RESEARCH ARTICLE
Age-dependent gene expression in the inner
ear of big brown bats (Eptesicus fuscus)
Beatrice Mao1¤*, Cynthia F. Moss2,3, Gerald S. Wilkinson1
1 Department of Biology, College of Computer, Mathematical, and Natural Sciences, University of Maryland,
College Park, Maryland, United States of America, 2 Department of Psychological and Brain Sciences,
Zanvyl Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, Maryland, United States of
America, 3 The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of
Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America
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OPEN ACCESS
Citation: Mao B, Moss CF, Wilkinson GS (2017)
Age-dependent gene expression in the inner ear of
big brown bats (Eptesicus fuscus). PLoS ONE 12
(10): e0186667. https://doi.org/10.1371/journal.
pone.0186667
Editor: Michael Smotherman, Texas A&M
University College Station, UNITED STATES
Received: June 4, 2017
Accepted: October 5, 2017
Published: October 26, 2017
Copyright: © 2017 Mao et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: B.M. was supported on training grant No.
T32 DC000046 from the National Institute of
Deafness and Communicative Disorders of the
National Institutes of Health (https://www.nidcd.
nih.gov/), awarded to A. N. Popper. Partial funding
for open access provided by the UMD Libraries’
Open Access Publishing Fund. The funders had no
role in study design, data collection and analysis,
¤ Current address: National Institute on Deafness and Communication Disorders, National Institutes of
Health, Bethesda, Maryland, United States of America
* beatrice.mao@nih.gov
Abstract
For echolocating bats, hearing is essential for survival. Specializations for detecting and processing high frequency sounds are apparent throughout their auditory systems. Recent
studies on echolocating mammals have reported evidence of parallel evolution in some
hearing-related genes in which distantly related groups of echolocating animals (bats and
toothed whales), cluster together in gene trees due to apparent amino acid convergence.
However, molecular adaptations can occur not only in coding sequences, but also in the regulation of gene expression. The aim of this study was to examine the expression of hearingrelated genes in the inner ear of developing big brown bats, Eptesicus fuscus, during the
period in which echolocation vocalizations increase dramatically in frequency. We found
that seven genes were significantly upregulated in juveniles relative to adults, and that the
expression of four genes through development correlated with estimated age. Compared to
available data for mice, it appears that expression of some hearing genes is extended in
juvenile bats. These results are consistent with a prolonged growth period required to
develop larger cochlea relative to body size, a later maturation of high frequency hearing,
and a greater dependence on high frequency hearing in echolocating bats.
Introduction
Echolocating bats have among the highest frequency hearing in the animal kingdom [1].
While high frequency hearing confers a survival benefit to many animals, it is essential for the
survival of bats, because they rely on echolocation to avoid obstacles, obtain food, and find
roosts and conspecifics. High frequencies also allow bats to control the directionality of calls
[2], [3], determine distance to targets [4], reject non-target echo clutter [5], and resolve fine
spatial details such as shape, size, and texture [6–8]. Furthermore, bats are exceptionally longlived for their size, with individuals of some species living more than 30 years [9]. The need for
echolocation throughout life suggests that the ability to hear high frequencies without severe
age-related deterioration may have been under positive selection in echolocating bats. This
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Age-dependent gene expression in the inner ear of big brown bats
decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
stands in contrast with the occurrence of age-related hearing loss (presbycusis) in humans,
which has been estimated to be 40% among those over 70 [10].
The importance of hearing to echolocators has been illustrated by a number of recent studies examining the molecular evolution of genes involved in hearing in bats. Several genes
known from human and mouse studies to be crucial for normal hearing, such as transmembrane channel-like 1 (Tmc1) and Prestin/SLC26A5, exhibit convergence between the two distantly related groups of echolocating bats, or even between echolocating bats and whales, such
that gene trees sometimes group echolocators together to the exclusion of non-echolocators
[11–17]. While the results of these studies are compelling, the amount or timing of gene
expression may also contribute to different phenotypes without requiring changes in coding
sequence. Recent studies have shown that changes in gene regulation can influence the physical differences between bats and other mammals: transgenic mice possessing bat limb enhancers exhibit prolonged expression of limb elongation genes [18] and develop significantly
longer limbs than control mice [19].
The big brown bat (Eptesicus fuscus) is an insectivore that hunts in edge spaces between
open and cluttered environments [20]. This behavior requires the disambiguation of cascades
of echoes from multiple objects into separate percepts [21–22], which must occur quickly
enough to inform motor decisions in flight. Because echolocation and flight are critical for a
young bat’s survival, the development of hearing occurs concurrently with echolocation calls
and the motor skills involved in flight [23–28]. The echolocation calls of juvenile big brown
bats undergo significant changes between birth and three weeks of age, becoming shorter in
duration and higher in frequency [26–29]. These changes in echolocation call frequencies
likely coincide with changes in their hearing, because the frequency place map of the cochlea
changes as it matures, with higher frequency hearing developing later [30, 31]. Additionally,
the call frequencies of five species of bats were lower in the first year of life than later in adulthood, suggesting that fine-tuning of echolocation calls may occur well after the development
of hearing is complete (summarized in [32]).
Because of their dependence on hearing for survival and their relatively well-developed
auditory systems, echolocating bats provide a valuable opportunity to examine postnatal
hearing development in an auditory specialist. Laryngeally echolocating bats possess larger
cochlea [33] relative to basicranial width than non-echolocating or non-laryngeally echolocating bats [34]. Bats using constant-frequency calls also exhibit overrepresentation of dominant
call frequencies in basilar membrane (BM) dimensions and spiral ganglion density [35], and
extremely short hair cells and stereocilia [36]. A recent study showed that echolocating bats
sustain a high prenatal cochlear growth rate throughout development compared to non-echolocating bats and other mammals [37], but which genes change expression during bat cochlear
development is unknown. Here, we report on the expression of selected hearing-related genes
in the inner ears of young big brown bats over a two-week period during which their calls rapidly increase in frequency, becoming more similar to adult echolocation calls [26–29]. Because
these pronounced frequency shifts in vocalizations have been reported to coincide with frequency shifts in hearing in several bat species (e.g., [24, 38, 39]), examining gene expression
during this period may provide insight into the regulatory changes associated with the development of high frequency hearing.
Materials and methods
Subjects and sample preparation
Pregnant female Eptesicus fuscus were captured in the wild under a permit from the Maryland
Department of Natural Resources. All twelve juvenile subjects were born in captivity. Because
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Age-dependent gene expression in the inner ear of big brown bats
they were group-housed and cluster together, exact dates of birth could not be directly recorded.
Instead, forearm length was measured with calipers and used to estimate age [40]. Forearm
length is a more accurate age estimator than mass for big brown bats, and results from formulae
relating forearm length to age do not differ between wild and captive bats [28]. Estimated ages
ranged from postnatal day (PND) 9 to 19. Juveniles were weighed, anesthetized with isoflurane
and euthanized via decapitation. All procedures were in accordance with the National Institutes
of Health’s Guide for the Care and Use of Laboratory Animals, and were approved by the Johns
Hopkins University Institutional Animal Care and Use Committee (protocol BA14A111). Samples were also obtained from two adult individuals under a protocol approved by the University
of Maryland Institutional Animal Care and Use Committee (R-13-76).
Inner ear samples, consisting of the entire otic capsule (both cochleae and vestibular
organs), were collected immediately post-mortem and placed into liquid nitrogen prior to
storage at -80˚C until extraction. Both left and right cochleae from an individual were pooled
and processed together. Samples were homogenized with a mortar and pestle while submerged
in liquid nitrogen. RNA extraction was performed using a mirVana kit (Ambion), with added
proteinase K (Sigma Aldrich) to improve yield [41]. All samples were treated with TURBO
DNA-free DNAse (Ambion) and cleaned with isopropanol and ethanol. Sample quality was
checked on a Nanodrop spectrophotometer and reverse transcribed with M-MLV (Thermo
Fisher) using a 50/50 mix of oligo-dT and random primers to lower the risk of bias or truncated transcripts associated with a single priming method [42, 43].
Gene selection and primer design
Candidate genes were selected based on one or more of the following criteria: upregulated in
an echolocating bat vs. a non-echolocating bat (e.g., [44]); upregulated in an adult mouse relative to juvenile mouse (e.g., [45]); expressed in mid- to late- development (e.g., [46]); evidence
of parallel or convergent evolution between echolocating bats and whales (e.g., [16]); evidence
of parallel or convergent evolution between distantly related echolocating bats (e.g., [16]); or
involved in formation of essential cochlear structures (e.g., [47]; Table 1). For each gene, all
available mRNA transcripts from Eptesicus fuscus and all bats of the genus Myotis (another
genus in the same family, Vespertilionidae), were downloaded from GenBank (NCBI) and
aligned using Clustal Omega (EMBL-EBI). Sequences from Myotis spp. were included in order
to reduce the risk of designing primers in regions with polymorphic sites. All primer pairs
were designed within the same exon to permit preliminary testing on genomic DNA.
To identify exons in an Eptesicus fuscus transcript, exonic regions of the Myotis lucifugus
transcript, as identified in Ensembl, were blasted against the transcript for Eptesicus fuscus. If
the Myotis transcript was not available in Ensembl, the mouse (Mus musculus) transcript was
used instead. If the exonic region was conserved among Eptesicus and Myotis spp., it was
entered into Primer-BLAST (NCBI). Potential primer pairs were checked for specificity
against Eptesicus fuscus RefSeq data, potential for cross- and self-dimerization, and potential to
form hairpins using Beacon Designer (Premier Biosoft). Only primers that were 100% conserved across all known transcripts from Eptesicus and Myotis spp. were used for quantitative
PCR. Primer sequences are given in Table 2.
Five-point dilution series (1:3 or 1:4) were performed for each gene and only primer pairs
with efficiencies greater than 90% after exclusion of non-linear dilutions (typically at the highest or lowest concentration of template) were selected for use. Post-amplification melt curves
were checked to ensure each product consisted of a single, narrow peak, and gel electrophoresis was performed for each amplicon to ensure a single product of correct size was produced
during amplification.
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Age-dependent gene expression in the inner ear of big brown bats
qPCR and data analysis
For each primer pair, 20 μL reactions were prepared for each of the samples in triplicate using
SYBR Select Master Mix (Thermo Fisher). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was included as a reference gene on each 96-well plate. Fluorescence was measured
using a Roche 480 Lightcycler and melt curves were measured immediately after the completion of all amplification cycles. Technical replicates that reached threshold two or more cycles
earlier or later than the other two replicates were excluded from analyses.
Table 1. Criteria for inclusion and other relevant information for genes included in this study, and references. In the “Criteria for inclusion column,”
letter codes mean the following: A, upregulated in an echolocating bat vs. a non-echolocating bat; B, upregulated in an adult mouse relative to juvenile mouse;
C, expressed in mid- to late- development; D, exhibits signs of parallel or convergent evolution between echolocating bats and whales; E, exhibits signs of parallel or convergent evolution between distantly related echolocating bats; F, participates in forming essential cochlear structures. aMutations in Gjb6 may
cause hearing loss by inducing a downregulation of Gjb2. Gjb6 appears not be critical for hearing, unlike Gjb2 (see [64]).
Gene
symbol
Full name of gene
Criteria for Location of gene
inclusion
product
Morphological effects of deletion
or mutation in mouse models
Associated with
human deafness
(and loci if
applicable)
Sources
Bmp7
Bone morphogenic
protein 7
F
throughout cochlear duct
loss of position-specific sensory cell
morphology consistent with loss of
tonotopy
yes
[48, 49]
Ceacam16
Carcinoembryonic
antigen-related cell
adhesion molecule 16
A, F
tallest OHC stereocilia
tips; TM
disruption of normal striated-sheet
matrix of TM, Hensen’s stripe absent
DFNA4
[44, 50–
53]
Col11A2
Collagen type XI alpha 2
chain
A
TM, cartilaginous otic
capsule, spiral limbus,
lateral wall, cristae
ampullaris
enlarged TM containing disorganized DFNA13; DFNB53
collagen fibrils; reduced density of
radial collagen fibers in the TM
[44, 54–
56]
GFAP
Glial fibrillary acidic
protein
B
supporting cells,
Schwann cells in SG and
osseous spiral lamina
greater loss of OHCs after noise
exposure
[45, 57,
58]
Gjb2
Gap junction protein beta AF
2
gap junctions of
supporting cells
severe degeneration of the organ of
Corti and SGN loss
DFNB1
[44, 59–
61]
Gjb6 a
Gap junction beta protein A, F
6
gap junctions of
supporting cells
missing OHCs
DFNB1; DFNA3
[44, 62–
65]
LOXHD1
Lipoxygenase homology
domains 1
A, B
cochlear and vestibular
hair cell stereocilia
fused stereocilia and ruffled apical
DFNB77
cell surface at cochlear base, leading
to eventual hair cell and SGN loss
[44, 66]
Pou3F4
POU class 3
transcription factor 4
A
throughout otic capsule
radial bundle defasciculation;
abnormal gap junctions; malformed
stapes footplate; reduced cochlear
coiling; other abnormalities
DFNX2
[44, 67–
69]
Pou4f3
POU class 4
transcription factor 3
C
nuclei of cochlear and
vestibular hair cells
loss of auditory and vestibular hair
cells; failure of differentiated hair
cells to develop stereociliary
bundles; loss of spiral and vestibular
ganglion neurons
DFNA15
[46, 70,
71]
Tmc1
Transmembrane
channel-like 1
A, D, E, F
MET channels of hair
cells
none
DFNA36; DFNB7;
DFNB11
[16, 44,
47, 72–
74]
Tmc2
Transmembrane
channel-like 2
F
MET channels of hair
cells
none
Tspan1
Tetraspanin 1
B
in zebrafish, rostral
mantle cells within
neuromasts
Ush1C
USH1 protein network
component harmonin
A, B, C, F
Upper tip link density of
stereocilia bundles;
cochlear and vestibular
neurosensory epithelia
[72–74]
[45, 75]
splayed hair cell bundles;
progressive degeneration of hair
cells
DFNB18
[44, 76–
80]
https://doi.org/10.1371/journal.pone.0186667.t001
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Age-dependent gene expression in the inner ear of big brown bats
Table 2. Primers used to amplify Eptesicus fuscus cDNA and calculated efficiencies based on dilution series. Efficiencies greater than 100% typically indicate the presence of inhibitors, the effects of which decrease at lower dilutions.
Gene
Forward primer
Reverse primer
Efficiency (%)
Bmp7
CCTACAAGGCGGTCTTCAGC
CGTCGGTGAGGAAGTGGCTA
102.2
CGAAGTGCTCGTCCAGTGTTG
ATCCAGGATACGGGCACCAAA
Ceacam16
Col11A2
GAPDH
GFAP
Gjb2
Gjb6
LoxHD1
Pou3f4
Pou4f3
Tmc1
Tmc2
Tspan1
Ush1C
ACATCGTAAGCACAGGCGAC
GGGCTGCCCAGAACATCATC
CACCGGCTTCAAGGAGACAC
CAGAAGGTCCGAATTGAAGGGT
TTCATCGGGGGTGTGAACAAA
CGAGATCGTCATAGAAACGGGC
AGCGATCTAGGCTCTCACCA
TGGATATCGTCTCCCACGGC
CTCATCTTTTGGGCTGTGAAG
CAGGACTGGTGGGCATCAAC
GTGCTCTTGGCTCTCGGTTT
GCTGGAAGAGGTGAGGCAG
CTGAAGGATGTAGGTGCCCG
GCTCAGGGATGACCTTGCC
TTCTCGATGTAGCTGGCGAAG
AAGATGACCCGGAAGAAGATGC
CACGAGGATCATGACACGGAAG
TCTTTGGATCGGTTCTTCCTGC
CATCCGAGGTTGGTGTCTCC
TGGTATGGTAGGTGGCGTCG
CCCAAGGGTGTCAGGATCTT
GTTGGATCGGGAGGCTTTGA
AGGGCACACTTGTTCTCAGTG
CTTGTTGGACTCCATCGCCA
102.6
101.6
109.4
101.4
108.0
95.6
102.5
111.0
108.3
102.0
107.2
109.9
103.9
https://doi.org/10.1371/journal.pone.0186667.t002
For each sample-primer combination on a given plate, the comparative CT method [81] was
used to calculate relative expression. Briefly, delta CT was calculated as the average threshold
cycle of replicates from the gene of interest minus the average threshold cycle of the GAPDH
replicates. To control for any batch effects, delta CT values were adjusted by the difference in
mean delta CT between batches for each gene. Delta CT values were then normalized by subtracting the average delta CT for all juvenile samples for a given gene (yielding delta-delta CT).
Fold expression was calculated as the efficiency-adjusted amplification factor raised to the negative delta-delta CT. Average CT and calculated fold expression values are given in S1 Table.
We performed t-tests to determine whether the mean adjusted fold expression values of
juveniles differed from adults for 13 genes. We also fitted least squares regression lines between
estimated age and adjusted fold change to identify genes that exhibited age-dependent expression. All statistical analyses were performed in JMP 13.0.0 (SAS Institute). Figures were generated in JMP and MATLAB R2015a (The Mathworks).
Results
Adult vs. juvenile expression
Of the 13 genes tested, eight exhibited differential expression between juveniles and adults
(Table 3; Fig 1). Expression was higher in adults for six genes—bone morphogenic protein 7
(Bmp7), carcinoembryonic antigen-related cell adhesion molecule 16 (Ceacam16), collagen
type XI alpha 2 chain (Col11A2), POU class 4 transcription factor 3 (Pou4f3), transmembrane
channel-like 2 (Tmc2), and USH1 protein network component harmonin (Ush1C), and higher
in juveniles for the remaining two genes—gap junction protein beta 2 (Gjb2) and POU class 3
transcription factor 4 (Pou3f4).
Age-related gene expression
Linear fits of adjusted fold change to estimated age revealed that juvenile age over a two-week
period predicted expression for four genes: POU class 3 transcription factor 4 (Pou3f4), transmembrane channel-like 1 (Tmc1), and gap junction protein beta 2 (Gjb2) and 6 (Gjb6; Table 3;
Fig 2).
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Age-dependent gene expression in the inner ear of big brown bats
Table 3. Results of two-sided t-tests performed on adjusted fold change between adults and juveniles (left) and bivariate fits of adjusted fold
change by estimated age (right). For all t-tests, there were 13 degrees of freedom, and bivariate fits had 11 degrees of freedom. Fold change values were
adjusted to the mean of all juvenile samples and also to differences in mean juvenile expression between batches (see Materials and Methods). Asterisks
denote level of significance (*p0.05, **p0.01, ***p0.005).
Adult vs. juvenile t-test
t Ratio
p Value
Age vs. adjusted fold change bivariate fit
Mean ± SE,
adult
Mean ± SE, juvenile
F ratio
p Value
Adjusted R2
Bmp7
-3.25**
<0.01
6.60 ± 5.44
1.04 ± 0.09
1.75
0.22
0.06
Ceacam16
-3.22**
<0.01
6.79 ± 5.61
1.08 ± 0.12
1.88
0.20
0.07
Col11A2
-2.92*
0.01
7.70 ± 6.98
1.17 ± 0.20
0.84
0.38
-0.02
GFAP
-1.90
0.08
5.16 ± 4.68
1.55 ± 0.48
0.39
0.55
-0.06
Gjb2
2.21*
0.05
0.30 ± 0.18
1.12 ± 0.14
14.85***
<0.01
0.56
Gjb6
1.89
0.08
0.27 ± 0.23
1.25 ± 0.20
18.62***
<0.01
0.62
LoxHD1
-1.93
0.08
5.24 ± 4.76
1.56 ± 0.48
0.32
0.58
-0.07
Pou3f4
2.31*
0.04
0.28 ± 0.03
1.11 ± 0.14
7.32*
0.02
0.37
Pou4f3
-3.21**
<0.01
49.44 ± 48.20
1.15 ± 0.19
1.02
0.34
0
Tmc1
-1.88
0.08
3.03 ± 2.18
1.30 ± 0.24
5.82*
0.04
0.31
Tmc2
0.01
-3.97***
<0.01
6.18 ± 4.04
1.06 ± 0.11
1.14
0.31
Tspan1
1.98
0.07
0.41 ± 0.23
1.12 ± 0.14
3.75
0.08
0.2
Ush1C
-3.01*
0.01
7.98 ± 7.27
1.08 ± 0.14
1.49
0.25
0.04
https://doi.org/10.1371/journal.pone.0186667.t003
Discussion
Adult vs. juvenile expression
We found significant differences between juvenile and adult bat in inner ear expression of
eight genes. The most significantly upregulated gene in adults was Tmc2 (Fig 1). Tmc1 and
Fig 1. Log2-scaled means and standard errors of adult and juvenile expression relative to GAPDH. Values were adjusted to
remove the effect of batch and normalized to average juvenile expression (see Materials and Methods). Juvenile data are shown in
light grey, and adult data are shown in dark grey. Asterisks denote level of significance of associated t-tests (see Table 3; *p0.05,
**p0.01, ***p0.005).
https://doi.org/10.1371/journal.pone.0186667.g001
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Age-dependent gene expression in the inner ear of big brown bats
Fig 2. Genes for which the relationship between adjusted fold change and estimated age was significant for juvenile bats.
Values were normalized to average juvenile expression and adjusted to remove the effect of batch (see Materials and Methods).
Asterisks denote level of significance of associated t-tests (see Table 3; *p0.05, **p0.01, ***p0.005).
https://doi.org/10.1371/journal.pone.0186667.g002
Tmc2 are expressed in the cochlea and vestibular system [72, 74], and encode components of
the mechanoelectrotransduction (MET) channels of hair cells [73]. Their protein products
may form heteromeric assemblies that confer different electrophysiological properties to hair
cells along the BM [73]. Despite functional redundancy between Tmc1 and Tmc2, mice with a
targeted deletion of Tmc1 are deaf because Tmc2 does not persist in the cochlea beyond early
postnatal ages [72]. In the utricle, Tmc1 and Tmc2 expression continues through the first few
postnatal weeks [72]. These observations in postnatal mice suggest that continued Tmc2
expression into adulthood in bats may be restricted to the balance organs.
Bmp7, Ceacam16, Col11A2, and Ush1C were also upregulated in adults relative to juveniles
(Fig 1). Bmp7 is expressed in a gradient along the basilar papilla, and disruption of this gradient results in loss of tonotopy and morphological changes in sensory cells [49]. While we
found that it was upregulated in adult bats, another study reported that Bmp7 is downregulated
in the cochlear sensory epithelia of P60 mice relative to P1 mice [82]. Ush1C encodes a protein,
harmonin, that is a component of upper tip-link densities of stereocilia bundles [78]. Mutations in Ush1C are associated with Usher syndrome type 1C in humans [76], and mouse
mutants exhibit splayed stereocilia bundles and progressive loss of hair cells and spiral ganglion neurons [77]. Cochlear expression of Ush1C drops prior to birth and then increases into
adulthood in mice [79] and is similarly expressed at higher levels in adult than juvenile bats
(Table 3; Fig 1).
Both Ceacam16 and Col11A2 encode proteins that are components of the tectorial membrane (TM), and their deletion disrupts TM structure [53, 56], resulting in hearing loss [52,
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Age-dependent gene expression in the inner ear of big brown bats
54]. The TM acts as an inertial mass which allows the outer hair cells (OHCs) to amplify BM
motion [83]. Reducing its mass by deleting Tectb improved the frequency selectivity of the BM
and neural response at high frequencies [84]. Ceacam16 may stabilize interactions between
TM glycoproteins, such that cochlear amplification becomes unstable without it [53]. The
upregulation of Col11A2 and Ceacam16 may, therefore, result in a TM structure which allows
bat hair cells to effectively amplify high frequency sounds.
Pou4f3 showed the greatest difference in expression between age groups (Fig 1). Pou4f3 is a
transcription factor implicated in progressive non-syndromic hearing loss in humans [71].
Mice lacking Pou4f3 fail to develop stereocilia bundles [46], resulting in the loss of hair cells
and spiral ganglion neurons [70]. Pou4f3 is expressed into adulthood in mice [46, 70, 85] but is
downregulated in the P60 mouse cochlea compared to P1 [82]. Taken together, the upregulation of Tmc2, Bmp7, Ush1C, Ceacam16, Col11A2, and Pou4f3 in adult big brown bats may
reflect continued development or maturation of the inner ear that continues beyond the time
point at which bats can fly and produce adult-like echolocation calls. The two genes that were
significantly upregulated in juveniles relative to adult bats, Gjb2 and Pou3f4, are discussed in
further detail in the next section, as their expression also correlated with juvenile age.
Age-related gene expression
Four genes were significantly upregulated with age in juvenile bats. Of these, Gjb2, Gjb6, and
Pou3f4 were downregulated in adult bats relative to juvenile bats, perhaps because their roles
in inner ear development were complete (Table 3; Figs 1 and 2). The expression of the fourth
gene, Tmc1, did not differ significantly between juveniles and adults, although standard errors
for adult samples were high due in part to small sample size (Fig 1). While levels of the protein
products (Cx26 and Cx30) of Gjb2 and Gjb6 saturate at P15 in the mouse cochlea [86], we
found that Gjb2 and Gjb6 expression increased through the third postnatal week in the inner
ears of bats. In an earlier report, these genes were significantly upregulated in the inner ears of
an echolocating bat (Myotis ricketti) compared to a non-echolocating bat (Cynopterus sphinx)
[44]. Gjb2 appears critical for cochlear function and is implicated in the most common form of
congenital deafness in humans [59, 87]. Gjb6 has also been linked to human deafness [62],
although the deleterious effects of Gjb6 knockdown in mice are less severe than those of Gjb2
and may be partly caused by associated downregulation of Gjb2 [64, 88].
The upregulation of Gjb2 and Gjb6 may reflect greater numbers of gap junctions in the bat
cochlea. Both genes may participate in the recycling of potassium, the major charge carrier in
transduction (reviewed in [89]). Conditional knockdown of Gjb2 in early postnatal mice
impaired OHC amplification and high frequency hearing [90], consistent with gap junction
conductivity enabling OHCs to respond to higher frequencies [91–93]. The continued expression of Gjb2 and Gjb6 may also result from prolonged development of the cochleae, which are
relatively large in echolocating bats [33, 34]. A recent paper showed that the relative median
prenatal growth rate of echolocating bats’ cochleae was approximately two and four times
larger, respectively, than that of non-echolocating mammals and non-laryngeally echolocating
bats [37].
Gjb2 and Gjb6 upregulation may provide some protection against hearing loss in echolocating bats, which depend on hearing throughout their long lives. Conditional knockdown of
Gjb2 in mice at P18 resulted in greater susceptibility to noise-induced hearing loss at P30 and
P45 [94], and mice lacking Gjb6 exhibited abnormal epithelial repair after hair cell loss and
reduced intercellular communication between supporting cells [95]. Cx26 and Cx30 may be
targets of oxidative damage, contributing to age-related and noise-induced hearing loss [96].
The increase of Gjb2 and Gjb6 expression during juvenile development in bats may, therefore,
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Age-dependent gene expression in the inner ear of big brown bats
be associated with a system of gap junctions that facilitates cochlear protection or repair. After
an hour of broadband noise exposure at 152 dB SPL, adult big brown bats showed no significant threshold shifts [97, 98], increase in errors, or changes in echolocation behavior when flying through a cluttered corridor [99]. Additionally, bat echolocation calls can be as intense as
140 dB, although they last only milliseconds [100], and it is unclear whether wild bats encounter sounds that could damage their hearing.
Pou3f4 is a transcription factor that has been implicated in X-linked non-syndromic deafness [67]. Pou3f4 mouse mutants exhibit audiological and balance impairments, reduced coiling of the cochlea [68], and defects in gap junctions [101]. Deletion of Pou3f4 from otic
mesenchyme causes defasciculation of spiral ganglion neurons [69], which could disrupt coordination of hair cell and neuronal frequencies [102]. These studies suggest that the continued
upregulation of Pou3f4 in the developing bat inner ear may be linked to cochlear elongation
and functional organization. One report did not find evidence of positive selection on Pou3f4
among echolocating bats [103], suggesting that change in expression, rather than sequence,
has been more important in bats.
Tmc1 encodes a MET channel protein [73] that localizes to the tip-links of stereocilia [74]
and which is essential for mechanotransduction in cochlear hair cells [72]. Reports of its postnatal expression pattern conflict: one study found a slight increase, then decrease in Tmc1
expression in the inner ear of mice from P9 to P19, with a net decrease of approximately 8%
over the period [47]. Another study reported a 2-fold increase between P9 and P19 in the utricle and a much greater increase over the same time period in the apex of the cochlea [72]. The
increase in Tmc1 we observed in developing big brown bats is consistent with the latter study,
and with a transcriptomic comparison of the inner ears of bats which showed that 18 hearingrelated genes were upregulated in an echolocating bat compared to a non-echolocating bat,
including Tmc1, which was also upregulated in echolocating bats relative to mice and rats [44].
Although the nature of our samples (entire inner ears) did not permit examination of gene
expression specifically in the cochlea or its basal, high frequency region, the upregulation of
Tmc1 could reflect a greater number of MET channels per hair cell, which might increase sensitivity to high frequencies by strengthening the influx of calcium and reducing the adaptation
time of hair cells (reviewed in [104]). In midshipman fish (Porichthys notatus), fluctuations in
the expression of a calcium-activated potassium (BK) channel conferred greater hearing sensitivity during the breeding season [105], and knockdown of BK channel genes increased thresholds in zebrafish larvae [106]. Alternatively, bat MET channels may contain more Tmc1
subunits. Because mouse hair cells expressing only wildtype Tmc1 had faster adaptation times
than those expressing only Tmc2 or only a Tmc1 mutant [73], MET channels incorporating
more Tmc1 subunits might respond better at high frequencies.
Although only a small set of genes were examined in this study, and we did not manipulate
gene expression directly and monitor subsequent phenotypic effects, this study provides the
first insight into the developmental expression of hearing genes in echolocating animals. Without separation of the cochlea from the vestibular organs, it is not possible to ascribe expression
differences to one section of the inner ear or the other. However, Tmc1 and Gjb2 mouse
mutants exhibit hearing loss without vestibular dysfunction, illustrating their greater importance for audition [47, 60]. Furthermore, hearing genes exhibiting various degrees of convergence between echolocating bats and whales have been implicated in human deafness [11–17],
as have most of the genes we identified as being significantly upregulated with age in big
brown bats. In particular, Tmc1 exhibits both sequence convergence [16] and upregulation
([44], this report) in echolocators, suggesting that in some cases selection may act on both coding sequence and gene regulation to confer improved hearing in echolocating mammals.
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Age-dependent gene expression in the inner ear of big brown bats
Supporting information
S1 Table. Average CT and calculated fold expression values for all individuals and genes.
(XLSX)
Acknowledgments
Wei Xian provided logistical support in the procurement of samples, Josephine Reinhardt and
Kimberly Paczolt offered advice on RNA extraction and qPCR, and the Moss and Wilkinson
labs gave valuable feedback. Partial funding for open access provided by the UMD Libraries’
Open Access Publishing Fund.
Author Contributions
Conceptualization: Beatrice Mao, Cynthia F. Moss, Gerald S. Wilkinson.
Data curation: Beatrice Mao.
Formal analysis: Beatrice Mao, Gerald S. Wilkinson.
Funding acquisition: Beatrice Mao, Cynthia F. Moss, Gerald S. Wilkinson.
Investigation: Beatrice Mao.
Methodology: Beatrice Mao, Gerald S. Wilkinson.
Resources: Cynthia F. Moss, Gerald S. Wilkinson.
Supervision: Cynthia F. Moss, Gerald S. Wilkinson.
Validation: Beatrice Mao.
Visualization: Beatrice Mao, Gerald S. Wilkinson.
Writing – original draft: Beatrice Mao.
Writing – review & editing: Beatrice Mao, Cynthia F. Moss, Gerald S. Wilkinson.
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