Download MAP3K1 function is essential for cyto

DMM Advance Online Articles. Posted 23 October 2015 as doi: 10.1242/dmm.023077
Access the most recent version at http://dmm.biologists.org/lookup/doi/10.1242/dmm.023077
MAP3K1 function is essential for cyto-architecture of mouse organ of Corti
and survival of auditory hair cells
Rizwan Yousaf,1 Qinghang Meng,2 Robert B. Hufnagel,3 Ying Xia,2 Chandrakala Puligilla,4
Zubair M. Ahmed,1 Saima Riazuddin,1,*
1
Department of Otorhinolaryngology Head & Neck Surgery, School of Medicine, University
of Maryland, Baltimore, MD, 21201, USA.
2
Department of Environmental Health, University of Cincinnati, College of Medicine,
Cincinnati, OH 45267, USA.
3
Divisions of Pediatric Ophthalmology and Human Genetics, Cincinnati Children’s Hospital
Medical Center, University of Cincinnati, OH 45229, USA.
4
Department of Pathology & Laboratory Medicine, Medical University of South Carolina,
*To whom correspondence should be addressed to:
Saima Riazuddin, PhD
Department of Otorhinolaryngology Head & Neck Surgery,
School of Medicine, University of Maryland,
800 West Baltimore St, Room 404,
Baltimore, MD, 21201, USA
Tel: 410-706-6161Email: [email protected]
© 2015. Published by The Company of Biologists Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any
medium provided that the original work is properly attributed.
Disease Models & Mechanisms • DMM • Advance article
Charleston, SC 29425, USA.
Keywords:
Map3k1, Mekk1, Fgfr3, Fgf8, Fgf10, Hearing loss, Supernumerary outer hair cells, MAPK
pathway, Fgf signaling pathway
Summary Statement
Map3k1 mutant mice exhibit early-onset profound hearing loss and supernumerary outer hair
cells, along with dysregulation of Fgf signaling pathway, accentuating its function in otic
Disease Models & Mechanisms • DMM • Advance article
epithelium development and morphogenesis.
Abstract
MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its
effect through various downstream affecter molecules, including JNK, ERK1/2 and p38. In
humans, mutant alleles of MAP3K1 are associated with 46, XY sex reversal. Until recently,
the only phenotype observed in Map3k1tm1Yxia mutant mice was open eyelids at birth. Here,
we report that homozygous Map3k1tm1Yxia mice have early-onset profound hearing loss
accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner
ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close
proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic
junctions in the organ of Corti are grossly preserved, Map3k1tm1Yxia mutant mice have
supernumerary functional OHCs and Deiters’ cells. Loss of MAP3K1 function resulted in the
down-regulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and
Fgf10 have a role in the otic placode induction or in otic epithelium development in mice and
their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our
morphogenesis genes. Collectively, our data highlight the critical role of MAP3K1 in the
development and function of the mouse inner ear and hearing.
Disease Models & Mechanisms • DMM • Advance article
studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear
Introduction
Mitogen-activated protein kinases (MAPKs) are responsible for regulating a wide
array of cellular functions and processes. The MAPK signaling cascade consists of three
tiered phosphorylation steps, starting with phosphorylation, in response to a plethora of
stimuli, by MAPK kinase kinases (MAP3Ks, MEK kinases, or MKKKs), which in turn
phosphorylate the MAPK kinases (MAP2Ks, MEK, or MKKs) and then the MAPKs
(Kyriakis and Avruch, 2001; Uhlik et al., 2004). MAP3K1, a member of the MAPK kinase
kinases family, plays a diverse cell signaling function in various biological systems, including
immune system development and function (Gallagher et al., 2007; Labuda et al., 2006),
vasculature remodeling (Li et al., 2005), tumor progression (Cuevas et al., 2006),
cardiogenesis (Minamino et al., 2002) and injury repair (Deng et al., 2006).
MAP3K1 belongs to the serine/threonine kinase class that also participates in the
regulation of the MAPK cascade (Cuevas et al., 2007; Hagemann and Blank, 2001; Uhlik et
al., 2004). MAP3K1 protein contains an ubiquitin interaction motif (UIM), a caspase-3
2003). MAP3K1 is associated with the plasma membrane and is tethered by α-actinin to actin
stress fibers and by Protein Tyrosine Kinase 2 (PTK2) to focal adhesions in cells (Christerson
et al., 2002; Cuevas et al., 2003). Following an apoptotic signal, caspase-3 cleaves MAP3K1
at p.Asp874, resulting in the separation of N terminus UIM motif from the 91-kDa C-terminal
kinase domain, releasing it from the cell membrane into the cytosol (Bonvin et al., 2002;
Schlesinger et al., 2002). MAP3K1 is activated in response to a number of different stimuli
such as cold, growth factors, mild hyper-osmolarity, microtubule disruption, cell shape
disturbance, pro-inflammatory cytokines and other physiological stresses (Sadoshima et al.,
2002; Xia et al., 2000; Yujiri et al., 1998). Once activated, MAP3K1 exerts its effect through
Disease Models & Mechanisms • DMM • Advance article
cleavage site and a conserved kinase domain (Uhlik et al., 2004; Witowsky and Johnson,
the JNK, ERK1/2 and p38 MAPK pathways, as well as the transcription factors c-Jun and
NF-kappa-B (Bonvin et al., 2002; Guan et al., 1998; Xia et al., 2000; Yujiri et al., 1998).
In humans, mutant alleles of MAP3K1 are associated with 46, XY gonadal dysgenesis
(Pearlman et al., 2010). These gain of function alleles affect the downstream phosphorylation
of p38 and ERK1/2, as well as binding of MAP3K1 with the co-factors RHOA and MAP3K4
(Loke and Ostrer, 2012). Additionally, in vitro studies have suggested that these mutations
alter the sex-determination pathway by concomitantly up-regulating β-catenin expression and
down-regulating SOX9, SRY, FGF9 and FGFR2 genes (Loke et al., 2014). However, the
Map3k1 loss of function allele displayed a minor testicular deficit in the developing gonad
and had normal gross appearance besides open eye-lid phenotype (Warr et al., 2011).
MAP3K1-deficient mice display an eye open at birth (EOB) phenotype (Zhang et al., 2003)
and have immune system and wound healing deficits, abnormal retinal vascularization,
disintegration of retinal pigment epithelium, loss of photoreceptors and retinal degeneration
(Mongan et al., 2011). Additionally, cultured keratinocytes from these mutant mice display a
al., 2003).
Previous studies have demonstrated the role of MAPK-mediated FGF signaling in otic
induction and development (Urness et al., 2010). Hearing depends on the precise organization
of sensory hair cells and non-sensory supporting cells within the organ of Corti (OC). Any
significant alteration in the cellular number, alignment or patterning within the OC causes
hearing loss, underlining the necessity of defined cytoarchitecture in the OC for sound
perception. Here, we report that mice lacking the kinase domain of MAP3K1 (Map3k1tm1Yxia)
have an extra row of OHCs and supporting cells in the OC and suffer from early-onset
profound hearing loss. Our results demonstrate the indispensable role of MAP3K1 in mouse
inner ear development and function.
Disease Models & Mechanisms • DMM • Advance article
lack of actin stress fiber formation and deficient cell migration (Yujiri et al., 2000; Zhang et
Results
MAP3K1 is localized with basal bodies in supporting cells
To comprehend the role of Map3k1 in inner ear development and function, we used
previously generated Map3k1tm1Yxia mice (Xia et al., 2000; Zhang et al., 2003). In these mice,
the exons encoding the kinase domain of MAP3K1 have been replaced with the bacterial
LacZ gene, resulting in the expression of a MAP3K1-β-galactosidase fusion protein (Fig.
1A). Immuno-labeling of the whole-mount preparation for the OC from these mice revealed
punctate expression of the MAP3K1-β-galactosidase fusion protein at the apical surface of
supporting cells (Fig. 1B). Interestingly, the MAP3K1-β-galactosidase fusion protein and
basal bodies marker pericentrin co-localize with a very exquisite pattern (Fig. 1C). The
MAP3K1-β-galactosidase fusion protein is expressed as two puncta on either side of the
pericentrin-labeled basal cell bodies (Fig. 1C, inset). Basal bodies are known to direct planar
cell polarity (PCP) of sensory hair cells (Jones et al., 2008). Therefore, we investigated the
orientation of the OHC bundles in Map3k1 mutant mice and no significant deficit was
Additionally, weak diffused cytoplasmic expression of Map3k1 was also observed in
the inner hair cells and outer hair cells (Fig. 1C), in the marginal and intermediate cells of the
stria vascularis (see supplementary material Fig. S2A). Moreover, β-galactosidase staining in
adult Map3k1 heterozygous mice revealed expression in supporting cells of the lesser
epithelial ridge, greater epithelial ridge, stria vascularis, Reissner’s membrane and the spiral
ganglion neurons (see supplementary material Fig. S2B).
Disease Models & Mechanisms • DMM • Advance article
observed (see supplementary material Fig. S1).
Map3k1tm1Yxia/tm1Yxia mice have supernumerary outer hair cells and Deiters’ cells
Upon confocal imaging of the OC from the Map3k1tm1Yxia heterozygous and
homozygous mice, we observed supernumerary outer hair cells (OHCs) throughout
development (Fig. 2). The Map3k1tm1Yxia heterozygous mice have sparse 1-10 cell stretches of
an extra row of OHCs, whereas homozygous Map3k1tm1Yxia mice have a continuous extra row
of OHCs in the apical, middle and basal cochlear turns (Fig. 2A). These supernumerary
OHCs also have correctly polarized mechanosensitive hair bundles at their apical poles (Fig.
2B). Although, no difference was observed in the number of inner hair cells (IHCs) (Fig. 2C),
a statistically significant increase in OHCs was observed throughout the cochlear duct in
homozygous and heterozygous Map3k1tm1Yxia mice (Fig. 2C). However, in comparison with
homozygous mutant mice, fewer extra OHCs was observed throughout the cochlear duct in
heterozygous Map3k1tm1Yxia mice (Fig. 2C), suggesting a dose-dependent role of MAP3K1mediated signaling in the cytoarchitecture of the mouse OC.
In higher vertebrates, each hair cell in the OC is enveloped by supporting cells (Fig.
upon a single Deiters' cell, and inner and outer pillar cells separate these two types of sensory
hair cells (Fig. 3A). To determine the effect of extra rows of OHCs on the precise
cytoarchitecture of the OC, we immuno-labeled the cochlear sections from P0 control and
Map3k1
tm1Yxia
mutant mice with an anti-Prox1 antibody, a marker for pillar and Deiters’
supporting cells. In Map3k1tm1Yxia mutant mice, the extra row of OHCs is also supported by
an extra row of Deiters’ cells (Fig. 3B). However, the organization of the inner and outer
pillar cells, Claudius cells and the tunnel of Corti is not affected in Map3k1tm1Yxia mutant mice
(Fig. 3B,C). These findings suggest that MAP3K1 has a role in the development of controlled
cyto-architecture of mouse organ of Corti (OC).
Disease Models & Mechanisms • DMM • Advance article
3A). In the mouse OC, the IHCs rest on the inner phalangeal cells, whereas each OHC rests
To assess the functional status of supernumerary hair cells, we briefly exposed (15
sec) the OC explants from control and Map3k1tm1Yxia mutant mice to FM1-43, a styryl
pyridinium dye that enters the hair cells via partially open mechanotransduction channels at
rest (Gale et al., 2001; Meyers et al., 2003). Hair cells from control and Map3k1tm1Yxia mice
showed dye loading in all rows of sensory cells, including the extra row of OHCs (Fig. 3D).
We also performed synaptophysin immunostaining to identify postsynaptic endings of
efferent neurons forming axodendritic synapses with the dendrites of spiral ganglion neurons
in P12 control and mutant mice (Fig. 3E). Interestingly, the synaptophysin labeling was also
observed at the base of an extra row of OHCs (Fig. 3E, arrowhead). Furthermore, no obvious
difference in the neuronal wiring of the OC in the control and Map3k1tm1Yxia mutant mice was
observed at P12 (Fig. 3F). However, the width of area of neurons around the OHCs appears
to be wider in Map3k1tm1Yxia mutant mice, likely due to the wiring of extra row of OHCs (Fig.
3F). Collectively, we observed that Map3k1tmYxia mutant mice have a completely developed,
structurally supported, polarized and functional extra row of innervated OHCs and Deiters’
Map3k1tm1Yxia mutant mice have early onset hearing loss
Next, to assess the hearing function of Map3k1tm1Yxia mutant mice, we measured
auditory brainstem responses (ABRs) and distortion product otoacoustic emissions
(DPOAEs). Although the wild-type control and Map3k1tm1Yxia heterozygous mice had
comparable hearing thresholds across all the tested frequencies, no significant hearing was
observed in the homozygous Map3k1tm1Yxia mice at P16 and P30 (Fig. 4A,B). Similarly, the
DPOAEs of wild-type and Map3k1tm1Yxia heterozygous mice were comparable, whereas the
homozygous Map3k1tm1Yxia mice had no detectable DPOAE thresholds (Fig. 4C). Taken
together with ABRs, these results suggest that hearing loss in Map3k1tm1Yxia mutant mice is
Disease Models & Mechanisms • DMM • Advance article
cells.
likely to result from peripheral (cochlear) deficiencies. In contrast, Map3k1tm1Yxia mutant mice
did not exhibit any observable vestibular dysfunction phenotypes, such as hyperactivity,
head-tossing, or circling behavior.
Map3k1tm1Yxia mutant mice display rapid degeneration of OHCs and spiral ganglion
neurons
Next, we examined the morphology of the cochlear epithelium at various
developmental stages to determine the underlying cause of hearing loss observed in the
Map3k1tm1Yxia mutant mice. Confocal imaging of myosin VIIa-labeled OC of Map3k1tm1Yxia
mice revealed normal development of inner and outer hair cells that were indistinguishable
from those of wild-type and heterozygous mice until P12 (Fig. 5). By P14, Map3k1tm1Yxia
homozygous mice display obvious degeneration of OHCs in the basal turn, and by P16, no
intact OHCs were observed in the basal turn (Fig. 5L). Furthermore, by P16, a varying degree
of OHCs degeneration was observed along the length of the cochlea (Fig. 5J-L). However, by
and many OHCs in the apical coil were also degenerated (Fig. 5M-O). In contrast, the IHCs
appeared largely intact even at P75 (Fig. 5 and data not shown). The loss of OHCs in
Map3k1tm1Yxia mutant mice was followed by progressive degeneration of the spiral ganglion,
which is more pronounced in the basal region at P90 (Fig. 6). Thus the elevated hearing
thresholds observed in MAP3K1 deficient mice are likely due to the rapid degeneration of
OHCs. These results suggest that MAP3K1 function is essential for the maintenance of OHCs
in the mouse auditory system.
The FGF signaling pathway genes are down regulated in Map3k1 mutant mice
We reasoned that extra row of OHCs and Deiters’ cells and degeneration of hair cells in
Map3k1 mutant mice is likely due to impaired intracellular signaling during the development
Disease Models & Mechanisms • DMM • Advance article
P30, almost all of the OHCs in the basal and middle turn of the cochlea were degenerated,
and maturation of mouse organ of Corti. Therefore, we examined the expression of various
genes associated with Map3k1-mediated signaling pathway (Fig. 7 & see supplementary
material Fig. S3), transcriptional targets of FGF signaling as well as the development related
genes in Map3k1tm1Yxia mutant mice at P10 (before the onset of hearing and OHCs
degeneration). As expected, the heterozygous mice had reduced expression of Map3k1, while
the homozygous mice had no expression (Fig. 7). Among the forty-two genes analyzed, we
observed significant (P<0.001) down-regulation of the Atf3, Fgfr3, Fgf8 and Fgf10 in the
inner ear of Map3k1 mutant mice (Fig. 7). Previous studies have shown that Fgfr3, Fgf8 and
Fgf10 have a role in the otic placode induction or in otic epithelium development in mice and
their functional deficits cause defects in cochlear morphogenesis and hearing loss (Hayashi et
al., 2007; Mansour et al., 2009; Pannier et al., 2009). Our results suggest that MAP3K1 has
an essential role in the regulation of these key cochlear morphogenesis genes during
Disease Models & Mechanisms • DMM • Advance article
development.
Discussion
MAP3K1 is involved in the cellular response to a wide array of factors including
growth factors, pro-inflammatory cytokines, cell shape disturbance, microtubule disruption
and a variety of cell stress signals (Cardone et al., 1997; Deak et al., 1998; Nakagami et al.,
2001; Widmann et al., 1998; Xia et al., 2000; Yujiri et al., 1998), translating into the
activation of the downstream JNK, p38, and ERK-mediated MAPK pathways (Schlesinger et
al., 1998; Xu et al., 1996). The full-length MAP3K1 protein is tethered to insoluble structures
such as membranes or the cytoskeleton by the actin-bundling protein, α-actinin (Christerson
et al., 1999) and have also been found to be associated with actin fibers entering the focal
adhesions and controlling their turnover (Cuevas et al., 2003). MAP3K1 interacts directly
with the signaling molecule MEK1 (Karandikar et al., 2000), which, in the mouse oocyte, has
been associated with the control of microtubule organization and co-localizes with the
centrosomal protein ϒ-tubulin (Yu et al., 2007). Interestingly, in Map3k1tm1Yxia mice, the
MAP3K1-lacZ fusion protein in the supporting cells of the OC also co-localized with the
the inner ear non-sensory cells and planar cell polarity (PCP) (Jones et al., 2008). There are
many examples of PCP proteins, like Vangl2, which are not expressed in the inner ear
sensory hair cells, but still severely affect the stereocilia bundle orientation (Montcouquiol et
al., 2006). However, in the Map3k1tm1Yxia mutant mice we did not observe any significant
stereocilia bundle orientation deficit, which might reflect that Map3k1 has either no direct
role in PCP or the functional redundancy by other family members.
In a parallel study, a splice site mutation in the Map3k1 gene in goya mutant mice was
identified as part of an ENU mutagenesis-screening program (Parker et al., 2015). Goya mice
also exhibit a progressive hearing loss phenotype along with the supernumerary OHCs.
Interestingly, when maintained on the same genetic background (C3H), the goya and
Disease Models & Mechanisms • DMM • Advance article
centrosomal protein and, therefore, might also participate in the microtubule organization in
Map3k1tm1Yxia mutant mice had a similar progressive hearing loss phenotype and were
profoundly deaf by 9 weeks (Parker et al., 2015). Modulation of the phenotype of a given
allele by the genetic background of an inbred strain is a well-documented phenomenon
(Doetschman, 2009; Montagutelli, 2000). Intriguingly, beside the worse hearing loss we
observed, Map3k1 mice on C57BL/6J background also have shown to exhibit a drastic
decrease in the number of animals surviving to maturity (Warr et al., 2011), which further
supports the notion of a Map3k1 modifier gene.
In the OC, the expression of MAP3K1 is observed in the supporting cells, while the
Map3k1tm1Yxia mutant mice had four rows of OHCs. During development, extra outer hair
cells can be found in wild-type mice, but they are generally restricted in the apical turns of
the cochlea, and their frequency can vary depending on different inbred mouse strains (Lim
and Anniko, 1985). Whereas, in Map3k1tm1Yxia mutant mice, the fourth row of OHCs was
found throughout development and along the length of cochlea. A few possibilities,
explaining such a phenotype include the lack of apoptosis of a population of cells in the
Normally, full-length MAP3K1 acts as an anti-apoptotic protein, but when cleaved by
Caspase 3, becomes pro-apoptotic (Schlesinger et al., 2002). Re-localization of the MAP3K1
C-terminal 91-kDa fragment containing the kinase domain is necessary for its pro-apoptotic
function; however, in the case of Map3k1tm1Yxia mice, the kinase domain is replaced with the
lacZ domain, rendering it incapable of inducing apoptosis in the supporting cells of the
developing OC.
Alternatively, the supernumerary OHCs in Map3k1tm1Yxia could stem from the deficit
in the control of the prosensory domain size during embryogenesis. The sensory epithelial
cell differentiation initiates with specification of the prosensory domain in the otocyst. Many
signaling molecules, including the Sonic hedgehog, Notch and Wnt pathway genes, act in
Disease Models & Mechanisms • DMM • Advance article
prosensory domain that would otherwise have undergone degeneration during development.
concert to form a highly specialized patterned sensory epithelium. Canonical Wnt (Wnt/βcatenin) signaling is also essential for the specification of the otic placode because either
conditional deletion or activation of β-catenin in Pax2-positive ectodermal cells or Foxg1positive placodal cells results in a significant reduction or expansion of the size of the otic
placode, respectively (Freyer and Morrow, 2010; Ohyama et al., 2006). MAP3K1, via direct
interaction with Axin1, is known to modulate Wnt/β-catenin signaling pathway activity (Sue
Ng et al., 2010). Previously, fibroblasts from Map3k1tm1Yxia mutant mice displayed a 10-fold
increase in transcription factor Lef/Tcf reporter activity in response to Wnt3a expression
compared to an only 3-fold increase observed in wild-type cells (Jin et al., 2013). As Wnt
expression regulates proliferation in the early prosensory domain and hair cell differentiation
in the later stage, the level of Wnt/β-catenin signaling can affect hair cell formation in a dosedependent manner (Jacques et al., 2012). It is plausible that, during development, Wnt/βcatenin signaling might have resulted in relative over-activation of downstream transcription
factors, leading to the expansion of the prosensory domain and excessive differentiation of
tissue from various embryonic developmental stages will provide knowledge regarding the
role of MAP3K1 in sensory cell specification and differentiation.
Map3k1tm1Yxia mutant mice exhibit down-regulation of at least four genes, which are
known for their role in the inner ear development and function. ATF3 is a transcription
factor, which is induced in response to a number of stress stimuli (Hai et al., 1999; Kyriakis
et al., 1994; Liang et al., 1996), is significantly down regulated in Map3k1tm1Yxia mutant mice.
ATF3 is also implicated in the survival, repair and neurite outgrowth in association with heat
shock protein 27, Akt and c-Jun (Nakagomi et al., 2003; Pearson et al., 2003). Also, Fgfr3
mutant cochlea show neuronal wiring pattern disruption (Puligilla et al., 2007). However in
Map3k1tm1Yxia mutant mice, we did not observe any gross deficit in the neuronal wiring.
Disease Models & Mechanisms • DMM • Advance article
OHCs in Map3k1tm1Yxia mutant mice. Future work using cell-specific markers and cochlear
Furthermore, the DPOAE studies suggest the hair cells functional deficit in Map3k1tm1Yxia
mutant mice. These results suggest that down regulation of Atf3 is unlikely be the reason of
supernumerary OHCs, Deiters’ cells and hearing deficits observed in Map3k1tm1Yxia mutant
mice.
Before the onset of hair cells degeneration at P10, we found significant downregulation of Fgfr3, Fgf8 and Fgf10 expression in the Map3k1tm1Yxia mutant mouse OC cells.
Dysfunction of Fgfr3 in mice, either due to loss (Hayashi et al., 2007) or gain of function
(Mansour et al., 2009; Pannier et al., 2009), results in hearing loss. Interestingly, both Fgfr3
mutant alleles have supernumerary OHCs (Hayashi et al., 2007; Mansour et al., 2009) as
observed in Map3k1tm1Yxia mutant mice. This implies that, in inner ear development, Map3k1
and Fgfr3 might participate in the same signaling cascade to control the precise
cytoarchitecture of the OC. However, in contrast to Fgfr3 mutant mice, which have either
loss of pillar or Deiters’ supporting cells in the OC (Hayashi et al., 2007; Mansour et al.,
2009; Pannier et al., 2009), Map3k1tm1Yxia mutant mice do not exhibit any obvious deficit in
OHCs and loss of pillar cells phenotype reported for the gain of function allele of Fgfr3
phenotype is remarkably similar to Sprouty2, an antagonist of FGF signaling, mutant mice
(Shim et al., 2005). Intriguingly, the hearing loss phenotype in Sprouty2 mutant mice can be
partially rescued by genetically reducing the Fgf8 expression level (Shim et al., 2005).
Furthermore, reduction of Fgf10 expression reverted the fate-switched supporting cells back
and restored hearing in Fgfr3 mutant mice (Mansour et al., 2013). Besides Fgfr3, Fgf8 and
Fgf10 are also down regulated in the Map3k1tm1Yxia mutant mice, which could be cause of
grossly intact supporting cells in these mutant mice as compared to Fgfr3 mutant mice.
Our studies further highlight the complexity of the signaling pathway(s) required for
precise cyto-architecture of mouse OC and maintenance of OHCs. We also found necessity of
Disease Models & Mechanisms • DMM • Advance article
the differentiation of pillar cells, rather have an extra row of Deiters’ cells. Overproduction of
MAP3K1 function for the regulation of FGF-mediated pathway in the auditory system and
Disease Models & Mechanisms • DMM • Advance article
hearing in mice.
Materials and Methods
Map3k1 mutant mice
Map3k1tm1Yxia mice were generated as described previously (Xia et al., 2000; Zhang et
al., 2003). In summary, the targeting vector replaced the entire kinase domain in the Map3k1
locus, resulting in the formation of a MAP3K1-β-Galactosidase fusion protein. These
targeted ES cells (Map3k1+/tm1Yxia) were injected into mouse blastocysts, and the resulting
chimeras were crossed with C57BL/6 mice to obtain mice with germline transmission of the
Map3k1tm1Yxia allele. All experiments were approved by the Animal Care and Use
Committees at University of Maryland, School of Medicine in accordance with the NIH
Guide for the Care and Use of Laboratory Animals.
ABR and DPOAE measurements
Hearing function was evaluated by ABR analyses at two different time points, P16
and P30 for the mice of all the three genotypes i.e., wild-type, heterozygous and
Avertin (0.4-0.75 mg/g body weight, Sigma-Aldrich, St. Louis, MO). All recordings were
performed in a sound-attenuated chamber using an auditory-evoked potential diagnostic
system RZ6 (Tucker-Davis Technologies, Inc. Alachua, FL) as previously described (Nayak
et al., 2013). Experiments represent the mean and standard error of mean of three or more
animals. Significance was analyzed using Student’s t-test.
DPOAE recordings were performed at P30 with an acoustic probe (ER-10C, Etymotic
Research, Elk Grove Village, IL) using a DP2000 DPOAE measurement system version 3.0
(Starkey Laboratory, Eden Prairie, MN). Two primary tones, with a frequency ratio of f2/f1 =
1.2, where f1 represents the first tone and f2 represents the second, were presented at
intensity levels L1 = 65 dB SPL and L2 = 55 dB SPL. Also, f2 was varied in one-eighth
Disease Models & Mechanisms • DMM • Advance article
Map3k1tm1Yxia homozygous mice. Mice were anesthetized with intraperitoneal injections of
octave steps from 8 to 16 kHz. DP grams comprised 2f1–f2 DPOAE amplitudes as a function
of f2.
Confocal imaging
The temporal bones from the control and mutant mice were isolated and fixed for 1
hour at room temperature or overnight at 4°C in 4% paraformaldehyde (Electron Microscopy
Sciences, Hatfield, PA), followed by three washes with PBS. Finely dissected cochlear coils
were permeabilized and blocked in 5% BSA, 2% normal goat serum and 0.1% Triton X-100
in PBS for 1 hour. The tissue samples were washed and probed with primary antibody
overnight at 4°C. We used the following primary antibodies in our studies: β-galactosidase
(1:1000 dilution; MP Biomedicals, Solon, OH), Myosin VIIa (1:200; Proteus BioSciences,
Ramona, CA), Synaptophysin (1:200; Abcam, Cambridge, MA), NF-200 (1:200; Sigma
Aldrich), Prox1 (1:100; Millipore, Billerica, MA) and Pericentrin (1:200; Millipore, Billerica,
MA). After three washes with PBS, samples were probed with a fluorescently labeled Alexa
temperature. Rhodamine-phalloidin or Alexa Fluor 647 conjugated phalloidin (1:250; Life
Technologies, Grand Island, NY) was used to label actin. Samples were mounted using
ProLongGold (Life Technologies, Grand Island, NY) and viewed under a LSM 700 confocal
microscope (Zeiss Microimaging Inc., Thornwood, NY) using a ×63, 1.4 N.A. oil-immersion
lens.
Disease Models & Mechanisms • DMM • Advance article
Fluor secondary antibody (1:500; Life Technologies, Grand Island, NY) for 1 hour at room
Scanning Electron Microscopy (SEM)
For SEM studies, the inner ears were isolated at P14 and fixed for 1.5 hrs in a fixative,
containing 2.5% glutaraldehyde and 2 mM CaCl2 in 0.1 M sodium cacodylate buffer, and
later washed three times with 0.1 M sodium cacodylate buffer. The inner ears were post-fixed
in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 hour at room temperature.
After washing three times with PBS buffer, inner ears were decalcified by incubating in 0.25
M EDTA for 1-2 days at 4°C. The samples were then finely dissected to expose the sensory
epithelium and to remove the tectorial membrane and the stria vascularis. The cochlear
tissues were then dehydrated in gradient alcohol changes, critical point dried, sputter coated
with platinum, and imaged on a field emission SEM.
Cochlear explants & FM1-43 dye uptake
OC explants from P3 wild-type and Map3k1tm1Yxia mutant mice were finely dissected
and cultured on collagen-coated glass-bottom Petri dishes (MatTek Corporation, Ashland,
Grand Island, NY) and 10 mg/ml ampicillin (Millipore, Billerica, MA) at 37°C and 5% CO2.
The explants were kept in vitro for 2 days to allow for complete adhesion to the dish. To test
the FM1-43
dye [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]-styryl)pyridinium
dibromide] (Life Technologies, Grand Island, NY) uptake by the hair cells, the culture was
exposed to 3.0 µM FM1-43 in Hank’s Balanced Salt solution (HBSS) for 15 s and then
quickly washed three times with HBSS. The culture was mounted with Fluoro-Gel (Electron
Microscopy Sciences, Crofton, MD) and immediately imaged under a LSM 700 confocal
microscope (Zeiss Microimaging Inc.).
Disease Models & Mechanisms • DMM • Advance article
MA), in DMEM medium supplemented with 10% fetal bovine serum (Life Technologies,
Cryosections
Hemisected heads of P0 mice or inner ears of P10 and older mice were collected and
fixed in 4% PFA at 4°C overnight. For older mice, the temporal bones were decalcified in
0.25 M EDTA for 1 to 2 days at 4°C. The inner ears were then equilibrated with 30% sucrose
in PBS overnight at 4°C, embedded in OCT, and immediately frozen by placing the block on
an ethanol/dry ice mix. The frozen tissue blocks were sectioned with a cryostat at 14 μm
thickness.
RT-PCR and real-time PCR
Total RNA was isolated from fine dissected OC isolated from P10 inner ear tissue of
wild-type, heterozygous and Map3k1tm1Yxia homozygous mice (5 mice each) using the
RiboPure RNA isolation kit (Life Technologies, Grand Island, NY) and cDNA was prepared
using an oligo-dT primer and SMARTScribe Reverse Transcriptase enzymes (Clontech,
Mountain View, CA). To determine the differential expression of various genes, SYBR-
DNA
Technologies
online
PrimeTime
qPCR
assay
design
tool
(http://www.idtdna.com/Scitools/Applications/RealTimePCR/). The real-time PCR assays
were performed in triplicate using an ABI StepOnePlus Real-time thermalcycler (Life
Technologies, Grand Island, NY). CT values were normalized using Gapdh and Actin as an
endogenous control, and fold changes of different genes were calculated using
SABiosciences
online
software
(http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The genes with a 2-fold
change and with a p value less than 0.05 based on a Student’s t test analysis were considered
significant.
Disease Models & Mechanisms • DMM • Advance article
Green-based real-time primers (available upon request) were designed using the Integrated
X-gal Staining
The inner ears were isolated from wild-type and Map3k1tm1Yxia heterozygous mice at P30 and
were fixed for 10 min at room temperature in LacZ fixative (1% formaldehyde, 0.2%
Glutaraldehyde & 0.02% NP-40 in PBS). Then, the samples were washed twice with PBS
containing 0.02% NP-40 and 2 mM MgCl2, 10 min each, followed by incubation at 37°C
overnight in a staining solution (1 mg/ml X-Gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 & 0.1
M MgCl2). Following staining, samples were washed in PBS, decalcified and cryosectioned
as described above. The sections were counterstained with hematoxylin and eosin and imaged
Disease Models & Mechanisms • DMM • Advance article
using a ×40 oil immersion lens on a Zeiss Axioplan Apotome-equipped microscope.
Acknowledgements
We thank Drs. A.P. Giese and E.M. Richard for their technical assistance, discussion of the
results and review of the manuscript.
Competing Interests
The authors declare no competing interests.
Authors' contributions
RY, RH, ZA, and SR designed the research. RY, YX, ZA, and SR designed and/or
contributed new reagents and performed research. RY, QM, RH, CP, YX, ZA, and SR
analyzed data. Every author drafted or revised the manuscript, and all authors approved the
final version.
This study was supported by National Institute on Deafness and Other Communication
Disorders (NIDCD/NIH) research grants R01DC011803 and R01DC011748 to S.R. and
R01DC012564 to Z.M.A. This work was also supported by the Action on Hearing Loss grant
to S.R.
Disease Models & Mechanisms • DMM • Advance article
Funding
References
Bonvin, C., Guillon, A., van Bemmelen, M. X., Gerwins, P., Johnson, G. L. and
Widmann, C. (2002). Role of the amino-terminal domains of MEKKs in the activation of
NF kappa B and MAPK pathways and in the regulation of cell proliferation and apoptosis.
Cell Signal 14, 123-31.
Cardone, M. H., Salvesen, G. S., Widmann, C., Johnson, G. and Frisch, S. M.
(1997). The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell 90,
315-23.
Christerson, L. B., Gallagher, E., Vanderbilt, C. A., Whitehurst, A. W., Wells,
C., Kazempour, R., Sternweis, P. C. and Cobb, M. H. (2002). p115 Rho GTPase activating
protein interacts with MEKK1. J Cell Physiol 192, 200-8.
Christerson, L. B., Vanderbilt, C. A. and Cobb, M. H. (1999). MEKK1 interacts
with alpha-actinin and localizes to stress fibers and focal adhesions. Cell Motil Cytoskeleton
43, 186-98.
protein kinase kinase kinases in signal integration. Oncogene 26, 3159-71.
Cuevas, B. D., Abell, A. N., Witowsky, J. A., Yujiri, T., Johnson, N. L., Kesavan,
K., Ware, M., Jones, P. L., Weed, S. A., DeBiasi, R. L. et al. (2003). MEKK1 regulates
calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of
migrating fibroblasts. EMBO J 22, 3346-55.
Cuevas, B. D., Winter-Vann, A. M., Johnson, N. L. and Johnson, G. L. (2006).
MEKK1 controls matrix degradation and tumor cell dissemination during metastasis of
polyoma middle-T driven mammary cancer. Oncogene 25, 4998-5010.
Disease Models & Mechanisms • DMM • Advance article
Cuevas, B. D., Abell, A. N. and Johnson, G. L. (2007). Role of mitogen-activated
Deak, J. C., Cross, J. V., Lewis, M., Qian, Y., Parrott, L. A., Distelhorst, C. W.
and Templeton, D. J. (1998). Fas-induced proteolytic activation and intracellular
redistribution of the stress-signaling kinase MEKK1. Proc Natl Acad Sci U S A 95, 5595-600.
Deng, M., Chen, W. L., Takatori, A., Peng, Z., Zhang, L., Mongan, M.,
Parthasarathy, R., Sartor, M., Miller, M., Yang, J. et al. (2006). A role for the mitogenactivated protein kinase kinase kinase 1 in epithelial wound healing. Mol Biol Cell 17, 344655.
Doetschman, T. (2009). Influence of genetic background on genetically engineered
mouse phenotypes. Methods Mol Biol 530, 423-33.
Freyer, L. and Morrow, B. E. (2010). Canonical Wnt signaling modulates Tbx1,
Eya1, and Six1 expression, restricting neurogenesis in the otic vesicle. Dev Dyn 239, 170822.
Gale, J. E., Marcotti, W., Kennedy, H. J., Kros, C. J. and Richardson, G. P.
(2001). FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer
Gallagher, E., Enzler, T., Matsuzawa, A., Anzelon-Mills, A., Otero, D., Holzer,
R., Janssen, E., Gao, M. and Karin, M. (2007). Kinase MEKK1 is required for CD40dependent activation of the kinases Jnk and p38, germinal center formation, B cell
proliferation and antibody production. Nat Immunol 8, 57-63.
Guan, Z., Buckman, S. Y., Pentland, A. P., Templeton, D. J. and Morrison, A. R.
(1998). Induction of cyclooxygenase-2 by the activated MEKK1 --> SEK1/MKK4 --> p38
mitogen-activated protein kinase pathway. J Biol Chem 273, 12901-8.
Hagemann, C. and Blank, J. L. (2001). The ups and downs of MEK kinase
interactions. Cell Signal 13, 863-75.
Disease Models & Mechanisms • DMM • Advance article
channel. J Neurosci 21, 7013-25.
Hai, T., Wolfgang, C. D., Marsee, D. K., Allen, A. E. and Sivaprasad, U. (1999).
ATF3 and stress responses. Gene Expr 7, 321-35.
Hayashi, T., Cunningham, D. and Bermingham-McDonogh, O. (2007). Loss of
Fgfr3 leads to excess hair cell development in the mouse organ of Corti. Dev Dyn 236, 52533.
Jacques, B. E., Puligilla, C., Weichert, R. M., Ferrer-Vaquer, A., Hadjantonakis,
A. K., Kelley, M. W. and Dabdoub, A. (2012). A dual function for canonical Wnt/betacatenin signaling in the developing mammalian cochlea. Development 139, 4395-404.
Jin, C., Chen, J., Meng, Q., Carreira, V., Tam, N. N., Geh, E., Karyala, S., Ho, S.
M., Zhou, X., Medvedovic, M. et al. (2013). Deciphering gene expression program of
MAP3K1 in mouse eyelid morphogenesis. Dev Biol 374, 96-107.
Jones, C., Roper, V. C., Foucher, I., Qian, D., Banizs, B., Petit, C., Yoder, B. K.
and Chen, P. (2008). Ciliary proteins link basal body polarization to planar cell polarity
regulation. Nat Genet 40, 69-77.
ERK2 cascade components. J Biol Chem 275, 40120-7.
Kyriakis, J. M. and Avruch, J. (2001). Mammalian mitogen-activated protein kinase
signal transduction pathways activated by stress and inflammation. Physiol Rev 81, 807-69.
Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F.,
Avruch, J. and Woodgett, J. R. (1994). The stress-activated protein kinase subfamily of cJun kinases. Nature 369, 156-60.
Labuda, T., Christensen, J. P., Rasmussen, S., Bonnesen, B., Karin, M.,
Thomsen, A. R. and Odum, N. (2006). MEK kinase 1 is a negative regulator of virusspecific CD8(+) T cells. Eur J Immunol 36, 2076-84.
Disease Models & Mechanisms • DMM • Advance article
Karandikar, M., Xu, S. and Cobb, M. H. (2000). MEKK1 binds raf-1 and the
Li, Y., Minamino, T., Tsukamoto, O., Yujiri, T., Shintani, Y., Okada, K.,
Nagamachi, Y., Fujita, M., Hirata, A., Sanada, S. et al. (2005). Ablation of MEK kinase 1
suppresses intimal hyperplasia by impairing smooth muscle cell migration and urokinase
plasminogen activator expression in a mouse blood-flow cessation model. Circulation 111,
1672-8.
Liang, G., Wolfgang, C. D., Chen, B. P., Chen, T. H. and Hai, T. (1996). ATF3
gene. Genomic organization, promoter, and regulation. J Biol Chem 271, 1695-701.
Lim, D. J. and Anniko, M. (1985). Developmental morphology of the mouse inner
ear. A scanning electron microscopic observation. Acta Otolaryngol Suppl 422, 1-69.
Loke, J. and Ostrer, H. (2012). Rapidly screening variants of uncertain significance
in the MAP3K1 gene for phenotypic effects. Clin Genet 81, 272-7.
Loke, J., Pearlman, A., Radi, O., Zuffardi, O., Giussani, U., Pallotta, R.,
Camerino, G. and Ostrer, H. (2014). Mutations in MAP3K1 tilt the balance from
SOX9/FGF9 to WNT/beta-catenin signaling. Hum Mol Genet 23, 1073-83.
syndrome model hearing loss reveals prolonged FGF-dependent plasticity in cochlear
supporting cell fates. Genes Dev 27, 2320-31.
Mansour, S. L., Twigg, S. R., Freeland, R. M., Wall, S. A., Li, C. and Wilkie, A.
O. (2009). Hearing loss in a mouse model of Muenke syndrome. Hum Mol Genet 18, 43-50.
Meyers, J. R., MacDonald, R. B., Duggan, A., Lenzi, D., Standaert, D. G.,
Corwin, J. T. and Corey, D. P. (2003). Lighting up the senses: FM1-43 loading of sensory
cells through nonselective ion channels. J Neurosci 23, 4054-65.
Minamino, T., Yujiri, T., Terada, N., Taffet, G. E., Michael, L. H., Johnson, G.
L. and Schneider, M. D. (2002). MEKK1 is essential for cardiac hypertrophy and
dysfunction induced by Gq. Proc Natl Acad Sci U S A 99, 3866-71.
Disease Models & Mechanisms • DMM • Advance article
Mansour, S. L., Li, C. and Urness, L. D. (2013). Genetic rescue of Muenke
Mongan, M., Wang, J., Liu, H., Fan, Y., Jin, C., Kao, W. Y. and Xia, Y. (2011).
Loss of MAP3K1 enhances proliferation and apoptosis during retinal development.
Development 138, 4001-12.
Montagutelli, X. (2000). Effect of the genetic background on the phenotype of mouse
mutations. J Am Soc Nephrol 11 Suppl 16, S101-5.
Montcouquiol, M., Sans, N., Huss, D., Kach, J., Dickman, J. D., Forge, A.,
Rachel, R. A., Copeland, N. G., Jenkins, N. A., Bogani, D. et al. (2006). Asymmetric
localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in
mammals. J Neurosci 26, 5265-75.
Nakagami, H., Morishita, R., Yamamoto, K., Yoshimura, S. I., Taniyama, Y.,
Aoki, M., Matsubara, H., Kim, S., Kaneda, Y. and Ogihara, T. (2001). Phosphorylation
of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell
death induced by high D-glucose in human endothelial cells. Diabetes 50, 1472-81.
Nakagomi, S., Suzuki, Y., Namikawa, K., Kiryu-Seo, S. and Kiyama, H. (2003).
neuronal death by promoting heat shock protein 27 expression and Akt activation. J Neurosci
23, 5187-96.
Nayak, G., Lee, S. I., Yousaf, R., Edelmann, S. E., Trincot, C., Van Itallie, C. M.,
Sinha, G. P., Rafeeq, M., Jones, S. M., Belyantseva, I. A. et al. (2013). Tricellulin
deficiency affects tight junction architecture and cochlear hair cells. J Clin Invest 123, 403649.
Ohyama, T., Mohamed, O. A., Taketo, M. M., Dufort, D. and Groves, A. K.
(2006). Wnt signals mediate a fate decision between otic placode and epidermis.
Development 133, 865-75.
Disease Models & Mechanisms • DMM • Advance article
Expression of the activating transcription factor 3 prevents c-Jun N-terminal kinase-induced
Pannier, S., Couloigner, V., Messaddeq, N., Elmaleh-Berges, M., Munnich, A.,
Romand, R. and Legeai-Mallet, L. (2009). Activating Fgfr3 Y367C mutation causes
hearing loss and inner ear defect in a mouse model of chondrodysplasia. Biochim Biophys
Acta 1792, 140-7.
Parker, A., Cross, S., Jackson, I. J., Hardisty-Hughes, R., Morse, S., Nicholson,
G., Coghill, E. and Brown, S. D. M. (2015). The splice site mutation goya in Map3k1
causes deafness and eye defects in the mouse. Dis Model Mech In this Issue.
Pearlman, A., Loke, J., Le Caignec, C., White, S., Chin, L., Friedman, A., Warr,
N., Willan, J., Brauer, D., Farmer, C. et al. (2010). Mutations in MAP3K1 cause 46,XY
disorders of sex development and implicate a common signal transduction pathway in human
testis determination. Am J Hum Genet 87, 898-904.
Pearson, A. G., Gray, C. W., Pearson, J. F., Greenwood, J. M., During, M. J. and
Dragunow, M. (2003). ATF3 enhances c-Jun-mediated neurite sprouting. Brain Res Mol
Brain Res 120, 38-45.
Fritzsch, B. and Kelley, M. W. (2007). Disruption of fibroblast growth factor receptor 3
signaling results in defects in cellular differentiation, neuronal patterning, and hearing
impairment. Dev Dyn 236, 1905-17.
Disease Models & Mechanisms • DMM • Advance article
Puligilla, C., Feng, F., Ishikawa, K., Bertuzzi, S., Dabdoub, A., Griffith, A. J.,
Sadoshima, J., Montagne, O., Wang, Q., Yang, G., Warden, J., Liu, J., Takagi,
G., Karoor, V., Hong, C., Johnson, G. L. et al. (2002). The MEKK1-JNK pathway plays a
protective role in pressure overload but does not mediate cardiac hypertrophy. J Clin Invest
110, 271-9.
Schlesinger, T. K., Bonvin, C., Jarpe, M. B., Fanger, G. R., Cardinaux, J. R.,
Johnson, G. L. and Widmann, C. (2002). Apoptosis stimulated by the 91-kDa caspase
cleavage MEKK1 fragment requires translocation to soluble cellular compartments. J Biol
Chem 277, 10283-91.
Schlesinger, T. K., Fanger, G. R., Yujiri, T. and Johnson, G. L. (1998). The TAO
of MEKK. Front Biosci 3, D1181-6.
Shim, K., Minowada, G., Coling, D. E. and Martin, G. R. (2005). Sprouty2, a
mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by
antagonizing FGF signaling. Dev Cell 8, 553-64.
Sue Ng, S., Mahmoudi, T., Li, V. S., Hatzis, P., Boersema, P. J., Mohammed, S.,
canonical Wnt signalling pathway. Biol Chem 391, 171-80.
Uhlik, M. T., Abell, A. N., Cuevas, B. D., Nakamura, K. and Johnson, G. L.
(2004). Wiring diagrams of MAPK regulation by MEKK1, 2, and 3. Biochem Cell Biol 82,
658-63.
Urness, L. D., Paxton, C. N., Wang, X., Schoenwolf, G. C. and Mansour, S. L.
(2010). FGF signaling regulates otic placode induction and refinement by controlling both
ectodermal target genes and hindbrain Wnt8a. Dev Biol 340, 595-604.
Disease Models & Mechanisms • DMM • Advance article
Heck, A. J. and Clevers, H. (2010). MAP3K1 functionally interacts with Axin1 in the
Warr, N., Bogani, D., Siggers, P., Brixey, R., Tateossian, H., Dopplapudi, A.,
Wells, S., Cheeseman, M., Xia, Y., Ostrer, H. et al. (2011). Minor abnormalities of testis
development in mice lacking the gene encoding the MAPK signalling component, MAP3K1.
PLoS One 6, e19572.
Widmann, C., Gerwins, P., Johnson, N. L., Jarpe, M. B. and Johnson, G. L.
(1998). MEK kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxininduced apoptosis. Mol Cell Biol 18, 2416-29.
Witowsky, J. A. and Johnson, G. L. (2003). Ubiquitylation of MEKK1 inhibits its
phosphorylation of MKK1 and MKK4 and activation of the ERK1/2 and JNK pathways. J
Biol Chem 278, 1403-6.
Xia, Y., Makris, C., Su, B., Li, E., Yang, J., Nemerow, G. R. and Karin, M.
(2000). MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by
proinflammatory stimuli and growth factor-induced cell migration. Proc Natl Acad Sci U S A
97, 5243-8.
Cobb, M. H. (1996). Cloning of rat MEK kinase 1 cDNA reveals an endogenous membraneassociated 195-kDa protein with a large regulatory domain. Proc Natl Acad Sci U S A 93,
5291-5.
Yu, L. Z., Xiong, B., Gao, W. X., Wang, C. M., Zhong, Z. S., Huo, L. J., Wang,
Q., Hou, Y., Liu, K., Liu, X. J. et al. (2007). MEK1/2 regulates microtubule organization,
spindle pole tethering and asymmetric division during mouse oocyte meiotic maturation. Cell
Cycle 6, 330-8.
Yujiri, T., Sather, S., Fanger, G. R. and Johnson, G. L. (1998). Role of MEKK1 in
cell survival and activation of JNK and ERK pathways defined by targeted gene disruption.
Science 282, 1911-4.
Disease Models & Mechanisms • DMM • Advance article
Xu, S., Robbins, D. J., Christerson, L. B., English, J. M., Vanderbilt, C. A. and
Yujiri, T., Ware, M., Widmann, C., Oyer, R., Russell, D., Chan, E., Zaitsu, Y.,
Clarke, P., Tyler, K., Oka, Y. et al. (2000). MEK kinase 1 gene disruption alters cell
migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect
in NF-kappa B activation. Proc Natl Acad Sci U S A 97, 7272-7.
Zhang, L., Wang, W., Hayashi, Y., Jester, J. V., Birk, D. E., Gao, M., Liu, C. Y.,
Kao, W. W., Karin, M. and Xia, Y. (2003). A role for MEK kinase 1 in TGF-beta/activin-
Disease Models & Mechanisms • DMM • Advance article
induced epithelium movement and embryonic eyelid closure. EMBO J 22, 4443-54.
Fig. 1.
Localization of MAP3K1 in the inner ear. A, MAP3K1 has two domains, a regulatory
domain and a kinase domain, whereas in the Map3k1 mutant mice, the kinase domain has
been replaced with a β-galactosidase reporter cassette, resulting in a MAP3K1-β-
Disease Models & Mechanisms • DMM • Advance article
Figures
galactosidase fusion protein. B, Confocal imaging of the whole mount preparation of organ of
Corti (OC) from P16 mice labeled with the β-galactosidase antibody (green) and phollidin
(red). As anticipated, no β-galactosidase labeling was observed in control (Map3k1+/+) mice,
whereas in the Map3k1tm1Yxia mutant mice, a distinct expression of the MAP3K1-βgalactosidase fusion protein was observed at the apical surface of supporting cells of the OC.
Scale bar: 10 μm. C, In sections of P0 mice, the MAP3K1-β-galactosidase fusion protein
(green) co-localizes with pericentin (red), a marker for basal cell bodies. In addition, weak
diffused cytoplasmic expression of fusion protein was also observed in inner and outer hair
cells. Scale bar: 10 μm. Inset, showing higher magnification of β-galactosidase and
Disease Models & Mechanisms • DMM • Advance article
pericentrin localization. Scale bar: 1 μm.
Map3k1tm1Yxia mutant mice have supernumerary outer hair cells. A, Whole-mount preparation
of the OC from P12 mice labeled with myosin VIIa (green) and phalloidin (red). In contrast
Disease Models & Mechanisms • DMM • Advance article
Fig. 2.
to three rows of OHCs observed in wild-type mice, Map3k1tm1Yxia mutant mice have an extra
row of OHCs along the length of the cochlea, while sparse patches of a fourth row of OHCs
was observed in Map3k1tm1Yxia heterozygous mice. Scale bar: 10 μm. B, Scanning electron
micrographs of P14 mice revealed a characteristic polarized “V” shaped stereocilia bundles at
the apical surface of supernumerary OHCs present in Map3k1tm1Yxia heterozygous and
homozygous mutant mice. Scale bar: 10 μm. C, Quantitation of IHCs and OHCs in control
and Map3k1tm1Yxia mice at P12. For quantification purposes, organ of Corti were isolated from
4 wild-type and Map3k1tm1Yxia mutant mice each and hair cells were counted in the apical
middle and basal coil regions. No significant difference was observed in the IHC number.
Statistically significant (*p<0.05, ** p< 0.01, ***p<0.001) increases in the OHC number were
observed in the Map3k1tm1Yxia homozygous mutant and heterozygous mice, with a gradient
Disease Models & Mechanisms • DMM • Advance article
from apex to base (mean ± standard error of mean).
Cytoarchitecture of the organ of Corti is preserved in Map3k1tm1Yxia mutant mice. A,
Schematic representation of the developing mouse OC at P0 and P10. OHC, outer hair cell;
IHC, inner hair cell; DC, Deiters’ cell; OPC, outer pillar cell; IPC, inner pillar cell; HC,
Disease Models & Mechanisms • DMM • Advance article
Fig. 3.
Hensen’s cells; IPhC, inner phalangeal supporting cells. B, Cross-section of Map3k1tm1Yxia
mutant and wild-type control mice at P0, immunostained with Prox1 (green) and myosin VIIa
(red). The arrows point to the pillar cells, whereas Deiters’ cells are marked by the
arrowheads. Map3k1tm1Yxia mutant mice have an extra row of OHCs accompanied by an extra
row of Deiters’ cells. C, Immunostaining with the anti-CD44 antibody, a marker for outer
pillars cells including Claudius cells, revealed an intact gross cytoarchitecture of the OC in
Map3k1tm1Yxia mutant mice at P0 and P10. D, No apparent difference in the FM1-43 dye
uptake was observed among control and Map3k1tm1Yxia mutant explants. FM1-43 dye was also
taken up by the supernumerary OHCs present in Map3k1tm1Yxia mutant mice. Scale bar: 10
μm. E, Supernumerary OHCs in Map3k1tm1Yxia mutant mice are innervated and have synaptic
junctions (arrowheads), immunolabeled with synaptophysin (green). F, Map3k1tm1Yxia mutant
mice have grossly intact neuronal wiring. Neurofilament (NF-200) protein immunostaining of
wild-type control and Map3k1tm1Yxia mutant mice revealed grossly intact neuronal wiring of
Disease Models & Mechanisms • DMM • Advance article
an extra row of OHCs. Scale bar: 10 μm.
Map3k1tm1Yxia mutant mice have elevated hearing thresholds. Hearing thresholds of 3 wildtype, 7 heterozygous and 8 Map3k1tm1Yxia homozygous mice were evaluated at P16, whereas 8
wild-type, 11 heterozygous and 7 Map3k1tm1Yxia homozygous mice were tested at P30. A,
Averaged ABR thresholds of wild-type, heterozygous and homozygous Map3k1tm1Yxia mice at
P16 and P30 in response to click stimulus. The Map3k1tm1Yxia mutant mice showed
significantly (***p<0.001) elevated thresholds compared with heterozygous and wild-type
Disease Models & Mechanisms • DMM • Advance article
Fig. 4.
mice at both ages (mean ± standard error of mean). B, Averaged ABR thresholds of wild-type
(white diamonds), heterozygous (grey squares) and homozygous Map3k1tm1Yxia mutant mice
(black triangles) at P16 (solid lines) and P30 (dashed lines), in response to 8-kHz, 16-kHz,
and 32-kHz tone-bursts. At both developmental stages, Map3k1tm1Yxia mutant mice showed
significantly (***p<0.001) elevated thresholds compared with the wild-type control and
heterozygous mice at all frequencies tested (mean ± standard error of mean). C, DPOAEs of
Map3k1tm1Yxia mutant (black triangles), heterozygous (grey squares) and wild-type control
mice (white diamonds) at P30, represented as a function of f2 stimulus frequencies.
Map3k1tm1Yxia mutant mice showed no responses, with values close to the noise floor,
Disease Models & Mechanisms • DMM • Advance article
indicating that the residual OHCs were non-functional.
OHCs in Map3k1tm1Yxia mutant mice degenerate as early as P14. Maximum intensity
projections of confocal Z-stacks of whole-mount cochleae labeled with the myosin VIIa
antibody (green) and phalloidin (red) are shown. A-C, Representative images from the apical,
Disease Models & Mechanisms • DMM • Advance article
Fig. 5.
middle and basal turns of the OC of a wild-type control mouse at P30. D-O, Images of the
OC from the three turns of the cochlea of Map3k1tm1Yxia mutant mice at P12 D-F, P14 G-I,
P16 J-L and P30 M-O. The hair cells appear to have normal development and morphology at
P12 in the apical and middle cochlear turns in Map3k1tm1Yxia mutant mice. Initial signs of
OHC degeneration are evident in the basal turn F. At P14, obvious degeneration of OHCs
was observed in Map3k1tm1Yxia mutant mice. K-L, Severe OHC degeneration can be observed
by P16 in the middle K, and basal turns L. The OHC loss progresses rapidly, and by P30,
severe degeneration is evident in all three cochlear turns. In contrast, IHCs remained intact
Disease Models & Mechanisms • DMM • Advance article
along the length of the cochlea. Scale bar: 10 μm.
Fig. 6.
Map3k1tm1Yxia mice at P90 displayed the degeneration of spiral ganglions in the basal turn.
Cochlear sections from control and Map3k1tm1Yxia mutant mice were stained with hematoxylin
Disease Models & Mechanisms • DMM • Advance article
and eosin. Scale bar: 20 μm.
Fig. 7.
Fgfr3, Fgf8, Fgf10 and Atf3 genes are down regulated in the organ of Corti of Map3k1tm1Yxia
mutant mice. Semi-quantification expression analysis of various developmental and
MAP3K1-mediated signaling pathways genes normalized against Gapdh and Actin
(endogenous controls) at P10. As anticipated, a dose-dependent, statistically significant
reduction in the expression of Map3k1 was observed in heterozygous and homozygous
significantly abolished (Student's t-test, *** p<0.001), suggesting the role of Map3k1 in
regulating their expression in the inner ear (mean ± standard error of mean).
Disease Models & Mechanisms • DMM • Advance article
mutant mice. Furthermore, the expression of Fgfr3, Fgf8, Fgf10 and Atf3 was also
Translational Impact
Clinical Issue
Hearing loss is one of the most common neurosensory disorders. Hearing depends on
the precise organization of sensory hair cells and non-sensory supporting cells within
the organ of Corti (OC). Any significant alteration in the cellular number, alignment
or patterning within the OC causes hearing loss, underlining the necessity of defined
cytoarchitecture in the OC for sound perception. Thus, understanding the molecular
signaling cascades that lead to inner ear sensory cells differentiation and function is
important for hearing restoration.
Results
In this study, to determine the role of Map3k1 in the mouse inner ear development and
function, we characterized the kinase null Map3k1 mutant mice. In the mouse inner
proximity to basal bodies. Hearing test of the homozygous Map3k1 mutant mice
revealed early-onset profound hearing loss, along with progressive degeneration of
outer hair cells (OHC). In the organ of Corti, loss of MAP3K1 function results in extra
row of functional outer hair cells (OHCs) and Deiters’ cells. Furthermore, loss of
MAP3K1 function resulted in the down-regulation of Fgfr3, Fgf8, Fgf10 and Atf3
expression in the inner ear. Previous studies have shown that Fgfr3, Fgf8 and Fgf10
have a role in the otic placode induction or in otic epithelium development in mice.
Current study speculates that MAP3K1 has an essential role in the regulation of these
key molecular factors required for cochlear morphogenesis.
Disease Models & Mechanisms • DMM • Advance article
ear, MAP3K1 is localized at the apical surface of the supporting cells with close
Implications and future directions
Our results elucidate the regulatory control of FGF signaling pathway that is required
for the development, function and survival of inner ear hair cells, and a novel role for
the MAP3K1 kinase protein, which may be applicable to controlled regeneration of
Disease Models & Mechanisms • DMM • Advance article
inner ear sensory cells for hearing restoration.