Molecular bases of k+ secretory cells in the inner ear: shared and distinct features between birds and mammals

ABSTRACT In the cochlea, mammals maintain a uniquely high endolymphatic potential (EP), which is not observed in other vertebrate groups. However, a high [K+] is always present in the inner

ear endolymph. Here, we show that Kir4.1, which is required in the mammalian stria vascularis to generate the highly positive EP, is absent in the functionally equivalent avian tegmentum

vasculosum. In contrast, the molecular repertoire required for K+ secretion, specifically NKCC1, KCNQ1, KCNE1, BSND and CLC-K, is shared between the tegmentum vasculosum, the vestibular dark

cells and the marginal cells of the stria vascularis. We further show that in barn owls, the tegmentum vasculosum is enlarged and a higher EP (~+34 mV) maintained, compared to other birds.

Our data suggest that both the tegmentum vasculosum and the stratified stria vascularis evolved from an ancestral vestibular epithelium that already featured the major cell types of the

auditory epithelia. Genetic recruitment of Kir4.1 specifically to strial melanocytes was then a crucial step in mammalian evolution enabling an increase in the cochlear EP. An increased EP

may be related to high-frequency hearing, as this is a hallmark of barn owls among birds and mammals among amniotes. SIMILAR CONTENT BEING VIEWED BY OTHERS DEVELOPMENTAL DIFFERENTIATION OF

MOUSE INNER EAR NEURON SUBPOPULATIONS RESOLVED WITH A PERIPHERIN-PROMOTER REPORTER WITHIN THE _GRM8_ LOCUS Article Open access 25 March 2025 SINGLE-CELL ATLAS COMPARISON ACROSS VERTEBRATES

REVEALS AUDITORY CELL EVOLUTION AND MECHANISMS FOR HAIR CELL REGENERATION Article Open access 19 December 2024 _TBX2_ IS A MASTER REGULATOR OF INNER VERSUS OUTER HAIR CELL DIFFERENTIATION

Article 04 May 2022 INTRODUCTION Mechanotransduction in the vertebrate inner ear, both auditory and vestibular, is mediated by K+ and Ca2+ influx into hair cells. The apical part of hair

cells, their mechanosensitive hair bundle, is bathed in a very unusual extracellular fluid, the endolymph of scala media1,2. This fluid resembles in its ionic composition an intracellular

milieu, as it features a high concentration of K+ and a low concentration of Na+ (elasmobranchs3, frogs4, turtles4, lizards3,4, birds4, mammals1). Furthermore, this endolymph shows a

positive potential (endolymphatic potential, EP) compared to other extracellular spaces5. The EP provides an enhanced electrical gradient for positively charged ions to flow into the hair

cells during stimulation when transduction channels open. The ionic composition, with K+ as the dominant cation, then determines that the transduction current is mostly carried by K+, with

Ca2+ being the second most important contributor6. Since the driving force for K+ to enter hair cells is predominantly electrical, the EP critically contributes to the exquisite sensitivity

of hair-cell mechanotransduction2. Although the basic mechanism of hair-cell mediated mechanotransduction is conserved between all vertebrates, an important difference concerns the extent of

the positive EP. Frogs, turtles, snakes, lizards and crocodilians typically maintain only +2 to +7 mV5. Birds show somewhat higher EP levels, typically between +10 and +20 mV7,8. Finally,

high values are observed in therian mammals with an EP of typically +80 to +90 mV1,5. The large difference in the EP between non-mammalian and mammalian tetrapods is paralleled by changes in

the secretory epithelia that generate the endolymph in scala media. In non-mammalian tetrapods, this epithelium overlies the basilar papilla (BP) and separates scala media and scala

vestibuli9. In birds, it is composed of a single layer of cells that form a series of folds into the scala media and has been termed tegmentum vasculosum9. In mammals, the functionally

equivalent structure is situated in the lateral cochlear wall and composed of two tissues. The spiral ligament contains five distinct types of fibrocytes and connective tissue, whereas the

epithelial stria vascularis is a stratified tissue comprising marginal, intermediate and basal cells2. In the mammalian cochlea, the molecular and cellular mechanisms generating the

endolymph and the EP have been investigated in great detail1,2. In a first step, K+ is taken up by fibrocytes of the spiral ligament and transported to intermediate and basal cells of the

stria vascularis, via a syncytial gap-junction network. Intermediate cells then release K+ into the intrastrial space, from where it is taken up by marginal cells. These cells form a single

layer and release K+ at their apical surface into scala media. The two-step process ultimately results in an EP of +80 to +90 mV and approximately 150 mM [K+] in scala media1,2. Recent years

have witnessed the identification of major molecular players in this K+ secretion route in the lateral cochlear wall. It comprises connexins 26 and 30, the plasma membrane transporters

Na+-K+-ATPase and NKCC1, the K+ channels Kir4.1, Kir 5.1, KCNQ1 and KCNE1, as well as the Cl- channel proteins CLC-Ka, CLC-Kb and Barttin1,2. Mutation in most of these genes is associated

with deafness in humans or mice10,11. In summary, there are marked differences concerning both the EP and the cellular basis of the generation of the endolymph between mammals and other

tetrapods. This raises two important questions regarding proximate and ultimate causes12: (1) what evolutionary changes caused the emergence of the high mammalian EP (“how” question) and (2)

what are the selective pressures that may have led to the exceptionally high EP in mammals (“why” question)? Here we addressed these questions by performing molecular and

electrophysiological studies in birds that show a more moderate but still significantly enhanced EP. We chose two avian species with distinct auditory capabilities, the chicken as an

auditory generalist and the barn owl as a known high-frequency specialist13. RESULTS An unusually highly positive EP is a hallmark of the mammalian cochlear endolymph. To investigate whether

this uniquely high EP is associated with evolutionary differences in the molecular underpinnings, we decided to characterize in the chicken the expression pattern of genes that are known to

be involved in mammals. For our study, 14 mammalian genes were selected: _ATP1A1_, _ATP1B1, ATP1A2_, _ATP1A3 encoding_ Na+-K+-ATPase subunits, _BSND, CLCNKA and CLCNKB,_ encoding Cl−

channel subunits, _KCNJ10_, _KCNJ16_, _KCNQ1_, _KCNE1_ encoding K+-channels and the gap junction genes _GJB2_ and _GJB6_ and _SLC12A2,_ encoding the Na+-K+-Cl− cotransporter NKCC1. For most

of them, chicken orthologs could be identified in _GenBank_ or _ensemble._ The only exceptions were the three genes encoding Cl−-channel proteins, as no reliable database entries were

detected when we initiated this study. PHYLOGENETIC ANALYSIS OF CLC-K AND BSND To identify the chicken orthologs of _BSND_ and _CLC-K_ channel proteins, phylogenetic analyses were performed

using available protein sequences from different vertebrate species (Tables 1 and 2). For Barttin, a single protein was identified in all vertebrate species (Fig. 1A). The CLC chloride

channel family comprises three subfamilies: CLC-1, -2 and CLC-K; CLC3, -4, -5 and CLC-6 and CLC7. Phylogenetical analyses of CLC-K rooted with the human CLC-1 sequence indicate that all

vertebrate groups except for amphibians (_Xenopus laevis_) contain at least one CLC-K protein sequence (Fig. 1B). Independent gene duplication events resulted in two paralogous CLC-K protein

sequences in _Homo sapiens, Rattus norvegicus_ and _Monodelphis domesticus_. These channels were named CLC-Ka and CLC-Kb in human, CLC-K1 and CLC-K2 in rat and CLC-Kx1 and CLC-Kx2 in

_Monodelphis domesticus_. Similar conclusions were obtained in a previous, more limited, analysis which also revealed that the amino acid sequence identity between the paralogous CLC-K

proteins of human and rat (_Homo sapiens_ CLC-Ka and CLC-Kb, _Rattus norvegicus_ CLC-K1 and CLC-K2) is larger than between CLC-K proteins across species (see also Table 3)14. RNA _IN SITU_

HYBRIDIZATION IN THE TEGMENTUM VASCULOSUM To study the expression pattern of the genes in the avian inner ear, RNA _in situ_ hybridization with DIG labeled antisense probes was performed on

cross sections of the cochlea isolated from chickens aged 12 to 17 days posthatching (P12 to P17). At this age, the chicken EP is mature15. PLASMA MEMBRANE TRANSPORTERS We first focused on

members of the Na+, K+-ATPase, as they are part of the K+ uptake apparatus in fibrocytes of the spiral ligament and marginal cells of the stria vascularis1,11. _ATP1A1,_ encoding the Na+,

K+-ATPase α1 was shown to be the sole alpha Na+, K+-ATPase isoform present in the stria vascularis in adult mice16 and rats17 and in the spiral ligament of adult mice16. During the first

month of life, the mouse spiral ligament expresses in addition _ATP1A2_ and _ATP1A3_16. We therefore analyzed the expression pattern of _ATP1A1, ATP1A2_ and _ATP1A3_ in the chicken inner

ear. All three genes were expressed throughout the tegmentum vasculosum (Fig. 2A–C). We could not distinguish between light cells and dark cells in the tegmentum vasculosum, a distinction

mainly made in electron microscopy studies through the different electron density of the cells (osmiophilic dark cells, osmiophobic light cells) or using enzymatic reactions. Cytological

differences, such as the apical microvilli or the extended plasma membrane infoldings throughout the sides and the basolateral part of dark cells9, were not discernable, likely due to the

rather harsh RNA _in situ_ hybridization conditions. The expression pattern of the three genes in the tegmentum vasculosum resembled that of fibrocytes in the spiral ligament. We therefore

included in our analysis _ATP1B1,_ encoding the Na+, K+-ATPase β1. This isoform is also strongly expressed in mammalian fibrocytes but only poorly in the stria vascularis16,17. Again, clear

expression of the gene was observed throughout the tegmentum vasculosum (Fig. 2D). Taken together, all four subunits of the Na+, K+-ATPase were expressed in the tegmentum vasculosum with no

obvious difference in their expression pattern. The Na+, K+, Cl−-cotransporter NKCC1 mediates cellular uptake of K+ and is present in mammals in both fibrocytes and in marginal cells of the

lateral wall. In the chicken inner ear, expression of the corresponding gene _SLC12A2_ was observed throughout the tegmentum vasculosum (Fig. 2E). Previous pharmacological experiments in

pigeon had suggested that NKCC1 is not involved in the generation of the EP in the avian scala media18. We therefore analyzed expression of _SLC12A2_ in another bird, the barn owl. Again, we

observed clear expression in the tegmentum vasculosum (Fig. 2F). This result confirmed that NKCC1 is part of the molecular repertoire of the avian tegmentum vasculosum. PLASMA MEMBRANE

CHANNELS Two _KCNJ_ genes, _KCNJ10_ and _KCNJ16_, play important roles in the generation of the endolymph. _KCNJ16_, encoding the inwardly rectifying K+ channel Kir5.1, is expressed in

fibrocytes of the spiral ligament where it likely controls cochlear K+ circulation11. Kir4.1, encoded by _KCNJ10_, resides in the apical membrane of intermediate cells in the stria

vascularis and is crucial for the generation of the EP19. In the chicken inner ear, neither the tegmentum vasculosum nor any other tissue was labeled using _KCNJ10_ and _KCNJ16 in situ_

probes (Fig. 3A,B). Labeling of neural populations such as the Nucleus reticularis lateralis in coronal sections of the brain and of cross sections of the kidney with the _KCNJ10_ and

_KCNJ16_ probes, respectively, demonstrated that the absence of labeling in the inner ear was not due to inappropriate probes (data not shown). Thus, neither inwardly rectifying K+ channels

Kir4.1 nor Kir5.1 are present in the chicken tegmentum vasculosum. The absence of Kir5.1 which resides in fibrocytes of the mammalian spiral ligament is in agreement with the lack of such a

tissue in the avian cochlea. As the barn owl exhibits an unusually high EP (see below), we tested the expression of _KCNJ10_ also in this bird. Again, no expression was noted in the

tegmentum vasculosum (data not shown). The CLC-K channel and its accessory protein BSND reside in the basolateral membrane of strial marginal cells. They likely provide an efflux system to

recycle Cl− back to the intrastrial space, which is required for sustained NKCC1 activity11. Both genes were expressed throughout the tegmentum vasculosum (Fig. 3C,D). Finally, the K+

channel proteins KvLQT1and IsK form a complex in the apical membrane of strial marginal cells that mediates final secretion of K+ to the endolymph. Both _KCNQ1_ and _KCNE1,_ encoding KvLQT1

and IsK, respectively, were expressed throughout the tegmentum vasculosum (Fig. 3E,F). RNA _IN SITU_ HYBRIDIZATION IN THE CRISTA AMPULLARIS Both the auditory and the vestibular hair-cell

organs of vertebrates are housed within the inner-ear labyrinth. Similar to their auditory counterparts, vestibular hair cells are bathed in endolymph. This vestibular endolymph displays a

high concentration of K+ and an endovestibular potential of +1 mV in the semicircular canals20,21, similar to the endolymph in the scala media of non-mammalian tetrapods. In the ampullae of

the three semicircular canals, endolymph is believed to be generated by vestibular dark cells flanking the neurosensory epithelium11,21. To examine whether these vestibular dark cells and

the tegmentum vasculosum share critical molecular components, we also analyzed the expression of _ATPA1, ATPB1, KCNJ10, KCNJ16, KCNQ1 and KCNE1,_ in the epithelia of the ampullae. In

addition, we included _GJB2_ and _GJB6,_ which were previously shown to be expressed in the tegmentum vasculosum22. Probes against _ATPA1, ATPB1, KCNQ1, KCNE1, GJB2_ and _GJB6_ clearly

labeled the vestibular dark cells (Fig. 4A,B,E,F), shown for _ATPB1, GJB2, KCNQ1, KCNE1_). In contrast, no expression of _KCNJ10_ and _KCNJ16_ was observed in the crista (Fig. 4C,D). Thus,

tegmentum vasculosum and the vestibular dark cells of the ampulla show a congruent expression pattern of genes involved in the generation of endolymph. TEGMENTUM VASCULOSUM AND EP IN THE

BARN OWL The highly positive EP in mammals may be associated with high frequency hearing, as hearing >10 kHz is a unique feature of therian mammals23. Among birds, barn owls show a unique

extension of the sensitive hearing range up to 12 kHz13,24. We therefore decided to investigate the tegmentum vasculosum and the EP in barn owls, to examine whether they evolved unique

features. Labeling of the tegmentum vasculosum using immunohistochemistry against Na+, K+ ATPase revealed that the tegmentum vasculosum is thicker and more folded than in other birds so far

investigated, e.g. chicken (Fig. 5). The enlargement is most prominent in the basal regions of the basilar papilla (Fig. 5B) where high frequency sounds are transduced13. Measurements of the

EP were obtained from 5 ears of 5 chickens and from 7 ears of 4 barn owls. The EP appeared as a sudden jump in the recorded potential to positive values, associated with a forward step of

the electrode. Fig. 6 shows a typical recording. EP values for the chicken ranged from 13.5 to 17.5 mV (median 14.3 mV), confirming previous measurements7,25. Values for the barn owl ranged

from 30.1 to 44.3 mV (median 33.8 mV). The EP of the barn owl was thus significantly higher than that of the chicken (Mann-Whitney U-Test, p = 0.003). DISCUSSION The auditory systems of

extant birds and mammals reflect 300 million years of parallel evolution26. This has resulted in marked differences such as a coiled cochlea, a three-ossicle middle ear, an increased EP,

prestin-based electromotility and an extended hearing range in mammals26,27,28. The evolutionary processes and selective pressure(s) for many of the mammalian-specific features remain

enigmatic. Here, we identified shared and distinct molecular features for the generation of the endolymph between birds and mammals. Our data pinpoint recruitment of Kir4.1 to the mammalian

secretory epithelium as a crucial evolutionary event on the molecular level, enabling the generation of a highly positive EP. Otherwise, the molecular toolkit for K+ secretion into the

auditory endolymph of the two vertebrate groups is rather similar. Finally, we demonstrate that the barn owl, a hearing specialist with an extended high-frequency hearing range among birds,

has the highest EP measured in any non-mammalian animal. Thus, an increased EP in this specialized bird and in mammals generally correlates with the evolution of higher-frequency hearing.

PROXIMATE CAUSE: EVOLUTION OF THE K+ SECRETION APPARATUS To answer the question what in tetrapod evolution caused the emergence of the high mammalian EP, we characterized the molecular

repertoire underlying the generation of the more moderate but still significantly positive endocochlear potential in the avian scala media. This analysis revealed that two of the tested

genes, _KCNJ10 and KCNJ16_, were not expressed in the tegmentum vasculosum. Mutations in _KCNJ10_ cause deafness in mice29 and humans30. The encoded protein Kir4.1 is expressed in the apical

membrane of intermediate cells of the stria vascularis and is thought to be a molecular key to the generation of the highly positive mammalian EP31. The apical membrane of intermediate

cells is separated from the basolateral membranes of the marginal cells by an electrically isolated intrastrial space. The fluid of this space exhibits a low K+ concentration (1–2 mM)

whereas the cytosol of intermediate cells has the usual high K+ concentration (140–150 mM)32,33,34. This constellation allows Kir4.1 to generate approximately +100 to +110 mV transmembrane

K+-diffusion potential2,19,32,35 which is the basis for the high mammalian EP. The lack of this gene in the avian tegmentum vasculosum is in full agreement with such a role, as the avian EP

is considerably lower. Furthermore, Kir4.1 is also absent in the mammalian vestibular system31, which also exhibits a low positive endovestibular potential of +1 mV20. Importantly,

vestibular dark cells closely resemble marginal cells with respect to the expression and localization of channels and transporters (see below). This supports the notion that recruitment of

Kir4.1 to the strial epithelium was important for the evolution of the high mammalian EP. We note, however, that recruitment of Kir4.1 alone is insufficient to explain the high mammalian EP,

as the layered structure of the cochlear lateral wall, generating an electrically isolated intrastrial space, is similarly crucial2,35. The lateral wall consists of two functional layers:

(1) a syncytium of intermediate and basal cells of the stria vascularis and fibrocytes of the spiral ligament, which are connected via gap junctions and (2) the marginal cells facing the

endolymph of scala media. These two layers are separated by the intrastrial space. The EP is based on two K+-diffusion potentials, the first being established in the intrastrial space by

Kir4.1 and maintained by Na+, K+-ATPases and NKCC1 in the basolateral membrane of the marginal cell layer and the second one depending on KCNQ1/KCNE1 channels at the apical surface of

marginal cells2. Thus, integration of Kir4.1 into a single layered epithelium such as the tegmentum vasculosum would be insufficient to generate a high EP in birds by this mechanism. The

role of Kir5.1, encoded by K_CNJ16_, is less clear. The gene is strongly expressed in fibrocytes of the spiral ligament36, which suggests an important role in K+ cycling in the inner ear.

Yet, no hearing deficits were reported for two independent _KCNJ16_ knockout mouse lines37,38. Note, however, that hearing was not explicitly tested. Its absence in the tegmentum vasculosum

is consistent with the lack of a spiral ligament in birds. In contrast to the two K+-channels, the other genes that are essential for K+ secretion in mammals, i.e. _ATP1A1_, _ATP1A2_,

_ATP1A3, ATP1B1, BSND, CLCK_, _KCNQ1_, _KCNE1, SLC4A11_ and _SLC12A2,_ are all present in the avian tegmentum vasculosum. The molecular complement for K+ secretion thus appears to be highly

conserved between birds and mammals. Yet, we note that our analysis lacks information on subcellular localization of these proteins, which is also important for their proper function.

Enzymatic analysis of the Na+, K+ ATPase already demonstrated its expression on the basolateral membrane39, similar to marginal cells. It will therefore be interesting to determine the

subcellular localization of the other transporters and channels as well. Due to the probable close evolutionary relationship between vestibular dark cells, marginal cells and the cells of

the tegmentum vasculosum (see below), we expect concordant subcellular localization of these proteins. Conflicting with conflicting with our findings, the involvement of NKCC1, encoded by

_SLC12A2,_ in the generation of the avian endolymph was previously questioned, based on pharmacological experiments in pigeon. Application of furosemide, a blocker of NKCC1, had neither an

effect on the EP nor on the spontaneous or tone-evoked discharge of single auditory-nerve fibers18, despite being a strong diuretic40. Yet, our RNA _in situ_ data from both chicken and barn

owl demonstrated clear expression of this gene in the avian tegmentum vasculosum. Reasons for this discrepancy remain unclear at present. It is conceivable that the intravenously applied

furosemide18 was unable to reach its targets in the dark cells that are separated from the vascular capillaries of the tegmentum vasculosum by a basement membrane9. Indeed, the unique

closeness of vascular capillaries and the basolateral membranes of marginal cells in the mammalian stria vascularis was suggested to be crucial for the effectiveness of intravenous

administration of diuretics, because, in contrast, application of furosemide directly into scala media has little effect on the EP41. We note that the tegmentum vasculosum and the marginal

cells of the cochlear stria vascularis share similar expression patterns also with vestibular dark cells, the presumed secretory elements in the cristae ampullares42,43. Both the tegmentum

vasculosum and the vestibular dark cells express _ATP1A1, ATP1B1, KCNQ1_, _KCNE1_, _BSDN_, _SLC12A2_ and _GJB2_ and _GJB6_. Except for _ATP1A2_, _ATP1A3 and ATP1B1_, all genes are expressed

in the strial marginal cells as well2. They hence share the molecular repertoire for K+ secretion. This signature adds to the previously described shared cytological, molecular and

physiological properties between these three epithelia9,21,42. They all present with a dense cytoplasm with tightly packed mitochondria, an infolded plasma membrane at the basolateral pole,

numerous microvilli at the luminal surface9 and Na+, K+-ATPase and Cl−-conductance in the basolateral membrane21. Additionally, K+ conductance is present in the apical membrane of vestibular

dark cells and strial marginal cells and K+ secretion is regulated via purinergic receptors, cAMP21 and ß1 adrenergic receptors44,45. Additionally, marginal cells and vestibular dark cells

share the tight junction proteins claudin-1, -3, -8, -9, -12, -14 and -1846 and the expression of the atrial natriuretic peptide47. These data bear important implications for the evolution

of the epithelia involved in the generation of the endolymph in scala media. Collectively, the many molecular and ultrastructural similarities suggest that dark cells of the ancestral

vestibular system, which arose at least 400 million years ago48 gave rise to those of the tegmentum vasculosum and the strial marginal cells. This is supported by their common origin from

the otocyst epithelium (tegmentum vasculosum49, dark cells50, marginal cells51). Furthermore, dark cells of the tegmentum vasculosum and the vestibular cristae are separated from the

underlying connective tissue by a basal lamina9,50,52, which is also present beneath the marginal cells early in development51. Most of the additional cell types in the mammalian spiral

ligament are also present in the vestibular epithelium, but are absent from the avian tegmentum vasculosum. Strial intermediate cells are melanocytes, which are also present in the

subepithelial area of the vestibular organ. Interestingly, the same gene regulatory network module, consisting of SOX10 and the endothelin-3/EDNRB signaling pathway is involved in the

generation of melanocytes in both the stria vascularis and the vestibular organ53. Fibrocytes, another constituent of the mammalian spiral ligament, are also present in the vestibular organ.

It thus appears that the organization of these cell types into a layered system with an intrastrial space and the recruitment of Kir4.1 to the melanocytes giving rise to the intermediate

cells of the mammalian stria vascularis were crucial steps in increasing the level of the EP in mammals. The avian tegmentum vasculosum displays neither of those two crucial characteristics,

yet maintains a significantly positive EP of up to +35 mV (in the owl). It remains an interesting challenge for future studies to explain how this is achieved. ULTIMATE CAUSE: POSSIBLE

SELECTIVE PRESSURES TOWARDS EVOLUTION OF AN INCREASED POSITIVE EP The selective pressure(s) resulting in the unique, highly positive EP in mammals currently remain unclear. It is commonly

stated2 that the EP serves to increase the sensitivity of hearing by augmenting the electrical gradient as the driving force for the influx of K+ and Ca2+ during transduction. This implies

that a higher EP conveys superior sensitivity. The best hearing thresholds of mammals are, however, not consistently lower than those of other vertebrates23. For example, an analysis of

terrestrial mammals revealed an average lowest behavioral threshold of 0.4 dB SPL54. This is easily met by a range of bird species with behavioral thresholds close to or even below 0 dB SPL

(starling -1 dB SPL, bullfinch -4.9 dB SPL, cockatiel -9.5, barn owl -18.5 dB SPL and great horned owl -15 dB SPL23). If anything, the avian cochlea is thus the more sensitive, as most birds

– in contrast to mammals - do not benefit from peripherally amplifying pinnae. Conversely, marsupial mammals have relatively high hearing thresholds of >20 dB SPL, despite the fact that

they maintain the typically mammalian, highly positive, cochlear EP5. Together, these data reveal a poor correlation between hearing threshold and the EP; mammals and non-mammalian tetrapods

do not divide along this line. Increased sensitivity _per se_ thus cannot explain the selective pressure driving the evolution of an increased EP. A unique feature of mammalian hearing is

the extension of the upper frequency limit >10 kHz. We report here a similar correlation in the barn owl between an increased EP and extended high-frequency hearing compared to other

birds. Although this does not immediately identify a mechanism, it suggests some benefit of an increased EP at higher auditory frequencies. Indeed, the EP is known to vary significantly even

along the tonotopic gradient of the mammalian cochlea, from about +70 mV in the most apical, low-frequency turn to about +90 mV basally55. CONCLUSION Our data shed light on the evolution of

the molecular repertoire in the vertebrate inner ear. The recruitment of Kir4.1 to the mammalian inner ear was a crucial step in generating the high EP, which has no equivalent in other

vertebrate groups. Furthermore, our study shows that the large-scale genomic data available for all important vertebrate groups will greatly assist in deciphering evolutionary processes in

the auditory system at the molecular level. This will ultimately result in a detailed understanding of the evolution of the auditory system, which is tied to human social evolution like no

other sensory system. MATERIAL AND METHODS ANIMALS Chickens (_Gallus gallus domesticus_), egg-layer breed, aged 18 to 36 days posthatching and barn owls (_Tyto alba guttata_) aged 34 to 966

days posthatching were used. All protocols were in accordance with the German Animal Protection law and approved by local animal care and use committees (Government of Upper Bavaria and

Laves, Oldenburg, Germany). Protocols also followed the NIH guide for the care and use of laboratory animals. TISSUE FIXATION Animals were injected with a lethal dose of sodium pentobarbital

(Narkodorm, CP-Pharma GmbH, Burgdorf, Germany) and perfused transcardially with phosphate-buffered saline (PBS) containing (mM): 130 NaCl, 7 Na2HPO4, 3 NaH2PO4, pH 7.4, followed by fixation

by 4% paraformaldehyde (PFA) in PBS. Animals were then decapitated and the head stored in PFA at 4°C overnight. For dissection of the cochlea, the middle ear cavity was opened and bony

structures were removed. For brain and kidney tissue, samples were collected after perfusion of the animal and stored in 30% sucrose and 4% PFA at 4°C overnight. EVOLUTIONARY BIOINFORMATICAL

ANALYSIS The human protein sequences of Barttin (NM_057176.2), CLC-Ka (NM_001042704.1) and CLC-Kb (NM_000085.4) were subjected to Blast analyses (Blastp, NCBI) against the databases of

following organisms: _Rattus norvegicus_, _Monodelphis domestica_, _Danio rerio_, _Takifugu rubripes_, _Latimeria chalumnae_, _Xenopus (Silurana_) _tropicalis_, _Gallus gallus_, _Taeniopygia

guttata_, _Anolis carolinensis_, _Strongylocentrotus purpuratus_ and _Ciona intestinalis_. Sequences with an E-value of at least 10−2 were saved and reverse blasted against _Homo sapiens_

protein database56. Protein sequences that showed in the reverse blast the same Barttin, CLC-Ka and CLC-Kb protein sequences of _Homo sapiens_ as a best hit were used for further analyses.

The multiple sequence alignments were generated by using the default settings in MUSCLE57 as implemented in SeaView v4.4.2 and manually improved by eye thereafter. The phylogenetic tree of

Barttin and CLC-Ka/b were constructed using maximum-likelihood analyses with a bootstrap analysis of 1,000 replicates (PhyML). The final tree was edited using FigTree. IMMUNOHISTOCHEMISTRY

Isolated barn owl cochleae were soaked in 20% gelatin in PBS at 40 °C for 30 minutes and the block then hardened by cooling. The embedded cochlea was cut with a razorblade into three pieces

which could then be oriented and cut separately. This was necessary to compensate for the pronounced curvature of the barn owl basilar papilla, in order to obtain approximate cross-sections

along its full length. The individual blocks were further hardened in 4% PFA in PBS for a day and then cut at 40 μm with a vibratome (Leica VT-1000S, Wetzlar, Germany). Isolated chicken

cochleae were cryprotected in 30% sucrose for a minimum of 30 minutes. They were then cut into two pieces, each of them mounted in TissueTek (VWR, Darmstadt, Germany), oriented for later

cross-sectioning. Cross sections of 20 μm thickness were cut on a cryostat (CM1950, Leica Biosystems). Sections were mounted on gelatinized slides, dried and subsequently immunoreacted

according to standard protocols, using 3% bovine serum albumin +0.2% TritonX-100 as blocking solution. The primary antibody was anti-Na+, K+ ATPase (Sigma-Aldrich, A276, Darmstadt, Germany)

at a concentration of 1:100, coupled to a biotinylated secondary anti-mouse antibody (Vector Laboratories, BA-2000, Eching, Germany) applied at 1:100 and subsequently to CY3-labelled

streptavidin (Jackson ImmunoResearch, 016-160-084, Hamburg, Germany), applied at 1:600. Control slides were treated in parallel, but omitting the primary antibody. Slides were finally

coverslipped under Vectashield (Vector Laboratories) and documented using an epifluorescence microscope (Nikon Diaphot-TMD or Nikon 90i) with a digital camera attached. RNA ISOLATION AND

REVERSE TRANSCRIPTION Total RNA was isolated from brain and kidney of chicken by the guanidine thiocyanate method58. The quality and quantity of RNA samples were assessed by gel

electrophoresis and optical density measurements, respectively. Reverse transcription of total RNA (10 μg) was performed using standard protocols with a mixture of random hexanucleotide and

poly-T primers and Revert AidTM M-MuLV reverse transcriptase (Thermo Scientific, St. Leon-Rot, Germany) as the enzyme59. RNA _IN SITU_ HYBRIDIZATION Probes for _ATP1A1_, _ATP1A2_, _ATP1A3_,

_ATP1B2, KCNJ10_, _KCNJ16_, _KCNQ1_, _KCNE1_, _GJB2_, _GJB6_ and _SLC12A2_ were generated from chicken brain cDNA and probes for _BSND_ and _CLCK_ from chicken kidney cDNA. The barn owl

_SLC12A2_ and _KCNJ10_ probes were generated from owl brain cDNA. Primers and Genbank accession numbers of the corresponding genes are given in Table 4. For barn owl _KCNJ10_, no public

sequence was available and primers were therefore based on conserved regions of a multiple sequence alignment of sauropsid _KCNJ10_ sequences. PCR products were cloned into the pGEM-T easy

vector (Promega, Mannheim, Germany) and transcribed by T7 or SP6 polymerases in the presence of digoxigenin-11-UTP (Roche, Mannheim, Germany)60. Transcription by T7 polymerase leads to the

generation of antisense probes, while transcription by SP6 polymerase leads to the generation of sense probes. Cochleae were cryoprotected in 15% or 30% sucrose in PBS for a minimum of 30 

minutes. They were then cut into 2 (chicken) or 4–5 (barn owl) pieces harboring approximately linear segments of basilar papilla each. The pieces were transferred to custom molds filled with

TissueTek (VWR, Darmstadt, Germany), oriented for later cross-sectioning and rapidly frozen on a metal platform cooled by liquid nitrogen. Specimens from the cochlea, brain, or kidney were

stored at −80 °C until needed. Cross sections of 20 μm thickness were cut on a cryostat (CM1950, Leica Biosystems) and sections stored at −80 °C until use. On-slide _in situ_ hybridization

was performed at 50–60 °C overnight in hybridization buffer (50% formamide, 5 × SSC, 2% blocker (Roche), 0.02% SDS, 0.1% N-Lauryl sarcosine60. Bound probes were detected with an

anti-Digoxigenin antibody conjugated to alkaline phosphatase (Roche). SP6 (sense) probes served as negative controls and yielded no staining. Slides were documented using DIC optics on a

Nikon Eclipse 90i microsope with a digital camera attached. EP MEASUREMENT All electrophysiological measurements were carried out under anaesthesia, with animal homeostasis as previously

described61,62. Briefly, anaesthesia was induced by intramuscular (i.m.) injection of ketamine (20 mg/kg for chickens, 10 mg/kg for owls) and xylazine (3 mg/kg). Cloacal temperature was

maintained at 41.5 °C (chickens) or 39 °C (owls) and an electrocardiogram recorded to monitor anaesthesia. Anaesthesia was mostly maintained by supplementary half-doses of ketamine and

xylazine or, in two chickens, by 1.5% isofluorane in carbogen, delivered via a custom respiration mask. All animals breathed unaided but most were intubated through a tracheal cut, in order

to prevent complications arising from salivation. All animals also received an additional, single dose of 20 mg/kg metamizole i.m. The head was held firmly in a custom device. In owls, an

opening in the posterior skull provided a view of the columella (middle ear ossicle) and the oval and round windows of the inner ear. In chickens, the external ear canal was widened and the

eardrum removed to achieve a similar view. The columella was clipped and the columellar footplate was carefully lifted from the oval window, which was then slit to open scala vestibuli and

expose the tegmentum vasculosum. Borosilicate glass electrodes filled with 3 M KCl and a typical impedance of 20–50 MOhms were placed under visual control and then advanced remotely by a

precision microdrive (Burleigh inchworm 6000ULN, Burleigh Park, USA) through the tegmentum into scala media. The potential relative to a grounded Ag/AgCl pellet electrode, placed under the

skin in the head region, was amplified (World Precision Instruments 767 electrometer, Berlin Germany) and fed to a National Instruments interface card (BNC 2110) mounted in a personal

computer. Custom written LabView software (National Instruments) was used to continuously monitor and store the measured potential. We only accepted measurements that satisfied the following

criteria: 1. Positive potential stable upon advancing the electrode a further 40–50 μm, 2. potential then stable over several minutes without moving the electrode, 3. potential returned to

within a few mV of the original zero point upon retreat of the electrode. The value of the EP was taken as the potential difference on retreat (see example in Fig. 6). At the conclusion of

measurements, animals were euthanised by an overdose of sodium pentobarbital. In three chickens, this was administered while an electrode was still in scala media and the change upon death

monitored. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Wilms, V. _et al._ Molecular bases of K+ secretory cells in the inner ear: shared and distinct features between birds and mammals.

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(2015). Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Carl Christel, Walter Hermes, Karla Peters and Daniela Thiele who participated in the EP measurements as part

of their undergraduate studies. Furthermore, we would like to acknowledge the help of Heiner Hartwich in cloning avian genes. English language services were provided by www.stels-ol.de. This

work was supported by the Deutsche Forschungsgemeinschaft grants No. 428/18-1 and HA 6338/2-1. The funding organization had no influence on the design of the study and collection, analysis

and interpretation of data and in writing the manuscript. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Neurogenetics group, Cluster of Excellence “Hearing4All”, School of Medicine and

Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, 26111, Germany Viviane Wilms, Chris Söffgen, Anna-Maria Hartmann & Hans Gerd Nothwang * Cochlea and Auditory

Brainstem Physiology, Cluster of Excellence “Hearing4All”, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, 26111, Germany Christine Köppl *

Research Center for Neurosensory Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, 26111, Germany Christine Köppl & Hans Gerd Nothwang * Systematics and Evolutionary Biology

Group, Institute for Biology and Environmental Sciences, Carl von Ossietzky University, Oldenburg, 26111, Germany Anna-Maria Hartmann Authors * Viviane Wilms View author publications You can

also search for this author inPubMed Google Scholar * Christine Köppl View author publications You can also search for this author inPubMed Google Scholar * Chris Söffgen View author

publications You can also search for this author inPubMed Google Scholar * Anna-Maria Hartmann View author publications You can also search for this author inPubMed Google Scholar * Hans

Gerd Nothwang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS C.K., A.-M.H. and H.G.N. designed experiments, V.W., C.K., C.S. and A.-M.H.

performed experiments, H.G.N. wrote the manuscript with the help of V.W., C.K. and A.-M.H. All authors analyzed and discussed the data. ETHICS DECLARATIONS COMPETING INTERESTS The authors

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wilms, V., Köppl, C., Söffgen, C. _et al._ Molecular bases of K+ secretory cells in the inner ear: shared and distinct features between

birds and mammals. _Sci Rep_ 6, 34203 (2016). https://doi.org/10.1038/srep34203 Download citation * Received: 27 April 2016 * Accepted: 08 September 2016 * Published: 29 September 2016 *

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