© 2015. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

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medium provided that the original work is properly attributed.

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,

Charleston, SC 29425, USA.

*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: sriazuddin@smail.umaryland.edu

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.023077Access the most recent version at DMM Advance Online Articles. Posted 23 October 2015 as doi: 10.1242/dmm.023077

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

epithelium development and morphogenesis.

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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

studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear

morphogenesis genes. Collectively, our data highlight the critical role of MAP3K1 in the

development and function of the mouse inner ear and hearing.

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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

cleavage site and a conserved kinase domain (Uhlik et al., 2004; Witowsky and Johnson,

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

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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

lack of actin stress fiber formation and deficient cell migration (Yujiri et al., 2000; Zhang et

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.

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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

observed (see supplementary material Fig. S1).

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).

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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 MAP3K1-

mediated signaling in the cytoarchitecture of the mouse OC.

In higher vertebrates, each hair cell in the OC is enveloped by supporting cells (Fig.

3A). In the mouse OC, the IHCs rest on the inner phalangeal cells, whereas each OHC rests

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).

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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’

cells.

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

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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

P30, almost all of the OHCs in the basal and middle turn of the cochlea were degenerated,

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

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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

development.

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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

centrosomal protein and, therefore, might also participate in the microtubule organization in

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

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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

prosensory domain that would otherwise have undergone degeneration during development.

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

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concert to form a highly specialized patterned sensory epithelium. Canonical Wnt (Wnt/β-

catenin) 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 Foxg1-

positive 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 dose-

dependent manner (Jacques et al., 2012). It is plausible that, during development, Wnt/β-

catenin signaling might have resulted in relative over-activation of downstream transcription

factors, leading to the expansion of the prosensory domain and excessive differentiation of

OHCs in Map3k1tm1Yxia mutant mice. Future work using cell-specific markers and cochlear

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.

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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 down-

regulation 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

the differentiation of pillar cells, rather have an extra row of Deiters’ cells. Overproduction of

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

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MAP3K1 function for the regulation of FGF-mediated pathway in the auditory system and

hearing in mice.

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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

Map3k1tm1Yxia homozygous mice. Mice were anesthetized with intraperitoneal injections of

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

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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

Fluor secondary antibody (1:500; Life Technologies, Grand Island, NY) for 1 hour at room

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.

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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,

MA), in DMEM medium supplemented with 10% fetal bovine serum (Life Technologies,

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.).

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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-

Green-based real-time primers (available upon request) were designed using the Integrated

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.

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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

using a ×40 oil immersion lens on a Zeiss Axioplan Apotome-equipped microscope.

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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.

Funding

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.D

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Figures

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-

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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-β-

galactosidase 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

pericentrin localization. Scale bar: 1 μm.

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Fig. 2.

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

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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

from apex to base (mean ± standard error of mean).

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Fig. 3.

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,

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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

an extra row of OHCs. Scale bar: 10 μm.

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Fig. 4.

Map3k1tm1Yxia mutant mice have elevated hearing thresholds. Hearing thresholds of 3 wild-

type, 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

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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,

indicating that the residual OHCs were non-functional.

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Fig. 5.

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,

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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

along the length of the cochlea. Scale bar: 10 μm.

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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

and eosin. Scale bar: 20 μm.

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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

mutant mice. Furthermore, the expression of Fgfr3, Fgf8, Fgf10 and Atf3 was also

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).

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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

ear, MAP3K1 is localized at the apical surface of the supporting cells with close

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.

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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

inner ear sensory cells for hearing restoration.

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