Cell-Type–Specific Roles of Na /K Atpase Subunits in Drosophila Auditory Mechanosensation

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

Madhuparna Roy, Elena Sivan-Loukianova, and Daniel F. Eberl1

Department of Biology, The University of Iowa, Iowa City, IA 52242

Edited by Charalambos P. Kyriacou, University of Leicester, Leicester, United Kingdom, and accepted by the Editorial Board November 22, 2012 (received for review May 27, 2012)

Ion homeostasis is a fundamental cellular process particularly a proximal axon (Fig. 1A). The scolopale cell, a principal support important in excitable cell activities such as hearing. It relies on cell, encloses the neuronal dendrites in a fluid-filled lumen, the + + the Na /K ATPase(alsoreferredtoastheNapump),whichis scolopale space. This fluid, the receptor lymph, resembles co- α β chlear endolymph and, like the endolymph, is believed to be rich composed of a catalytic subunit and a subunit required for its + transport to the plasma membrane and for regulating its activity. in K ions (7). The scolopale cells are structurally enforced with We show that α and β subunits are expressed in Johnston’sorgan actin-based scolopale rods. Auditory mechanosensation involves (JO), the Drosophila auditory organ. We knocked down expression the transduction of the mechanical sound stimulus (10) through of α subunits (ATPα and α-like) and β subunits (nrv1, nrv2,andnrv3) the rotation of distal antennal segments into a neuronal response individually in JO with UAS/Gal4-mediated RNAi. ATPα shows ele- in JO (11). Using electrophysiological techniques we can record vated expression in the ablumenal membrane of scolopale cells, sound-evoked potentials (SEPs) from the auditory nerve (9, 12). which enwrap JO neuronal dendrites in endolymph-like compart- The JO also mediates gravity and wind detection, in addition to auditory mechanosensation (13–15). ments. Knocking down ATPα,butnotα-like, in the entire JO or only fi We used the auditory mechanosensory system of Drosophila in scolopale cells using speci c drivers, resulted in complete deaf- melanogaster, with its molecular genetic and electrophysiological ness. Among β subunits, nrv2 is expressed in scolopale cells and nrv3 nrv2 techniques, to understand the role of the Na pump in maintaining in JO neurons. Knocking down in scolopale cells blocked Nrv2 auditory ion homeostasis. Our hypothesis is that the Na pump is α expression, reduced ATP expression in the scolopale cells, and important in maintaining the ion homeostasis of the auditory re- caused almost complete deafness. Furthermore, knockdown of ei- ceptor lymph. We show that ATPα is the sole Na pump α subunit in CELL BIOLOGY ther nrv2 or ATPα specifically in scolopale cells causes abnormal, JO and that it has elevated expression in scolopale cells. The β electron-dense material accumulation in the scolopale space. Simi- subunits show cell-type specific expression and functions in JO, larly, nrv3 functions in JO but not in scolopale cells, suggesting neu- with nrv2 being specific to scolopale cells and nrv3 specificto ron specificity that parallels nrv2 scolopale cell–specific support of neurons. We also show that ATPα preferentially localizes to the the catalytic ATPα. Our studies provide an amenable model to inves- scolopale cell ablumenal membrane. Such functional pump local- + tigate generation of endolymph-like extracellular compartments. ization is consistent with a role in pumping K ions into the scolopale cell en route to the receptor lymph, a role that resembles its receptor lymph | chordotonal organ | scolopidium | sensory cilium | contribution to generating vertebrate inner ear endolymph. scala media Results sing the energy of ATP hydrolysis, the Na pump extrudes cy- ATPα Is Expressed in JO Scolopidia and Required for Hearing. Dro- + + α α toplasmic Na (out) and extracellular K (in) in a 3:2 ratio and sophila has three subunit genes, ATP , JYalpha, and CG3701. U α maintains the gradient of these cations across the membrane (1, 2), ATP encodes at least nine mRNA isoforms (16). JYalpha is fi thus controlling the electrolytic and fluid balance in the cells and testes-speci c and has been linked to the mechanism for hybrid organs throughout the body (1). Among its other functions, the Na sterility between D. melanogaster and Drosophila simulans (17) pump helps maintain the resting potential of cells, regulates cellular and CG3701 is an α-like subunit with low expression in adult and volume, and facilitates transport of solutes in and out of cells. Ion pupal stages but moderate expression in testis (18). In adult flies, homeostasis of most biological systems depends on the Na pump. ATPα is expressed in the eye and brain (Fig. S1A), consistent In the auditory system, this pump has been linked to the mainte- with other studies (19), and in JO (Fig. 1B). nance of the inner ear osmotic balance (3). The scala media of the ATPα is expressed in the plasma membrane of JO neurons and + inner ear is filled with a K -rich extracellular fluid known as en- much more abundantly in scolopale cells (Fig. 1 B and C). To dolymph, which is essential for preserving the sensory structures determine if ATPα is functionally important in JO scolopidia, we and supporting transduction. Maintaining the endolymph homeo- wanted to test hearing in flies carrying ATPα mutations. However, stasis is critical to sustain auditory functions. Loss of endolymphatic ATPα mutants are homozygous lethal at early larval stages (20, balance causes collapse of the endolymphatic compartment, lead- 21). To circumvent these limitations, we used RNAi to knock + ing to hearing loss in mammals (4). K channels and pumps, in- down ATPα using the Gal4/UAS system (22, 23). We used ato- + cluding the Na pump, ensure proper cycling and secretion of K Gal4 (Fig. S2A) to drive expression of double-stranded RNA ions in the stria vascularis cells of the cochlea. The Na pump has under UAS control in the JO sense organ precursor cells to knock also been linked to age-related hearing loss (5) and Ménière dis- down ATPα only in these cells and their progeny. Knockdown ease (6). A detailed functional analysis of this pump is therefore animals were deaf, with complete loss of SEPs (Fig. 2). necessary to gain insight into the molecular physiology of hearing loss resulting from loss of auditory ionic homeostasis. Although vertebrate and invertebrate auditory systems differ Author contributions: M.R., E.S.-L., and D.F.E. designed research; M.R., E.S.-L., and D.F.E. structurally, they evolved from the same primitive mechanosensors performed research; M.R., E.S.-L., and D.F.E. analyzed data; and M.R. and D.F.E. wrote (7, 8), and there are striking developmental genetic similarities the paper. between the two lineages. The fly auditory organ, Johnston’s organ The authors declare no conflict of interest. (JO), is a chordotonal organ (cho) housed in the second antennal This article is a PNAS Direct Submission. C.P.K. is a guest editor invited by the segment (9). The JO comprises an array of ∼250 auditory units or Editorial Board. scolopidia. Each scolopidium comprises two to three ciliated sen- 1To whom correspondence should be addressed. E-mail: [email protected]. sory neurons associated with several support cells. These bipolar This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. neurons are monodendritic with a single distal cilium and 1073/pnas.1208866110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1208866110 PNAS Early Edition | 1of6 Downloaded by guest on October 4, 2021 required in the scolopale cells. To distinguish whether this requirement is developmental or physiological, we used Gal80ts,a temperature-sensitive repressor of Gal4, for temporal control. Conditions that prevent RNAi knockdown during development (18 °C) but allow it for 3 d at the adult stage (30 °C) resulted in significant hearing reduction compared with genotypically identical flies raised and maintained at 18 °C (Fig. S3). Thus, ATPα function is required at the adult stage after development is complete. ATPα is also required during development as flies raised at 30 °C and switched to 18 °C at adulthood are not rescued (Fig. S3). EM indicated abnormal accumulation of electron-dense material in the JO scolopale space in knockdown animals (Fig. 3 D and G). This material included membrane-bound organelles such α as mitochondria. To determine if such morphological defects af- Fig. 1. ATP is expressed in JO, with highest expression in scolopale cells. fected extracellular proteins known to be present in the scolopale (A) Diagram of a single JO scolopidium showing sensory neurons and support cells with important structural features. (B) Confocal image showing space, we examined Eyeshut/Spacemaker (Eys) protein (25, 26), ATPα protein expression in JO, stained with α5 monoclonal antibody (green). which localizes in the scolopale space (27), but found no differ- ATPα is specifically expressed in the neuron (N) cell body and the scolopale ences compared with control (Fig. S4). cell (S) as indicated by the white brackets. (C) Magnified image of JO scolopidia showing ATPα expression specifically in scolopale cells (white arrows) Functional Importance of ATPα. Campaniform and bristle organ and in the plasma membrane of neurons (white arrowhead). α5 (green) receptor lymph, and likely that of cho organs, is thought to be rich + counterstained with Texas Red phalloidin (magenta) labeling the actin in in K , resembling vertebrate inner ear endolymph (7). Therefore, + scolopale rods. (Scale bars: B and C, 10 and 5 μm, respectively.) our model is that the Na pump actively transports K ions toward the JO scolopale space. However, the Na pump is also essential for septate junction formation in a pump-independent manner Next we used UAS-RNAi against the α-like subunit (CG3701) (28, 29). Septate junctions are critical to maintain luminal integrity using the same ato-Gal4 driver. Driving CG3701 knockdown in in embryonic salivary glands and trachea. The scolopale cell has the JO sense organ precursor cell lineage had no effect on extensive septate junctions (30, 31), likely to seal the scolopale hearing (Fig. 2). This is consistent with CG3701 expression pri- space. Morphological defects such as accumulation of organelles marily in testis. JYalpha expression is also testis-specific (18). including mitochondria may indicate putative defects at the septate Therefore, ATPα is likely the only α subunit gene required for junctions between scolopale cell and cap cell or between the scolo- hearing in Drosophila. pale cell and the neuronal cell bodies. Accumulation of mitochondria in the scolopale cell could also imply metabolic stress. When ATPα Is Necessary for Scolopale Cell Function in Hearing. To investigate we stained knockdown (NompAGal4/+; UASRNAi-α/UASDicer2) if the elevated expression of ATPα in the scolopale cell has and control (NompAGal4/+; +/CyO) JO with Lucifer Yellow (32) physiological relevance for auditory function, we wanted to in conjunction with Texas Red Phalloidin, a marker for the remove it only from the scolopale cells. We used nompA-Gal4 actin-rich scolopale rods, we found that the Lucifer Yellow (24) (Fig. S2B) to drive ATPα RNAi only in JO scolopale cells. remained excluded from the scolopale space to the same extent Staining with an ATPα antibody showed that the RNAi knocks in knockdown and control animals (Fig. S5). This result suggests down ATPα protein almost completely in JO scolopale cells that ATPα may not be a major contributor to junctional integrity (Fig. 3B) compared with controls (Fig. 3A). However, ATPα of scolopale cells. Alternatively, the RNAi knockdown effect may expression is retained in other cell types, including cap cells and be insufficient to compromise the junctional complex while still neurons. Electrophysiological recordings revealed these knock- disrupting hearing physiology. down animals to be deaf (Fig. 2), indicating that ATPα function is Expression of β Subunits in JO. Flies have three Na-pump β-subunit genes encoded by nervana genes nrv1, nrv2,andnrv3. All three nrv genes have tissue-specific expression. nrv1 is present in the eye, muscle, heart, and fat body as well as digestive and excretory tissues (18), whereas nrv2 is important in the tracheal system, where it is required for pump-independent septate junction integrity (28, 29). The nrv3 gene is expressed in the brain, the eye, and the JO in adults (Fig. S1B) and in the embryonic CNS and cho sensory cells (Fig. S1D) (29). In addition, nrv3 is the principal β subunit in adult photoreceptor cells (19). To determine which of these three genes participate in JO function, we first tested their expression in JO. We stained cryosections of adult head with attached antennae using pan-Nrv monoclonal antibody Nrv5F7 (33) and a polyclonal antibody against Nrv3. Staining with these antibodies largely overlaps in the brain, eye, and the JO neurons. However, in the scolopale cell, there is specific staining only with Nrv5F7, but not with Nrv3 Fig. 2. ATPα, but not α-like, is required in JO for hearing. Histogram of SEPs α antibody (Fig. S6), indicating either or both Nrv1 and Nrv2 but from the antennal nerves of subunit knockdown (black bars) and control not Nrv3 are present in scolopale cells. Subsequent immuno- animals (white bars). The control genotypes are ato-Gal4/UAS-Dicer2 and NompA-Gal4/FM4; +/CyO, whereas the knockdown animals are ato-Gal4/ staining with a polyclonal antibody against Nrv1 (29) revealed no UAS-ATPα (or α-like)-RNAi; UAS-Dicer2/+ and nompA-Gal4; UAS-ATPα (or Nrv1 protein expression in the JO neurons and scolopale cells, α-like)-RNAi/CyO;+. Knockdown experiments are shown for two α subunit only a low level of Nrv1 expression in the cap cells (Fig. S7). genes (ATPα and α-like) with ato-Gal4 (drives expression in entire JO lineage) Previously, Nrv1 protein was found to express at low levels in the and nompA-Gal4 (drives expression in scolopale cells). Hearing loss in ATPα R7–R8 adult photoreceptor cells, but to be absent in brain tissue knockdown flies is highly significant with both drivers (two-tailed t test with (19). From the differential staining pattern of Nrv5F7 and Nrv3 Welch’s correction for ato-Gal4 (P < 0.001, n = 5 per genotype) and for (Fig. S6) and the Nrv1 pattern (Fig. S7), it is likely that only the nrv2 nompA-Gal4 (P < 0.0001, n = 15 per genotype.) gene has a scolopale cell–specific expression. Indeed, staining JO

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1208866110 Roy et al. Downloaded by guest on October 4, 2021 To reconcile the enriched Nrv5F7 antibody staining of scolopale cells and Nrv2 staining with the lack of functional defects upon RNAi-mediated knockdown of nrv1 or nrv2 in the JO sense organ lineage, we used nompA-Gal4 to test for scolopale cell– specific effects. Knockdown of nrv2, but not nrv1, resulted in almost complete hearing loss (Fig. 5). Thus, nrv2 has a strong requirement in scolopale cells. Failure of ato-Gal4 to reveal this function may reflect differences in expression level and timing compared with nompA-Gal4. Knocking down nrv1 specifically in the cap cells with the cap cell–specific pyx-Gal4 driver (13) had no effect on hearing (Fig. S7).

nrv2 Mediates Scolopale Cell-Specific Expression of ATPα. To confirm that hearing loss resulting from nrv2 knockdown is coupled with loss or decrease of Nrv2 protein from scolopale cells and to determine the effect on ATPα, we used the Nrv2-specific polyclonal antibody (29) and costained with α-5, a monoclonal antibody against ATPα (Fig. 4, Fig. S8). In control animals, Nrv2 and ATPα colocalized in the scolopale cell. A partial overlap was also observed in the ligament cells, another type of JO support cell. As seen previously, ATPα expressed most robustly in the scolopale cells, but also in the JO neurons and the antennal nerves (Fig. 4A). The Nrv2 protein stained only in the support cells with highest expression in the scolopale cells. In nrv2 knockdown animals, Nrv2 staining was completely lost from the scolopale cells, but the remaining supporting cells retained staining (Fig. 4B, Fig. S8). We also observed reduced ATPα expression in the scolopale cells with an almost complete loss in the cell body of these cells. To quantify

the decrease in ATPα and Nrv2 protein levels in the scolopale cells, CELL BIOLOGY we measured their fluorescent intensity ratios in scolopale cells versus neurons (Fig. 4) under identical imaging conditions using the neuronal signal from ATPα as the universal reference. Rel- ative Nrv2 expression is greatly reduced in scolopale cells in nrv2 knockdown animals. Similarly, relative scolopale cell ATPα expression is also strongly reduced in nrv2 knockdown animals, although still detectable (Fig. 4). Thus, nrv2 is required for ATPα Fig. 3. ATPα knockdown in JO scolopale cells disrupts the scolopale space. expression, localization, or stability in scolopale cells. Confocal images showing ATPα (α-5 mAb; green) and Nrv3 (αNrv3; magenta) EM analysis of JO in nrv2 knockdown animals revealed ab- expression in adult antenna in control animals (A) and in ATPα knockdown normal electron-dense material accumulation in the scolopale animals (B) in which ATPα expression is abolished in scolopale cells. Geno- space and presence of organelles, particularly mitochondria (Fig. types as in Fig. 2. (Scale bars: A and B,10μm). EM cross-sectional view 6), similar to our observations in ATPα knockdown animals (Fig. showing three adult JO scolopidia in control (C) and in scolopale cell ATPα 3 D and G). Furthermore, scolopidia in nrv2 knockdown animals knockdown animals (D). (E) Diagram of a scolopidium, with dotted line in- appear narrower than those in controls. These findings support dicating the level of cross sections (C and D). EM showing longitudinal-sec- Nrv2 as the specific β subunit partner of ATPα in scolopale cells. tions of adult JO control (F) and scolopale cell ATPα knockdown animals (G). The block arrows with black border show the electron light area in the Model for Subcellular Localization of ATPα. Our results indicate that scolopale space (SS) (C and F); the white block arrows show the electron- ATPα is present in the scolopale cell membrane (Fig. 1C). We dense material (D and G). Abnormal mitochondria present in the scolopale μ propose a model in which the Na pump is present preferentially in space as shown in (D) (asterisk). (Scale bar: C, D, F, and G,1 m.) the ablumenal (outer) plasma membrane of the scolopale cell, where + it pumps K ions into the scolopale cell cytoplasm in exchange for + + Na ions (Fig. 7A). This position will ensure the K enrichment of the with a polyclonal antibody against Nrv2 protein (29) indicates + scolopale cell cytoplasm with subsequent K movement to the sco- strong scolopale cell expression (Fig. 4A), although some staining lopale space receptor lymph by an unknown mechanism. In support is also seen in cap cells and ligament cells. Thus, among Na-pump fi β – fi of this model, we found in high-magni cation deconvolution images -subunit genes, there is a cell type speci c expression pattern with that ATPα is found primarily outside the scolopale rods (Fig. 7B). nrv3 expressed only in JO neurons, nrv2 expressed primarily in Furthermore, a transgenic flylinethathasachimericATPα–GFP scolopale cells, and nrv1 expressed at a low level in cap cells. fusion protein (34) shows similar localization. This protein (which may not reflect expression of all ATPα splice forms) accumulates β Subunit Functional Requirements in the JO. We wanted to test primarily around the junction between scolopale cell and cap cell or if lack of any of the nrv genes also had a physiological effect on between the scolopale cell and the neuronal inner dendritic segments hearing. Although we generated two intragenic nrv3 deletion 15 47 (Fig. 7C). This also implies a putative septate junction function of the mutants, nrv3 and nrv3 , they are lethal at early larval stages. Na pump, although our dye-exclusion approach revealed no junc- Similarly, nrv2 mutants are homozygous lethal (28), whereas no tional compromise. Although further experiments will be required to nrv1 mutant alleles are available. Therefore, to conduct adult fully address this question, our data are most consistent with a role hearing studies, we used RNAi to knock down expression of these β for the Na pump in receptor lymph homeostasis. subunit genes in the JO sense organ precursors using the ato-Gal4 driver. Knocking down expression of nrv1 or nrv2 with this driver Discussion hadnosignificant effect on hearing (Fig. 5). However, knocking The Na pump is important for sustaining JO auditory transduction down nrv3 with ato-Gal4 resulted in nearly complete deafness by mediating ion homeostasis. Our study revealed that the Na (Fig. 5). Thus, nrv3 is required in the JO, most likely in the JO pump is localized preferentially to the scolopale cell ablumenal + neurons based on its expression pattern. plasma membrane, from where it likely pumps K ions into the

Roy et al. PNAS Early Edition | 3of6 Downloaded by guest on October 4, 2021 Fig. 4. Scolopale cell-specific nrv2 gene knockdown abolishes Nrv2 protein and reduces ATPα protein. (A) Control JO section showing colocalization of ATPα subunit (α-5 mAb; green) and Nrv2 (α-Nrv2 Ab; red) in the scolopale cell. The scolopale cell and the neuronal cell body regions are indicated with white brackets. N or S represents neuronal or scolopale cell region (A and B), as indicated by the white boxes from which fluorescent intensities were measured. Genotypes as in Fig. 5. (B) Scolopale cell-specific nrv2 RNAi knockdown shows a complete absence of Nrv2 protein from the scolopale cell region in JOs processed side by side with the control JOs and imaged with identical settings. ATPα also appears reduced in these cells. (C) Bar

graph showing fluorescent intensity ratio (FS/FN) for Nrv2 and ATPα protein expression. FS represents the fluorescent intensity of the subunit proteins under investigation in the scolopale cell region (box labeled S in A and B)andFN represents ATPα expression in neurons (box labeled N in A and B). Data are from five control antennae and six nrv2 knockdown antennae. Using t tests, P = 0.0008 for Nrv2 protein expression, with Welch’s correction, and P < 0.0001 for ATPα expression. (Scale bar: A and B,10μm.)

scolopale cell cytoplasm en route to the scolopale space. Several The Na pump has a pump-independent cell junctional activity re- lines of evidence support this conclusion. First, we observed that sponsible for maintaining the epithelial barrier function in the ATPα, the principal JO α subunit, has a strikingly high expression in Drosophila tracheal system, mediated by the Nrv2 β subunit (28, the scolopale cell. This suggested that the ATPα gene has a scolo- 29). In the fly auditory system, failure to preserve junctional in- pale cell–specific specialized role. Second, most ATPα protein tegrity of the scolopale cells because of a lack of functional pumps localizes outside the scolopale rods, supporting an ablumenal may also cause fluid retention inside the scolopale space, which plasma membrane localization. Third, ATPα knockdown in the could manifest as the morphological abnormalities we saw when scolopale cell resulted in deafness, loss of scolopale cell integrity, either ATPα or nrv2 were knocked down in the scolopale cell. and morphological defects such as presence of distended cilia, However, our Lucifer Yellow dye exclusion assay, in animals in implying ionic imbalance in the scolopale space. Taken together, which ATPα has been knocked down with nompA-Gal4,argues the Na pump is likely to be involved in maintaining JO receptor against such a junctional role of the Na pump in scolopale cells, lymph ion homeostasis. Other molecular players must work in although one cannot absolutely rule out a junctional role of the conjunction with the Na pump to maintain the ion homeostasis of pump because the Lucifer Yellow molecules may be too large to the system. However, their identification must await further study. detect a mild junctional compromise, or RNAi may not completely The Na pump may also have alternative functions that are not inhibit the pump. Furthermore, septate junctions require numerous dependent on pump activity. Our results show subcellular locali- other components, so loss of only one component may not com- zation of the ATPα–GFP fusion protein accumulating near the pletely dismantle the septate junctions in these cells. Future genetic scolopale cell–cap cell junction and the scolopale cell–neuron inner rescue experiments using constructs with inactivated pump func- dendritic segment junction (Fig. 7B). Septate junctions are known tion in an ATPα knockdown background would indicate whether to be present between these cell types. Insect septate junctions the observed knockdown phenotype is a pump-independent func- form a transepithelial diffusion barrier that limits solute passage tion of the Na pump. However, because of the early requirement of through the spaces between adjacent cells in an epithelium (35). the Na pump during development and cross-reactivity of the RNAi to both the endogenous and rescue construct gene copies, such experiments are currently not feasible. Our finding of organelles such as mitochondria in the scolopale space, often devoid of plasma membrane enclosure, raises the question of their origin. The most likely source of these is the scolopale cell itself. One possibility is that concomitantly com- promised ion homeostasis and osmotic balance results in scolopale cell membrane rupture, releasing cellular contents. Torn + membranes can rapidly reseal themselves through a Ca2 -dependent process (36). In Drosophila embryos, cells undergoing such a cell membrane tear form a membrane plug to reseal the gap in the lipid bilayer through a coordinated activity of the cell membrane and the cytoskeleton (37). A second possibility is that the extraneous material in the scolopale space results primarily from a developmental requirement of the Na pump. The cho cell lineage for each scolopidium comes from a single sense organ– precursor cell, specified in the imaginal disk epithelium. Lineage cell division occurs early, before massive changes in cell shape, with enormous subsequent elongation. Loss of ion homeostasis during this process may prevent the high developmental fidelity required for these cell shape changes. A third possible explana- β fi tion may be partial apoptosis of the scolopale cell. Ultrastruc- Fig. 5. subunit genes nrv2 and nrv3 are required in speci c JO cell types turally, it is clear that the scolopale cell is still alive in the for hearing. Histogram of SEPs of β subunit knockdown (black bars) and control animals (white bars). RNAi knockdown of the three β subunit genes knockdown animals because the nuclei are not heteropyknotic (nrv1, nrv2, and nrv3) with ato-Gal4 and nompA-Gal4 drivers for the entire and we see no cell shrinkage; both of which are hallmarks of JO lineage and for the scolopale cell only, respectively, are shown. Geno- apoptotic cells. Nevertheless, ionic or osmotic imbalances in the types as in Fig. 2 except that the UAS-RNAi constructs are against β subunits, cell may trigger a subset of apoptotic features such as blebbing of and the ato-Gal4 genotypes also include UAS-Dicer2. Hearing loss is highly the plasma membrane. Such blebs into the scolopale space would significant in ato-Gal4–mediated knockdown of nrv3 (P < 0.0001, n = 15 per initially be membrane-bound, but this membrane may be un- genotype) and in nompA-Gal4–mediated knockdown of nrv2 (P < 0.0001, stable and break down in the context of the scolopale space. The n = 10). All P values are based on two-tailed t tests with Welch’s correction. mitochondria found in the scolopale space also frequently

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1208866110 Roy et al. Downloaded by guest on October 4, 2021 + K ions into the scolopale cell en route to the scolopale space to help + maintain its K -rich ionic composition (Fig. 7A). RNAi-mediated knockdown of Na pump subunits results in deafness and loss of morphological integrity. All of these findings are consistent with our model. However, to absolutely confirm the relevance of this model, additional experiments are required. First, it would be informative to measure the receptor lymph ionic concentration and directly + demonstrate the high K concentration within the scolopale space. However, such experiments may prove to be technically challenging because of the small size of the scolopidium. We also need to identify other molecules that work in combination with the Na pump to maintain the receptor lymph for efficient auditory transduction. In the vertebrate inner ear, the endocochlear potential is ach- ieved by maintaining the ionic composition of the endolymph in the fluid compartment into which the stereocilia project (42). De- regulation of the ion concentration or fluid volume in the endolymph, mediated by cells in the stria vascularis and the lateral walls of the organ of Corti, is thought to underlie hearing disorders such as Ménière disease (43), and may contribute to age-related hearing loss (6). Although the Na pump is thought to participate in enriching + K intheendolymphandtocontributetofluid volume homeostasis in the endolymph, the precise mechanisms of Ménière and related diseases are not well understood. In this article, we have taken ad- vantage of our rapidly advancing understanding of the Drosophila auditory system to systematically investigate the expression and functional roles of each Na pump subunit in the auditory organ. This study defines the cell-type specificity of Na pump subunits as well as the functional and morphological consequences of cell type–specific Fig. 6. Knocking down nrv2 in JO scolopale cells disrupts the scolopale

loss of function of these subunits. It also sets the stage for future CELL BIOLOGY space. (A and C) Control sections. (A) Cross- and (C) longitudinal sections studies to elucidate the detailed pathways and mediators of ion showing the normal structure of scolopidia with electron-light scolopale fl space in a scolopidium as shown by the white block arrow with black border. transport and uid regulation. Our model of the Na pump sub- The control image in C is the same as in Fig. 3F.(B and D) Experimental cellular localization in the ablumenal membrane of the scolopale sections. (B) Cross- and (D) longitudinal sections showing abnormal accu- cell provides a useful system to investigate endocochlear potential mulation of electron-dense material, shown by the white block arrow, in- generation in endolymph-like extracellular compartments and its cluding membrane bound bodies such as mitochondria (asterisks in B)inthe malfunctions in connection to inner ear disorders such as age-re- scolopale space. (Scale bar: 1 μm.) Genotypes as in Fig. 5. lated hearing loss and Ménière disease. Materials and Methods display swelling or disrupted cristae, observations that are also Drosophila Stocks. The following fly stocks were used: nompA-Gal4, Sp/CyO, consistent with apoptotic features in Drosophila (38). and pyx-Gal4 are previously described in (13) and (24), ato-Gal4 was a gift Neuronal receptor currents resulting from auditory transduction from B. Hassan (Katholieke Universiteit, Leuven, Belgium) via G. Boekhoff- likely cause depletion of ions in the receptor lymph; therefore, Falk (University of Wisconsin, Madison, WI). UAS-ATPα-RNAi (v12330), UAS- α a mechanism must exist by which the receptor lymph is replenished like-RNAi (v10737), UAS-nrv1-RNAi (v46542), UAS-nrv2-RNAi (v2660), UAS- + with a constant supply of K ions. The receptor lymph filling the nrv3-RNAi (v44486), and UAS-Dicer2 were supplied by the Vienna Drosophila + RNAi Centre. The GFP fusion line ATPα (34) was donated by W. Chia (National scolopale space in JO is likely to be highly enriched in K ions in University of Singapore, Singapore). w1118 was used for normal expression of analogy to the bristle receptors and campaniform sensilla in insects ts + Na pump subunits in JO. UAS-GFP and tub-Gal80 were obtained from the that have been shown to be rich in K ions (39, 40). In addition, + Drosophila Stock Center in Bloomington, IN. vertebrate endolymph is K enriched (41). Our model of the Na pump in the fly auditory system is that it is present in the ablumenal RNAi and UAS/Gal4 System. RNAi was used to knock down Na pump subunit plasma membrane of the scolopale cell where it actively transports genes in specific spatiotemporal patterns using the Gal4/UAS system (22, 23).

Fig. 7. Model of ablumenal localization of ATPα in the scolopale cell. (A) Diagram showing a single scolopidium with predicted locations for Na pump. Red arrows represent the direction of K+ transport through the Na pump (green bar) in the plasma membrane. (B) Cross-section of JO scolopidia showing presence of ATPα protein stained with α5 mAb (green), indicated by white arrows on the scolopale cell ablumenal plasma membrane surrounding the scolopale rods (magenta). Texas Red phalloidin, which stains the scolopale cell actin rods, is used as a marker for the boundary between the luminal and ablumenal plasma membranes of the scolopale cell. (C) Longitudinal sectional view of the scolopidium shows ATPα-GFP fusion protein at the scolopale cell-cap cell junctions (white brackets), in the apical epithelium (asterisk) and in neuron cell bodies (N). ATPα-GFP expression in the scolopale cell outside the scolopale rods (brackets) is consistent with preferential ablumenal localization.

Roy et al. PNAS Early Edition | 5of6 Downloaded by guest on October 4, 2021 The Gal4 drivers ato-Gal4 and nompA-Gal4 have JO-specific and scolopale gifts from G. Beitel (Northwestern University, Chicago, IL). Monoclonal anti- cell–specific expression patterns (Fig. S7). UAS-Dicer2 was used in conjuga- bodies α5 recognizing ATPα [1:100 diluted in PBS + 0.1% (vol/vol) Triton X-100 tion with the Gal4 driver in certain cases to enhance the knockdown effect. (PBT) + BSA], 5F7 recognizing all three Nrv isoforms and 21A6 recognizing Eys/ Spam (1:200 diluted in PBT+BSA) were obtained from The University of Iowa Electrophysiology. Auditory recordings were conducted in experimental Developmental Studies Hybridoma Bank. Secondary antibodies labeled with and control flies as described elsewhere (9, 12). The fly was mounted in a 200-μL Alexa Fluor-488 or TRITC were obtained from Sigma (1:200 diluted in PBT+BSA). pipette tip trimmed such that only the head protruded. The neck was immo- Texas Red phalloidin (1:200 diluted in PBT+BSA) was used to stain the scolopale bilized with plasticine. The computer-generated pulse component of the Dro- rods for 1 h. All confocal images were taken using a Leica SP2 Confocal Mi- sophila courtship song was played through a speaker and the sound was α transported through a Tygon tube (Fisher Scientific) placed at a distance of 1 croscope except for the ATP ablumenal localization images, for which a Del- mm from the fly’s head. The sound stimulus intensity was measured at 5.3 mm/s taVision Deconvolution microscope was used. at the position of the antennae, using a calibrated Emkay NR3158 particle ve- locity microphone (Knowles). Two tungsten electrodes were used; the recording Statistics. Statistical analyses of electrophysiology data and fluorescence ratio electrode was inserted at the joint between the first and second antennal data were performed in GraphPad Prism software using the Student t test, segment from a dorsofrontal direction and the reference electrode was inserted with Welch’s correction for unequal variances where needed. The relative fi in the head cuticle. The signals were ampli ed by a DAM50 differential ampli- fluorescent signals for Nrv2 and ATPα in the scolopale cells were calculated fi er (WPI) and digitized and normalized using Superscope II software (GW using ATPα signal in neurons as the reference. Instruments). Details about this assay are available elsewhere (9, 12). ACKNOWLEDGMENTS. We thank Sara Paul and Greg Beitel for generously α EM. The heads of nompA-Gal4; UAS-ATP -RNAi and nompA-Gal4; UAS-nrv2- providing the Nrv antibodies and Sarit Smolikov for use of the DeltaVision RNAi and corresponding controls were fixed, processed, and embedded in Deconvolution Microscope. Lydia Morris and Ryan Kavlie participated in Epon resin according to the protocol described in (44). Transmission EM was generation of nrv3 deletion mutations. The calibrated microphone was kindly conducted with a JEOL 1230 instrument. provided by Martin Göpfert. We also thank the Developmental Studies Hy- bridoma Bank at The University of Iowa for monoclonal antibodies. Thanks to the Carver Center for Imaging and Central Microscopy Research Facilities at Immunohistochemistry and Microscopy. Antennae were dissected in PBS, fixed The University of Iowa for use of confocal and electron microscopes and the in 4% (vol/vol) paraformaldehyde in PBS for 30 min, embedded in OCT (Ted Carver Center for Genomics at The University of Iowa for sequencing support. μ Pella) and then cut into 25- m sections in a cryostat. The antennal cryosections This work was supported by National Institutes of Health Grant DC004848 (to were stained with primary polyclonal antibodies against Nrv2 generated in D.F.E.) and facilitated by the Iowa Center for Molecular Auditory Neurosci- rabbit and Nrv1 and Nrv3 generated in guinea pig, respectively (29), generous ence, supported by P30 DC010362 (to Steven Green).

1. Blanco G, Mercer RW (1998) Isozymes of the Na-K-ATPase: Heterogeneity in structure, 24. Chung YD, Zhu J, Han Y, Kernan MJ (2001) nompA encodes a PNS-specific, ZP domain diversity in function. Am J Physiol 275(5 Pt 2):F633–F650. protein required to connect mechanosensory dendrites to sensory structures. Neuron 2. Chow DC, Forte JG (1995) Functional significance of the beta-subunit for hetero- 29(2):415–428. dimeric P-type ATPases. J Exp Biol 198(Pt 1):1–17. 25. Cook B, Hardy RW, McConnaughey WB, Zuker CS (2008) Preserving cell shape under 3. Bartolami S, et al. (2011) Critical roles of transitional cells and Na/K-ATPase in the environmental stress. Nature 452(7185):361–364. formation of vestibular endolymph. J Neurosci 31(46):16541–16549. 26. Husain N, et al. (2006) The agrin/perlecan-related protein eyes shut is essential for 4. Lang F, Vallon V, Knipper M, Wangemann P (2007) Functional significance of channels epithelial lumen formation in the Drosophila retina. Dev Cell 11(4):483–493. and transporters expressed in the inner ear and kidney. Am J Physiol Cell Physiol 293 27. Lee E, Sivan-Loukianova E, Eberl DF, Kernan MJ (2008) An IFT-A protein is required to (4):C1187–C1208. delimit functionally distinct zones in mechanosensory cilia. Curr Biol 18(24):1899–1906. 5. Diaz RC, et al. (2007) Conservation of hearing by simultaneous mutation of Na,K- 28. Paul SM, Ternet M, Salvaterra PM, Beitel GJ (2003) The Na+/K+ ATPase is required for ATPase and NKCC1. J Assoc Res Otolaryngol 8(4):422–434. septate junction function and epithelial tube-size control in the Drosophila tracheal – 6. Wangemann P (2006) Supporting sensory transduction: Cochlear fluid homeostasis system. Development 130(20):4963 4974. and the endocochlear potential. J Physiol 576(Pt 1):11–21. 29. Paul SM, Palladino MJ, Beitel GJ (2007) A pump-independent function of the Na,K- 7. Eberl DF (1999) Feeling the vibes: Chordotonal mechanisms in insect hearing. Curr ATPase is required for epithelial junction function and tracheal tube-size control. – Opin Neurobiol 9(4):389–393. Development 134(1):147 155. 8. Boekhoff-Falk G (2005) Hearing in Drosophila: Development of Johnston’s organ and 30. Baumgartner S, et al. (1996) A Drosophila neurexin is required for septate junction – emerging parallels to vertebrate ear development. Dev Dyn 232(3):550–558. and blood-nerve barrier formation and function. Cell 87(6):1059 1068. 9. Eberl DF, Hardy RW, Kernan MJ (2000) Genetically similar transduction mechanisms 31. Carlson SD, Hilgers SL, Juang JL (1997) First developmental signs of the scolopale (glial) cell and neuron comprising the chordotonal organ in the Drosophila embryo. for touch and hearing in Drosophila. J Neurosci 20(16):5981–5988. Glia 19(3):269–274. 10. Albert JT, Nadrowski B, Göpfert MC (2007) Mechanical signatures of transducer 32. Todi SV, Franke JD, Kiehart DP, Eberl DF (2005) Myosin VIIA defects, which underlie the gating in the Drosophila ear. Curr Biol 17(11):1000–1006. Usher 1B syndrome in humans, lead to deafness in Drosophila. Curr Biol 15(9):862–868. 11. Göpfert MC, Robert D (2001) Biomechanics. Turning the key on Drosophila audition. 33. Sun B, Salvaterra PM (1995) Characterization of nervana, a Drosophila melanogaster Nature 411(6840):908. neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase an- 12. Eberl DF, Kernan MJ (2011) Recording sound-evoked potentials from the Drosophila tibodies. J Neurochem 65(1):434–443. antennal nerve. Cold Spring Harb Protoc (1):prot5576. 34. Morin X, Daneman R, Zavortink M, Chia W (2001) A protein trap strategy to detect 13. Sun Y, et al. (2009) TRPA channels distinguish gravity sensing from hearing in John- GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl ston’s organ. Proc Natl Acad Sci USA 106(32):13606–13611. Acad Sci USA 98(26):15050–15055. 14. Kamikouchi A, et al. (2009) The neural basis of Drosophila gravity-sensing and 35. Tepass U, Tanentzapf G, Ward R, Fehon R (2001) Epithelial cell polarity and cell hearing. Nature 458(7235):165–171. junctions in Drosophila. Annu Rev Genet 35:747–784. 15. Yorozu S, et al. (2009) Distinct sensory representations of wind and near-field sound 36. McNeil PL (2002) Repairing a torn cell surface: Make way, lysosomes to the rescue. J in the Drosophila brain. Nature 458(7235):201–205. Cell Sci 115(Pt 5):873–879. 16. McQuilton P, St Pierre SE, Thurmond J; FlyBase Consortium (2012) FlyBase 101—the 37. Abreu-Blanco MT, Verboon JM, Parkhurst SM (2011) Single cell wound repair: Dealing – basics of navigating FlyBase. Nucleic Acids Res 40(Database issue):D1):D706 D714. with life’s little traumas. BioArchitecture 1(3):114–121. 17. Masly JP, Jones CD, Noor MA, Locke J, Orr HA (2006) Gene transposition as a cause of 38. Abdelwahid E, et al. (2007) Mitochondrial disruption in Drosophila apoptosis. Dev Cell – hybrid sterility in Drosophila. Science 313(5792):1448 1450. 12(5):793–806. 18. Chintapalli VR, Wang J, Dow JA (2007) Using FlyAtlas to identify better Drosophila 39. Küppers J (1974) Measurements on the ionic milieu of the receptor terminal in mecha- – melanogaster models of human disease. Nat Genet 39(6):715 720. noreceptive sensilla of insects. Symposium on Mechanoreception, ed Schwartzkopf J 19. Baumann O, Salvaterra PM, Takeyasu K (2010) Developmental changes in beta-subunit (Westdeutscher Verlag, Opladen, Germany), pp 387–394. – composition of Na,K-ATPase in the Drosophila eye. Cell Tissue Res 340(2):215 228. 40. Thurm U, Küppers J (1980) Epitheilal physiology of insect sensilla. Insect Biology in the 20. Palladino MJ, Bower JE, Kreber R, Ganetzky B (2003) Neural dysfunction and neuro- Future, eds Locke M, Smith DS (Academic Press, New York), pp 735–763. degeneration in Drosophila Na+/K+ ATPase alpha subunit mutants. J Neurosci 23(4): 41. Corey DP, Hudspeth AJ (1979) Ionic basis of the receptor potential in a vertebrate hair 1276–1286. cell. Nature 281(5733):675–677. 21. Feng Y, Huynh L, Takeyasu K, Fambrough DM (1997) The Drosophila Na,K-ATPase 42. Hibino H, Nin F, Tsuzuki C, Kurachi Y (2010) How is the highly positive endocochlear alpha-subunit gene: Gene structure, promoter function and analysis of a cold-sensi- potential formed? The specific architecture of the stria vascularis and the roles of the tive recessive-lethal mutation. Genes Funct 1(2):99–117. ion-transport apparatus. Pflugers Arch 459(4):521–533. 22. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell 43. Ross MD, Ernst SA, Kerr TP (1982) Possible functional roles of Na+,K+-ATPase in the fates and generating dominant phenotypes. Development 118(2):401–415. inner ear and their relevance to Ménière’s disease. Am J Otolaryngol 3(5):353–360. 23. Dietzl G, et al. (2007) A genome-wide transgenic RNAi library for conditional gene 44. Sarpal R, et al. (2003) Drosophila KAP interacts with the kinesin II motor subunit KLP64D inactivation in Drosophila. Nature 448(7150):151–156. to assemble chordotonal sensory cilia, but not sperm tails. Curr Biol 13(19):1687–1696.

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