of the ion er (1) are transferred to the neurons of the Johnston’s organ (2), which then convert the vibration in- channels and the resulting motor move- to an electric signal (3). The transmission is realized by mechanically activated ion channels. The cou- ments is sufficient to explain active am- pling of the mechanical stimulus to the ion channels occurs via “gating springs”. Together with motors plification in the fly ear. This implies that (4), opening of the ion channels causes an active movement of the neurons, which amplify the sound- induced vibrations of the sound receiver. b Structure of the NOMPC protein. Besides a channel pore re- active amplification and transduction are gion, NOMPC contains 29 ankyrin residues that possibly act as a “gating spring”. Adapted from Göpfert coupled. The same transduction-driven [4] (a) and Zanini und Göpfert [10] (b) amplification mechanism explains the active motility of the sensory hair bundles cellular cap. The vibrations of the funic- Auditory mechanisms: active of vertebrate hair cells, which, alongside ulus are converted into electrical signals, amplification and transduction Prestin-driven somatic motility, contrib- which are then encoded as action poten- utes to active amplification in the verte- tials by the neurons and transmitted via The Drosophila ear does not contain a brate ear. their axons into the antennal mechano- middle ear and sensory cells are directly sensory motor center in the fly brain. coupled to the sound receiver. Therefore, Channels, gating Regarding their axonal projection tar- the neurons directly influence the mech- springs and motors get, the approximately 500 mechano- anistic behavior of the sound receiver, sensory neurons can be subdivided in- which can be measured noninvasively via Several ion channels of the transient re- to five classes, of which only the first two laser Doppler vibrometry. Analyses of the ceptor potential (TRP) family are in- are apparently true “hearing cells”: Cal- vibration characteristics of the antennal volved in transduction and amplification cium imaging showed that the 250 neu- receiver have shown that it exhibits all fea- in the fly ear, including the TRPN1 chan- rons of classes A and B are very sensitive tures of the cochlear receiver in vertebrate nel “No mechanoreceptor potential C” to vibrations of the funiculus, whereas the ears, i.e. it is an active amplifier based on (NOMPC), and the TRPV channels Nan- 200 neurons of classes C and E preferen- the active movement of the hair cells. Ge- chung (Nan) and Inactive (Iav). tially respond to sustained mechanical de- netic manipulations showed that corre- NOMPC is a mechanically activated flection of the funiculus and detect gravity sponding active deflection of the mech- mechanotransduction channel that lo- and wind. The remaining 50 class D neu- anosensory neurons in the Johnston’s or- calizes to the distal ciliary region of the rons are activated by prolonged deflection gan cause active amplification in the fly mechanosensory neurons. Loss of the and vibration of the funiculus, although ear and that only class A and B auditory channel leads to a complete loss of ac- large deflection and vibration amplitudes neurons are necessary: without these neu- tive amplification and sensitive hearing are required. rons, amplification is disrupted, whereas response. Without NOMPC, only loud

e-Neuroforum 3 · 2014 | 73 Abstract · Zusammenfassung

sounds can evoke an electric response yet been identified on a molecular level. e-Neuroforum 2014 · 5:72–76 in the fly ear. The latter responses likely NOMPC itself seems to be a good candi- DOI 10.1007/s13295-014-0063-7 © Springer-Verlag 2014 originate from the gravity and wind-sensi- date, as its N-terminus contains a molec- tive class C and E neurons, since they per- ular spring composed of 29 ankyrin res- M. Kittelmann · M.C. Göpfert sist when class A and B neurons are ab- idues. Recent findings suggest that this Mechanisms and genes lated and correlate with the calcium sig- spring couples the NOMPC channel to in Drosophila hearing nals in class C and E neurons. Signatures microtubules. Manipulation of the num- of the channel opening mechanics in the ber of ankyrin residues will be needed to Abstract The fruit fly Drosophila melanogaster com- antennal receiver provide evidence that show if these act as a gating spring. It is municates acoustically and hears with its an- NOMPC could be the transduction chan- also possible that the cell membrane em- tennae. Fundamental aspects of hearing can nel of the class A and B auditory neurons: bedding these channels acts as the gating be studied in these antennal ears, the audi- analyzing the mechanical responses of the spring—a possibility that is currently dis- tory sensory cells of which are evolutionarily sound receiver over a broad range of stim- cussed for hair cells. related to vertebrate hair cells and are spec- ulus amplitudes, the opening signatures of Similarly, the motors that apparently ified developmentally by homologous transcription factors. Like vertebrate hair cells, at least two ion channel types are visible. drive active amplification in the fly ear to- Drosophila auditory sensory cells are also mo- One channel type is very sensitive and gether with NOMPC have also not been tile and actively amplify the mechanical vi- mainly associates with class A and B au- molecularly identified, whereby the cil- brations they transduce. The transduction ditory neurons. The second channel type ia of the mechanosensory neurons in the and amplification mechanisms rely on the in- is rather insensitive and mainly affiliat- fly ear show dynein-like arms and axone- terplay between mechanically activated ion ed with the gravity- and wind-sensitive mal dyneins are therefore excellent can- channels and motor proteins, whose movement impacts upon the macroscopic perfor- class C and E neurons. Class C and E neu- didates. A genetic screen recently iden- mance of the ear. The first molecular trans- rons function in a NOMPC-independent tified several axonemal dynein genes ex- ducer components have been identified and manner; however, mechanical opening of pressed in the fly ear. Initial results sug- various auditory system-relevant proteins the sensitive channels requires NOMPC. gest that these genes are required for ac- have been described. Several of these pro- Without NOMPC, these channels are tive oscillation amplification. If dynein teins are conserved components of cilia, suggesting the fly’s ear as a model for human cil- mechanistically decoupled from the an- motors do drive active amplification in iopathies. The evolution of sensory signaling tennal receiver, suggesting that NOMPC the fly ear, the amplification and trans- cascades can also be studied using the fly’s forms the sensitive channels or transfers duction mechanisms used by the sensory ear, as the fly employs key chemo- and pho- the vibrations of the sound receiver to the cells in the fly would be similar to verte- toreceptor proteins to hear. Evidence is al- channels. brate hair cells, but the molecules involved so accumulating that the fly’s ear is a multi- Both TRPV channels Nan and Iav are would be different. While TRP channels functional sensory organ, which, in addition to mediating hearing, serves to detect wind, located in the proximal ciliary region of function as auditory transduction chan- gravity and presumably temperature. the mechanosensory neurons, possi- nels in the fly, transmembrane channel- bly forming heteromeric Nan–Iav chan- like (TMC) proteins are discussed as au- Keywords nels. Loss of Nan–Iav leads to complete ditory transduction channels in hair cells. Antennae · Mechanotransduction · Cilia · TRP loss of electric responses in the neurons; In addition, hair cells use prestin and my- ion channels · Signaling however, active amplification persists and osin motors for active amplification. Al- becomes excessive. The complete abol- though prestin is present in the fly ear, it ishment of electrical responses indicates remains unclear whether it plays a role in several of these genes cause primary cil- that Nan–Iav could form the transduc- the amplification process. iary dyskinesia and Kartagener syndrome tion channel. NOMPC would then act as in humans. These inheritable diseas- preamplifier and, together with the mo- Ciliary genes and chemo- and es are caused by defects in primary cilia, tors, generate the amplification, whereas photoreceptor proteins the same type of cilia that play a role in Nan–Iav would generate the gross trans- fly hearing. New genes causing these dis- duction current. Alternatively, Nan–Iav The auditory sensory cells of flies and eases were identified using the fly ear, thus could propagate the electric signals of vertebrates are evolutionarily related, al- proving it to be an interesting model sys- NOMPC and the as yet unidentified less though they evidently use different pro- tem and not just for studying hearing. sensitive transduction channels. Howev- teins for amplification and transduction. Surprisingly, some of the 274 genes er, as long as it remains unclear whether This is mirrored by the use of a similar identified in the fly hearing organ encode Nan–Iav channels are mechanically acti- genetic toolkit: of 274 recently identified chemo- and photoreceptor proteins. One vated mechanotransduction channels, we genes expressed in the fly ear, every fifth such example is the new family of iono- cannot discriminate between these two has a counterpart in humans that is in- tropic receptors, chemoreceptor proteins possibilities. volved in hearing defects. Many of these present in certain chemosensory cells of Gating springs, the elastic components genes encode motors and channels. More- the fly. Additionally, nearly all compo- that transmit mechanical force to the over, many conserved ciliary genes are in- nents of the phototransduction cascade mechanotransduction channels, have not volved in fly hearing and mutations in from the fly eye, including visual rhodop-

74 | e-Neuroforum 3 · 2014 sin and the TRP channels forming the cells, but were already present and played Prof. Dr. M.C. Göpfert Abteilung Zelluläre Neurobiologie, Universität phototransduction channels, were found a sensory role. Analyzing the function of Göttingen in the fly ear. Functional analyses have these proteins in the fly ear promises to Julia-Lermontowa-Weg 3, 37077 Göttingen shown that mutations in most of these provide insights into the evolution of sen- Germany genes lead to hearing defects in flies. Even sory signaling cascades and possibly the [email protected] the visual rhodopsins, known to collect original sensory function of chemo- and Martin Göpfert studied biology at the Univer- photons in the fly eye, have proven to be photoreceptor proteins. sity of Erlangen and received his doctorate essential in fly hearing. Several visual rho- on acoustic detection of bats by nocturnal in- dopsins that differ in their sensitivity to The fly ear as sects in 1998. After postdocs at the University of spectral light are expressed in the audito- multifunctional organ Zürich (1998–2001) and the University of Bris- ry sensory cells in the fly ear. The rhodop- tol (2001–2003) in the lab of Prof. Daniel Rob- ert, Martin Göpfert became group leader of the sins localize to the sensory cilia of the cells As mentioned above, in addition to hear- Volkswagenstiftungs Nachwuchsgruppe “ac- and increase the sensitivity of the trans- ing, the hearing organ of the fly serves the tive auditory mechanics in insects” in the Animal duction channels, possibly by modulating detection of gravity and wind. New find- Physiological Institute at the University of Co- the stiffness of the cell membrane. Similar ings indicate that the auditory sensory logne. He was awarded a fellowship from Leop- rhodopsin-dependent stiffness modifica- cells are also important for sensing tem- oldina and a Royal Society University Research Fellowship, and received the Walther Arndt tions were recently described in fly pho- perature. Temperature sets the circadi- Award of the German Zoological Association toreceptors, where these modifications an clock and an important gene involved (2005) and the Biology award of the Göttingen are likely to activate the phototransduc- in this process is expressed specifically in Academy of Sciences and Humanities (2006) for tion channels. In contrast, the function of chordotonal stretch receptors, including his research on hearing in mosquitoes and Dro- rhodopsins in the fly ear seems to be in- those in the fly ear. Similarly, a connec- sophila. Since 2008, Martin Göpfert has been a full professor at the University of Göttingen, dependent of light, raising the question of tion between temperature sensation and where he is head of the Department for Cellular which stimulus activates the rhodopsins chordotonal stretch receptors was found Neurobiology. His major research interests are in the fly ear. in fly larva. Additionally, TRP channels, the genetic and functional basis of hearing and which play a key role in temperature sen- mechanosensory signal transduction. Evolution of sensory sation, are expressed in a subset of senso- signaling cascades ry cells in the flies Johnston’s organ. It is as Acknowledgments. Our work is funded by the yet unclear how the fly ear detects temper- Deutsche Forschungsgemeinschaft DFG (SPP 1608 The function of chemo- and photorecep- ature and how it encodes mechanical as and SFB 889). tor proteins in the auditory sensory cells of well as thermal stimuli. Considering the the fly is interesting in terms of their evo- functional diversity and the multiple key Compliance with lution: fly chemo- and photoreceptors de- components yet to be discovered, research ethical guidelines tecting chemical or light stimuli via iono- on the fly ear remains fascinating. tropic receptors or visual rhodopsins, re- Conflict of interest. M. Kittelmann and M.C. Göpfert state that there are no conflicts of interest. All nation- spectively, as well as auditory sensory cells Corresponding address al guidelines on the care and use of laboratory animals and vertebrate hair cells are specified by have been followed and the necessary approval was the transcription factor atonal. This sug- M. Kittelmann obtained from the relevant authorities. gests that these cells are evolutionarily re- Abteilung Zelluläre Neurobiologie, Universität lated and originate from a common an- Göttingen References Julia-Lermontowa-Weg 3, 37077 Göttingen cestral cell. These ancestral or protosen- Germany 1. Boekhoff-Falk G, Eberl DF (2014) The Drosophi- sory cells were presumably present in ev- la auditory system. Wiley Interdiscip Rev Dev Biol ery segment of the fly. This serial arrange- Maike Kittelmann studied biology at the Univer- 3:179–191 ment is still conserved in the chordotonal sity of Göttingen and received her doctor’s de- 2. Effertz T, Nadrowski B, Piepenbrock D et al (2012) Direct gating and mechanical integrity of Dro- stretch receptors used in fly hearing, indi- gree in 2012, investigating the synaptic ultra- structure and regulation of synaptic transmis- sophila auditory transducers require TRPN1. Nat Neurosci 15:1198–1200 cating that these receptors are very simi- sion in Caenorhabditis elegans. During her doc- lar to these protosensory cells. During the 3. Gong Z, Son W, Chung YD et al (2004) Two interde- torate Maike Kittelmann was awarded a scholar- pendent TRPV channel subunits, Inactive and Nan- course of segment specialization mediat- ship from the Education Abroad Program (EAP) chung, mediate hearing in Drosophila. J Neurosci ed by Hox genes, these protosensory cells for graduate students and was able to advance 24:9059–9066 evolved into chemo- and photoreceptor her electron microscopic studies for 1 year at 4. Göpfert MC (2007) Amplification and feedback in the University of California, San Diego. During invertebrates. In: Dallos P, Oertel D (eds) The sens- cells in certain body parts; whereas cells in her postdoc in the lab of Prof. Martin Göpfert es, a comprehensive reference, Vol. I. Elsevier, Am- other body regions retained their function (2012–2014) she focused on the ultrastructur- sterdam, pp 293–299 as chordotonal stretch receptors. The fact al components of the Drosophila ear. She was 5. Kamikouchi A., Inagaki HK, Effertz T et al (2009) The neural basis of Drosophila gravity-sensing and funded by the SPP 1608. that these stretch receptors use chemo- hearing. Nature 458:165–171 and phototransduction proteins suggests that these proteins did not arise with the evolution of chemo- and photoreceptor

e-Neuroforum 3 · 2014 | 75 Review article

6. Moore DJ, Onoufriadis A, Shoemark A et al (2013) Mutations in ZMYND10, a gene essential for prop- er axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia. Am J Hum Genet 93:346–356 7. Nadrowski B, Albert JT, Göpfert MC (2008) Trans- ducer-based force generation explains active process in Drosophila hearing. Curr Biol 18:1365–1372 8. Senthilan PR, Piepenbrock D, Ovezmyradov G et al (2012) Drosophila auditory organ genes and genetic hearing defects. Cell 150:1042–1054 9. Sehadova H, Glaser FT, Gentile C et al (2009) Tem- perature entrainment of Drosophila’s circadian clock involves the gene nocte and signaling from peripheral sensory tissues to the brain. Neuron 64:251–266 10. Zanini D, Göpfert MC (2013) Mechanosensation: tethered ion channels. Curr Biol 23:R349–R351

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