Departmental Senator, Medical School Faculty Senate (2008 - 2011)
Director of Research, Otolaryngology - Stanford (2005 - Present)
Provost's Advisory Committee on Postdoctoral Affairs, Stanford (2009 - 2017)
Vice Chair for Research, Otolaryngology - Stanford (2014 - Present)
Honors & Awards
Juergen Tonndorf Award, Deafness Research Foundation (2001)
Basil O'Connor Starter Scholar Research Award, March of Dimes (2001-2003)
Franklin M. Rizer Lectureship, The American Neurotology Society (2004)
Burt Evans Young Investigator Award, National Organization for Hearing Research Foundation (2005)
James Wiggins Associate Professor, Harvard Medical School (4/2005)
Albert and Ellen Grass Faculty Grant Award, Marine Biological Laboratory, Woods Hole, MA (Summer 2004, Summer 2005)
Frontiers of Science Scholar (US/Japan 2006), Kavli Foundation (2006)
McKnight Neuroscience of Brain Disorders Award, McKnight Endowment Fund for Neuroscience (2005-2007)
Edward C. and Amy H. Sewall Professor, Stanford School of Medicine (7/ 2010)
Member, Collegium Oto-Rhino-Laryngologicum Amicitiae Sacrum (9/2011)
Boards, Advisory Committees, Professional Organizations
Auditory System Study Section (AUD), member, NIDCD/NIH (2014 - Present)
Award of Merit Committee, Association for Research in Otolaryngology Midwinter Meeting (2014 - Present)
Scientific Advisor, Inception 3, Inc (2012 - Present)
Communication Disorders Review Committee (CDRC study section), chair, NIDCD/NIH (2012 - 2013)
Workshop on hair cell regeneration and future therapies, chair and co-organizer, NIDCD (2012 - 2012)
Hearing Restoration Project, collaborative research group member, Hearing Health Foundation (2011 - Present)
Research Advisory Board, American Otological Society (2010 - 2015)
Program Committee Member, Association for Research in Otolaryngology Midwinter Meeting (2010 - 2013)
Scientific Advisor, Otonomy (2009 - 2012)
Council of Scientific Trustees, Deafness Research Foundation / Hearing Health Foundation (2008 - 2014)
Communication Disorders Review Committee (CDRC study section), member, NIDCD/NIH (2008 - 2013)
Stem Cell Policy Working Group, Harvard University (2004 - 2005)
Postdoctoral Fellow, The Rockefeller University, New York, NY, Sensory Neuroscience (2000)
Dr rer nat (Ph.D.), Johannes Gutenberg University, Mainz, Germany, Genetics (1994)
Dipl Biol (M.S.), Johannes Gutenberg University, Mainz, Germany, Biological Sciences (1990)
Community and International Work
Biology of the Inner Ear Course, http://www.mbl.edu/bie/
Course Co-director (2013, 2015, 2017)
Marine Biological Laboratory, Woods Hole, MA
Opportunities for Student Involvement
9th Molecular Biology of Hearing & Deafness Conference, Stanford University
Organizer, June 22-25
Opportunities for Student Involvement
Current Research and Scholarly Interests
We are interested how the inner ear forms from an early anlage called the otic placode. Our goal is to describe the otic lineage from an early placodal progenitor until it splits up into multiple cell types making up the sensory epithelia, innervating ganglia, and accessory structures.
In parallel, we apply knowledge we gained from guiding embryonic and induced pluripotent stem cells along the otic lineage to find ways for treatment of hearing loss. This involves identification of mechanisms of sensory hair cell regeneration in animals such as chickens that recover from hearing gloss, screening for potential regenerative targets that can be activated with drugs, and exploring reprograming as well as cell transplantation strategies.
Independent Studies (13)
- Directed Investigation
BIOE 392 (Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Sum)
- Directed Reading in Otolaryngology
OTOHNS 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Win, Spr)
- Graduate Research
NEPR 399 (Sum)
- Graduate Research
OTOHNS 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr)
- Medical Scholars Research
OTOHNS 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Out-of-Department Graduate Research
BIO 300X (Aut, Win)
- Undergraduate Research
OTOHNS 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Win, Spr)
- Directed Investigation
Graduate and Fellowship Programs
Biology (School of Humanities and Sciences) (Phd Program)
Biomedical Informatics (Phd Program)
Human Genetics and Genetic Counseling (Masters Program)
Neurotology (Fellowship Program)
Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids.
2018; 22 (1): 242–54
The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.
View details for DOI 10.1016/j.celrep.2017.12.007
View details for PubMedID 29298425
Aminoglycoside Damage and Hair Cell Regeneration in the Chicken Utricle.
Journal of the Association for Research in Otolaryngology : JARO
In this study, we present a systematic characterization of hair cell loss and regeneration in the chicken utricle in vivo. A single unilateral surgical delivery of streptomycin caused robust decline of hair cell numbers in striolar as well as extrastriolar regions, which in the striola was detected very early, 6 h post-insult. During the initial 12 h of damage response, we observed global repression of DNA replication, in contrast to the natural, mitotic hair cell production in undamaged control utricles. Regeneration of hair cells in striolar and extrastriolar regions occurred via high rates of asymmetric supporting cell divisions, accompanied by delayed replenishment by symmetric division. While asymmetric division of supporting cells is the main regenerative response to aminoglycoside damage, the detection of symmetric divisions supports the concept of direct transdifferentiation where supporting cells need to be replenished after their phenotypic conversion into new hair cells. Supporting cell divisions appear to be well coordinated because total supporting cell numbers throughout the regenerative process were invariant, despite the initial large-scale loss of hair cells. We conclude that a single ototoxic drug application provides an experimental framework to study the precise onset and timing of utricle hair cell regeneration in vivo. Our findings indicate that initial triggers and signaling events occur already within a few hours after aminoglycoside exposure. Direct transdifferentiation and asymmetric division of supporting cells to generate new hair cells subsequently happen largely in parallel and persist for several days.
View details for DOI 10.1007/s10162-017-0646-4
View details for PubMedID 29134476
Small Molecules for Early Endosome-Specific Patch Clamping.
Cell chemical biology
2017; 24 (7): 907–16.e4
To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages.
View details for DOI 10.1016/j.chembiol.2017.05.025
View details for PubMedID 28732201
Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin.
Frontiers in synaptic neuroscience
2017; 9: 13
Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKIIδ in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKIIδ also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKIIδ revealed ten phosphorylation sites, five of which are located within C2-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca2+ affinity of the recombinant C2F domain 10-fold, and increases the Ca2+ affinity of the C2C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKIIδ in IHCs.
View details for DOI 10.3389/fnsyn.2017.00013
View details for PubMedID 29046633
View details for PubMedCentralID PMC5632675
Identification of novel MYO18A interaction partners required for myoblast adhesion and muscle integrity.
2016; 6: 36768-?
The unconventional myosin MYO18A that contains a PDZ domain is required for muscle integrity during zebrafish development. However, the mechanism by which it functions in myofibers is not clear. The presence of a PDZ domain suggests that MYO18A may interact with other partners to perform muscle-specific functions. Here we performed double-hybrid screening and co-immunoprecipitation to identify MYO18A-interacting proteins, and have identified p190RhoGEF and Golgin45 as novel partners for the MYO18A PDZ domain. We have also identified Lurap1, which was previously shown to bind MYO18A. Functional analyses indicate that, similarly as myo18a, knockdown of lurap1, p190RhoGEF and Golgin45 by morpholino oligonucleotides disrupts dystrophin localization at the sarcolemma and produces muscle lesions. Simultaneous knockdown of myo18a with either of these genes severely disrupts myofiber integrity and dystrophin localization, suggesting that they may function similarly to maintain myofiber integrity. We further show that MYO18A and its interaction partners are required for adhesion of myoblasts to extracellular matrix, and for the formation of the Golgi apparatus and organization of F-actin bundles in myoblast cells. These findings suggest that MYO18A has the potential to form a multiprotein complex that links the Golgi apparatus to F-actin, which regulates muscle integrity and function during early development.
View details for DOI 10.1038/srep36768
View details for PubMedID 27824130
Single-cell analysis delineates a trajectory toward the human early otic lineage
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (30): 8508-8513
Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Single-cell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types.
View details for DOI 10.1073/pnas.1605537113
View details for Web of Science ID 000380346200056
View details for PubMedID 27402757
View details for PubMedCentralID PMC4968758
Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture.
2016; 11 (9)
Stem cell-derived inner ear sensory epithelia are a promising source of tissues for treating patients with hearing loss and dizziness. We recently demonstrated how to generate inner ear sensory epithelia, designated as inner ear organoids, from mouse embryonic stem cells (ESCs) in a self-organizing 3D culture. Here we improve the efficiency of this culture system by elucidating how Wnt signaling activity can drive the induction of otic tissue. We found that a carefully timed treatment with the potent Wnt agonist CHIR99021 promotes induction of otic vesicles-a process that was previously self-organized by unknown mechanisms. The resulting otic-like vesicles have a larger lumen size and contain a greater number of Pax8/Pax2-positive otic progenitor cells than organoids derived without the Wnt agonist. Additionally, these otic-like vesicles give rise to large inner ear organoids with hair cells whose morphological, biochemical and functional properties are indistinguishable from those of vestibular hair cells in the postnatal mouse inner ear. We conclude that Wnt signaling plays a similar role during inner ear organoid formation as it does during inner ear development in the embryo.
View details for DOI 10.1371/journal.pone.0162508
View details for PubMedID 27607106
Applications for single cell trajectory analysis in inner ear development and regeneration
CELL AND TISSUE RESEARCH
2015; 361 (1): 49-57
Single cell trajectory analysis is a computational approach that orders cells along a pseudotime axis. This temporal modeling approach allows the characterization of transitional processes such as lineage development, response to insult, and tissue regeneration. The concept can also be applied to resolve spatial organization of cells within the originating tissue. Known as temporal and spatial transcriptomics, respectively, these methods belong to the most powerful analytical techniques for quantitative gene expression data currently available. Here, we discuss three different approaches: principal component analysis, the 'Monocle' algorithm, and self-organizing maps. We use a previously published qRT-PCR dataset of single neuroblast cells isolated from the developing mouse inner ear to highlight the basic features of the three methods and their individual limitations, as well as the distinct advantages that make them useful for research on the inner ear. The complex developmental morphogenesis of the inner ear and its specific challenges such as the paucity of cells as well as important open questions such as sensory hair cell regeneration render this organ a prime target for single cell trajectory analysis strategies.
View details for DOI 10.1007/s00441-014-2079-2
View details for Web of Science ID 000357115200005
View details for PubMedID 25532874
Quantitative High-Resolution Cellular Map of the Organ of Corti
2015; 11 (9): 1385-1399
The organ of Corti harbors highly specialized sensory hair cells and surrounding supporting cells that are essential for the sense of hearing. Here, we report a single cell gene expression data analysis and visualization strategy that allows for the construction of a quantitative spatial map of the neonatal organ of Corti along its major anatomical axes. The map displays gene expression levels of 192 genes for all organ of Corti cell types ordered along the apex-to-base axis of the cochlea. Statistical interrogation of cell-type-specific gene expression patterns along the longitudinal gradient revealed features of apical supporting cells indicative of a propensity for proliferative hair cell regeneration. This includes reduced expression of Notch effectors, receptivity for canonical Wnt signaling, and prominent expression of early cell-cycle genes. Cochlear hair cells displayed expression gradients of genes indicative of cellular differentiation and the establishment of the tonotopic axis.
View details for DOI 10.1016/j.celrep.2015.04.062
View details for Web of Science ID 000356069500007
View details for PubMedID 26027927
- Changes in the regulation of the Notch signaling pathway are temporally correlated with regenerative failure in the mouse cochlea FRONTIERS IN CELLULAR NEUROSCIENCE 2015; 9
3D computational reconstruction of tissues with hollow spherical morphologies using single-cell gene expression data.
2015; 10 (3): 459-474
Single-cell gene expression analysis has contributed to a better understanding of the transcriptional heterogeneity in a variety of model systems, including those used in research in developmental, cancer and stem cell biology. Nowadays, technological advances facilitate the generation of large gene expression data sets in high-throughput format. Strategies are needed to pertinently visualize this information in a tissue structure-related context, so as to improve data analysis and aid the drawing of meaningful conclusions. Here we describe an approach that uses spatial properties of the tissue source to enable the reconstruction of hollow sphere-shaped tissues and organs from single-cell gene expression data in 3D space. To demonstrate our method, we used cells of the mouse otocyst and the renal vesicle as examples. This protocol presents a straightforward computational expression analysis workflow, and it is implemented on the MATLAB and R statistical computing and graphics software platforms. Hands-on time for typical experiments can be <1 h using a standard desktop PC or Mac.
View details for DOI 10.1038/nprot.2015.022
View details for PubMedID 25675210
Identification and characterization of mouse otic sensory lineage genes.
Frontiers in cellular neuroscience
2015; 9: 79-?
Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells.
View details for DOI 10.3389/fncel.2015.00079
View details for PubMedID 25852475
Cisplatin Exposure Damages Resident Stem Cells of the Mammalian Inner Ear
2014; 243 (10): 1328-1337
Background: Cisplatin is a widely used chemotherapeutic agent that can also cause ototoxic injury. One potential treatment for cisplatin-induced hearing loss involves the activation of endogenous inner ear stem cells, which may then produce replacement hair cells. In this series of experiments, we examined the effects of cisplatin exposure on both hair cells and resident stem cells of the mouse inner ear. Results: Treatment for 24 hr with 10 µM cisplatin caused significant loss of hair cells in the mouse utricle, but such damage was not evident until 4 days after the cisplatin exposure. In addition to killing hair cells, cisplatin treatment also disrupted the actin cytoskeleton in remaining supporting cells, and led to increased histone H2AX phosphorylation within the sensory epithelia. Finally, treatment with 10 µM cisplatin appeared to have direct toxic effects on resident stem cells in the mouse utricle. Exposure to cisplatin blocked the proliferation of isolated stem cells and prevented sphere formation when those cells were maintained in suspension culture. Conclusion: The results suggest that inner ear stem cells may be injured during cisplatin ototoxicity, thus limiting their ability to mediate sensory repair. Developmental Dynamics, 2014. © 2014 Wiley Periodicals, Inc.
View details for DOI 10.1002/DVDY.24150
View details for Web of Science ID 000342809200015
View details for PubMedID 24888499
a-Tubulin K40 acetylation is required for contact inhibition of proliferation and cell-substrate adhesion.
Molecular biology of the cell
2014; 25 (12): 1854-1866
Acetylation of α-tubulin on lysine 40 marks long-lived microtubules in structures such as axons and cilia, and yet the physiological role of α-tubulin K40 acetylation is elusive. Although genetic ablation of the α-tubulin K40 acetyltransferase αTat1 in mice did not lead to detectable phenotypes in the developing animals, contact inhibition of proliferation and cell-substrate adhesion were significantly compromised in cultured αTat1(-/-) fibroblasts. First, αTat1(-/-) fibroblasts kept proliferating beyond the confluent monolayer stage. Congruently, αTat1(-/-) cells failed to activate Hippo signaling in response to increased cell density, and the microtubule association of the Hippo regulator Merlin was disrupted. Second, αTat1(-/-) cells contained very few focal adhesions, and their ability to adhere to growth surfaces was greatly impaired. Whereas the catalytic activity of αTAT1 was dispensable for monolayer formation, it was necessary for cell adhesion and restrained cell proliferation and activation of the Hippo pathway at elevated cell density. Because α-tubulin K40 acetylation is largely eliminated by deletion of αTAT1, we propose that acetylated microtubules regulate contact inhibition of proliferation through the Hippo pathway.
View details for DOI 10.1091/mbc.E13-10-0609
View details for PubMedID 24743598
View details for PubMedCentralID PMC4055265
Inner ear hair cell-like cells from human embryonic stem cells.
Stem cells and development
2014; 23 (11): 1275-1284
In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.
View details for DOI 10.1089/scd.2014.0033
View details for PubMedID 24512547
View details for PubMedCentralID PMC4028088
Reconstruction of the Mouse Otocyst and Early Neuroblast Lineage at Single-Cell Resolution
2014; 157 (4): 964-978
The otocyst harbors progenitors for most cell types of the mature inner ear. Developmental lineage analyses and gene expression studies suggest that distinct progenitor populations are compartmentalized to discrete axial domains in the early otocyst. Here, we conducted highly parallel quantitative RT-PCR measurements on 382 individual cells from the developing otocyst and neuroblast lineages to assay 96 genes representing established otic markers, signaling-pathway-associated transcripts, and novel otic-specific genes. By applying multivariate cluster, principal component, and network analyses to the data matrix, we were able to readily distinguish the delaminating neuroblasts and to describe progressive states of gene expression in this population at single-cell resolution. It further established a three-dimensional model of the otocyst in which each individual cell can be precisely mapped into spatial expression domains. Our bioinformatic modeling revealed spatial dynamics of different signaling pathways active during early neuroblast development and prosensory domain specification. PAPERFLICK:
View details for DOI 10.1016/j.cell.2014.03.036
View details for Web of Science ID 000335765500022
View details for PubMedID 24768691
Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus.
Proceedings of the National Academy of Sciences of the United States of America
2013; 110 (35): 14456-14461
In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFβ/BMP signaling, in which low levels of TGFβ/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/β-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.
View details for DOI 10.1073/pnas.1307376110
View details for PubMedID 23940359
View details for PubMedCentralID PMC3761577
- Special issue on inner ear development and regeneration HEARING RESEARCH 2013; 297: 1-2
Tympanic border cells are Wnt-responsive and can act as progenitors for postnatal mouse cochlear cells
2013; 140 (6): 1196-1206
Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.
View details for DOI 10.1242/dev.087528
View details for Web of Science ID 000315445800006
View details for PubMedID 23444352
View details for PubMedCentralID PMC3585657
A Novel Ion Channel Formed by Interaction of TRPML3 with TRPV5
2013; 8 (2)
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.
View details for DOI 10.1371/journal.pone.0058174
View details for Web of Science ID 000315524900242
View details for PubMedID 23469151
FCHSD1 and FCHSD2 Are Expressed in Hair Cell Stereocilia and Cuticular Plate and Regulate Actin Polymerization In Vitro
2013; 8 (2)
Mammalian FCHSD1 and FCHSD2 are homologous proteins containing an amino-terminal F-BAR domain and two SH3 domains near their carboxyl-termini. We report here that FCHSD1 and FCHSD2 are expressed in mouse cochlear sensory hair cells. FCHSD1 mainly localizes to the cuticular plate, whereas FCHSD2 mainly localizes along the stereocilia in a punctuate pattern. Nervous Wreck (Nwk), the Drosophila ortholog of FCHSD1 and FCHSD2, has been shown to bind Wsp and play an important role in F-actin assembly. We show that, like its Drosophila counterpart, FCHSD2 interacts with WASP and N-WASP, the mammalian orthologs of Drosophila Wsp, and stimulates F-actin assembly in vitro. In contrast, FCHSD1 doesn't bind WASP or N-WASP, and can't stimulate F-actin assembly when tested in vitro. We found, however, that FCHSD1 binds via its F-BAR domain to the SH3 domain of Sorting Nexin 9 (SNX9), a well characterized BAR protein that has been shown to promote WASP-Arp2/3-dependent F-actin polymerization. FCHSD1 greatly enhances SNX9's WASP-Arp2/3-dependent F-actin polymerization activity. In hair cells, SNX9 was detected in the cuticular plate, where it colocalizes with FCHSD1. Our results suggest that FCHSD1 and FCHSD2 could modulate F-actin assembly or maintenance in hair cell stereocilia and cuticular plate.
View details for DOI 10.1371/journal.pone.0056516
View details for Web of Science ID 000315184200089
View details for PubMedID 23437151
A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.
2013; 8 (6)
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.
View details for DOI 10.1371/journal.pone.0066026
View details for PubMedID 23750277
View details for PubMedCentralID PMC3672136
- Regenerative Medicine for the Special Senses: Restoring the Inputs JOURNAL OF NEUROSCIENCE 2012; 32 (41): 14053-14057
- Constitutive Activity of TRPML2 and TRPML3 Channels versus Activation by Low Extracellular Sodium and Small Molecules JOURNAL OF BIOLOGICAL CHEMISTRY 2012; 287 (27): 22701-22708
Oriented collagen as a potential cochlear implant electrode surface coating to achieve directed neurite outgrowth
EUROPEAN ARCHIVES OF OTO-RHINO-LARYNGOLOGY
2012; 269 (4): 1111-1116
In patients with severe to profound hearing loss, cochlear implants (CIs) are currently the only therapeutic option when the amplification with conventional hearing aids does no longer lead to a useful hearing experience. Despite its great success, there are patients in which benefit from these devices is rather limited. One reason may be a poor neuron-device interaction, where the electric fields generated by the electrode array excite a wide range of tonotopically organized spiral ganglion neurons at the cost of spatial resolution. Coating of CI electrodes to provide a welcoming environment combined with suitable surface chemistry (e.g. with neurotrophic factors) has been suggested to create a closer bioelectrical interface between the electrode array and the target tissue, which might lead to better spatial resolution, better frequency discrimination, and ultimately may improve speech perception in patients. Here we investigate the use of a collagen surface with a cholesteric banding structure, whose orientation can be systemically controlled as a guiding structure for neurite outgrowth. We demonstrate that spiral ganglion neurons survive on collagen-coated surfaces and display a directed neurite growth influenced by the direction of collagen fibril deposition. The majority of neurites grow parallel to the orientation direction of the collagen. We suggest collagen coating as a possible future option in CI technology to direct neurite outgrowth and improve hearing results for affected patients.
View details for DOI 10.1007/s00405-011-1775-8
View details for Web of Science ID 000301978000007
View details for PubMedID 21952794
Concise Review: Inner Ear Stem Cells-An Oxymoron, but Why?
2012; 30 (1): 69-74
Hearing loss, caused by irreversible loss of cochlear sensory hair cells, affects millions of patients worldwide. In this concise review, we examine the conundrum of inner ear stem cells, which obviously are present in the inner ear sensory epithelia of nonmammalian vertebrates, giving these ears the ability to functionally recover even from repetitive ototoxic insults. Despite the inability of the mammalian inner ear to regenerate lost hair cells, there is evidence for cells with regenerative capacity because stem cells can be isolated from vestibular sensory epithelia and from the neonatal cochlea. Challenges and recent progress toward identification of the intrinsic and extrinsic signaling pathways that could be used to re-establish stemness in the mammalian organ of Corti are discussed.
View details for DOI 10.1002/stem.785
View details for Web of Science ID 000298598400012
View details for PubMedID 22102534
Serial Analysis of Gene Expression in the Chicken Otocyst
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2011; 12 (6): 697-710
The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few immigrating neural crest-derived cells. The otocyst plays a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory and prosensory domains, giving rise to individual vestibular and auditory organs and their associated ganglia. The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known. The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method. Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors. We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as SOX4, SOX5, SOX7, SOX8, SOX11, and SOX18, which previously were not known to be expressed in the developing inner ear. Despite its limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies.
View details for DOI 10.1007/s10162-011-0286-z
View details for Web of Science ID 000297135900004
View details for PubMedID 21853378
Gpr126 is essential for peripheral nerve development and myelination in mammals
2011; 138 (13): 2673-2680
In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.
View details for DOI 10.1242/dev.062224
View details for Web of Science ID 000291348700005
View details for PubMedID 21613327
Intrinsic regenerative potential of murine cochlear supporting cells
The lack of cochlear regenerative potential is the main cause for the permanence of hearing loss. Albeit quiescent in vivo, dissociated non-sensory cells from the neonatal cochlea proliferate and show ability to generate hair cell-like cells in vitro. Only a few non-sensory cell-derived colonies, however, give rise to hair cell-like cells, suggesting that sensory progenitor cells are a subpopulation of proliferating non-sensory cells. Here we purify from the neonatal mouse cochlea four different non-sensory cell populations by fluorescence-activated cell sorting (FACS). All four populations displayed proliferative potential, but only lesser epithelial ridge and supporting cells robustly gave rise to hair cell marker-positive cells. These results suggest that cochlear supporting cells and cells of the lesser epithelial ridge show robust potential to de-differentiate into prosensory cells that proliferate and undergo differentiation in similar fashion to native prosensory cells of the developing inner ear.
View details for DOI 10.1038/srep00026
View details for Web of Science ID 000296046900002
View details for PubMedID 22355545
View details for PubMedCentralID PMC3216513
Genetic Inactivation of Trpml3 Does Not Lead to Hearing and Vestibular Impairment in Mice
2010; 5 (12)
TRPML3, a member of the transient receptor potential (TRP) family, is an inwardly rectifying, non-selective Ca2+-permeable cation channel that is regulated by extracytosolic Na+ and H+ and can be activated by a variety of small molecules. The severe auditory and vestibular phenotype of the TRPML3(A419P) varitint-waddler mutation made this protein particularly interesting for inner ear biology. To elucidate the physiological role of murine TRPML3, we conditionally inactivated Trpml3 in mice. Surprisingly, lack of functional TRPML3 did not lead to circling behavior, balance impairment or hearing loss.
View details for DOI 10.1371/journal.pone.0014317
View details for Web of Science ID 000285246900022
View details for PubMedID 21179200
View details for PubMedCentralID PMC3001452
Characterization of stem cells derived from the neonatal auditory sensory epithelium
2010; 58 (11): 1056-?
In contrast to regenerating hair cell-bearing organs of nonmammalian vertebrates the adult mammalian organ of Corti appears to have lost its ability to maintain stem cells. The result is a lack of regenerative ability and irreversible hearing loss following auditory hair cell death. Unexpectedly, the neonatal auditory sensory epithelium has recently been shown to harbor cells with stem cell features. The origin of these cells within the cochlea's sensory epithelium is unknown.We applied a modified neurosphere assay to identify stem cells within distinct subregions of the neonatal mouse auditory sensory epithelium. Sphere cells were characterized by multiple markers and morphologic techniques.Our data reveal that both the greater and the lesser epithelial ridge contribute to the sphere-forming stem cell population derived from the auditory sensory epithelium. These self-renewing sphere cells express a variety of markers for neural and otic progenitor cells and mature inner ear cell types.Stem cells can be isolated from specific regions of the auditory sensory epithelium. The distinct features of these cells imply a potential application in the development of a cell replacement therapy to regenerate the damaged sensory epithelium.
View details for DOI 10.1007/s00106-010-2155-1
View details for Web of Science ID 000284254700001
View details for PubMedID 20632158
A helix-breaking mutation in the epithelial Ca2+ channel TRPV5 leads to reduced Ca2+-dependent inactivation
2010; 48 (5): 275-287
TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.
View details for DOI 10.1016/j.ceca.2010.09.007
View details for Web of Science ID 000285537200005
View details for PubMedID 21035851
View details for PubMedCentralID PMC3780571
PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23
BMC CELL BIOLOGY
The atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown.PIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of cadherin 23. By binding to cadherin 23, PIST retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and harmonin, PIST was detected in mouse inner ear sensory hair cells.PIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1 and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST's retention. Our finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate the plasma membrane expression of cadherin 23.
View details for DOI 10.1186/1471-2121-11-80
View details for Web of Science ID 000283656300001
View details for PubMedID 20958966
Curing hearing loss: Patient expectations, health care practitioners, and basic science
19th Annual ASHA/NIH/NIDCD Research Symposium on Neural Regeneration and Communication Processes
ELSEVIER SCIENCE INC. 2010: 311–18
Millions of patients are debilitated by hearing loss, mainly caused by degeneration of sensory hair cells in the cochlea. The underlying reasons for hair cell loss are highly diverse, ranging from genetic disposition, drug side effects, traumatic noise exposure, to the effects of aging. Whereas modern hearing aids offer some relief of the symptoms of mild hearing loss, the only viable option for patients suffering from profound hearing loss is the cochlear implant. Despite their successes, hearing aids and cochlear implants are not perfect. Particularly frequency discrimination and performance in noisy environments and general efficacy of the devises vary among individual patients. The advent of regenerative medicine, the publicity of stem cells and gene therapy, and recent scientific achievements in inner ear cell regeneration have generated an emerging spirit of optimism among scientists, health care practitioners, and patients. In this review, we place the different points of view of these three groups in perspective with the goal of providing an assessment of patient expectations, health care reality, and potential future treatment options for hearing disorders.(1) Readers will be encouraged to put themselves in the position of a hearing impaired patient or family member of a hearing impaired person. (2) Readers will be able to explain why diagnosis of the underlying pathology of hearing loss is difficult. (3) Readers will be able to list the main directions of current research aimed to cure hearing loss. (4) Readers will be able to understand the different viewpoints of patients and their relatives, health care providers, and scientists with respect to finding novel treatments for hearing loss.
View details for DOI 10.1016/j.jcomdis.2010.04.002
View details for Web of Science ID 000279199700006
View details for PubMedID 20434163
Mechanosensitive Hair Cell-like Cells from Embryonic and Induced Pluripotent Stem Cells
2010; 141 (4): 704-716
Mechanosensitive sensory hair cells are the linchpin of our senses of hearing and balance. The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the permanence of hearing loss and certain balance disorders. Here, we present a stepwise guidance protocol starting with mouse embryonic stem and induced pluripotent stem cells, which were directed toward becoming ectoderm capable of responding to otic-inducing growth factors. The resulting otic progenitor cells were subjected to varying differentiation conditions, one of which promoted the organization of the cells into epithelial clusters displaying hair cell-like cells with stereociliary bundles. Bundle-bearing cells in these clusters responded to mechanical stimulation with currents that were reminiscent of immature hair cell transduction currents.
View details for DOI 10.1016/j.cell.2010.03.035
View details for Web of Science ID 000277623600022
View details for PubMedID 20478259
Small Molecule Activators of TRPML3
CHEMISTRY & BIOLOGY
2010; 17 (2): 135-148
We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel, which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na(+)]. This synergism revealed the existence of distinct and cooperative activation mechanisms and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed the absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are nonresponsive to the activators identified in this screen.
View details for DOI 10.1016/j.chembiol.2009.12.016
View details for Web of Science ID 000275897400008
View details for PubMedID 20189104
Twinfilin 2 Regulates Actin Filament Lengths in Cochlear Stereocilia
JOURNAL OF NEUROSCIENCE
2009; 29 (48): 15083-15088
Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50-100 actin-filled stereocilia, which are organized in three rows in a staircase manner. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day 5 (P5) onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.
View details for DOI 10.1523/JNEUROSCI.2782-09.2009
View details for Web of Science ID 000272361700007
View details for PubMedID 19955359
The tissue-specific expression of TRPML2 (MCOLN-2) gene is influenced by the presence of TRPML1
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
2009; 459 (1): 79-91
Mucolipidosis type IV is a lysosomal storage disorder caused by the loss or dysfunction of the mucolipin-1 (TRPML1) protein. It has been suggested that TRPML2 could genetically compensate (i.e., become upregulated) for the loss of TRPML1. We thus investigated this possibility by first studying the expression pattern of mouse TRPML2 and its basic channel properties using the varitint-waddler (Va) model. Here, we confirmed the presence of long variant TRPML2 (TRPML2lv) and short variant (TRPML2sv) isoforms. We showed for the first time that, heterologously expressed, TRPML2lv-Va is an active, inwardly rectifying channel. Secondly, we quantitatively measured TRPML2 and TRPML3 mRNA expressions in TRPML1-/- null and wild-type (Wt) mice. In wild-type mice, the TRPML2lv transcripts were very low while TRPML2sv and TRPML3 transcripts have predominant expressions in lymphoid and kidney organs. Significant reductions of TRPML2sv, but not TRPML2lv or TRPML3 transcripts, were observed in lymphoid and kidney organs of TRPML1-/- mice. RNA interference of endogenous human TRPML1 in HEK-293 cells produced a comparable decrease of human TRPML2 transcript levels that can be restored by overexpression of human TRPML1. Conversely, significant upregulation of TRPML2sv transcripts was observed when primary mouse lymphoid cells were treated with nicotinic acid adenine dinucleotide phosphate, or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline sulfonamide, both known activators of TRPML1. In conclusion, our results indicate that TRPML2 is unlikely to compensate for the loss of TRPML1 in lymphoid or kidney organs and that TRPML1 appears to play a novel role in the tissue-specific transcriptional regulation of TRPML2.
View details for DOI 10.1007/s00424-009-0716-5
View details for Web of Science ID 000271028700008
View details for PubMedID 19763610
Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells
Neural differentiation of embryonic stem (ES) cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming.Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of Oct4 and NANOG and increased expression of nestin. ES cells containing a GFP gene under the control of the Sox1 regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents.Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.
View details for DOI 10.1186/1471-2202-10-122
View details for Web of Science ID 000271323100002
View details for PubMedID 19778451
View details for PubMedCentralID PMC2761926
Quo vadis, hair cell regeneration?
2009; 12 (6): 679-685
Hearing loss is a global health problem with profound socioeconomic impact. We contend that acquired hearing loss is mainly a modern disorder caused by man-made noise and modern drugs, among other causes. These factors, combined with increasing lifespan, have exposed a deficit in cochlear self-regeneration that was irrelevant for most of mammalian evolution. Nevertheless, the mammalian cochlea has evolved from phylogenetically older structures, which do have the capacity for self-repair. Moreover, nonmammalian vertebrates can regenerate auditory hair cells that restore sensory function. We will offer a critical perspective on recent advances in stem cell biology, gene therapy, cell cycle regulation and pharmacotherapeutics to define and validate regenerative medical interventions for mammalian hair cell loss. Although these advances are promising, we are only beginning to fully appreciate the complexity of the many challenges that lie ahead.
View details for DOI 10.1038/nn.2311
View details for Web of Science ID 000266380900007
View details for PubMedID 19471265
Stem/Progenitor Cells Derived from the Cochlear Sensory Epithelium Give Rise to Spheres with Distinct Morphologies and Features
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2009; 10 (2): 173-190
Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair cell-like cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.
View details for DOI 10.1007/s10162-009-0161-3
View details for Web of Science ID 000265568200003
View details for PubMedID 19247714
Life and Death of Sensory Hair Cells Expressing Constitutively Active TRPML3
JOURNAL OF BIOLOGICAL CHEMISTRY
2009; 284 (20): 13823-13831
The varitint-waddler mutation A419P renders TRPML3 constitutively active, resulting in cationic overload, particularly in sustained influx of Ca(2+). TRPML3 is expressed by inner ear sensory hair cells, and we were intrigued by the fact that hair cells are able to cope with expressing the TRPML3(A419P) isoform for weeks before they ultimately die. We hypothesized that the survival of varitint-waddler hair cells is linked to their ability to deal with Ca(2+) loads due to the abundance of plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2 significantly reduced [Ca(2+)](i) increase and apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform of PMCA2, operating at 30% efficacy, showed a significantly decreased ability to rescue the Ca(2+) loading of cells expressing TRPML3(A419P). When we combined mice heterozygous for the varitint-waddler mutant allele with mice heterozygous for the deaf-waddler mutant allele, we found severe hair bundle defects as well as increased hair cell loss compared with mice heterozygous for each mutant allele alone. Furthermore, 3-week-old double mutant mice lacked auditory brainstem responses, which were present in their respective littermates containing single mutant alleles. Likewise, heterozygous double mutant mice exhibited severe circling behavior, which was not observed in mice heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a molecular rationale for the delayed hair cell loss in varitint-waddler mice. They also show that hair cells are able to survive for weeks with sustained Ca(2+) loading, which implies that Ca(2+) loading is an unlikely primary cause of hair cell death in ototoxic stress situations.
View details for DOI 10.1074/jbc.M809045200
View details for Web of Science ID 000265877300059
View details for PubMedID 19299509
Rethinking How Hearing Happens
2009; 62 (3): 305-307
Inner ear hair cells convert hair bundle deflection into mechanical force sensed by ion channels via extracellular tip links between adjacent stereocilia. In this Neuron issue, Grillet and colleagues show the protein harmonin mechanically reinforces tip link upper insertion sites. Harmonin loss at this site reduces mechanotransduction kinetics and sensitivity.
View details for DOI 10.1016/j.neuron.2009.04.019
View details for Web of Science ID 000266146100001
View details for PubMedID 19447085
Isolation of sphere-forming stem cells from the mouse inner ear.
Methods in molecular biology (Clifton, N.J.)
2009; 493: 141-162
The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.
View details for DOI 10.1007/978-1-59745-523-7_9
View details for PubMedID 18839346
Diverse Expression Patterns of LIM-Homeodomain Transcription Factors (LIM-HDs) in Mammalian Inner Ear Development
2008; 237 (11): 3305-3312
LIM-homeodomain transcription factors (LIM-HDs) are essential in tissue patterning and differentiation. But their expression patterns in the inner ear are largely unknown. Here we report on a study of twelve LIM-HDs, by their tempo-spatial patterns that imply distinct yet overlapping roles, in the developing mouse inner ear. Expression of Lmx1a and Isl1 begins in the otocyst stage, with Lmx1a exclusively in the non-sensory and Isl1 in the prosensory epithelia. The second wave of expression at E12.5 includes Lhx3, 5, 9, Isl2, and Lmx1b in the differentiating sensory epithelia with cellular specificities. With the exception of Lmx1a and Lhx3, all LIM-HDs are expressed in ganglion neurons. Expression of multiple LIM-HDs within a cell type suggests their redundant function.
View details for DOI 10.1002/dvdy.21735
View details for Web of Science ID 000260930400020
View details for PubMedID 18942141
MAGI-1, A Candidate Stereociliary Scaffolding Protein, Associates with the Tip-Link Component Cadherin 23
JOURNAL OF NEUROSCIENCE
2008; 28 (44): 11269-11276
Inner ear hair-cell mechanoelectrical transduction is mediated by a largely unidentified multiprotein complex associated with the stereociliary tips of hair bundles. One identified component of tip links, which are the extracellular filamentous connectors implicated in gating the mechanoelectrical transduction channels, is the transmembrane protein cadherin 23 (Cdh23), more specifically, the hair- cell-specific Cdh23(+68) splice variant. Using the intracellular domain of Cdh23(+68) as bait, we identified in a cochlear cDNA library MAGI-1, a MAGUK (membrane-associated guanylate kinase) protein. MAGI-1 binds via its PDZ4 domain to a C-terminal PDZ-binding site on Cdh23. MAGI-1 immunoreactivity was detectable throughout neonatal stereocilia in a distribution similar to that of Cdh23. As development proceeded, MAGI-1 occurred in a punctate staining pattern on stereocilia, which was maintained into adulthood. Previous reports suggest that Cdh23 interacts via an internal PDZ-binding site with the PDZ1 domain of the stereociliary protein harmonin, and potentially via a weaker binding of its C terminus with harmonin's PDZ2 domain. We propose that MAGI-1 has the ability to replace harmonin's PDZ2 binding at Cdh23's C terminus. Moreover, the strong interaction between PDZ1 of harmonin and Cdh23 is interrupted by a 35 aa insertion in the hair-cell-specific Cdh23(+68) splice variant, which puts forward MAGI-1 as an attractive candidate for an intracellular scaffolding partner of this tip-link protein. Our results consequently support a role of MAGI-1 in the tip-link complex, where it could provide a sturdy connection with the cytoskeleton and with other components of the mechanoelectrical transduction complex.
View details for DOI 10.1523/JNEUROSCI.3833-08.2008
View details for Web of Science ID 000260502400018
View details for PubMedID 18971469
Transient receptor potential vanilloid 4 deficiency suppresses unloading-induced bone loss
JOURNAL OF CELLULAR PHYSIOLOGY
2008; 216 (1): 47-53
Mechanosensing is one of the crucial components of the biological events. In bone, as observed in unloading-induced osteoporosis in bed ridden patients, mechanical stress determines the levels of bone mass. Many molecules have been suggested to be involved in sensing mechanical stress in bone, while the full pathways for this event has not yet been identified. We examined the role of TRPV4 in unloading-induced bone loss. Hind limb unloading induced osteopenia in wild-type mice. In contrast, TRPV4 deficiency suppressed such unloading-induced bone loss. As underlying mechanism for such effects, TRPV4 deficiency suppressed unloading-induced reduction in the levels of mineral apposition rate and bone formation rate. In these mice, unloading-induced increase in the number of osteoclasts in the primary trabecular bone was suppressed by TRPV4 deficiency. Unloading-induced reduction in the longitudinal length of primary trabecular bone was also suppressed by TRPV4 deficiency. TRPV4 protein is expressed in both osteoblasts and osteoclasts. These results indicated that TRPV4 plays a critical role in unloading-induced bone loss.
View details for DOI 10.1002/jcp.21374
View details for Web of Science ID 000256600800006
View details for PubMedID 18264976
Stimulus-specific modulation of the cation channel TRPV4 by PACSIN 3
JOURNAL OF BIOLOGICAL CHEMISTRY
2008; 283 (10): 6272-6280
TRPV4, a member of the vanilloid subfamily of the transient receptor potential (TRP) channels, is activated by a variety of stimuli, including cell swelling, moderate heat, and chemical compounds such as synthetic 4alpha-phorbol esters. TRPV4 displays a widespread expression in various cells and tissues and has been implicated in diverse physiological processes, including osmotic homeostasis, thermo- and mechanosensation, vasorelaxation, tuning of neuronal excitability, and bladder voiding. The mechanisms that regulate TRPV4 in these different physiological settings are currently poorly understood. We have recently shown that the relative amount of TRPV4 in the plasma membrane is enhanced by interaction with the SH3 domain of PACSIN 3, a member of the PACSIN family of proteins involved in synaptic vesicular membrane trafficking and endocytosis. Here we demonstrate that PACSIN 3 strongly inhibits the basal activity of TRPV4 and its activation by cell swelling and heat, while leaving channel gating induced by the synthetic ligand 4alpha-phorbol 12,13-didecanoate unaffected. A single proline mutation in the SH3 domain of PACSIN 3 abolishes its inhibitory effect on TRPV4, indicating that PACSIN 3 must bind to the channel to modulate its function. In line herewith, mutations at specific proline residues in the N terminus of TRPV4 abolish binding of PACSIN 3 and render the channel insensitive to PACSIN 3-induced inhibition. Taken together, these data suggest that PACSIN 3 acts as an auxiliary protein of TRPV4 channel that not only affects the channel's subcellular localization but also modulates its function in a stimulus-specific manner.
View details for DOI 10.1074/jbc.M706386200
View details for Web of Science ID 000253779500034
View details for PubMedID 18174177
Stem-cell-based approaches for treating inner ear diseases
2008; 56 (1): 21-26
The capacity of stem cells to regenerate lost tissue cells has gained recognition among physicians. Despite the successful use of blood stem cells for treating blood cancers, other stem cell types have not yet been widely introduced into clinical practice. Therapy options involving stem cells for inner ear diseases consequently have not been implemented. Nonetheless, several reports have recently been published describing the generation of morphologically and immunologically distinctive inner ear cell types-such as hair cells, supporting cells, and spiral ganglion neurons-from stem cells. Although promising, all of these studies still lack functional results regarding hearing restoration or vestibular function.
View details for DOI 10.1007/s00106-007-1652-3
View details for Web of Science ID 000252715900005
View details for PubMedID 18231694
Emerging Strategies for Restoring the Cochlea
Auditory Trauma, Protection, and Repair
View details for DOI 10.1007/978-0-387-72561-1_11
A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (49): 19583-19588
Homozygote varitint-waddler (Va) mice, expressing a mutant isoform (A419P) of TRPML3 (mucolipin 3), are profoundly deaf and display vestibular and pigmentation deficiencies, sterility, and perinatal lethality. Here we show that the varitint-waddler isoform of TRPML3 carrying an A419P mutation represents a constitutively active cation channel that can also be identified in native varitint-waddler hair cells as a distinct inwardly rectifying current. We hypothesize that the constitutive activation of TRPML3 occurs as a result of a helix-breaking proline substitution in transmembrane-spanning domain 5 (TM5). A proline substitution scan demonstrated that the inner third of TRPML3's TM5 is highly susceptible to proline-based kinks. Proline substitutions in TM5 of other TRP channels revealed that TRPML1, TRPML2, TRPV5, and TRPV6 display a similar susceptibility at comparable positions, whereas other TRP channels were not affected. We conclude that the molecular basis for deafness in the varitint-waddler mouse is the result of hair cell death caused by constitutive TRPML3 activity. To our knowledge, our study provides the first direct mechanistic link of a mutation in a TRP ion channel with mammalian hearing loss.
View details for DOI 10.1073/pnas.0709846104
View details for Web of Science ID 000251525800074
View details for PubMedID 18048323
Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
2007; 293 (5): F1699-F1713
Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4alpha-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.
View details for DOI 10.1152/ajprenal.00462.2006
View details for Web of Science ID 000250548000032
View details for PubMedID 17699550
LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells
BMC DEVELOPMENTAL BIOLOGY
Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation.LIF-treatment led to a dose-dependent increase of the number of neurons and glial cells in cultures of sphere-derived cells. Based on the detection of developmental and progenitor cell markers that are maintained in LIF-treated cultures and the increase of cycling nestin-positive progenitors, we propose that LIF maintains a pool of neural progenitor cells. We further provide evidence that LIF increases the number of nestin-positive progenitor cells directly in a cell cycle-independent fashion, which we interpret as an acceleration of neurogenesis in sphere-derived progenitors. This effect is further enhanced by an anti-apoptotic action of LIF. Finally, LIF and the neurotrophins BDNF and NT3 additively promote survival of stem cell-derived neurons.Our results implicate LIF as a powerful tool to control neural differentiation and maintenance of stem cell-derived murine spiral ganglion neuron precursors. This finding could be relevant in cell replacement studies with animal models featuring spiral ganglion neuron degeneration. The additive effect of the combination of LIF and BDNF/NT3 on stem cell-derived neuronal survival is similar to their effect on primary spiral ganglion neurons, which puts forward spiral ganglion-derived neurospheres as an in vitro model system to study aspects of auditory neuron development.
View details for DOI 10.1186/1471-213X-7-112
View details for Web of Science ID 000251607800001
View details for PubMedID 17935626
TRP channels as candidates for hearing and balance abnormalities in vertebrates
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE
2007; 1772 (8): 1022-1027
In this review, we summarize the potential functional roles of transient receptor potential (TRP) channels in the vertebrate inner ear. The history of TRP channels in hearing and balance is characterized at great length by the hunt for the elusive transduction channel of sensory hair cells. Such pursuit has not resulted in unequivocal identification of the transduction channel, but nevertheless revealed a number of candidates, such as TRPV4, TRPN1, TRPA1, and TRPML3. Much of the circumstantial evidence indicates that these TRP channels potentially play significant roles in inner ear physiology. Based on mutations in the corresponding mouse genes, TRPV4 and TRPML3 are possible candidates for human hearing, and potentially also balance disorders. We further discuss the role of the invertebrate TRP channels Nanchung, Inactive, and TRPN1 and how the functional analysis of these channels provides a link to vertebrate hearing and balance. In summary, only a few TRP channels have been analyzed thus far for a prospective role in the inner ear, and this makes the search for additional TRPs associated with inner ear function quite a tantalizing endeavor.
View details for DOI 10.1016/j.bbadis.2007.01.002
View details for Web of Science ID 000249187400021
View details for PubMedID 17300924
Robust postmortem survival of murine vestibular and cochlear stem cells
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2007; 8 (2): 194-204
Potential treatment strategies of neurodegenerative and other diseases with stem cells derived from nonembryonic tissues are much less subjected to ethical criticism than embryonic stem cell-based approaches. Here we report the isolation of inner ear stem cells, which may be useful in cell replacement therapies for hearing loss, after protracted postmortem intervals. We found that neonatal murine inner ear tissues, including vestibular and cochlear sensory epithelia, display remarkably robust cellular survival, even 10 days postmortem. Similarly, isolation of sphere-forming stem cells was possible up to 10 days postmortem. We detected no difference in the proliferation and differentiation potential between stem cells isolated directly after death and up to 5 days postmortem. At longer postmortem intervals, we observed that the potency of sphere-derived cells to spontaneously differentiate into mature cell types diminishes prior to the cells losing their potential for self-renewal. Three-week-old mice also displayed sphere-forming stem cells in all inner ear tissues investigated up to 5 days postmortem. In summary, our results demonstrate that postmortem murine inner ear tissue is suited for isolation of stem cells.
View details for DOI 10.1007/s10162-007-0079-6
View details for Web of Science ID 000246339800004
View details for PubMedID 17334849
The potential role of endogenous stem cells in regeneration of the inner ear
Inner Ear Biology Workshop Symposium on Terminal Differentiation - A Challenge in Regeneration
ELSEVIER SCIENCE BV. 2007: 48–52
Stem cells in various mammalian organs retain the capacity to renew themselves and may be able to restore damaged tissue. Their existence has been proven by genetic tracer studies that demonstrate their differentiation into multiple tissue types and by their ability to self-renew through proliferation. Stem cells from the adult nervous system proliferate to form clonal floating colonies called spheres in vitro, and recent studies have demonstrated sphere formation by cells in the cochlea in addition to the vestibular system and the auditory ganglia, indicating that these tissues contain cells with stem cell properties. The presence of stem cells in the inner ear raises the hope of regeneration of mammalian inner ear cells but is difficult to correlate with the lack of spontaneous regeneration seen in the inner ear after tissue damage. Loss of stem cells postnatally in the cochlea may correlate with the loss of regenerative capacity and may limit our ability to stimulate regeneration. Retention of sphere forming ability in adult vestibular tissues suggests that the limited capacity for repair may be attributed to the continued presence of progenitor cells. Future strategies for regeneration must consider the distribution of endogenous stem cells in the inner ear and whether the tissue retains cells with the capacity for regeneration.
View details for DOI 10.1016/j.heares.2006.12.015
View details for Web of Science ID 000246630900008
View details for PubMedID 17321086
Differential distribution of stem cells in the auditory and vestibular organs of the inner ear
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2007; 8 (1): 18-31
The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.
View details for DOI 10.1007/s10162-006-0058-3
View details for Web of Science ID 000244389200003
View details for PubMedID 17171473
View details for PubMedCentralID PMC2538418
Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells
MOLECULAR AND CELLULAR NEUROSCIENCE
2007; 34 (1): 59-68
Stem cells have been demonstrated in the inner ear but they do not spontaneously divide to replace damaged sensory cells. Mesenchymal stem cells (MSC) from bone marrow have been reported to differentiate into multiple lineages including neurons, and we therefore asked whether MSCs could generate sensory cells. Overexpression of the prosensory transcription factor, Math1, in sensory epithelial precursor cells induced expression of myosin VIIa, espin, Brn3c, p27Kip, and jagged2, indicating differentiation to inner ear sensory cells. Some of the cells displayed F-actin positive protrusions in the morphology characteristic of hair cell stereociliary bundles. Hair cell markers were also induced by culture of mouse MSC-derived cells in contact with embryonic chick inner ear cells, and this induction was not due to a cell fusion event, because the chick hair cells could be identified with a chick-specific antibody and chick and mouse antigens were never found in the same cell.
View details for DOI 10.1016/j.mcn.2006.10.003
View details for Web of Science ID 000243598200007
View details for PubMedID 17113786
Engraftment and differentiation of embryonic stem cell-derived neural progenitor cells in the cochlear nerve trunk: Growth of processes into the organ of corti
JOURNAL OF NEUROBIOLOGY
2006; 66 (13): 1489-1500
Hearing loss in mammals is irreversible because cochlear neurons and hair cells do not regenerate. To determine whether we could replace neurons lost to primary neuronal degeneration, we injected EYFP-expressing embryonic stem cell-derived mouse neural progenitor cells into the cochlear nerve trunk in immunosuppressed animals 1 week after destroying the cochlear nerve (spiral ganglion) cells while leaving hair cells intact by ouabain application to the round window at the base of the cochlea in gerbils. At 3 days post transplantation, small grafts were seen that expressed endogenous EYFP and could be immunolabeled for neuron-specific markers. Twelve days after transplantation, the grafts had neurons that extended processes from the nerve core toward the denervated organ of Corti. By 64-98 days, the grafts had sent out abundant processes that occupied a significant portion of the space formerly occupied by the cochlear nerve. The neurites grew in fasciculating bundles projecting through Rosenthal's canal, the former site of spiral ganglion cells, into the osseous spiral lamina and ultimately into the organ of Corti, where they contacted hair cells. Neuronal counts showed a significant increase in neuronal processes near the sensory epithelium, compared to animals that were denervated without subsequent stem cell transplantation. The regeneration of these neurons shows that neurons differentiated from stem cells have the capacity to grow to a specific target in an animal model of neuronal degeneration.
View details for DOI 10.1002/neu.20310
View details for Web of Science ID 000241903700008
View details for PubMedID 17013931
PACSINs bind to the TRPV4 cation channel - PACSIN 3 modulates the subcellular localization of TRPV4
JOURNAL OF BIOLOGICAL CHEMISTRY
2006; 281 (27): 18753-18762
TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mammalian PACSINs 1 and 3. The PACSINs are a protein family consisting of three members that have been implicated in synaptic vesicular membrane trafficking and regulation of dynamin-mediated endocytotic processes. In biochemical interaction assays we found that all three murine PACSIN isoforms can bind to the amino terminus of rodent TRPV4. No member of the PACSIN protein family was able to biochemically interact with TRPV1 and TRPV2. Co-expression of PACSIN 3, but not PACSINs 1 and 2, shifted the ratio of plasma membrane-associated versus cytosolic TRPV4 toward an apparent increase of plasma membrane-associated TRPV4 protein. A similar shift was also observable when we blocked dynamin-mediated endocytotic processes, suggesting that PACSIN 3 specifically affects the endocytosis of TRPV4, thereby modulating the subcellular localization of the ion channel. Mutational analysis shows that the interaction of the two proteins requires both a TRPV4-specific proline-rich domain upstream of the ankyrin repeats of the channel and the carboxyl-terminal Src homology 3 domain of PACSIN 3. Such a functional interaction could be important in cell types that show distribution of both proteins to the same subcellular regions such as renal tubule cells where the proteins are associated with the luminal plasma membrane.
View details for DOI 10.1074/jbc.M602452200
View details for Web of Science ID 000238687300056
View details for PubMedID 16627472
Reinnervation of hair cells by auditory neurons after selective removal of spiral ganglion neurons
JOURNAL OF NEUROBIOLOGY
2006; 66 (4): 319-331
Hearing loss can be caused by primary degeneration of spiral ganglion neurons or by secondary degeneration of these neurons after hair cell loss. The replacement of auditory neurons would be an important step in any attempt to restore auditory function in patients with damaged inner ear neurons or hair cells. Application of beta-bungarotoxin, a toxin derived from snake venom, to an explant of the cochlea eradicates spiral ganglion neurons while sparing the other cochlear cell types. The toxin was found to bind to the neurons and to cause apoptotic cell death without affecting hair cells or other inner ear cell types as indicated by TUNEL staining, and, thus, the toxin provides a highly specific means of deafferentation of hair cells. We therefore used the denervated organ of Corti for the study of neuronal regeneration and synaptogenesis with hair cells and found that spiral ganglion neurons obtained from the cochlea of an untreated newborn mouse reinnervated hair cells in the toxin-treated organ of Corti and expressed synaptic vesicle markers at points of contact with hair cells. These findings suggest that it may be possible to replace degenerated neurons by grafting new cells into the organ of Corti.
View details for DOI 10.1002/neu.20232
View details for Web of Science ID 000235587000001
View details for PubMedID 16408287
Sonic hedgehog promotes mouse inner ear progenitor cell proliferation and hair cell generation in vitro
2006; 17 (2): 121-124
Sonic hedgehog (Shh) signaling is essential for auditory cell fate determination and inner ear dorsal/ventral patterning during development. Here, we show that Shh accelerates inner ear progenitor cell proliferation, and the inhibitor of Shh signaling cyclopamine reduces mitotic growth of otocyst cells in vitro. The number of hair cells in cultures of inner ear progenitor cells that were treated with Shh was significantly higher than that in control cultures. When Shh signaling was blocked with cyclopamine, hair cell generation was largely inhibited. Our results suggest that Shh may be a regulator of inner ear progenitor cell growth and hair cell generation.
View details for Web of Science ID 000235349800003
View details for PubMedID 16407756
Generation of inner ear cell types from embryonic stem cells.
Methods in molecular biology (Clifton, N.J.)
2006; 330: 71-92
The senses of hearing and balance are mediated by hair cells located in the cochlea and in the vestibular organs of the vertebrate inner ear. Loss of hair cells and other cell types of the inner ear results in hearing and balance disorders that substantially diminish the quality of life. The irreversibility of hearing loss in mammals is caused by the inability of the cochlea to replace lost hair cells. No drugs are available that stimulate inner ear cell regeneration. We describe here protocols to generate inner ear progenitor cells from murine ES cells and to differentiate these progenitors into hair cells and potentially into other inner ear cell types. In addition, we provide a modification of the protocol describing culture conditions in which human ES cells express a similar set of inner ear markers. Inner ear progenitor cells, generated from ES cells, may be used for the development of cell replacement therapy for the diseased inner ear, for high-throughput drug screening, and for the study of inner ear development.
View details for PubMedID 16846017
The mechanosensitive nature of TRPV channels
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
2005; 451 (1): 193-203
Transient receptor potential vanilloid (TRPV) channels are widely expressed in both sensory and nonsensory cells. Whereas the channels display a broad diversity to activation by chemical and physical stimuli, activation by mechanical stimuli is common to many members of this group in both lower and higher organisms. Genetic screening in Caenorhabditis elegans has demonstrated an essential role for two TRPV channels in sensory neurons. OSM-9 and OCR-2, for example, are essential for both osmosensory and mechanosensory (nose-touch) behaviors. Likewise, two Drosophila TRPV channels, NAN and IAV, have been shown to be critical for hearing by the mechanosensitive chordotonal organs located in the fly's antennae. The mechanosensitive nature of the channels appears to be conserved in higher organisms for some TRPV channels. Two vertebrate channels, TRPV2 and TRPV4, are sensitive to hypotonic cell swelling, shear stress/fluid flow (TRPV4), and membrane stretch (TRPV2). In the osmosensing neurons of the hypothalamus (circumventricular organs), TRPV4 appears to function as an osmoreceptor, or part of an osmoreceptor complex, in control of vasopressin release, whereas in inner ear hair cells and vascular baroreceptors a mechanosensory role is suggestive, but not demonstrated. Finally, in many nonsensory cells expressing TRPV4, such as vascular endothelial cells and renal tubular epithelial cells, the channel exhibits well-developed local mechanosensory transduction processes where both cell swelling and shear stress/fluid flow lead to channel activation. Hence, many TRPV channels, or combinations of TRPV channels, display a mechanosensitive nature that underlies multiple mechanosensitive processes from worms to mammals.
View details for DOI 10.1007/s00424-005-1424-4
View details for Web of Science ID 000232372800023
View details for PubMedID 15909178
BMP4 signaling is involved in the generation of inner ear sensory epithelia
BMC DEVELOPMENTAL BIOLOGY
The robust expression of BMP4 in the incipient sensory organs of the inner ear suggests possible roles for this signaling protein during induction and development of auditory and vestibular sensory epithelia. Homozygous BMP4-/- animals die before the inner ear's sensory organs develop, which precludes determining the role of BMP4 in these organs with simple gene knockout experiments.Here we use a chicken otocyst culture system to perform quantitative studies on the development of inner ear cell types and show that hair cell and supporting cell generation is remarkably reduced when BMP signaling is blocked, either with its antagonist noggin or by using soluble BMP receptors. Conversely, we observed an increase in the number of hair cells when cultured otocysts were treated with exogenous BMP4. BMP4 treatment additionally prompted down-regulation of Pax-2 protein in proliferating sensory epithelial progenitors, leading to reduced progenitor cell proliferation.Our results implicate BMP4 in two events during chicken inner ear sensory epithelium formation: first, in inducing the switch from proliferative sensory epithelium progenitors to differentiating epithelial cells and secondly, in promoting the differentiation of hair cells within the developing sensory epithelia.
View details for DOI 10.1186/1471-213X-5-16
View details for Web of Science ID 000234278900001
View details for PubMedID 16107213
- Sound from silence NATURE MEDICINE 2005; 11 (3): 249-250
Identification of chicken transmembrane channel-like (TMC) genes: Expression analysis in the cochlea
2005; 132 (4): 1115-1122
Mutations of the human gene encoding transmembrane channel-like protein (TMC)1 cause dominant and recessive nonsyndromic hearing disorders, suggesting that this protein plays an important role in the inner ear. In this study, we cloned chicken Tmc2 (GgTmc2) from a cochlear cDNA library and we annotated four additional TMC family members: GgTmc1, GgTmc3, GgTmc6, and GgTmc7. All chicken TMCs possess the defining TMC signature motif and display high conservation of their genomic structure when compared with other vertebrate TMC genes. GgTmc1 is localized on the chicken sex chromosome Z at a locus that displays conserved synteny with the loci of mammalian orthologues residing on autosomes. In contrast, the locus of GgTmc2 does not exhibit conserved synteny with its mammalian orthologues. Because murine TMC1 and TMC2 are restrictively expressed in cochlear hair cells, we determined the expression of the chicken orthologues in the basilar papilla, the avian equivalent of the organ of Corti. While GgTmc2 was present throughout the basilar papilla and in other tissues, GgTmc1 transcript was detected specifically in the basal portion of the basilar papilla and was not detectable in any other tissue or organ studied. GgTmc3 and GgTmc6 were detectable in all organs analyzed. Antibody labeling revealed that GgTmc2 is predominantly associated with the lateral membranes of hair and supporting cells. The expression of GgTmc2 by both cell types was further confirmed by RT-PCR using isolated cells. This expression and subcellular localization of GgTmc2 is in agreement with the proposed potential role of this novel class of transmembrane proteins in ion transport.
View details for DOI 10.1016/j.neuroscience.2005.01.046
View details for Web of Science ID 000228986400022
View details for PubMedID 15857715
Islet-1 expression in the developing chicken inner ear
JOURNAL OF COMPARATIVE NEUROLOGY
2004; 477 (1): 1-10
The cell types of the inner ear originate from the otic placode, a thickened layer of ectoderm adjacent to the developing hindbrain. The placode invaginates and forms the otic pit, which pinches off as a small vesicle called the otocyst. Presumptive cochleovestibular neurons delaminate from the anterior ventral part of the otocyst and form the cochleovestibular ganglion of the inner ear. Here we show that the LIM/homeodomain protein islet-1 is expressed in cells of the ventral part of the otic placode and that this ventral expression is maintained at the otic pit and the otocyst stages. Auditory and vestibular neurons originate from this islet-1-positive zone of the otocyst, and these neurons maintain islet-1 expression until adulthood. We also demonstrate that islet-1 becomes up-regulated in the presumptive sensory epithelia of the inner ear in regions that are defined by the expression domains of BMP4. The up-regulation of islet-1 in developing inner ear hair and supporting cells is accompanied by down-regulation of Pax-2 in these cell types. Islet-1 expression in hair and supporting cells persists until early postnatal stages, when the transcriptional regulator is down-regulated in hair cells. Our data is consistent with a role for islet-1 in differentiating inner ear neurons and sensory epithelia cells, perhaps in the specification of cellular subtypes in conjunction with other LIM/homeodomain proteins.
View details for DOI 10.1002/cne.20190
View details for Web of Science ID 000223160400001
View details for PubMedID 15281076
Correlation of Pax-2 expression with cell proliferation in the developing chicken inner ear
JOURNAL OF NEUROBIOLOGY
2004; 60 (1): 61-70
In vertebrates, the paired-box transcription factor Pax-2 is one of the earliest markers of the developing inner ear and is robustly expressed in the otic placode and the otic vesicle. Mutations in the Pax-2 gene result in developmental defects of the vestibular and auditory apparatus. We set out to investigate whether regions of Pax-2 expression in the developing otic vesicle correlate with areas of cell proliferation or cell death, which would indicate a possible role of Pax-2 in these processes. Regionalized proliferation and local apoptosis are the principal mechanisms that lead to the complex morphogenesis of the highly compartmentalized inner ear starting from a simple vesicle. We found a high correlation of Pax-2 expression with proliferating cells in the walls of the early otic vesicle. Apoptotic cells were mostly localized outside of the Pax-2-expressing regions. At later stages, we found the highest intensity of proliferating and Pax-2-positive cells in areas of the developing sensory epithelia. When hair cells begin to differentiate, they maintain a lower level of Pax-2 expression than neighboring cells for a brief period, before they completely down-regulate expression of this transcription factor. We conclude that a significant proportion of proliferating cells in the developing otocyst express Pax-2, in particular in regions that include developing sensory patches. This implicates Pax-2 as a marker for proliferating hair and supporting cell progenitors. Furthermore, the likelihood that Pax-2-expressing cells in the otocyst die by apoptosis is much lower when compared with cells residing in Pax-2-negative regions.
View details for DOI 10.1002/neu.20013
View details for Web of Science ID 000222102000007
View details for PubMedID 15188273
Stem cells as therapy for hearing loss
TRENDS IN MOLECULAR MEDICINE
2004; 10 (7): 309-315
One of the greatest challenges in the treatment of inner-ear disorders is to find a cure for the hearing loss that is caused by the loss of cochlear hair cells or spiral ganglion neurons. The recent discovery of stem cells in the adult inner ear that are capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatment regimens. Here, we propose different approaches for using stem cells to regenerate the damaged inner ear and we describe the potential obstacles that translational approaches must overcome for the development of stem-cell-based cell-replacement therapies for the damaged inner ear.
View details for DOI 10.1016/j.molmed.2004.05.008
View details for Web of Science ID 000223020700004
View details for PubMedID 15242678
Absence of the RGS9 center dot G beta 5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (3): 1581-1584
Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.
View details for DOI 10.1074/jbc.C300456200
View details for Web of Science ID 000188005700003
View details for PubMedID 14625292
Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear
JOURNAL OF COMPARATIVE NEUROLOGY
2004; 468 (1): 125-134
The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.
View details for DOI 10.1002/cne.10944
View details for Web of Science ID 000186988300008
View details for PubMedID 14648695
The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo
JOURNAL OF NEUROSCIENCE
2003; 23 (32): 10175-10181
DEP (for Disheveled, EGL-10, Pleckstrin) homology domains are present in numerous signaling proteins, including many in the nervous system, but their function remains mostly elusive. We report that the DEP domain of a photoreceptor-specific signaling protein, RGS9 (for regulator of G-protein signaling 9), plays an essential role in RGS9 delivery to the intracellular compartment of its functioning, the rod outer segment. We generated a transgenic mouse in which RGS9 was replaced by its mutant lacking the DEP domain. We then used a combination of the quantitative technique of serial tangential sectioning-Western blotting with electrophysiological recordings to demonstrate that mutant RGS9 is expressed in rods in the normal amount but is completely excluded from the outer segments. The delivery of RGS9 to rod outer segments is likely to be mediated by the DEP domain interaction with a transmembrane protein, R9AP (for RGS9 anchoring protein), known to anchor RGS9 on the surface of photoreceptor membranes and to potentiate RGS9 catalytic activity. We show that both of these functions are also abolished as the result of the DEP domain deletion. These findings indicate that a novel function of the DEP domain is to target a signaling protein to a specific compartment of a highly polarized neuron. Interestingly, sequence analysis of R9AP reveals the presence of a conserved R-SNARE (for soluble N-ethylmaleimide-sensitive factor attachment protein receptor) motif and a predicted overall structural homology with SNARE proteins involved in vesicular trafficking and fusion. This presents the possibility that DEP domains might serve to target various DEP-containing proteins to the sites of their intracellular action via interactions with the members of extended SNARE protein family.
View details for Web of Science ID 000186536100001
View details for PubMedID 14614075
Generation of hair cells by stepwise differentiation of embryonic stem cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (23): 13495-13500
The increase in life expectancy is accompanied by the growing burden of chronic diseases. Hearing loss is perhaps the most prevalent of all chronic diseases. In addition to age-related hearing loss, a substantial number of cases of audiological impairment are either congenital in nature or acquired during childhood. The permanence of hearing loss is mainly due to the inability of the cochlear sensory epithelium to replace lost mechanoreceptor cells, or hair cells. Generation of hair cells from a renewable source of progenitors that can be transplanted into damaged inner ears is a principal requirement for potential cell replacement therapy in this organ. Here, we present an experimental protocol that enables us to routinely create inner ear progenitors from murine embryonic stem cells in vitro. These progenitors express a comprehensive set of marker genes that define the developing inner ear, in particular the organ's developing sensory patches. We further demonstrate that cells that express markers characteristic of hair cells differentiate from embryonic stem cell-derived progenitors. Finally, we show that these progenitors integrate into the developing inner ear at sites of epithelial injury and that integrated cells start expressing hair cell markers and display hair bundles when situated in cochlear or vestibular sensory epithelia in vivo.
View details for Web of Science ID 000186573700069
View details for PubMedID 14593207
Expression patterns of the RGS9-1 anchoring protein R9AP in the chicken and mouse suggest multiple roles in the nervous system
MOLECULAR AND CELLULAR NEUROSCIENCE
2003; 24 (3): 687-695
In retinal photoreceptors, the duration of G protein signalling is tightly regulated by the GTPase-activating protein RGS9-1. RGS9-1 is anchored to the disk membranes of photoreceptor outer segments by association with the membrane-spanning protein R9AP. Here we report the cloning of chicken R9AP from an inner ear cDNA library and the isolation of a murine R9AP cDNA from a retinal library. In the chicken, R9AP appears to be expressed in a variety of neuronal tissues, particularly in sensory cells including inner ear hair cells, photoreceptors, and dorsal root ganglion neurons. In the mouse, R9AP is detectable predominantly in photoreceptors, but it is also weakly expressed in other areas of the central nervous system. The expression of R9AP beyond photoreceptors led us to examine potential alternative roles for R9AP besides anchoring RGS9-1 and we found sequence homology and structural similarity of the protein with members of the SNARE protein family. Expression of chicken and mouse R9AP interfered with intracellular trafficking of an indicator protein in an in vitro assay, suggesting a more active role of the protein, possibly in targeting. GTPase-activating proteins to specific membranous compartments.
View details for DOI 10.1016/S1044-7431(03)00231-8
View details for Web of Science ID 000187117700014
View details for PubMedID 14664818
Expression of Frizzled genes in the developing chick eye
GENE EXPRESSION PATTERNS
2003; 3 (5): 659-662
Frizzleds are transmembrane receptors that can transduce signals dependent upon binding of Wnts, a large family of secreted glycoproteins homologous to the Drosophila wingless (wg) gene product and critical for a wide variety of normal and pathological developmental processes. In the nervous system, Wnts and Frizzleds play an important role in anterior-posterior patterning, cell fate decisions, proliferation, and synaptogenesis. However, little is known about the role of Frizzled signaling in the developing eye. We isolated cDNAs for ten chick Frizzleds and analyzed the spatial and temporal expression patterns during eye development in the chick embryo. Frizzled-1 to -9 are specifically expressed in the eye at various stages of development and show a complex and partially overlapping pattern of expression.
View details for DOI 10.1016/S1567-133X(03)00107-8
View details for Web of Science ID 000185582300016
View details for PubMedID 12972002
Pluripotent stem cells from the adult mouse inner ear
2003; 9 (10): 1293-1299
In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.
View details for DOI 10.1038/nm925
View details for Web of Science ID 000185669700041
View details for PubMedID 12949502
TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins
Mutations in the transmembrane cochlear expressed gene 1 (TMC1) cause deafness in human and mouse. Mutations in two homologous genes, EVER1 and EVER2 increase the susceptibility to infection with certain human papillomaviruses resulting in high risk of skin carcinoma. Here we report that TMC1, EVER1 and EVER2 (now TMC6 and TMC8) belong to a larger novel gene family, which is named TMC for trans membrane channel-like gene family.Using a combination of iterative database searches and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments we assembled contigs for cDNA encoding human, murine, puffer fish, and invertebrate TMC proteins. TMC proteins of individual species can be grouped into three subfamilies A, B, and C. Vertebrates have eight TMC genes. The majority of murine TMC transcripts are expressed in most organs; some transcripts, however, in particular the three subfamily A members are rare and more restrictively expressed.The eight vertebrate TMC genes are evolutionary conserved and encode proteins that form three subfamilies. Invertebrate TMC proteins can also be categorized into these three subfamilies. All TMC genes encode transmembrane proteins with intracellular amino- and carboxyl-termini and at least eight membrane-spanning domains. We speculate that the TMC proteins constitute a novel group of ion channels, transporters, or modifiers of such.
View details for Web of Science ID 000184265300001
View details for PubMedID 12812529
Vertebrate and invertebrate TRPV-like mechanoreceptors
2003; 33 (5-6): 471-478
Our senses of touch, hearing, and balance are mediated by mechanosensitive ion channels. In vertebrates, little is known about the molecular composition of these mechanoreceptors, an example of which is the transduction channel of the inner ear's receptor cells, hair cells. Members of the TRP family of ion channels are considered candidates for the vertebrate hair cell's mechanosensitive transduction channel and here we review the evidence for this candidacy. We start by examining the results of genetic screens in invertebrates that identified members of the TRP gene family as core components of mechanoreceptors. In particular, we discuss the Caenorhabditis elegans OSM-9 channel, an invertebrate TRPV channel, and the Drosophila melanogaster TRP channel NOMPC. We then evaluate basic features of TRPV4, a vertebrate member of the TRPV subfamily, which is gated by a variety of physical and chemical stimuli including temperature, osmotic pressure, and ligands. Finally, we compare the characteristics of all discussed mechanoreceptive TRP channels with the biophysical characteristics of hair cell mechanotransduction, speculating about the possible make-up of the elusive inner ear mechanoreceptor.
View details for DOI 10.1016/S0143-4160(03)00062-9
View details for Web of Science ID 000183673700018
View details for PubMedID 12765692
Parvalbumin 3 is an abundant Ca2+ buffer in hair cells
JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY
2002; 3 (4): 488-498
Ca2+ signaling serves distinct purposes in different parts of a hair cell. The Ca2+ concentration in stereocilia regulates adaptation and, through rapid transduction-channel reclosure, underlies amplification of mechanical signals. In presynaptic active zones, Ca2+ mediates the exocytotic release of afferent neurotransmitter. At efferent synapses, Ca2+ activates the K+ channels that dominate the inhibitory postsynaptic potential. A copious supply of diffusible protein buffer isolates the three signals by restricting the spread of free Ca2+ and limiting the duration of its action. Using cDNA subtraction and a gene expression assay based on in situ hybridization, we detected abundant expression of mRNAs encoding the Ca2+ buffer parvalbumin 3 in bullfrog saccular and chicken cochlear hair cells. We cloned cDNAs encoding this protein from the corresponding inner-ear libraries and raised antisera against recombinant bullfrog parvalbumin 3. Immunohistochemical labeling indicated that parvalbumin 3 is a prominent Ca2+-binding protein in the compact, cylindrical hair cells of the bullfrog's sacculus, and occurs as well in the narrow, peanut-shaped hair cells of that organ. Using quantitative Western blot analysis, we ascertained that the concentration of parvalbumin 3 in saccular hair cells is approximately 3 mM. Parvalbumin 3 is therefore a significant mobile Ca2+ buffer, and perhaps the dominant buffer, in many types of hair cell. Moreover, parvalbumin 3 provides an early marker for developing hair cells in the frog, chicken, and zebrafish.
View details for DOI 10.1007/s10162-002-2050-x
View details for Web of Science ID 000180010300009
View details for PubMedID 12072915
Molecular screens for inner ear genes
JOURNAL OF NEUROBIOLOGY
2002; 53 (2): 265-275
Identification of the genes that encode proteins that are important for proper function of specific inner ear cell types is central to our understanding of the molecular basis of hearing and balance. Whereas the combination of electrophysiology and biophysics has resulted in an exquisite understanding of inner ear function, little is known about the proteins that confer these properties at the cellular level. Furthermore, the genes that control inner ear development, susceptibility to wear and tear, regeneration from damage, and age-related degeneration, are largely unknown. This review discusses tools that have been developed during the past few years to address this imbalance between a thorough physiologic characterization of inner ear function and a detailed understanding at a molecular level of the proteins involved in these functions. Creation of inner ear cDNA libraries has laid the foundation for the discovery of genes that are specifically expressed by cell types of the inner ear and that encode proteins that are important for molecular processes in these cells. In conjunction with expressed sequence tag database analysis, cDNA subtraction, and DNA arrays, functionally important genes, whose specific expression patterns are usually verified by gene expression analysis, can be identified. Discussion of these techniques takes into account the specific characteristics of the inner ear in relation to its study using molecular biological approaches.
View details for DOI 10.1002/neu.10122
View details for Web of Science ID 000178846600012
View details for PubMedID 12382280
Application of physiological genomics to the study of hearing disorders
JOURNAL OF PHYSIOLOGY-LONDON
2002; 543 (1): 3-12
Although the biophysical principles of how the ear operates are reasonably well understood, little is known about the specific genes that confer normal function to the inner ear. Nevertheless, the recent implementation of genomic tools has led to extraordinary progress in the identification of mutated genes that cause non-syndromic and syndromic forms of deafness. Part of this success is directly related to the sequencing of the human and mouse genomes and improved gene annotation methods. This review discusses how physiological genomic tools, such as genomic databases, expressed sequence tag databases and DNA arrays have been applied to find candidate genes for important molecular processes in the inner ear. It also illustrates, using the discovery of genes encoding essential components of cochlear K+ homeostasis as an example, how the combination of physiological genomic tools with physiological and morphological information has led to an in-depth understanding of cochlear ion homeostasis. Finally, it discusses how the use of applied genomic tools, such as gene arrays, will further advance our knowledge of how the inner ear works, develops, ages and regenerates.
View details for DOI 10.1113/jphysiol.2002.018911
View details for Web of Science ID 000177848300002
View details for PubMedID 12181277
- A unified nomenclature for the superfamily of TRP cation channels MOLECULAR CELL 2002; 9 (2): 229-231
Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor
2000; 103 (3): 525-535
The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nervous system, the channel is expressed in neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells.
View details for Web of Science ID 000090144100017
View details for PubMedID 11081638
A novel conserved cochlear gene, OTOR: Identification, expression analysis, and chromosomal mapping
2000; 66 (3): 242-248
We have identified a novel cochlear gene, designated OTOR, from a comparative sequence analysis of over 4000 clones from a human fetal cochlear cDNA library. Northern blot analysis of human and chicken organs shows strong OTOR expression only in the cochlea; very low levels are detected in the chicken eye and spinal cord. Otor and Col2A1 are coexpressed in the cartilaginous plates of the neural and abneural limbs of the chicken cochlea, structures analogous to the mammalian spiral limbus, osseous spiral lamina, and spiral ligament, and not in any other tissues in head and body sections. The human OTOR gene localizes to chromosome 20 in bands p11.23-p12.1 and more precisely to STS marker WI-16380. We have isolated cDNAs orthologous to human OTOR in the mouse, chicken, and bullfrog. The encoded protein, designated otoraplin, has a predicted secretion signal peptide sequence and shows a high degree of cross-species conservation. Otoraplin is homologous to the protein encoded by CDRAP/MIA (cartilage-derived retinoic acid sensitive protein/melanoma inhibitory activity), which is expressed predominantly by chondrocytes, functions in cartilage development and maintenance, and has growth-inhibitory activity in melanoma cell lines.
View details for DOI 10.1006/geno.2000.6224
View details for Web of Science ID 000088039000002
View details for PubMedID 10873378
The specification of sympathetic neurotransmitter phenotype depends on gp130 cytokine receptor signaling
1998; 125 (23): 4791-4802
Sympathetic ganglia are composed of noradrenergic and cholinergic neurons. The differentiation of cholinergic sympathetic neurons is characterized by the expression of choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP), induced in vitro by a subfamily of cytokines, including LIF, CNTF, GPA, OSM and cardiotrophin-1 (CT-1). To interfere with the function of these neuropoietic cytokines in vivo, antisense RNA for gp130, the common signal-transducing receptor subunit for neuropoietic cytokines, was expressed in chick sympathetic neurons, using retroviral vectors. A strong reduction in the number of VIP-expressing cells, but not of cells expressing ChAT or the adrenergic marker tyrosine hydroxylase (TH), was observed. These results reveal a physiological role of neuropoietic cytokines for the control of VIP expression during the development of cholinergic sympathetic neurons.
View details for Web of Science ID 000077742200020
View details for PubMedID 9806927
A transient role for ciliary neurotrophic factor in chick photoreceptor development
JOURNAL OF NEUROBIOLOGY
1998; 37 (4): 672-683
Previous studies suggest that ciliary neurotrophic factor (CNTF) may represent one of the extrinsic signals controlling the development of vertebrate retinal photoreceptors. In dissociated cultures from embryonic chick retina, exogenously applied CNTF has been shown to act on postmitotic rod precursor cells, resulting in an two- to fourfold increase in the number of cells acquiring an opsin-positive phenotype. We now demonstrate that the responsiveness of photoreceptor precursors to CNTF is confined to a brief phase between their final mitosis and their terminal differentiation owing to the temporally restricted expression of the CNTF receptor (CNTFR alpha). As shown immunocytochemically, CNTFR alpha expression in the presumptive photoreceptor layer of the chick retina starts at embryonic day 8 (E8) and is rapidly down-regulated a few days later prior to the differentiation of opsin-positive photoreceptors, both in vivo and in dissociated cultures from E8. We further show that the CNTF-dependent in vitro differentiation of rods is followed by a phase of photoreceptor-specific apoptotic cell death. The loss of differentiated rods during this apoptotic phase can be prevented by micromolar concentrations of retinol. Our results provide evidence that photoreceptor development depends on the sequential action of different extrinsic signals. The time course of CNTFR alpha expression and the in vitro effects suggest that CNTF or a related molecule is required during early stages of rod differentiation, while differentiated rods depend on additional protective factors for survival.
View details for Web of Science ID 000077193000014
View details for PubMedID 9858267
Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction
1998; 20 (3): 299-303
DFNA9 is an autosomal dominant, nonsyndromic, progressive sensorineural hearing loss with vestibular pathology. Here we report three missense mutations in human COCH (previously described as Coch5b2), a novel cochlear gene, in three unrelated kindreds with DFNA9. All three residues mutated in DFNA9 are conserved in mouse and chicken Coch, and are found in a region containing four conserved cysteines with homology to a domain in factor C, a lipopolysaccharide-binding coagulation factor in Limulus polyphemus. COCH message, found at high levels in human cochlear and vestibular organs, occurs in the chicken inner ear in the regions of the auditory and vestibular nerve fibres, the neural and abneural limbs adjacent to the cochlear sensory epithelium and the stroma of the crista ampullaris of the vestibular labyrinth. These areas correspond to human inner ear structures which show histopathological findings of acidophilic ground substance in DFNA9 patients.
View details for Web of Science ID 000076698100028
View details for PubMedID 9806553
Differential regulation of ciliary neurotrophic factor receptor-alpha expression in all major neuronal cell classes during development of the chick retina
JOURNAL OF COMPARATIVE NEUROLOGY
1998; 400 (2): 244-254
Ciliary neurotrophic factor (CNTF) exerts a multiplicity of effects on a broad spectrum of target cells, including retinal neurons. To investigate how this functional complexity relates to the regulation of CNTF receptor alpha (CNTFR alpha) expression, we have studied the developmental expression of the receptor protein in chick retina by using immunocytochemistry. During the course of development, the receptor is expressed in all retinal layers, but three levels of specificity can be observed. First, the expression is regulated temporally with immunoreactivity observed in ganglion cells (embryonic day 8 [E8] to adult), photoreceptor precursors (E8-E12), amacrine cells (E10 to adult), bipolar cells (E12-E18), differentiated rods (E18 to adult), and horizontal cells (adult). Second, expression is restricted to distinct subpopulations of principal retinal neurons: preferentially, large ganglion cells; subpopulations of amacrine cells, including a particular type of cholinergic neuron; a distinctly located type of bipolar cell; and rod photoreceptors. Third, expression exhibits subcellular restriction: it is confined largely to dendrites in mature amacrine cells and is restricted entirely to outer segments in mature rods. These data correlate with CNTF effects on the survival of ganglion cells and mature photoreceptors, the in vitro differentiation of photoreceptor precursors and cholinergic amacrine cells, and the number of bipolar cells in culture described here or in previous studies. Thus, our results demonstrate an exceptional degree of complexity with respect to the regulation of neuronal CNTFR alpha expression in a defined model system. This suggests that the same signaling pathway is used to mediate a variety of regulatory influences, depending on the developmental stage and cell type.
View details for Web of Science ID 000076106100006
View details for PubMedID 9766402
Molecular markers for cell types of the inner ear and candidate genes for hearing disorders
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (19): 11400-11405
To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted in situ hybridization on slides bearing cryosections of late embryonic chicken heads, bodies, and cochleae. One hundred and twenty of the 196 clones analyzed encode 12 proteins whose mRNAs are specifically or highly expressed in the chicken's inner ear; the remainder encode proteins that occur more widely. We identified proteins that have been described previously as expressed in the inner ear, such as beta-tectorin, calbindin, and type II collagen. A second group of proteins abundant in the inner ear includes five additional types of collagens. A third group, including Coch-5B2 and an ear-specific connexin, comprises proteins whose human equivalents are candidates to account for hearing disorders. This group also includes proteins expressed in two unique cell types of the inner ear, homogene cells and cells of the tegmentum vasculosum.
View details for Web of Science ID 000075957100071
View details for PubMedID 9736748
- Two deaf mice, two deaf mice ... NATURE MEDICINE 1998; 4 (5): 560-561
Onset of CNTFR alpha expression and signal transduction during neurogenesis in chick sensory dorsal root ganglia
1997; 191 (1): 1-13
The expression of ciliary neurotrophic factor receptor alpha (CNTFRalpha) was investigated in the developing chick dorsal root ganglion (DRG) using affinity-purified anti-CNTFRalpha antibodies. At thoracic levels, CNTFRalpha-immunoreactivity (CNTFRalpha-IR) was first observed at stage 19 (E3) in cells with neuronal morphology. CNTFRalpha-IR is restricted to the neuronal lineage in the DRG throughout development. CNTFRalpha expression precedes that of neuron-specific beta tubulin, Hu antigen, and Q211 antigen, which are markers expressed in developing sensory neurons. [3H]Thymidine-labeling studies showed the onset of CNTFRalpha expression during terminal mitosis of sensory neuron precursors, making CNTFRalpha the earliest known neuronal marker in the DRG. CNTFRalpha-mediated signal transduction was demonstrated in E7 and E11 DRG neuron cultures by CNTF-induced STAT3 phosphorylation. Although low ligand concentrations (5 pM) elicit STAT3 phosphorylation in E7 and E11 DRG neurons, a survival response is only observed in neurons from E11 DRG. This implicates a complex readout mechanism downstream of STAT3 phosphorylation leading to different cellular responses that depend on the age of the DRG neuron. These results argue against a role of CNTFRalpha ligands in the control of early neuron survival but are compatible with other functions in neurogenesis and sensory neuron development.
View details for Web of Science ID A1997YG86600001
View details for PubMedID 9356167
Distribution of Ca2+-activated K+ channel isoforms along the tonotopic gradient of the chicken's cochlea
1997; 19 (5): 1061-1075
In some cochleae, the number and kinetic properties of Ca2+-activated K+ (KCa) channels partly determine the characteristic frequency of each hair cell and thus help establish a tonotopic map. In the chicken's basilar papilla, we found numerous isoforms of KCa channels generated by alternative mRNA splicing at seven sites in a single gene, cSlo. In situ polymerase chain reactions demonstrated cSlo expression in hair cells and revealed differential distributions of KCa channel isoforms along the basilar papilla. Analysis of single hair cells by the reverse transcription polymerase chain reaction confirmed the differential expression of channel variants. Heterologously expressed cSlo variants differed in their sensitivities to Ca2+ and voltage, suggesting that the distinct spatial distributions of cSlo variants help determine the tonotopic map.
View details for Web of Science ID A1997YH59400013
View details for PubMedID 9390519
Extrinsic signals in the developing nervous system: The role of neurokines during neurogenesis
PERSPECTIVES ON DEVELOPMENTAL NEUROBIOLOGY
1996; 4 (1): 19-34
Vertebrate neurogenesis involves many distinct differentiation stages that are regulated by extrinsic signals. Survival and differentiation effects on cultured neurons of several lineages are elicited by members of the neurokine family of growth factors, ciliary neurotrophic factor (CNTF) and the related avian factor, growth promoting activity (GPA). The selective actions of these factors are mediated through the activation of heteromeric receptor complexes and depend on the presence of the ligand-binding receptor subunits CNTFR alpha and GPAR alpha. The in vivo localization of CNTFR alpha and GPAR alpha is consistent with the previously assigned biological functions but also suggest novel functions for these receptors and their ligands during neurogenesis.
View details for Web of Science ID A1996WV03000003
View details for PubMedID 9169916
ANALYSIS OF FUNCTION AND EXPRESSION OF THE CHICK GPA RECEPTOR (GPAR-ALPHA) SUGGESTS MULTIPLE ROLES IN NEURONAL DEVELOPMENT
1995; 121 (8): 2681-2693
Growth promoting activity (GPA) is a chick growth factor with low homology to mammalian ciliary neurotrophic factor (CNTF) (47% sequence identity with rat CNTF) but displays similar biological effects on neuronal development. We have isolated a chick cDNA coding for GPA receptor (GPAR alpha), a GPI-anchored protein that is 70% identical to hCNTFR alpha. Functional analysis revealed that GPAR alpha mediates several biological effects of both GPA and CNTF. Soluble GPAR alpha supports GPA- and CNTF-dependent survival of human TF-1 cells. In sympathetic neurons, GPAR alpha mediates effects of both GPA and CNTF on the expression of vasoactive intestinal peptide (VIP) as shown by the inhibition of GPA- and CNTF-mediated VIP induction upon GPAR alpha antisense RNA expression. These results demonstrate that GPAR alpha is able to mediate effects of two neurokines that are only distantly related. GPAR alpha mRNA expression is largely restricted to the nervous system and was detected in all neurons that have been shown to respond to GPA or CNTF by increased survival or differentiation, i.e. ciliary, sympathetic, sensory dorsal root, motoneurons, retinal ganglion cells and amacrine cells. Interestingly, GPAR alpha mRNA was additionally found in neuronal populations and at developmental periods not known to be influenced by GPA or CNTF, suggesting novel functions for GPAR alpha and its ligands during neurogenesis and neuron differentiation.
View details for Web of Science ID A1995RN70300038
View details for PubMedID 7671828
GPA AND CNTF PRODUCE SIMILAR EFFECTS IN SYMPATHETIC NEURONS BUT DIFFER IN RECEPTOR-BINDING
1993; 5 (3): 357-360
The effects of growth promoting activity (GPA) on sympathetic neurone development were investigated in vitro and compared with the effects of ciliary neurotrophic factor (CNTF). GPA interfered with sympathetic neurone proliferation and induced the expression of vasoactive intestinal peptide (VIP) in neurones from 7-day-old (E7) chick embryos. The biological effects observed with saturating levels of GPA are indistinguishable from the effects of CNTF. The effects on VIP expression suggest that GPA may be involved in the specification of sympathetic neurone phenotypes. Whereas half maximal effects are achieved at lower concentrations of GPA than CNTF, GPA competes less efficiently than CNTF for the binding of 125I-labelled CNTF. This suggests similar, but not identical interactions of CNTF and GPA with receptors on chick sympathetic neurones.
View details for Web of Science ID A1993MP00200045
View details for PubMedID 8298104
Bioluminescence-based detection of genetically engineered microorganisms in nonsterile river water.
Microbial releases : viruses, bacteria, fungi
1992; 1 (1): 35-39
The luminescence genes of the marine bacterium Vibrio fischeri were cloned into a lac expression vector and introduced into Escherichia coli and Pseudomonas putida. Survival of the cells in river water samples was monitored by light measurements. Whereas E. coli survived in sterilized river water for more than 29 days, it died off in nonsterile river water after 9 to 13 days. The engineered P. putida cells survived in nonsterile river water for more than 137 days. The detection limit for E. coli was 11 cells/ml.
View details for PubMedID 1341987