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1.  A null mutation of mouse Kcna10 causes significant vestibular and mild hearing dysfunction 
Hearing research  2013;300:1-9.
KCNA10 is a voltage gated potassium channel that is expressed in the inner ear. The localization and function of KCNA10 was studied in a mutant mouse, B6-Kcna10TM45, in which the single protein coding exon of Kcna10 was replaced with a beta-galactosidase reporter cassette. Under the regulatory control of the endogenous Kcna10 promoter and enhancers, beta-galactosidase was expressed in hair cells of the vestibular organs and the organ of Corti. KCNA10 expression develops in opposite tonotopic gradients in the inner and outer hair cells. Kcna10TM45 homozygotes display only a mild elevation in pure tone hearing thresholds as measured by auditory brainstem response (ABR), while heterozygotes are normal. However, Kcna10TM45 homozygotes have absent vestibular evoked potentials (VsEPs) or elevated VsEP thresholds with prolonged peak latencies, indicating significant vestibular dysfunction despite the lack of any overt imbalance behaviors. Our results suggest that Kcna10 is expressed primarily in hair cells of the inner ear, with little evidence of expression in other organs. The Kcna10TM45 targeted allele may be a model of human nonsyndromic vestibulopathy.
PMCID: PMC3684051  PMID: 23528307
KCNA10; inner ear; vestibular dysfunction; Kcna10 knockout mouse
2.  Paxillin mutations affect focal adhesions and lead to altered mitochondrial dynamics 
Cancer Biology & Therapy  2013;14(7):679-691.
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.
PMCID: PMC3742497  PMID: 23792636
mitochondria; paxillin; gene mutation; cell motility; fission; fusion; mitochondrial dynamics
3.  Essential role of Cenexin1, but not Odf2, in ciliogenesis 
Cell Cycle  2013;12(4):655-662.
Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2−/− cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.
PMCID: PMC3594266  PMID: 23343771
Cenexin1; Odf2; Rab8a; Chibby; primary cilia; ciliogenesis
4.  Phylogenomic analyses of KCNA gene clusters in vertebrates: why do gene clusters stay intact? 
Gene clusters are of interest for the understanding of genome evolution since they provide insight in large-scale duplications events as well as patterns of individual gene losses. Vertebrates tend to have multiple copies of gene clusters that typically are only single clusters or are not present at all in genomes of invertebrates. We investigated the genomic architecture and conserved non-coding sequences of vertebrate KCNA gene clusters. KCNA genes encode shaker-related voltage-gated potassium channels and are arranged in two three-gene clusters in tetrapods. Teleost fish are found to possess four clusters. The two tetrapod KNCA clusters are of approximately the same age as the Hox gene clusters that arose through duplications early in vertebrate evolution. For some genes, their conserved retention and arrangement in clusters are thought to be related to regulatory elements in the intergenic regions, which might prevent rearrangements and gene loss. Interestingly, this hypothesis does not appear to apply to the KCNA clusters, as too few conserved putative regulatory elements are retained.
We obtained KCNA coding sequences from basal ray-finned fishes (sturgeon, gar, bowfin) and confirmed that the duplication of these genes is specific to teleosts and therefore consistent with the fish-specific genome duplication (FSGD). Phylogenetic analyses of the genes suggest a basal position of the only intron containing KCNA gene in vertebrates (KCNA7). Sistergroup relationships of KCNA1/2 and KCNA3/6 support that a large-scale duplication gave rise to the two clusters found in the genome of tetrapods. We analyzed the intergenic regions of KCNA clusters in vertebrates and found that there are only a few conserved sequences shared between tetrapods and teleosts or between paralogous clusters. The orthologous teleost clusters, however, show sequence conservation in these regions.
The lack of overall conserved sequences in intergenic regions suggests that there are either other processes than regulatory evolution leading to cluster conservation or that the ancestral regulatory relationships among genes in KCNA clusters have been changed together with their regulatory sites.
PMCID: PMC1978502  PMID: 17697377
5.  Overexpression of paxillin induced by miR-137 suppression promotes tumor progression and metastasis in colorectal cancer 
Carcinogenesis  2012;34(4):803-811.
The deregulation of paxillin (PXN) has been involved in the progression and metastasis of different malignancies including colorectal cancer (CRC). miR-137 is frequently suppressed in CRC. PXN is predicted to be a direct target of miR-137 in CRC cells. On this basis, we hypothesized that overexpression of PXN induced by suppression of miR-137 may promote tumor progression and metastasis and predicts poor prognosis. We detected the expression of PXN and miR-137 in clinical tumor tissues by immunohistochemical analysis and real-time PCR, positive PXN staining was observed in 198 of the 247 (80.1%) cases, whereas no or weak PXN staining was observed in the adjacent non-cancerous area. Higher level of PXN messenger RNA (mRNA) and lower level of miR-137 was observed in cancer tissues than adjacent non-cancerous tissues. High expression of PXN and low expression of miR-137 was associated with aggressive tumor phenotype and adverse prognosis. Moreover, the expression of PXN was negatively correlated with miR-137 expression. A dual-luciferase reporter gene assay validated that PXN was a direct target of miR-137. The use of miR-137 mimics or inhibitor could decrease or increase PXN mRNA and protein levels in CRC cell lines. Knockdown of PXN or ectopic expression of miR-137 could markedly inhibit cell proliferation, migration and invasion in vitro and repress tumor growth and metastasis in vivo. Taken together, these results demonstrated that overexpression of PXN induced by suppression of miR-137 promotes tumor progression and metastasis and could serve as an independent prognostic indicator in CRC patients.
PMCID: PMC3616669  PMID: 23275153
6.  Structural Consequences of Kcna1 Gene Deletion and Transfer in the Mouse Hippocampus 
Epilepsia  2007;48(11):2023-2046.
Mice lacking the Kv1.1 potassium channel α subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1−/− (“knock-out”) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons.
Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1−/− animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1−/− brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E.coli lacZ reporter gene. Vector-injected mice were were examined to determine the extent of neuronal infection.
Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1−/− mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed “ectopic” Kv1.1 α channel subunit expression.
Kcna1 deletion in mice results in a seizure disorder that resembles – electrographically and neuropathologically – the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection
PMCID: PMC2752664  PMID: 17651419
Epilepsy; Gene therapy; Hippocampal pathology; Knock-out; Potassium channel; Seizures
7.  Acute Hypoxia Selectively Inhibits KCNA5 Channels in Pulmonary Artery Smooth Muscle Cells 
Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K+ (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents (IK(V)) is selective to PASMC; hypoxia has little effect on IK(V) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or hetero-tetramers of pore-forming α subunits and regulatory β subunits. KCNA5 is a Kv channel α subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. Here, we show that acute hypoxia selectively inhibits IK(V) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased IK(V) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect IK(V) in KCNA5-transfected HEK-293 and COS-7. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of Po2 from 145 to 35 mmHg reduced IK(V) by ~40% in rat PASMC transfected with human KCNA5, but had no effect on IK(V) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential, and acute hypoxia selectively reduced KCNA5 channel activity in PASMC relative to MASMC and other cell types. Since Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on IK(V) in PASMC and MASMC may also be due to different expression levels of KCNA5 channels.
PMCID: PMC1363730  PMID: 16236819
membrane potential; K+ channels; vascular smooth muscle; pulmonary
8.  A Novel Atoh1 “Self-Terminating” Mouse Model Reveals the Necessity of Proper Atoh1 Level and Duration for Hair Cell Differentiation and Viability 
PLoS ONE  2012;7(1):e30358.
Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
PMCID: PMC3261193  PMID: 22279587
9.  HMGA2, the Architectural Transcription Factor High Mobility Group, Is Expressed in the Developing and Mature Mouse Cochlea 
PLoS ONE  2014;9(2):e88757.
Hmga2 protein belongs to the non-histone chromosomal high-mobility group (HMG) protein family. HMG proteins have been shown to function as architectural transcription regulators, facilitating enhanceosome formation on a variety of mammalian promoters. Hmga2 are expressed at high levels in embryonic and transformed cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, Hmga2. Our previous affymetrix array data showed that Hmga2 is expressed in the developing and adult mammalian cochleas. However, the spatio-temporal expression pattern of Hmga2 in the murine cochlea remained unknown. In this study, we report the expression of Hmga2 in developing and adult cochleas using immunohistochemistry and quantitative real time PCR analysis. Immunolabeling of Hmga2 in the embryonic, postnatal, and mature cochleas showed broad Hmga2 expression in embryonic cochlea (E14.5) at the level of the developing organ of Corti in differentiating hair cells, supporting cells, in addition to immature cells in the GER and LER areas. By postnatal stage (P0–P3), Hmga2 is predominantly expressed in the hair and supporting cells, in addition to cells in the LER area. By P12, Hmga2 immunolabeling is confined to the hair cells and supporting cells. In the adult ear, Hmga2 expression is maintained in the hair and supporting cell subtypes (i.e. Deiters’ cells, Hensen cells, pillar cells, inner phalangeal and border cells) in the cochlear epithelium. Using quantitative real time PCR, we found a decrease in transcript level for Hmga2 comparable to other known inner ear developmental genes (Sox2, Atoh1, Jagged1 and Hes5) in the cochlear epithelium of the adult relative to postnatal ears. These data provide for the first time the tissue-specific expression and transcription level of Hmga2 during inner ear development and suggest its potential dual role in early differentiation and maintenance of both hair and supporting cell phenotypes.
PMCID: PMC3925159  PMID: 24551154
10.  Polymorphism in the KCNA3 Gene Is Associated with Susceptibility to Autoimmune Pancreatitis in the Japanese Population 
Disease markers  2011;31(4):223-229.
Autoimmune pancreatitis (AIP), characterized by irregular narrowing of the main pancreatic duct, swelling of the pancreas, and histological evidence of lymphoplasmacytic inflammation by high serum immunoglobulin G4, is distinct from ordinary pancreatitis. However, genetic factors involved in the etiology and pathophysiology of AIP remain unclear. Sixty-four patients with autoimmune pancreatitis (53 men, 11 women; mean age, 62.4 years) and 104 healthy Japanese controls were enrolled in this study. We performed an association analysis using 400 microsatellite markers with an average spacing of 10.8 cM in the genome. We also evaluated the association of AIP with seven single nucleotide polymorphisms (SNPs) within the 20-kb region around the potassium voltage-gated channel, shaker-related subfamily, member 3 gene (KCNA3). We identified six statistically significant markers (D1S2726, D5S410, D6S460, D10S548, D15S128, and D20S186; P < 0.05) related to susceptibility. The surrounding region showing the strong association (P = 7.4 × 10−7, Pc = 0.0015) contained the KCNA3 gene. Further analysis by SNP genotyping in KCNA3 gene revealed that four SNPs (rs2840381, rs1058184, rs2640480, rs1319782) were significantly associated with the AIP susceptibility (P < 0.007). KCNA3 is known to be involved in immunomodulation of autoreactive effector and memory T cell–mediated autoimmune diseases. Our findings provide the first evidence that KCNA3 is associated with AIP and suggest that KCNA3 may influence the risk for AIP.
PMCID: PMC3826803  PMID: 22045429
AIP; autoimmune pancreatitis; SNPs; KCNA3; disease susceptibility
11.  Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti 
Hearing research  2010;275(1-2):66-80.
Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell deaths. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), most of the developing organ of Corti is lost through embryonic cell deaths, with the remaining cells transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, leaving only few basal and a more prominent apical innervation. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.
PMCID: PMC3065550  PMID: 21146598
hair cell differentiation; flat epithelium; organ of Corti; innervation of the ear; conditional deletion; mouse ear mutants
12.  Paxillin is an intrinsic negative regulator of platelet activation in mice 
Thrombosis Journal  2014;12:1.
Paxillin is a LIM domain protein localized at integrin-mediated focal adhesions. Although paxillin is thought to modulate the functions of integrins, little is known about the contribution of paxillin to signaling pathways in platelets. Here, we studied the role of paxillin in platelet activation in vitro and in vivo.
Methods and results
We generated paxillin knockdown (Pxn-KD) platelets in mice by transplanting bone marrow cells transduced with a lentiviral vector carrying a short hairpin RNA sequence, and confirmed that paxillin expression was significantly reduced in platelets derived from the transduced cells. Pxn-KD platelets showed a slight increased in size and augmented integrin αIIbβ3 activation following stimulation of multiple receptors including glycoprotein VI and G protein-coupled receptors. Thromboxane A2 biosynthesis and the release of α-granules and dense granules in response to agonist stimulation were also enhanced in Pxn-KD platelets. However, Pxn-KD did not increase tyrosine phosphorylation or intracellular calcium mobilization. Intravital imaging confirmed that Pxn-KD enhanced thrombus formation in vivo.
Our findings suggest that paxillin negatively regulates several common platelet signaling pathways, resulting in the activation of integrin αIIbβ3 and release reactions.
PMCID: PMC3904695  PMID: 24383745
Platelet; Glycoprotein; Platelet aggregation; Release reaction
13.  Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti 
Developmental cell  2009;16(1):58-69.
The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that – unlike other Hes/Hey factors – Hey2 expression is Notch-independent. Hey2 is activated by FGF and blocks hair cell differentiation, while mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch-independent, also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.
PMCID: PMC2696015  PMID: 19154718
14.  A Gene Trap Knockout of the Abundant Sperm Tail Protein, Outer Dense Fiber 2, Results in Preimplantation Lethality 
Genesis (New York, N.Y. : 2000)  2006;44(11):515-522.
Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a wide-spread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein–protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice.
PMCID: PMC3038656  PMID: 17078042
Odf2; sperm; gene trap; outer dense fibers
15.  A Quantitative Analysis of the Spatiotemporal Pattern of Transient Receptor Potential Gene Expression in the Developing Mouse Cochlea 
TRP genes encode a diverse family of ion channels which have been implicated in many sensory functions. Because several TRP channels have similar properties to the elusive hair cell transduction channel, recent attention has focused on TRP gene expression in the inner ear. At least four TRP genes are known to be expressed in hair cells: TRPC3, TRPV4, TRPA1, and TRPML3. However, there is little evidence supporting any of these as a component of the transduction complex. Other less well-characterized TRP channels are expressed in the inner ear, in particular, within the organ of Corti. Because of their potential role in sensory function, we investigated the developmental expression of RNA that encodes all 33 TRP subunits as well as several splice variants. We designed a quantitative PCR screen using cochlear samples acquired before, during, and after the time when mechanotransduction is acquired in sensory hair cells (embryonic day 17 to postnatal day 8). Cochleas, which included the organ of Corti, stria vascularis, and Reissner’s membrane, were subdivided into four equal quadrants which allowed for regional comparison during development. Expression of RNA transcripts that encoded 33 TRP subunits plus several splice forms and beta-actin were quantified in 28 samples for a total of 1,092 individual measurements, each done in triplicate. We detected RNA that encoded all TRP channels except two: TRPC7 and TRPM8. The largest changes in RNA expression were for TRPA1 (>100-fold), TRPP3 (>50-fold), and TRPC5.2 (>20-fold) which suggested that these subunits may contribute to normal cochlear function. Furthermore, the screen revealed TRPP3 and PKD1L3 RNA expression patterns that were correlated with the acquisition of sensory transduction in outer hair cells (Lelli et al., J Neurophysiol. 101:2961–2973, 2009). Numerous spatiotemporal expression gradients were identified many of which may contribute to the normal functional development of the mouse cochlea.
Electronic supplementary material
The online version of this article (doi:10.1007/s10162-009-0193-8) contains supplementary material, which is available to authorized users.
PMCID: PMC2820207  PMID: 19834762
mouse; inner ear; organ of corti; TRP channels; development; quantitative PCR
16.  The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs 
PLoS Genetics  2014;10(1):e1004055.
The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.
Author Summary
The organ of Corti is a sensory structure in the cochlea that mediates our sense of hearing. It consists of one row of inner hair cells and three rows of outer hair cells, together with an array of neighboring supporting cells. The precise arrangement of these different cell types is regulated very tightly by a number of signaling pathways during embryonic development, and mutations in genes that regulate this pattern often lead to deafness. We have generated a mouse mutant containing a lentiviral insertion in a gene encoding a putative RNA splicing factor called Sfswap. Homozygous mutant mice have hearing and balance defects, and have an abnormal arrangement of hair cells in their cochlea. These defects are consistent with defects in the Notch signaling pathway. We show that Sfswap mutants interact genetically with a mutation in Jagged1, which encodes a Notch ligand. We show that expression of some genes involved in Notch signaling is disrupted in Sfswap mutant mice. Our work is one of the first studies to show that a putative splicing factor has specific effects on Notch signaling pathway members and on inner ear development.
PMCID: PMC3879212  PMID: 24391519
17.  Requirement of hCenexin for Proper Mitotic Functions of Polo-Like Kinase 1 at the Centrosomes▿ †  
Molecular and Cellular Biology  2006;26(22):8316-8335.
Outer dense fiber 2 (Odf2) was initially identified as a major component of sperm tail cytoskeleton and later was suggested to be a widespread component of centrosomal scaffold that preferentially associates with the appendages of the mother centrioles in somatic cells. Here we report the identification of two Odf2-related centrosomal components, hCenexin1 and hCenexin1 variant 1, that possess a unique C-terminal extension. Our results showed that hCenexin1 is the major isoform expressed in HeLa cells, whereas hOdf2 is not detectably expressed. Mammalian polo-like kinase 1 (Plk1) is critical for proper mitotic progression, and its association with the centrosome is important for microtubule nucleation and function. Interestingly, depletion of hCenexin1 by RNA interference (RNAi) delocalized Plk1 from the centrosomes and the C-terminal extension of hCenexin1 was crucial to recruit Plk1 to the centrosomes through a direct interaction with the polo-box domain of Plk1. Consistent with these findings, the hCenexin1 RNAi cells exhibited weakened γ-tubulin localization and chromosome segregation defects. We propose that hCenexin1 is a critical centrosomal component whose C-terminal extension is required for proper recruitment of Plk1 and other components crucial for normal mitosis. Our results further suggest that the anti-Odf2 immunoreactive centrosomal antigen previously detected in non-germ line cells is likely hCenexin1.
PMCID: PMC1636773  PMID: 16966375
18.  Nongenetic factors influence severity of episodic ataxia type 1 in monozygotic twins(Video) 
Neurology  2010;75(4):367-372.
Episodic ataxia type 1 (EA1) is a monogenic channelopathy caused by mutations of the potassium channel gene KCNA1. Affected individuals carrying the same mutation can exhibit considerable variability in the severity of ataxia, neuromyotonia, and other associated features. We investigated the phenotypic heterogeneity of EA1 in 2 sets of identical twins to determine the contribution of environmental factors to disease severity. One of the mutations was also found in a distantly related family, providing evidence of the influence of genetic background on the EA1 phenotype.
We evaluated 3 families with an EA1 phenotype, 2 of which included monozygotic twins. We sequenced the KCNA1 gene and studied the biophysical consequences of the mutations in HEK cells.
We identified a new KCNA1 mutation in each pair of twins. Both pairs reported striking differences in the clinical severity of symptoms. The F414S mutation identified in one set of twins also occurred in a distantly related family in which seizures complicated the EA1 phenotype. The other twins had an R307C mutation, the first EA1 mutation to affect an arginine residue in the voltage-sensor domain. Both mutants when expressed exerted a dominant-negative effect on wild-type channels.
These results broaden the range of KCNA1 mutations and reveal an unexpectedly large contribution of nongenetic factors to phenotypic variability in EA1. The occurrence of epilepsy in 1 of 2 families with the F414S mutation suggests an interplay of KCNA1 with other genetic factors.
= episodic ataxia type 1.
PMCID: PMC2918890  PMID: 20660867
19.  Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival 
PLoS ONE  2012;7(1):e30853.
In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1KINeurog1) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1+/KINeurog1) and homozygous (Atoh1KINeurog1/KINeurog1) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete ‘flat epithelium’ in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1f/+), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1KINeurog1 can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons.
PMCID: PMC3265522  PMID: 22292060
20.  The Notch Ligand JAG1 Is Required for Sensory Progenitor Development in the Mammalian Inner Ear 
PLoS Genetics  2006;2(1):e4.
In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.
Deafness and adult-onset hearing loss are significant health problems. In most cases, deafness or vestibular dysfunction results when the sensory cells in the inner ear, known as hair cells, degenerate due to environmental or genetic causes. In the mammalian inner ear, the hair cells and their associated supporting cells can be found in six different patches that have particular functions related to hearing or balance. Unfortunately, unlike in birds or fish, mammalian hair cells show little ability to regenerate, resulting in a permanent hearing or balance disorder when damaged. Here, the authors show that a protein called JAG1, a ligand in the Notch signaling pathway, is required for the normal development of all six sensory regions in the mammalian inner ear. In ears that lacked JAG1, some of the sensory patches were missing completely, whereas others were small and lacked particular cell types. The authors showed that JAG1 is required by the sensory precursors, progenitor cells that give rise to both the hair cells and the supporting cells. By understanding how the sensory areas develop normally, it is hoped that molecular tools can be developed that will aid sensory regeneration in the mammalian inner ear.
PMCID: PMC1326221  PMID: 16410827
21.  Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis 
Cell and tissue research  2008;334(3):339-358.
At E8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1adr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal for the first time that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule and utricle, a “cochlear-gravistatic” endorgan. The enlarged anterior canal crista develops by fusion of horizontal and anterior crista whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti showed a loss of cochlear hair cells, suggesting that long term hair cell maintenance is also disrupted in these mutants.
PMCID: PMC2654344  PMID: 18985389
dreher; Lmx1a; ear; mouse; hair cell maintenance; sensory epithelium formation
22.  Sleep in Kcna2 knockout mice 
BMC Biology  2007;5:42.
Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3–4 h/day instead of 8–14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO) mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system.
Continuous (24 h) electroencephalograph (EEG), electromyogram (EMG), and video recordings were used to measure sleep and waking in Kcna2 KO, heterozygous (HZ) and wild-type (WT) pups (P17) and HZ and WT adult mice (P67). Sleep stages were scored visually based on 4-s epochs. EEG power spectra (0–20 Hz) were calculated on consecutive 4-s epochs. KO pups die by P28 due to generalized seizures. At P17 seizures are either absent or very rare in KO pups (< 1% of the 24-h recording time), and abnormal EEG activity is only present during the seizure. KO pups have significantly less non-rapid eye movement (NREM) sleep (-23%) and significantly more waking (+21%) than HZ and WT siblings with no change in rapid eye movement (REM) sleep time. The decrease in NREM sleep is due to an increase in the number of waking episodes, with no change in number or duration of sleep episodes. Sleep patterns, daily amounts of sleep and waking, and the response to 6 h sleep deprivation are similar in HZ and WT adult mice.
Kv1.2, a mammalian homologue of Shaker, regulates neuronal excitability and affects NREM sleep.
PMCID: PMC2151933  PMID: 17925011
23.  Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea 
PLoS ONE  2010;5(7):e11661.
At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation.
Methodology/Principal Findings
We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells.
Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 might actually be mediated in large part by Neurod1. We suggest that Neurod1 is regulated by both Neurog1 and Atoh1 and provides a negative feedback for either gene. Through this and other feedback, Neurod1 suppresses alternate fates of neurons to differentiate as hair cells and regulates hair cell subtypes.
PMCID: PMC2908541  PMID: 20661473
24.  Ion Channel Gene Expression in the Inner Ear 
The ion channel genome is still being defined despite numerous publications on the subject. The ion channel transcriptome is even more difficult to assess. Using high-throughput computational tools, we surveyed all available inner ear cDNA libraries to identify genes coding for ion channels. We mapped over 100,000 expressed sequence tags (ESTs) derived from human cochlea, mouse organ of Corti, mouse and zebrafish inner ear, and rat vestibular end organs to Homo sapiens, Mus musculus, Danio rerio, and Rattus norvegicus genomes. A survey of EST data alone reveals that at least a third of the ion channel genome is expressed in the inner ear, with highest expression occurring in hair cell-enriched mouse organ of Corti and rat vestibule. Our data and comparisons with other experimental techniques that measure gene expression show that every method has its limitations and does not per se provide a complete coverage of the inner ear ion channelome. In addition, the data show that most genes produce alternative transcripts with the same spectrum across multiple organisms, no ion channel gene variants are unique to the inner ear, and many splice variants have yet to be annotated. Our high-throughput approach offers a qualitative computational and experimental analysis of ion channel genes in inner ear cDNA collections. A lack of data and incomplete gene annotations prevent both rigorous statistical analyses and comparisons of entire ion channelomes derived from different tissues and organisms.
Electronic supplementary material
The online version of this article (doi:10.1007/s10162-007-0082-y) contains supplementary material, which is available to authorized users.
PMCID: PMC2538437  PMID: 17541769
hearing; ionic currents; ion channels; genome; EST mapping; expressed sequence tags; splice variants; alternative transcripts
25.  Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.  
In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.
PMCID: PMC2816938  PMID: 20134402

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