Stefan Heller

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.

The TRP superfamily includes a diversity of non-voltage-gated cation channels that vary significantly in their selectivity and mode of activation. Nevertheless, members of the TRP superfamily share significant sequence homology and predicted structural similarities. Currently, most of the genes and proteins that comprise the TRP superfamily have multiple names and, in at least one instance, two distinct genes belonging to separate subfamilies have the same name. Moreover, there are many cases in which highly related proteins that belong to the same subfamily have unrelated names. Therefore, to minimize confusion, we propose a unified nomenclature for the TRP superfamily.

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.

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.

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.

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.

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 .

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.

Journal of NeurobiologyVolume 66, Issue 13 p. 1489-1500 Research Article Engraftment and differentiation of embryonic stem cell–derived neural progenitor cells in the cochlear nerve trunk: Growth of processes into the organ of corti C. Eduardo Corrales, C. Eduardo Corrales Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114Search for more papers by this authorLuying Pan, Luying Pan Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114Search for more papers by this authorHuawei Li, Huawei Li Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114Search for more papers by this authorM. Charles Liberman, M. Charles Liberman Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard & MIT, Cambridge, MA 02139Search for more papers by this authorStefan Heller, Stefan Heller Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard & MIT, Cambridge, MA 02139Search for more papers by this authorAlbert S.B. Edge, Corresponding Author Albert S.B. Edge albert_edge@meei.harvard.edu Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard & MIT, Cambridge, MA 02139Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115Search for more papers by this author C. Eduardo Corrales, C. Eduardo Corrales Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114Search for more papers by this authorLuying Pan, Luying Pan Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114Search for more papers by this authorHuawei Li, Huawei Li Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114Search for more papers by this authorM. Charles Liberman, M. Charles Liberman Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard & MIT, Cambridge, MA 02139Search for more papers by this authorStefan Heller, Stefan Heller Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard & MIT, Cambridge, MA 02139Search for more papers by this authorAlbert S.B. Edge, Corresponding Author Albert S.B. Edge albert_edge@meei.harvard.edu Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115 Tillotson Unit for Cell Biology, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114 Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard & MIT, Cambridge, MA 02139Department of Otology and Laryngology, Harvard Medical School, Boston MA 02115Search for more papers by this author First published: 29 September 2006 https://doi.org/10.1002/neu.20310Citations: 140AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract 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. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 Citing Literature Supporting Information This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0022-3034/suppmat . Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume66, Issue13November 2006Pages 1489-1500 RelatedInformation

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. 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. The TRPV4 protein, initially described as OTRPC4 (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar), VR-OAC (2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar), TRP12 (3Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (257) Google Scholar), and VRL-2 (4Delany N.S. Hurle M. Facer P. Alnadaf T. Plumpton C. Kinghorn I. See C.G. Costigan M. Anand P. Woolf C.J. Crowther D. Sanseau P. Tate S.N. Physiol. Genomics. 2001; 4: 165-174Crossref PubMed Scopus (201) Google Scholar), is a member of the TRPV (vanilloid-type transient receptor potential) superfamily consisting of mainly nonspecific cation channels. Like many other transient receptor potential ion channels, TRPV4 contains three ankyrin-like repeat domains in its amino-terminal intracellular domain, six putative transmembrane-spanning domains, a pore loop region, and a transient receptor potential domain near its carboxyl terminus. TRPV4 is activated by exposure to hypotonicity (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar, 2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar), although it has recently been proposed that this activation is mediated by second messengers (5Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (799) Google Scholar, 6Nilius B. Watanabe H. Vriens J. Pflugers Arch. Eur. J. Physiol. 2003; 446: 298-303Crossref PubMed Scopus (126) Google Scholar). Osmosensation is a form of mechanosensation mediated by ion channels or associated structures that measure tension in membranes or in other elastic elements. In agreement with its proposed function in osmosensation, TRPV4 mRNA transcript is found in epithelial cells of kidney tubules, in the stria vascularis of the cochlea, in sweat glands, and in the osmosensory cells of the circumventricular organs of the brain (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (796) Google Scholar, 2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar). Interestingly, the distribution of TRPV4 protein in other tissues such as airway smooth muscle, oviduct, spleen, heart, liver, testis, keratinocyte, inner ear hair cells, and dorsal root ganglion suggests that the role of this channel is not at all restricted to osmosensation. This notion is supported by reports of a variety of other stimuli that activate TRPV4. For example, increasing the temperature above 27 °C has been found to activate TRPV4 (2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 7Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar, 8Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 47044-47051Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Likewise, TRPV4 is gated by synthetic agonists such as the phorbol ester 4-α-phorbol-12,13-didecanoate (8Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 47044-47051Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 9Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar) and endogenous agonist precursors such as the endocannabinoid anandamide, or its hydrolysis product, arachidonic acid, which is metabolized into 5′,6′-epoxyeicosatrienoic acid by cytochrome P-450 epoxygenase (5Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (799) Google Scholar). Particularly, the 5′,6′-epoxyeicosatrienoic acid metabolite is a potent activator of TRPV4 (5Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (799) Google Scholar, 10Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (488) Google Scholar). The involvement of TRPV4 in sensing a variety of stimuli in vivo was shown by recent studies demonstrating: (i) that TRPV4 is necessary for a circumventricular organ-mediated osmosensation in the CNS (11Liedtke W. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13698-13703Crossref PubMed Scopus (632) Google Scholar); (ii) that thermosensation and nociception is mediated by TRPV4 in mouse skin keratinocytes (12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Biol. Chem. 2004; 279: 21569-21575Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and human mammary keratinocytes (13Gopinath P. Wan E. Holdcroft A. Facer P. Davis J.B. Smith G.D. Bountra C. Anand P. BMC Womens Health. 2005; 5: 2Crossref PubMed Scopus (138) Google Scholar), respectively; (iii) that TRPV4 functions as osmotically gated transducer in primary afferent nociceptive nerve fibers (12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Biol. Chem. 2004; 279: 21569-21575Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 14Alessandri-Haber N. Yeh J.J. Boyd A.E. Parada C.A. Chen X. Reichling D.B. Levine J.D. Neuron. 2003; 39: 497-511Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar); and (iv) that nociception through TRPV4 may well be transduced through a mechanosensory process (15Alessandri-Haber N. Dina O.A. Yeh J.J. Parada C.A. Reichling D.B. Levine J.D. J. Neurosci. 2004; 24: 4444-4452Crossref PubMed Scopus (269) Google Scholar, 16Suzuki M. Watanabe Y. Oyama Y. Mizuno A. Kusano E. Hirao A. Ookawara S. Neurosci. Lett. 2003; 353: 189-192Crossref PubMed Scopus (100) Google Scholar, 17Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar). It has been hypothesized that the mechanical/osmotic gating of TRPV4 by cell swelling is dependent on active phospholipase A2, an enzyme necessary for arachidonic acid synthesis (5Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (799) Google Scholar, 10Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (488) Google Scholar). A second messenger-based activation of TRPV4 is consistent with previous electrophysiological findings showing that, by switching from a cell-attached to a cell-detached patch clamp modus, the open probability of TRPV4 substantially decreases (2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar). This phenomenon could also be interpreted as a direct result of the essential requirement of the ion channel for a specific association with intracellular structures. Notwithstanding such penumbral structure-function relation of the TRPV4 gating mechanisms, we hypothesized that TRPV4 interacts with other proteins and that such association may determine the subcellular targeting of TRPV4, its retention in distinct membrane regions, or its withdrawal from the plasma membrane. As a first step toward the identification of interaction partners, we conducted a yeast two-hybrid screen for TRPV4-binding proteins, and we identified the PACSINs, a group of three proteins, each encoded by an individual gene, as TRPV4-binding partners. We provide evidence that this interaction happens between the carboxyl-terminal SH3 3The abbreviations used are: SH, Src homology; PRD, proline-rich domain; PBS, phosphate-buffered saline; DIP, dynamin inhibitory peptide; FITC, fluorescein isothiocyanate; 4α-PDD, 4α-phorbol 12,13-dideconate; RT, reverse transcription. domain that is present in all three PACSINs and a triple-proline motif within the amino-terminal proline-rich domain (PRD) of TRPV4. Recent studies suggest that PACSIN proteins functionally interact with the endocytotic machinery and participate in synaptic vesicular targeting (18Modregger J. Ritter B. Witter B. Paulsson M. Plomann M. J. Cell Sci. 2000; 113: 4511-4521Crossref PubMed Google Scholar). Here we report that co-expression of PACSIN 3, but not PACSINs 1 and 2, shifts the ratio between intracellular and plasma membrane-associated TRPV4 toward an apparent retention of the protein in or near the plasma membrane. Yeast Two-hybrid Screen and Two-hybrid Assays—Two-hybrid bait vectors were generated by PCR from chicken TRPV4 cDNA (accession number NM_204692) (2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar). We used the following primer pairs: the amino-terminal fragment (amino acids 1-453), forward, 5′-gaGAATTCatggcagaccccgaagacccccgtg-3′, and reverse, 5′-gagaGTCGACagcaccgaacttgcgc-3′; first intracellular loop (amino acids 514-539), forward, 5′-gaGAATTCaatatcaaagatctcttcatg-3′, and reverse, 5′-gagaGTCGACtcagtagagcagctgaaatgagcc-3′; second intracellular loop (amino acids 577-606), forward, 5′-gaGAATTCtacttcacgcgagggctcaagc-3′, and reverse, 5′-gagaGTCGACtcagaccaggaggaagcggaacag-3′; and carboxyl-terminal fragment (amino acids 699-852), forward, 5′-gaGAATTCatgctcatcgccctcatgggtg-3′, and reverse, 5′-gagaGTCGACctagagtggggagctgggggtc-3′. Restriction enzyme recognition sites for EcoRI and SalI (indicated in uppercase letters in the primer sequences) were used to subclone the amplified cDNA fragments in frame with the Gal4 binding domain into the yeast-Escherichia coli shuttle vector pBD-GAL4 Cam (Stratagene). The identities and integrity of the cDNA inserts were confirmed by sequencing. For screening, we first transformed yeast strain AH109 (Clontech) with each of the bait vectors, and then we individually transformed each bait-containing yeast cell population with plasmid DNA prepared from a chicken basilar papilla cDNA library in the HybriZAP two-hybrid bacteriophage λ vector (19Heller S. Sheane C.A. Javed Z. Hudspeth A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11400-11405Crossref PubMed Scopus (87) Google Scholar). Auto-activation in bait-containing yeast cells was suppressed by adding 5 mm of the competitive His3 protein inhibitor 3-amino-1,2,4-triazole to the dropout medium. For each bait, 2 × 107 transformants were selectively screened for Gal4-induced activation of the HIS3 and ADE2 genes by using the lithium acetate/single-stranded carrier DNA/polyethylene glycol method (20Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2077) Google Scholar). Clones were considered positive when they conferred histidine autotrophy to a deficient yeast strain and when they led to expression of the reporter enzyme β-galactosidase assayed by conversion of colorless 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to a blue precipitate. Only cDNAs that were isolated multiple times independently were further studied. Positive colonies were further tested for expression of the Gal-4-dependent reporter lacZ by filter lift assay (for details, see HybriZAP-2.1 two-hybrid cDNA synthesis kit manual (Stratagene) and Matchmaker Gal4 Two-Hybrid System 3 user manual (Clontech)). Library cDNA in pAD-GAL4 phagemid vector was recovered from individual yeast colonies via ampicillin selection, and cDNA inserts were sequence-verified. Antibodies—To generate antibodies to TRPV4, the amino terminus of rat TRPV4 (amino acids 1-233) lacking the ankyrin repeats was obtained by PCR with a hexahistidine moiety at its carboxyl terminus and subcloned into the pFastBac1 vector (Invitrogen). Recombinant TRPV4 amino terminus was expressed in insect cells (SF9; Invitrogen) and purified under native conditions with Ni2+-conjugated agarose beads (nickel-nitrilotriacetic acid; Qiagen). Two female rabbits were each initially immunized with 200 μg of the recombinant TRPV4 amino terminus; additional boost injections were given at 2-3-week intervals (Covance). The sera of both animals displayed strong reactivity against the immunogen in Western blots. All of the experiments described in this publication were done with the combined final bleed sera of both rabbits and were enriched by affinity purification on a column generated with recombinant TRPV4 amino-terminal protein (Ultralink; Pierce). Antibody specificity was shown by blocking the signal on Western blots via preincubating the anti-TRPV4 polyclonal sera with the amino-terminal TRPV4 polypeptide (Fig. 1A). Another control for antibody specificity was demonstrated by comparing Western blots of kidney and keratinocyte protein extracts from trpv4-null mice and wild-type littermates (11Liedtke W. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13698-13703Crossref PubMed Scopus (632) Google Scholar, 12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Biol. Chem. 2004; 279: 21569-21575Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). We also used previously characterized affinity-purified antibodies to glutathione S-transferase fusion proteins of PACSINs 1, 2, and 3 (18Modregger J. Ritter B. Witter B. Paulsson M. Plomann M. J. Cell Sci. 2000; 113: 4511-4521Crossref PubMed Google Scholar, 21Plomann M. Lange R. Vopper G. Cremer H. Heinlein U.A. Scheff S. Baldwin S.A. Leitges M. Cramer M. Paulsson M. Barthels D. Eur. J. Biochem. 1998; 256: 201-211Crossref PubMed Scopus (78) Google Scholar, 22Qualmann B. Kelly R.B. J. Cell Biol. 2000; 148: 1047-1062Crossref PubMed Scopus (252) Google Scholar). Polyclonal anti-TRPV1 and anti-TRPV2 antibodies were purchased from Calbiochem EMD Biosciences (San Diego, CA). The manufacturer demonstrates specificity of these antibodies by blocking the signal on Western blots through preincubation of the antibodies with excess antigen (see manufacturer's product description). 9E10 monoclonal antibody to c-Myc was obtained from the Developmental Studies Hybridoma Bank and immobilized on agarose beads from a commercial supplier (ProFound c-Myc-Tag immunoprecipitation kit; Pierce). Co-immunoprecipitation—For co-immunoprecipitation, we generated expression vectors for the interaction partners using the plasmid pcDNA3.1 (Invitrogen). We also used the TOPO-pCR8/pTREX Gateway vector systems (Invitrogen) to subclone and express the full-length murine cDNAs of TRPV1, TRPV2, and TRPV4 proteins. The corresponding primer pairs to amplify these cDNAs were: (i) TRPV1-forward, 5′-aagcttaccatggagaaatgggctagcttaga-3′, and TRPV1-reverse, 5′-tctagatttatttcattatttctcccctggggccatgga-3′; (ii) TRPV2-forward, 5′-gaattcaccatgacttcagcctccaacccc-3′, and TRPV2-reverse, 5′-ctcgagtttatttcattagtgggactggaggacctgaag-3′; and (iii) TRPV4-forward, 5′-aagcttaccatggcagatcctggtgatggtc-3′, and TRPV4-reverse, 5′-tctagatttatttcattacagtggggcatcgtccgtcct-3′. The deletion and point mutations were introduced by PCR, and the resulting amplification products were sub-cloned into the pCDNA3.1 expression vector and sequence-verified. The cell lines HEK293, HeLa, and NIH 3T3 were transfected with single plasmids or with combinations of the expression vectors using Lipofectamine 2000 (Invitrogen) or polyethylinimine (Polysciences, Inc.). Transfected cells were washed with cold PBS 48 h after transfection and lysed in ice-cold precipitation assay buffer consisting of 150 mm NaCl, 1% octylphenyl-polyethylene glycol (Igepal CA-630), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate in 50 mm Tris at pH 7.5. For immunoprecipitation, we used immobilized monoclonal anti-c-Myc agarose beads and performed the experiments according to the manufacturer's recommendation. Alternatively, we added 9E10 monoclonal antibody to c-Myc at a dilution of 1:250 to cell lysates. Following 2 h of incubation at 4 °C, immunoglobulins and bound proteins were precipitated from the lysate with protein A-agarose (Affi-Gel; Bio-Rad), washed four times with precipitation assay buffer, and the bound proteins were separated by polyacrylamide gel electrophoresis. All of the immunoprecipitation-bound proteins were subjected to high stringency washes with 500 mm NaCl in Tris-buffered saline plus 1% Tween 20. Each immunoprecipitation was controlled by mock experiments without the addition of antibody. The omission of the antibody and the use of cell lysates from control-transfected cells always confirmed the specificity of the results presented in this study. Western Blots and Immunocytochemistry—Western blots were incubated for 1 h at room temperature in 2.5% (v/v) Liquid Block (Amersham Biosciences) and 0.1% (v/v) Tween 20 in PBS (PBS-T). The blots were incubated overnight at 4 °C with antiserum diluted in PBS-T with 2.5% Liquid Block. The following antiserum dilutions were used: 1:5000 for monoclonal 9E10 anti-c-Myc antibody; 1:5000 for rabbit anti-PACSIN 3; 1:10000 rabbit anti-TRPV4; 1:1000 for rabbit anti-mouse TRPV1; and 1:1000 for rabbit anti-mouse TRPV2. The blots were washed four times for 10 min each at room temperature. Detection was performed with IRDye 700- and IRDye 800-conjugated secondary antibodies (Rockland Immunochemicals, Gilbertsville, PA) and scanning with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Transfected human cell lines HEK293, HeLa, and mouse NIH 3T3 cells were used for immunofluorescence studies. Kidney tissue was taken from wild-type C57BL/6 mouse (Charles River Labs, MA) and fixed with 4% paraformaldehyde-fixed for cryosections. The cultured cells were fixed with methanol. All of the specimens were blocked in 0.1% Triton-100, 1% bovine serum albumin (w/v), and 5% (w/v) heat-inactivated goat serum in PBS (PBT1). The slides or cells were then incubated overnight at 4 °C using the following antibodies: rabbit anti-TRPV4 (1:500 dilution), rabbit anti-PACSIN 1, 2, or 3 (1:250 dilution), mouse anti-c-Myc (9E10; 1:500), red fluorescent Alexa Fluor 594 wheat germ agglutinin (1:200 dilution, Invitrogen), rabbit anti-mouse TRPV1 (1:250 dilution), and rabbit anti-mouse TRPV2 (1:500). Unbound antibodies were removed by three PBT1 washes and one PBT2 (same formulation as PBT1 but without the serum) wash for 15 min each at room temperature. FITC- and Cy5-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch) were diluted 1:1000 in PBT2. TOTO-3 iodide (Invitrogen) was diluted 1:2000. A 2-h incubation period in the secondary antibody mixture preceded three washes for 15 min each in PBS. The coverslipped specimens were analyzed by fluorescence or confocal microscopy (Zeiss Axioskop 2, Leica TCS SP2 or Zeiss LSM Pascal). The dynamin inhibitory peptide (DIP) and the membrane-permeant myristoylated DIP (Tocris Bioscience, Cologne, Germany) were dissolved in Me2SO and added to the transfected cells at a concentration of 100 μm 16 h before fixation and immunostaining. The control cultures were treated with the appropriate amount of Me2SO. For statistical analysis of our quantitative data, we performed unpaired Student's t tests using the Kaleidagraph software (Synergy, Reading, PA). Electrophysiology—HEK293 cells were seeded 12-18 h after transfection onto poly-l-lysine-coated glass coverslips and incubated for 3 h before use. Whole cell currents utilizing ruptured patches were measured with an EPC-9 amplifier (HEKA Electronic, Lambrecht, Germany; sampling rate, 1 ms; 8-Pole Bessel filter 3kHz). Patch electrodes had a DC resistance of 2-4 MΩ when filled with intracellular solution. An Ag-AgCl wire was used as reference electrode. We used a ramp protocol, starting with a voltage step from 20 to -100 mV followed by a 400-ms linear ramp to +100 mV. This protocol was repeated every 5 s. The cell membrane capacitance values were used to calculate current densities. The standard extracellular solution contained 150 mm NaCl, 6 mm CsCl, 1 mm MgCl2, 5 mm CaCl2, 10 mm glucose, 10 mm HEPES, buffered at pH 7.4 (adjusted with NaOH). The pipette solution was composed of 20 mm CsCl, 100 mm Asp, 1 mm MgCl2, 10 mm HEPES, 4 mm Na2ATP, 10 mm BAPTA, 2.93 mm CaCl2. The free Ca2+ concentration of this solution was 50 nm. The non-protein kinase C-activating phorbol ester, 4α-phorbol 12,13-dideconate (4α-PDD; Sigma), was applied at a 1 μm concentration from a 10 mm stock solution in ethanol. Calcium Imaging—The cells were loaded with Fura-2 by adding 2 μm Fura-2 acetoxymethyl ester to the medium for 20 min at 37 °C. For imaging, the cells were perfused with an extracellular solution containing 150 mm NaCl, 6 mm CsCl, 1 mm MgCl2, 1.5 mm CaCl2, 10 mm HEPES, 10 mm glucose, buffered at pH 7.4, and 1 μm 4α-PDD was applied for activation. The intracellular [Ca2+]i was measured with an imaging system consisting of a Polychrome IV monochromator (TILL Photonics, Martinsried, Germany) and a Roper Scientific charge-coupled device camera connected to an Axiovert 200M inverted microscope (Zeiss, Germany). Monochromator and camera were controlled with Metafluor software (version 6.3; Universal Imaging, Downingtown, PA). Fluorescence was measured during alternating excitation at 357 and 380 nm and corrected for the individual background fluorescence. The absolute Ca2+ concentration was obtained from the fluorescence ratios using the equation [Ca2+] = Keff(R - R0)/(R1 - R), where Keff, R0, and R1 are calibration constants. R0 and R1 were estimated by perfusing cells with Ca2+-free solution and high Ca2+ containing solution, respectively. The effective binding constant, Keff, was calculated by the equation Keff = Kd(R1 + α)/(R0 + α), where Kd is the dissociation constant of Fura-2, and α is the isocoefficient. Kd value was taken from Paltauf-Doburzynska and Graier (23Paltauf-Doburzynska J. Graier W.F. Cell Calcium. 1997; 21: 43-51Crossref PubMed Scopus (26) Google Scholar). The isocoefficient α was obtained as described by Zhou and Neher (24Zhou Z. Neher E. Pflugers Arch. 1993; 425: 511-517Crossref PubMed Scopus (116) Google Scholar). RT-PCR—RT-PCR analysis of mouse tissues was performed using murine cDNA (mouse multiple tissue cDNA panel B; BD Biosciences) as template with the following oligonucleotides in standard PCRs: (i) PACSIN 3-forward, 5′-ttccgtaaagctcagaagccct-3′, and PACSIN 3-reverse, 5′-tgtcggtacaatgctggtcaga-3′; and (ii) TRPV4-forward, 5′-atcatcctcaccttcgtgctcctg-3′, and TRPV4-reverse, 5′-acaccggacaaatgcctaaatgta-3′. The amplification products were separated in a 1% agarose gel and documented using a digital camera system (Kodak). Identification of PACSINs as TRPV4-binding Partners—We generated four different yeast two-hybrid bait vectors representing Gal4-binding domain fusions with the chicken TRPV4 amino terminus, each of the two intracellular loops and the carboxyl terminus. Only the amino-terminal bait yielded positive clones in yeast two-hybrid screening of a chicken inner ear cDNA library. We identified two groups of clones, all of which represented multiple in-frame isolates of different lengths of the chicken homologues for mammalian PACSINs 1 and 3 (18Modregger J. Ritter B. Witter B. Paulsson M. Plomann M. J. Cell Sci. 2000; 113: 4511-4521Crossref PubMed Google Scholar). All Three PACSINs Bind to TRPV4—To confirm the yeast two-hybrid screening results, we sought to determine whether the mammalian isoforms of TRPV4 and PACSINs bind each other in biochemical assays. In co-immunoprecipitation experiments, we found that all three PACSINs were capable of precipitating co-expressed rat TRPV4 amino terminus (Fig. 1B). Co-expression and immunoprecipitation of the full-length rat TRPV4 as well as murine TRPV4 with all three PACSINs confirmed this observation (Fig. 1, C and D). Neither mouse TRPV1 nor mouse TRPV2 were able to co-precipitate with any of the PACSIN family members, an indication that the binding between PACSINs and TRPV4 is specific (Fig. 1, E and F). PACSIN 3, but Not PACSINs 1 and 2, Increases the Number of Cells Showing Predominant Plasma Membrane Association of TRPV4—Because the PACSINs have been implicated in endocytotic processes, we decided to investigate the subcellular distribution of TRPV4 in the presence of PACSINs. Therefore, we transfected HEK293, HeLa, and NIH 3T3 cells with expression vectors for each protein alone or in combinations. In all of the cell lines used for these studies, we obtained similar results: we noticed that the overall number of cells that displayed pronounced plasma membrane localization of TRPV4 was significantly increased from ∼20-33% to 68-77%, depending on the cell type studied, when PACSIN 3 was co-transfected (77 ± 3.3%, mean ± S.E., n = 3, in HEK293 and 67.5 ± 2.9%, n = 8, in NIH 3T3 cells). Co-transfection with PACSINs 1 and 2 did not alter the number of cells with pronounced localization of TRPV4 in the plasma membrane, which was 20-33% (19.5 ± 1.8%, n = 3, for PACSIN 1 and 30.3 ± 0.3%, n = 4, for PACSIN 2 in HEK293 and 24.2 ± 6.3%, n = 3, for PACSIN 1 and 28.6 ± 2.9%, n = 11, for PACSIN 2 in NIH 3T3 cells) (Fig. 2). Control vector co-expression also did not change the number of cells with pronounced TRPV4 plasma membrane localization (30.2 ± 1.6% in HEK293 and 32.7 ± 4.1% in NIH 3T3 cells). Our co-immunoprecipitation results suggest that neither TRPV1 nor TRPV2 interact with any PACSIN protein. Co-expression of these TRPV family members with PACSINs 2 and 3 revealed no effect on their subcellular distribution (8.9 ± 3.9%, n = 3, for PACSIN 2 and 8.1 ± 1.0%, n = 3, for PACSIN 3 co-expressed with TRPV1; 74.5 ± 1.2%, n = 3, for PACSIN 2 and 70.6 ± 3.8%, n = 4, for PACSIN 3 co-expressed with TRPV2) (Fig. 3), suggesting that the effect of PACSIN 3 is specifically targeting TRPV4 and is not the result of a general inhibition of endocytosis. PACSIN 3 Increases the Ratio of Plasma Membrane-associated TRPV4 versus Cytoplasmic TRPV4—To characterize the apparent effect of PAC

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.

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.

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.

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.

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.

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.

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.

Sensorineural hearing loss is most commonly caused by the death of hair cells in the organ of Corti, and once lost, mammalian hair cells do not regenerate. In contrast, other vertebrates such as birds can regenerate hair cells by stimulating division and differentiation of neighboring supporting cells. We currently know little of the genetic networks which become active in supporting cells when hair cells die and that are activated in experimental models of hair cell regeneration. Several studies have shown that neonatal mammalian cochlear supporting cells are able to trans-differentiate into hair cells when cultured in conditions in which the Notch signaling pathway is blocked. We now show that the ability of cochlear supporting cells to trans-differentiate declines precipitously after birth, such that supporting cells from six-day-old mouse cochlea are entirely unresponsive to a blockade of the Notch pathway. We show that this trend is seen regardless of whether the Notch pathway is blocked with gamma secretase inhibitors, or by antibodies against the Notch1 receptor, suggesting that the action of gamma secretase inhibitors on neonatal supporting cells is likely to be by inhibiting Notch receptor cleavage. The loss of responsiveness to inhibition of the Notch pathway in the first postnatal week is due in part to a down-regulation of Notch receptors and ligands, and we show that this down-regulation persists in the adult animal, even under conditions of noise damage. Our data suggest that the Notch pathway is used to establish the repeating pattern of hair cells and supporting cells in the organ of Corti, but is not required to maintain this cellular mosaic once the production of hair cells and supporting cells is completed. Our results have implications for the proposed used of Notch pathway inhibitors in hearing restoration therapies.

Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

In mammals, hearing loss is irreversible due to the lack of regenerative potential of non-sensory cochlear cells. Neonatal cochlear cells, however, can grow into organoids that harbor sensory epithelial cells, including hair cells and supporting cells. Here, we purify different cochlear cell types from neonatal mice, validate the composition of the different groups with single-cell RNA sequencing (RNA-seq), and assess the various groups’ potential to grow into inner ear organoids. We find that the greater epithelial ridge (GER), a transient cell population that disappears during post-natal cochlear maturation, harbors the most potent organoid-forming cells. We identified three distinct GER cell groups that correlate with a specific spatial distribution of marker genes. Organoid formation was synergistically enhanced when the cells were cultured at increasing density. This effect is not due to diffusible signals but requires direct cell-to-cell contact. Our findings improve the development of cell-based assays to study culture-generated inner ear cell types.

<h2>Summary</h2> Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry <i>in vivo</i> and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders.

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.

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 4α-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 4α-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. 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 4α-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 4α-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. TRPV4 2The abbreviations used are:TRPV4transient receptor potential vanilloid family member 4TMtransmembrane[Ca2+]iintracellular calcium concentrationHEK-293human embryonic kidney cell line 293HTShypotonic solutionAAarachidonic acid4α-PDD4α-phorbol 12,13-didecanoatepFpicofarad(s).2The abbreviations used are:TRPV4transient receptor potential vanilloid family member 4TMtransmembrane[Ca2+]iintracellular calcium concentrationHEK-293human embryonic kidney cell line 293HTShypotonic solutionAAarachidonic acid4α-PDD4α-phorbol 12,13-didecanoatepFpicofarad(s). is a Ca2+- and Mg2+-permeable non-selective cation channel of the vanilloid-type transient receptor potential (TRPV) channel subfamily (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (791) Google Scholar, 2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. 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A. 2004; 101: 396-401Crossref PubMed Scopus (486) Google Scholar). transient receptor potential vanilloid family member 4 transmembrane intracellular calcium concentration human embryonic kidney cell line 293 hypotonic solution arachidonic acid 4α-phorbol 12,13-didecanoate picofarad(s). transient receptor potential vanilloid family member 4 transmembrane intracellular calcium concentration human embryonic kidney cell line 293 hypotonic solution arachidonic acid 4α-phorbol 12,13-didecanoate picofarad(s). Several studies have provided evidence for regulation of TRPV4 expression and/or function by auxiliary proteins such as the AIP4 ubiquitin ligases, “With-No-K” kinase, PACSIN 3, and OS-9 (31Fu Y. Subramanya A. Rozansky D. Cohen D.M. Am. J. Physiol. 2006; 290: F1305-F1314Crossref PubMed Scopus (73) Google Scholar, 32Sidhaye V.K. Guler A.D. Schweitzer K.S. D'Alessio F. Caterina M.J. King L.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4747-4752Crossref PubMed Scopus (88) Google Scholar, 33Liu X. Bandyopadhyay B. Nakamoto T. Singh B. Liedtke W. Melvin J.E. Ambudkar I. J. Biol. Chem. 2006; 281: 15485-15495Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 34Cuajungco M.P. Grimm C. Oshima K. D'Hoedt D. Nilius B. Mensenkamp A.R. Bindels R.J. Plomann M. Heller S. J. Biol. Chem. 2006; 281: 18753-18762Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 35Wegierski T. Hill K. Schaefer M. Walz G. EMBO J. 2006; 25: 5659-5669Crossref PubMed Scopus (72) Google Scholar). The PACSIN family consists of three members, PACSIN 1–3, which have been implicated in vesicle trafficking and endocytosis, although their exact biological relevance is unclear (36Plomann M. Lange R. Vopper G. Cremer H. Heinlein U.A. Scheff S. Baldwin S.A. Leitges M. Cramer M. Paulsson M. Barthels D. Eur. J. Biochem. 1998; 256: 201-211Crossref PubMed Scopus (78) Google Scholar, 37Ritter B. Modregger J. 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In this study we reveal that PACSIN 3 not only affects the subcellular localization of TRPV4 but also modulates the sensitivity of TRPV4 to distinct stimuli. PACSIN 3 reduces the basal activity of TRPV4 and prevents activation by heat, cell swelling, and AA, whereas activation by 4α-PDD remains unaffected. Mutagenesis experiments further reveal that the modulation of TRPV4 activity requires binding of PACSIN 3 to a proline-rich region in the channel's N terminus. Our results highlight the importance of the N terminus of TRPV4 in channel gating, and suggest that PACSIN 3 has a function comparable to that of the β-subunits of voltage-gated Ca2+ and Na+ channels (40Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar, 41Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (422) Google Scholar, 42Richards M.W. Butcher A.J. Dolphin A.C. Trends Pharmacol. Sci. 2004; 25: 626-632Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Cell Culture and Transfection—HEK-293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, 2 mm l-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity controlled incubator with 10% (v/v) CO2. HEK-293 cells were transiently co-transfected with expression vectors encoding murine TRPV4 (Ensembl Gene ID: ENSMUSG00000014158) (30Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (486) Google Scholar) or murine TRPV4 mutants and PACSIN 1 (ENSMUSG00000040276), PACSIN 2 (ENSMUSG00000016664), or PACSIN 3 (ENS-MUSG00000027257) vectors in a 1:5 ratio. 12–18 h after transfections, the cells were seeded onto poly-l-lysine (Sigma)-coated coverslips and were further incubated for another 3 h before use. Measurement of [Ca2+]i—Cells were loaded with 2 μm Fura-2 acetoxymethyl ester for 20 min at 37 °C, and [Ca2+]i was measured with a monochromator-based imaging system consisting of a Polychrome IV monochromator (TILL Photonics, Martinsreid, Germany) and a Roper Scientific charge-coupled device camera connected to an Axiovert 200M inverted microscope (Zeiss, Germany). Monochromator and camera were controlled by Metafluor software (Version 6.3, Universal Imaging, Downingtown, PA). Fluorescence was measured during alternating excitation at 340 and 380 nm and corrected for the individual background fluorescence. The absolute Ca2+ concentration was obtained from the fluorescence ratios using the equation [Ca2+] = Keff(R - R0)/(R1 - R), where Keff (Keff = 2930), R0 (R0 = 0.18), and R1 (R1 = 6.0) are calibration constants. R0 and R1 were estimated by perfusing cells with Ca2+-free solution and high Ca2+ containing solution in the presence of 1 μm ionomycin, respectively. The effective binding constant, Keff, was calculated by the equation, Keff = Kd(R1 + α)/(R0 + α) with Kd, the dissociation constant of Fura-2, and α, the isocoefficient. The Kd value was taken from Paltauf-Doburzynska and Graier (43Paltauf-Doburzynska J. Graier W.F. Cell Calcium. 1997; 21: 43-51Crossref PubMed Scopus (26) Google Scholar). The isocoefficient α was obtained as described by Zhou and Neher (44Zhou Z. Neher E. J. Physiol. 1993; 469: 245-273Crossref PubMed Scopus (355) Google Scholar). The temperature of bath solutions was warmed by using a water jacket device (Warner Instruments); additionally a second external temperature sensor was used to control the bath solution. Electrophysiology—Whole cell currents were measured with an EPC-10 patch-clamp amplifier (HEKA Electronic, Lambrecht, Germany, at a sampling rate, 1 ms; 8-Pole Bessel filter, 3 kHz) using ruptured patches. Patch electrodes had a DC resistance of 2–4 MΩ when filled with intracellular solution. An Ag-AgCl wire was used as reference electrode. Capacitance and access resistance were monitored continuously. Between 50 and 70% of the series resistance (Rs = 6.3 ± 0.3 mΩ before compensation) was electronically compensated to minimize voltage errors. A ramp protocol, consisting of a voltage step from a holding of 0 mV to -100 mV followed by a 400-ms linear ramp to +100 mV, was applied. This protocol was repeated every 5 s. Cell membrane capacitance (Cs = 6.0 ± 0.3 picofarads (pF)) values were used to calculate current densities. Solutions—For electrophysiological measurements, the standard extracellular solution contained (in mm): 150 NaCl, 6 CsCl, 1 MgCl2, 5 CaCl2, 10 glucose, 10 HEPES, buffered at pH 7.4 with NaOH. The Ca2+-free pipette solution was composed of (in mm): 20 CsCl, 100 Cs-Asp, 1 MgCl2, 10 HEPES, 4 Na2ATP, 10 BAPTA, buffered at pH 7.2 with CsOH. For measuring cell swelling-activated currents, we used a isotonic solution containing (in mm): 80 NaCl, 6 CsCl, 1.5 CaCl2, 1 MgCl2, 10 Hepes, 90 d-mannitol, 10 glucose, pH 7.4, resulting in 320 ± 10 mosmol. Cell swelling was induced by removing d-mannitol from the solution (giving 245 ± 10 mosm, a 25% reduction in osmolarity). The standard solution for calcium imaging experiments consisted of (in mm): 150 NaCl, 6 CsCl, 1 MgCl2, 1.5 CaCl2, 10 Hepes, 10 glucose, buffered at a pH 7.4 with NaOH. The non-protein kinase C-activating phorbol ester, 4α-phorbol 12,13-dideconate (4α-PDD, Sigma) was applied at a 1 μm concentration from a 10 mm stock solution in ethanol. Arachidonic acid (Sigma) was used at a final concentration of 10 μm froma 10 mm stock solution in Me2SO. Capsaicin was applied at a 100 nm concentration of a 1 mm stock solution in ethanol. Data Analysis—Electrophysiological data were analyzed by using Patchmaster software (HEKA Elektronic, Lambrecht, Germany). Origin 6.1 software was used for statistical analyses and data display of electrophysiological and calcium imaging experiments. Data are expressed as mean ± S.E. Statistical analysis was performed with the Student's t test. ROBETTA Modeling—The N-terminal 470 amino acids of TRPV4 were used for fully automated prediction of the three-dimensional structure using the ROBETTA server (45Chivian D. Kim D.E. Malmstrom L. Bradley P. Robertson T. Murphy P. Strauss C.E. Bonneau R. Rohl C.A. Baker D. Proteins. 2003; 53: 524-533Crossref PubMed Scopus (246) Google Scholar, 46Kim D.E. Chivian D. Baker D. Nucleic Acids Res. 2004; 32: W526-W531Crossref PubMed Scopus (1317) Google Scholar). The ROSETTA fragment insertion method was used to provide both ab initio and comparative models of protein domains (47Simons K.T. Kooperberg C. Huang E. Baker D. J. Mol. Biol. 1997; 268: 209-225Crossref PubMed Scopus (1113) Google Scholar). Comparative models were built from structures detected by PSI-BLAST or 3DJury-A1 and aligned by the K*Sync alignment method (48Chivian D. Baker D. Nucleic Acids Res. 2006; 34: e112Crossref PubMed Scopus (90) Google Scholar). The domain parsing and -fold detection were achieved using the Ginzu method (45Chivian D. Kim D.E. Malmstrom L. Bradley P. Robertson T. Murphy P. Strauss C.E. Bonneau R. Rohl C.A. Baker D. Proteins. 2003; 53: 524-533Crossref PubMed Scopus (246) Google Scholar, 49Kim D.E. Chivian D. Malmstrom L. Baker D. Proteins. 2005; 61: 193-200Crossref PubMed Scopus (75) Google Scholar). Loop regions were assembled from fragments and optimized to fit the aligned template structure (50Rohl C.A. Strauss C.E. Chivian D. Baker D. Proteins. 2004; 55: 656-677Crossref PubMed Scopus (276) Google Scholar). PACSIN 3 Affects Heat-induced Activation of TRPV4—In a previous study it was shown that interaction with PACSIN 3 affects the subcellular distribution of TRPV4, which results in an apparent increase of the relative plasma membrane association of the channel (34Cuajungco M.P. Grimm C. Oshima K. D'Hoedt D. Nilius B. Mensenkamp A.R. Bindels R.J. Plomann M. Heller S. J. Biol. Chem. 2006; 281: 18753-18762Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). It remained untested whether PACSIN 3 affects channel gating. In this study, we show that PACSIN 3 co-immunoprecipitates endogenous TRPV4 from mouse kidney lysates, and we assess the interaction of various TRPV4 mutants with either PACSIN 2 or PACSIN 3 (supplemental Figs. S1 and S2). Finally, we used intracellular Ca2+ ([Ca2+]i) measurements and whole cell patch clamp recordings to evaluate the effects of PACSIN 3 on basal TRPV4 activity and on the response to moderate heat, hypotonic cell swelling, and AA. Transient expression of TRPV4 in HEK-293 cells results in spontaneous channel activity, which has been attributed to partial heat-activation at room temperature, and which leads to a significant but variable increase in basal [Ca2+]i (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (791) Google Scholar, 2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 12Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 47044-47051Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 30Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (486) Google Scholar). Interestingly, co-expression of PACSIN 3 together with TRPV4 resulted in basal [Ca2+]i levels that were significantly lower than in cells expressing TRPV4 alone, and similar to the level observed in non-transfected HEK-293 cells, suggesting that PACSIN 3 inhibits spontaneous activity of TRPV4. Heating the bath solution from 22 to 42 °C resulted in a robust increase of [Ca2+]i in TRPV4-expressing cells, which reverted to the baseline level when the temperature was brought back to 22 °C (Fig. 1A, for average [Ca2+]i values see Fig. 4), which is in agreement with previous studies (12Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 47044-47051Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 22Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar, 30Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (486) Google Scholar). Subsequent application of 1 μm 4α-PDD to the same cells induced a second increase in [Ca2+]i. In contrast, co-transfection of PACSIN 3 fully abolished the [Ca2+]i response to heat, whereas the response to a subsequent 4α-PDD application remained unchanged (Fig. 1B, for average [Ca2+]i values see Fig. 4, B and E). Similar results were obtained in whole cell patch clamp experiments. In TRPV4-transfected cells, heating to 42 °C evoked typical TRPV4 currents (amplitude (n = 8): -47 ± 10 pA/pF (-80 mV) and 95 ± 6 pA/pF (80 mV); Fig. 1, C and E), whereas no heat-activated currents could be detected when PACSIN 3 was co-expressed with TRPV4 (amplitude (n = 6): -14.5 ± 4.5 pA/pF (-80 mV) and 25.1 ± 3.2 pA/pF (80 mV), p < 0.05, Fig. 1, D and F). TRPV1, TRPV2, and TRPV3 are closely related heat-activated channels, but lack the prolinerich domain required for the interaction of TRPV4 with PAC-SIN 3. In line therewith, we found that co-expression of PAC-SIN 3 had no effect on the sensitivity of TRPV1 to heat or capsaicin (100 nm) (Fig. 2). These results demonstrate that the effect of PACSIN 3 on heat-induced activation of TRPV4 is not a general property of heat-activated TRP channels.FIGURE 4Effect of various stimuli on intracellular calcium in TRPV4 alone and TRPV4 co-expressed with PACSIN 3-expressing cells. A, basal [Ca2+]i levels in non-transfected cells, and cells transfected with TRPV4 and TRPV4 with PACSIN3. B–E, average [Ca2+]i increases induced by 4α-PDD (B), HTS (C), AA (D), and heat (E) in non-transfected cells and in cells transfected with TRPV4, or TRPV4 with PACSIN 3 (n = 30–50). ***, p < 0.005, significant difference compared with WT TRPV4-expressing cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2Effect of PACSIN 3 on TRPV1-expressing cells. A and B, [Ca2+]i response to heat and capsaicin (100 nm) in cells expressing TRPV1 (A) and TRPV1 with PACSIN 3 (B). C, basal [Ca2+]i level in non-transfected cells (n = 40), cells expressing TRPV1 (n = 36) and TRPV1 with PACSIN 3 (n = 32). D, average [Ca2+]i increase in response to heat in non-transfected cells, TRPV1, and TRPV1 with PACSIN 3-transfected cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activation of TRPV4 by Cell Swelling Is Affected by PACSIN 3—Both in [Ca2+]i measurement and in whole cell recordings, application of a 25% hypotonic solution (HTS) evoked a robust response in TRPV4-expressing cells (Figs. 3A, 3B, and 4C). Strikingly, co-expression of PACSIN 3 completely abolished the response to HTS (Figs. 3D, 3E, and 4C) (after HTS stimulation; [Ca2+]i for TRPV4 (n = 20): 316 ± 20 nm, for TRPV4 plus PACSIN 3 (n = 30): 100 ± 19 nm, p < 0.005; amplitude for TRPV4 (n = 5): -52 ± 15 pA/pF (-80 mV) and 68 ± 14 pA/pF (80 mV), for TRPV4 plus PACSIN 3 (n = 5): -7 ± 2 pA/pF (-80 mV) and 13 ± 2 pA/pF (80 mV), p < 0.05). The mechanism for TRPV4 activation by cell swelling is distinct from heat- and 4α-PDD-induced channel activation. We have previously shown that swelling-induced activation of TRPV4 occurs via the PLA2-induced production of AA, which is further metabolized to epoxyeicosatrienoic acids that activate the channel in a membrane-delimited manner (28Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (795) Google Scholar, 29Vriens J. Owsianik G. Fisslthaler B. Suzuki M. Janssens A. Voets T. Morisseau C. Hammock B.D. Fleming I. Busse R. Nilius B. Circ. Res. 2005; 97: 908-915Crossref PubMed Scopus (305) Google Scholar, 30Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (486) Google Scholar). Therefore, one possible explanation of the lack of HTS-induced TRPV4 activation could be a direct inhibitory effect of PACSIN 3 on the generation of AA. However, direct extracellular application of AA, which evokes robust [Ca2+]i in TRPV4-expressing cells (28Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (795) Google Scholar, 29Vriens J. Owsianik G. Fisslthaler B. Suzuki M. Janssens A. Voets T. Morisseau C. Hammock B.D. Fleming I. Busse R. Nilius B. Circ. Res. 2005; 97: 908-915Crossref PubMed Scopus (305) Google Scholar, 30Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (486) Google Scholar) (Figs. 3C and 4D), was without effect when PACSIN 3 was co-expressed (Figs. 3F and 4D). These experiments demonstrate that PACSIN 3 has a strong inhibitory effect on the basal TRPV4 activity and on its response to heat and cell swelling, whereas the responsiveness to 4α-PDD remains conserved. Previous studies have shown that [Ca2+]i has a strong modulatory effect on TRPV4 function and can cause both potentiation and inhibition of TRPV4 function (51Watanabe H. Vriens J. Janssens A. Wondergem R. Droogmans G. Nilius B. Cell Calcium. 2003; 33: 489-495Crossref PubMed Scopus (102) Google Scholar, 52Strotmann R. Schultz G. Plant T.D. J. Biol. Chem. 2003; 278: 26541-26549Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). This raised the question whether the strong inhibition of HTS-induced activation of TRPV4 in cells co-expressing PACSIN 3 would be related to the reduced basal [Ca2

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.

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.

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.

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.

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.

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 Axin2lacZ 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 Axin2lacZ 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.

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.

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.

Abstract Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.

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.

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.

Sensorineural hearing loss is the most common sensory deficit in humans. Despite the global scale of the problem, only limited treatment options are available today. The mammalian inner ear is a highly specialized postmitotic organ, which lacks proliferative or regenerative capacity. Since the discovery of hair cell regeneration in non-mammalian species however, much attention has been placed on identifying possible strategies to reactivate similar responses in humans. The development of successful regenerative approaches for hearing loss strongly depends on a detailed understanding of the mechanisms that control human inner ear cellular specification, differentiation and function, as well as on the development of robust in vitro cellular assays, based on human inner ear cells, to study these processes and optimize therapeutic interventions. We summarize here some aspects of inner ear development and strategies to induce regeneration that have been investigated in rodents. Moreover, we discuss recent findings in human inner ear development and compare the results with findings from animal models. Finally, we provide an overview of strategies for in vitro generation of human sensory cells from pluripotent and somatic progenitors that may provide a platform for drug development and validation of therapeutic strategies in vitro.

The utricle is a vestibular sensory organ that requires mechanosensitive hair cells to detect linear acceleration. In neonatal mice, new hair cells are derived from non-sensory supporting cells, yet cell type diversity and mechanisms of cell addition remain poorly characterized. Here, we perform computational analyses on single-cell transcriptomes to categorize cell types and resolve 14 individual sensory and non-sensory subtypes. Along the periphery of the sensory epithelium, we uncover distinct groups of transitional epithelial cells, marked by Islr, Cnmd, and Enpep expression. By reconstructing de novo trajectories and gene dynamics, we show that as the utricle expands, Islr+ transitional epithelial cells exhibit a dynamic and proliferative phase to generate new supporting cells, followed by coordinated differentiation into hair cells. Taken together, our study reveals a sequential and coordinated process by which non-sensory epithelial cells contribute to growth of the postnatal mouse sensory epithelium.

In contrast to mammals, birds recover naturally from acquired hearing loss, which makes them an ideal model for inner ear regeneration research. Here, we present a validated single-cell RNA sequencing resource of the avian cochlea. We describe specific markers for three distinct types of sensory hair cells, including a previously unknown subgroup, which we call superior tall hair cells. We identify markers for the supporting cells associated with tall hair cells, which represent the facultative stem cells of the avian inner ear. Likewise, we present markers for supporting cells that are located below the short cochlear hair cells. We further infer spatial expression gradients of hair cell genes along the tonotopic axis of the cochlea. This resource advances neurobiology, comparative biology, and regenerative medicine by providing a basis for comparative studies with non-regenerating mammalian cochleae and for longitudinal studies of the regenerating avian cochlea.

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.

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 β subunit (Gβ5). RGS9·Gβ5 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 Gβ5 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·Gβ5 complex, and therefore all three proteins, RGS9, Gβ5, and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP. 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 β subunit (Gβ5). RGS9·Gβ5 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 Gβ5 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·Gβ5 complex, and therefore all three proteins, RGS9, Gβ5, and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP. Timely termination of the light response in retinal photoreceptors is essential for normal vision (reviewed in Refs. 1.Burns M.E. Baylor D.A. Annu. Rev. Neurosci. 2001; 24: 779-805Crossref PubMed Scopus (334) Google Scholar and 2.Arshavsky V.Y. Lamb T.D. Pugh Jr, E.N. Annu. Rev. Physiol. 2002; 64: 153-187Crossref PubMed Scopus (506) Google Scholar). On the molecular level, the normal time course of the light response requires rapid deactivation of the G protein transducin, which relays the visual signal to the effector, cyclic GMP phosphodiesterase. Deactivation of transducin occurs when the transducin α subunit hydrolyzes its bound GTP. In normal rods, GTP hydrolysis is catalyzed by the complex of the regulator of G protein signaling protein (RGS9) 1The abbreviations used are: RGS9, regulator of G protein signaling protein; Gβ5, type 5 G protein β subunit; R9AP, RGS9 anchor protein; DEP, disheveled/Egl-10/pleckstrin. with type 5 G protein β subunit (Gβ5) (reviewed in Refs. 2.Arshavsky V.Y. Lamb T.D. Pugh Jr, E.N. Annu. Rev. Physiol. 2002; 64: 153-187Crossref PubMed Scopus (506) Google Scholar and 3.Cowan C.W. He W. Wensel T.G. Prog. Nucleic Acids Res. Mol. Biol. 2000; 65: 341-359Crossref Google Scholar). Recent studies have demonstrated that photoreceptors lacking RGS9 or Gβ5 produce light responses that recover at an abnormally slow rate (4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar, 5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar). In photoreceptors, the RGS9·Gβ5 complex is tightly associated with the transmembrane protein R9AP (RGS9 anchor protein), which anchors RGS9·Gβ5 on the surface of the disc membranes of the outer segment, which is the subcellular compartment where visual transduction occurs (6.Hu G. Wensel T.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9755-9760Crossref PubMed Scopus (144) Google Scholar, 7.Lishko P.V. Martemyanov K.A. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2002; 277: 24376-24381Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 8.Hu G. Zhang Z. Wensel T.G. J. Biol. Chem. 2003; 278: 14550-14554Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). R9AP is a 25-kDa protein structurally related to members of the SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) protein family, which are involved in vesicular trafficking and exocytosis (8.Hu G. Zhang Z. Wensel T.G. J. Biol. Chem. 2003; 278: 14550-14554Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9.Keresztes G. Mutai H. Hibino H. Hudspeth A.J. Heller S. Mol. Cell. Neurosci. 2003; 24: 687-695Crossref PubMed Scopus (26) Google Scholar, 10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar). In mammals, R9AP is expressed predominantly in the retina (6.Hu G. Wensel T.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9755-9760Crossref PubMed Scopus (144) Google Scholar, 9.Keresztes G. Mutai H. Hibino H. Hudspeth A.J. Heller S. Mol. Cell. Neurosci. 2003; 24: 687-695Crossref PubMed Scopus (26) Google Scholar), whereas in chicken it is also present in cochlear hair cells and dorsal root ganglion neurons (9.Keresztes G. Mutai H. Hibino H. Hudspeth A.J. Heller S. Mol. Cell. Neurosci. 2003; 24: 687-695Crossref PubMed Scopus (26) Google Scholar). R9AP dramatically enhances the ability of RGS9·Gβ5 to stimulate transducin GTPase (7.Lishko P.V. Martemyanov K.A. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2002; 277: 24376-24381Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 8.Hu G. Zhang Z. Wensel T.G. J. Biol. Chem. 2003; 278: 14550-14554Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar) and participates in the delivery of RGS9·Gβ5 to photoreceptor outer segment (10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar). In this study, we analyzed visual signaling in rods of R9AP knockout mice. The knockout did not affect the overall retinal morphology or photoreceptor development. However, light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gβ5 knockout rods. The effect of the R9AP knockout on the photoresponse recovery was explained by a complete absence of any detectable RGS9 in the retinas of 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 RGS9·Gβ5, and therefore R9AP should be considered an essential component of the GTPase-activating complex for transducin. Generation of the R9AP Knockout Mouse—Primers specific for the coding region of the mouse R9AP gene, Rgs9–1bp, (forward, 5′-GCGCGGCTCGTCTTGGAGAC-3′; reverse, 5′-CAGAGGTTTCAGAGCCTGGTTCC-3′) were used for PCR screen of a 129/SvJ mouse bacterial artificial chromosome genomic library (Genome Systems, St. Louis, MO) for a clone containing the complete Rgs9–1bp gene with the flanking sequences. The targeting vector contained 2 kb of PCR-amplified genomic sequence directly upstream of the Rgs9–1bp start codon followed by a 6.1-kb cassette containing the tau-lacZ reporter gene and the neomycin resistance gene, which are flanked by lox sites, and a 2.8-kb genomic sequence directly downstream of the stop codon of the gene. The vector was used to transfect E14 embryonic stem cells (11.Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (925) Google Scholar). The targeting of the Rgs9–1bp locus was confirmed by screening genomic the DraI-digested DNA of G418-resistant embryonic stem cell clones for homologous recombination using a Southern probe specific to sequence outside of the targeted region (Fig. 1A). After germ line transmission was obtained, animals heterozygous for the targeted allele were crossed to create R9AP knockout animals. Analysis was performed in mice that showed 100% penetrance of the retinal phenotypes described in a mixed (129/SvJ x C57BL/6) background. Southern Blot Analysis—Genomic DNA was extracted from mouse tails, and 10 μg of DNA was digested overnight with DraI, electrophoretically fractionated in 0.6% agarose gel, denatured, and transferred to a nylon membrane (Hybond-N, AP Biotech). Hybridization was done overnight at 62 °C in hybridization solution (ExpressHyb, Clontech) including 10 μg/ml denatured herring sperm DNA. The membrane was washed twice with 2× SSC, 0.1% SDS at 62 °C and twice with 0.2× SSC, 0.1% SDS at 62 °C for 10–15 min each. Primers for generating the Southern probe were: forward, 5′-CAAAATCATTGAGCGGCACC-3′; and reverse, 5′-AGTATTGGAGAGGTCACTTG-3′. Northern Blot Analysis—Denatured total RNA was isolated from retinas with Trizol reagent (Invitrogen). After separation on 0.8% formaldehyde-agarose gels, RNA was transferred to nylon membrane (Hybond-N, AP Biotech) and incubated at 60 °C with RGS9-specific probe in hybridization solution (ExpressHyb, Clontech). The probe was generated by PCR amplification of the 1–1300 region of the RGS9 gene and labeled with 32P by random priming. After the membranes were washed with 0.1% SDS in 0.2× SSC at 68 °C, the blots were exposed to XAR-5 film (Eastman Kodak). Western Blot Analysis—Two mouse retinas were removed from the eyes, placed in 100 μl of deionized water, and homogenized by sonication. Rhodopsin concentration in retinal homogenates was determined spectrophotometrically from the difference in the absorption at 500 nm before and after bleaching the sample using the extinction coefficient of ϵ500 = 40500. Samples containing 20 pmol of rhodopsin were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. For protein detection, membranes were incubated with one of the following antibodies: rabbit antibody against the R9AP-(102–144) fragment (10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar), sheep anti-RGS9c antibody (12.Makino E.R. Handy J.W. Li T. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1947-1952Crossref PubMed Scopus (195) Google Scholar), sheep anti-Gβ5 NTL antibody (12.Makino E.R. Handy J.W. Li T. Arshavsky V.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1947-1952Crossref PubMed Scopus (195) Google Scholar), and rabbit anti-Gαt1 antibody (Santa Cruz Biotechnology). After incubation with horseradish peroxidase-conjugated secondary antibodies, the signals were detected using the West Pico ECL Western blot detection system (Pierce). Preparation of Plastic-embedded Cross-sections of the Retina—Eyes were enucleated, cleaned of outside tissue, and fixed for 1 h in freshly prepared 2% paraformaldehyde with 2.5% glutaraldehyde in 0.1 m cacodylate buffer containing 2.5 mm CaCl2 (pH 7.4). The eye globe was then hemisected along the vertical meridian and allowed to fix overnight at the same buffer. The eye cup was rinsed with excess 0.1 m cacodylate buffer (pH 7.4) and placed into 2% osmium tetroxide. The eye cup was gradually dehydrated in an increasing ethanol series (25–100%) and embedded in Epon. 1-μm cross-sections were obtained and stained with alkaline toluidine blue for light microscopy. Suction Electrode Recordings—Mice were housed in 12-h cyclic light conditions and dark-adapted overnight before an experiment. Under infrared light, animals were anesthetized and euthanized, and the retinas were removed and stored in Leibovitz's L-15 medium (Invitrogen) with 10 mm glucose and 0.1 mg/ml bovine serum albumin on ice. Small pieces of retina were placed in the recording chamber and perfused with bicarbonate-buffered Locke's solution, bubbled with 95% O2, 5% CO2, and warmed to 35–37 °C (pH 7.4). Responses to flashes (500 nm, 10 ms) were recorded from individual rods using suction electrodes as described (5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar). Briefly, individual outer segments were drawn into a glass pipette containing 140 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl2, 1.2 mm CaCl2, 3 mm HEPES, 0.2 mm EDTA, and 10 mm glucose (pH 7.4). The bath and suction electrodes were connected to calomel half-cells by agar bridges, and the bath voltage was maintained at ground potential by an active feedback circuit. The rod membrane current was amplified (Axopatch 1B, Axon Instruments, Union City, CA) and filtered at 20 Hz with an 8-pole Bessel filter. Data were digitized continuously at 200 Hz using NiDAQ (National Instruments, Austin, TX) for IgorPro (Wavemetrics, Lake Oswego, OR). Light intensities were controlled with neutral density filters and calibrated daily (United Detector Technology, Baltimore, MD). The average response to a high number (>30) of flashes was considered to be a dim flash response (linear response) if its mean amplitude was less than 20% of the maximal response amplitude. These dim flash responses were used to estimate the form of the single photon response using the “variance to mean” method as described previously (13.Mendez A. Burns M.E. Roca A. Lem J. Wu L.W. Simon M.I. Baylor D.A. Chen J. Neuron. 2000; 28: 153-164Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Integration time, used as a measure of the duration of the dim flash response, is defined as the time integral of the average linear response divided by its peak amplitude (14.Baylor D.A. Hodgkin A.L. J. Physiol. 1973; 234: 163-198Crossref PubMed Scopus (293) Google Scholar). The time that a bright flash response remained in saturation was calculated as the time interval between the midpoint of the flash and the time at which the current recovered by 10%. Generation and Characterization of the R9AP Knockout Mouse—To test the function of R9AP in mice, we induced a null mutation into the R9AP gene (Rgs9–1bp) by gene targeting (Fig. 1A). We replaced the complete gene with a reporter gene and a neomycin resistance gene by homologous recombination in embryonic stem cells. The targeting of the Rgs9–1bp locus was confirmed by Southern blot analysis of DraI-digested genomic DNA (Fig. 1B) and by PCR (not shown). After germ line transmission was obtained, the animals heterozygous for the targeted allele were intercrossed to generate R9AP knockout mice. Mice that were homozygously lacking Rgs9–1bp were viable and fertile and displayed no obvious behavioral abnormalities. They also had normal retinal morphology up to at least 2 months of age (Fig. 2A). The total amount of rhodopsin in their retinas was also normal (384 ± 89 pmol/retina versus 406 ± 103 pmol/retina in wild type mice; S.E., n = 2). The Absence of RGS9 from the Retinas of R9AP Knockout Mice—We analyzed the effects of the R9AP knockout on the expression of the proteins constituting the GTPase-activating complex for transducin. No detectable amount of R9AP was present in the retinas of R9AP knockout animals, consistent with targeted disruption of the R9AP locus in the genome (Fig. 2B). Strikingly, the retinas lacking R9AP also lacked any detectable amount of RGS9, and had significantly reduced levels of Gβ5. The amounts of R9AP, RGS9, and Gβ5 in the retinas of R9AP+/– heterozygous animals bearing only one functional R9AP allele were about one-half of that present in their wild type littermates. In contrast, the amount of transducin in the retinas of knockout and heterozygous animals was normal. Our data indicate that R9AP is required for the expression of RGS9 and Gβ5. This is similar to the lack of Gβ5 expression in RGS9 knockout mice (4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar) and the reciprocal lack of RGS9 expression in Gβ5 knockouts (15.Chen C.K. Eversole-Cire P. Zhang H.K. Mancino V. Chen Y.J. He W. Wensel T.G. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6604-6609Crossref PubMed Scopus (177) Google Scholar). In both RGS9 and Gβ5 knockout mice, this regulation was not caused by a reduction of Gβ5 or RGS9 mRNA and was argued to occur on the posttranslational level. To test whether the absence of R9AP affects RGS9 expression in a similar fashion, we compared RGS9 mRNA levels in retinas of R9AP knockout mice and their wild type littermates. As shown in Fig. 2C, these mRNA levels were indeed similar. Electrophysiological Properties of the Rods from R9AP Knockout Mice—To study the effects of inactivating the R9AP gene on the electrophysiological properties of intact rods, we used suction electrodes to record rod responses to flashes of light. A representative family of responses from a R9AP knockout rod is shown in Fig. 3A. On average, R9AP knockout rods displayed normal maximal response amplitudes and normal sensitivity to light (Table I). However, the responses of R9AP knockout rods were slow to recover, similar to the responses of rods with known defects in transducin deactivation, such as those of RGS9 and Gβ5 knockout mice (4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar, 5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar).Table IElectrophysiological properties of R9AP knockout rodsStrainMaximal amplitudeIoIntegration timeDim flash τrecTime to peakElementary amplitudeBright flash τrecpAphotons/μm2ssmspAsR9AP-/-11.2 ± 1.0 (15)54 ± 92.2 ± 0.42.0 ± 0.5282 ± 620.54 ± 0.099.3 ± 0.5(10)(11)(11)(11)(11)(13)Wild type11.3 ± 0.5 (34)70 ± 50.26 ± 0.020.17 ± 0.0289 ± 30.34 ± 0.040.19 ± 0.01(27)(30)(28)(30)(28)(21) Open table in a new tab To quantify the effect on photoresponse kinetics, we measured the time to peak, the duration, and the recovery time constant of responses of many R9AP knockout rods. The average duration of the dim flash response, as measured by integration time (see “Experimental Procedures”), was on the order of 2 s (Table I). This is 10-fold slower than the integration time that has been observed for dim flash responses of wild type mouse rods but indistinguishable from the integration times measured for both RGS9 and Gβ5 knockout rods (4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar, 5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar). The recovery time constant was assessed by fitting a single exponential curve to the falling phases of the average dim flash response. The average time constant of this exponential recovery was 2.0 ± 0.5 s (Fig. 3; Table I), very similar to the time constant of recovery observed in RGS9 (2.6 ± 0.3 s, from Ref. 4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar) and Gβ5 (2.5 ± 0.2 s, from Ref. 5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar) knockout rods. It is well known that the recovery of responses of rods lacking RGS9 and Gβ5 slows further as the flash strength increases (4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar, 5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar, 10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar). R9AP knockout rods also showed this phenomenon. For bright flashes that produce saturating responses, the dominant time constant of recovery can be determined by measuring the slope of the dependence of saturation time (Tsat) on the natural log of the flash strength (16.Pepperberg D.R. Cornwall M.C. Kahlert M. Hofmann K.P. Jin J. Jones G.J. Ripps H. Visual Neurosci. 1992; 8: 9-18Crossref PubMed Scopus (161) Google Scholar). We found that in R9AP knockout rods, the dominant time constant of recovery, τD, was 9.3 ± 0.5 s (Fig. 3C; Table I), indistinguishable from the τD values reported for both RGS9 (8.99 ± 0.22 s, from Ref. 4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar) and Gβ5 knockout rods (8.8 ± 0.3 s, from Ref. 5.Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar). The striking similarity of the R9AP knockout responses to those of the RGS9 and Gβ5 knockouts indicates that inactivating the R9AP gene has the same functional consequences as inactivating either the catalyst, RGS9, or its binding partner, Gβ5. We conclude that all three proteins, RGS9, Gβ5, and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP. In this study we report that knocking out the gene for R9AP results in a functional knockout of RGS9, which is evidenced by the lack of RGS9 protein in photoreceptors and abnormally slow recovery of the light response. In addition, the reduction in the R9AP protein level in the retinas of heterozygous mice causes a proportional reduction of RGS9·Gβ5. Provided that the levels of RGS9 mRNA were not affected by the R9AP knockout, the most plausible explanation for these effects is that the association of RGS9 with R9AP plays a crucial role in stabilizing the entire GTPase-activating complex and that this interaction can determine the amount of functionally active RGS9·Gβ5 in the cell. These data also suggest that all RGS9·Gβ5 in rods is present as the complex with R9AP, making it unlikely that photoreceptors contain less R9AP than RGS9 as suggested previously (6.Hu G. Wensel T.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9755-9760Crossref PubMed Scopus (144) Google Scholar). In principle, one could argue that the absence of RGS9 in the knockout may be explained by a drastically reduced translation of RGS9 mRNA without R9AP. However, this is unlikely because RGS9 mRNA is efficiently translated without R9AP in several eukaryotic protein expression systems (17.He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18.Skiba N.P. Martemyanov K.A. Elfenbein A. Hopp J.A. Bohm A. Simonds W.F. Arshavsky V.Y. J. Biol. Chem. 2001; 276: 37365-37372Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19.Granneman J.G. Zhai Y. Zhu Z. Bannon M.J. Burchett S.A. Schmidt C.J. Andrade R. Cooper J. Mol. Pharmacol. 1998; 54: 687-694PubMed Google Scholar). Interestingly, the effect of the R9AP knockout on the expression level of RGS9 is not entirely reciprocal. RGS9 knockout causes only a modest reduction in the R9AP levels and does not affect the delivery of R9AP to rod outer segments (10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar). There is also a difference in the levels of Gβ5 in R9AP and RGS9 knockouts. Although undetectable in the photoreceptors of RGS9 knockout (4.Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (334) Google Scholar), an appreciable fraction of Gβ5 is present in the R9AP knockout photoreceptors (Fig. 2B). This is somewhat curious because the stability of the Gβ5 molecule is known to be dependent on its binding to the G protein γ subunit-like (GGL) domain of RGS9 (17.He W. Lu L.S. Zhang X. El Hodiri H.M. Chen C.K. Slep K.C. Simon M.I. Jamrich M. Wensel T.G. J. Biol. Chem. 2000; 275: 37093-37100Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18.Skiba N.P. Martemyanov K.A. Elfenbein A. Hopp J.A. Bohm A. Simonds W.F. Arshavsky V.Y. J. Biol. Chem. 2001; 276: 37365-37372Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). One possible explanation is that in the R9AP knockout, RGS9 and Gβ5 interact with one another prior to the degradation of RGS9 and that this early interaction somehow makes Gβ5 more resistant to subsequent degradation. To the contrary, no Gβ5 is ever formed in photoreceptors of RGS9 knockout mice, perhaps because the RGS9·Gβ5 complex is never formed. Along with the results reported in our other recent study (10.Martemyanov K.A. Lishko P.V. Calero N. Keresztes G. Sokolov M. Strissel K.J. Leskov I.B. Hopp J.A. Kolesnikov A.V. Chen C.-K. Lem J. Heller S. Burns M.E. Arshavsky V.Y. J. Neurosci. 2003; 23: 10175-10181Crossref PubMed Google Scholar), our data allow us to define the part of the RGS9 molecule that is primarily responsible for targeting unanchored RGS9 for degradation. Previously, we expressed RGS9 lacking the N-terminal DEP (disheveled/Egl–10/pleckstrin) domain in the rods of RGS9 knockout mice. This RGS9 mutant was not able to interact with R9AP (see also Refs. 7.Lishko P.V. Martemyanov K.A. Hopp J.A. Arshavsky V.Y. J. Biol. Chem. 2002; 277: 24376-24381Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar and 8.Hu G. Zhang Z. Wensel T.G. J. Biol. Chem. 2003; 278: 14550-14554Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) but was not degraded. The results of the present study suggest that the DEP domain per se plays a decisive role in the cellular fate of the RGS9 molecule. The DEP domain binding to R9AP allows RGS9 survival and delivery to the site of its function in the rod outer segment. Conversely, we suggest that the presence of exposed DEP domain lacking its R9AP partner affects RGS9 expression, most likely by leading to degradation of RGS9. Finally, the strong expression of R9AP in avian hair cells (9.Keresztes G. Mutai H. Hibino H. Hudspeth A.J. Heller S. Mol. Cell. Neurosci. 2003; 24: 687-695Crossref PubMed Scopus (26) Google Scholar) led us to address whether R9AP knockouts display deficiencies of the inner ear function. Our initial assessment of auditory brainstem responses and distortion product otoacoustic emissions (20.Maison S.F. Luebke A.E. Liberman M.C. Zuo J. J. Neurosci. 2002; 22: 10838-10846Crossref PubMed Google Scholar) did not reveal any significant differences between the knockout and wild type littermates (data not shown). This indicates that cochlear mechanics, transduction, and synaptic transmission at low and moderate sound levels are not significantly altered by the R9AP knockout. These results are consistent with our comparative analysis of R9AP expression, which is detectable in several neural cell types of the chicken but is restricted to photoreceptors in the mouse (9.Keresztes G. Mutai H. Hibino H. Hudspeth A.J. Heller S. Mol. Cell. Neurosci. 2003; 24: 687-695Crossref PubMed Scopus (26) Google Scholar). We are grateful to Dr. A. J. Hudspeth for supporting the initial stages of this work and Drs. P. Feinstein and C. Yang for help in generating the R9AP knockout mouse.

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 β-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.

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.

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.

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.

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.

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.

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.

<h2>Summary</h2> Protruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics. Utilizing quantitative gene-expression data for key bundle proteins from single cells of the developing chick utricle, we reconstructed <i>de novo</i> a bifurcating trajectory that spanned from progenitor cells to mature striolar and extrastriolar hair cells. Extraction and alignment of developmental trails and association of pseudotime with bundle length measurements linked expression dynamics of individual genes with bundle growth stages. Differential trail analysis revealed high-resolution dynamics of transcripts that control striolar and extrastriolar bundle development, including those that encode proteins that regulate [Ca²⁺]_i or mediate crosslinking and lengthening of actin filaments.

Sensory hair cells are prone to apoptosis caused by various drugs including aminoglycoside antibiotics. In mammals, this vulnerability results in permanent hearing loss because lost hair cells are not regenerated. Conversely, hair cells regenerate in birds, making the avian inner ear an exquisite model for studying ototoxicity and regeneration. Here, we use single-cell RNA sequencing and trajectory analysis on control and dying hair cells after aminoglycoside treatment. Interestingly, the two major subtypes of avian cochlear hair cells, tall and short hair cells, respond differently. Dying short hair cells show a noticeable transient upregulation of many more genes than tall hair cells. The most prominent gene group identified is associated with potassium ion conductances, suggesting distinct physiological differences. Moreover, the dynamic characterization of >15,000 genes expressed in tall and short avian hair cells during their apoptotic demise comprises a resource for further investigations toward mammalian hair cell protection and hair cell regeneration.

ABSTRACT The avian hearing organ is the basilar papilla that, in sharp contrast to the mammalian cochlea, can regenerate sensory hair cells and thereby recover from deafness within weeks. The mechanisms that trigger, sustain and terminate the regenerative response in vivo are largely unknown. Here, we profile the changes in gene expression in the chicken basilar papilla after aminoglycoside antibiotic-induced hair cell loss using RNA-sequencing. We identified changes in gene expression of a group of immune-related genes and confirmed with single-cell RNA-sequencing that these changes occur in supporting cells. In situ hybridization was used to further validate these findings. We determined that the JAK/STAT signaling pathway is essential for upregulation of the damage-response genes in supporting cells during the second day after induction of hair cell loss. Four days after ototoxic damage, we identified newly regenerated, nascent auditory hair cells that express genes linked to termination of the JAK/STAT signaling response. The robust, transient expression of immune-related genes in supporting cells suggests a potential functional involvement of JAK/STAT signaling in sensory hair cell regeneration.

Hearing starts, at the cellular level, with mechanoelectrical transduction by sensory hair cells. Sound information is then transmitted via afferent synaptic connections with auditory neurons. Frequency information is encoded by the location of hair cells along the cochlear duct. Loss of hair cells, synapses, or auditory neurons leads to permanent hearing loss in mammals. Birds, in contrast, regenerate auditory hair cells and functionally recover from hearing loss. Here, we characterized regeneration and reinnervation in sisomicin-deafened chickens and found that afferent neurons contact regenerated hair cells at the tips of basal projections. In contrast to development, synaptic specializations are established at these locations distant from the hair cells' bodies. The protrusions then contracted as regenerated hair cells matured and became functional 2 weeks post-deafening. We found that auditory thresholds recovered after 4-5 weeks. We interpret the regeneration-specific synaptic reestablishment as a location-preserving process that might be needed to maintain tonotopic fidelity.

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.

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.

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.

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.

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.

ABSTRACT Loss of inner ear hair cells leads to incurable balance and hearing disorders because these sensory cells do not effectively regenerate in humans. A potential starting point for therapy would be the stimulation of quiescent progenitor cells within the damaged inner ear. Inner ear progenitor/stem cells, which have been described in rodent inner ears, would be principal candidates for such an approach. Despite the identification of progenitor cell populations in the human fetal cochlea and in the adult human spiral ganglion, no proliferative cell populations with the capacity to generate hair cells have been reported in vestibular and cochlear tissues of adult humans. The present study aimed at filling this gap by isolating colony‐forming progenitor cells from surgery‐ and autopsy‐derived adult human temporal bones in order to generate inner ear cell types in vitro . Sphere‐forming and mitogen‐responding progenitor cells were isolated from vestibular and cochlear tissues. Clonal spheres grown from adult human utricle and cochlear duct were propagated for a limited number of generations. When differentiated in absence of mitogens, the utricle‐derived spheres robustly gave rise to hair cell‐like cells, as well as to cells expressing supporting cell‐, neuron‐, and glial markers, indicating that the adult human utricle harbors multipotent progenitor cells. Spheres derived from the adult human cochlear duct did not give rise to hair cell‐like or neuronal cell types, which is an indication that human cochlear cells have limited proliferative potential but lack the ability to differentiate into major inner ear cell types. Anat Rec, 303:461–470, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Abstract 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. J. Comp. Neurol. 477:1–10, 2004. © 2004 Wiley‐Liss, Inc.

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.

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.

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.

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.

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.

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. Learning outcomes: (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.

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.

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 243:1328–1337, 2014 . © 2014 Wiley Periodicals, Inc.

Amanda S. Janesick1,2 and Stefan Heller1,2 1Department of Otolaryngology—Head and Neck Surgery, Stanford University School of Medicine, Stanford, California 94315 2Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94315 Correspondence: janesick{at}stanford.edu; hellers{at}stanford.edu

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.

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.

Abstract 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. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 61–70, 2004

Abstract 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. Developmental Dynamics 237:3305–3312, 2008. © 2008 Wiley‐Liss, Inc.

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.

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.

The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels.Background: Physiological function(s) and activation mechanism(s) of TRPML2 and TRPML3 channels are largely unknown.Results: TRPML2 and TRPML3 channels are activated by different small chemical compounds and low extracellular sodium. Mutations in the first and second extracellular loops render TRPML3 constitutively active in high extracellular sodium.Conclusion: TRPML2 and TRPML3 display similar activation mechanisms.Significance: Novel insights into TRPML2 and TRPML3 activation are provided. The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels. Background: Physiological function(s) and activation mechanism(s) of TRPML2 and TRPML3 channels are largely unknown. Results: TRPML2 and TRPML3 channels are activated by different small chemical compounds and low extracellular sodium. Mutations in the first and second extracellular loops render TRPML3 constitutively active in high extracellular sodium. Conclusion: TRPML2 and TRPML3 display similar activation mechanisms. Significance: Novel insights into TRPML2 and TRPML3 activation are provided.

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.

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 α (CNTFRα) 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 CNTFRα 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. J. Comp. Neurol. 400:244–254, 1998. © 1998 Wiley-Liss, Inc.

ABSTRACT 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.

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.

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.

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.

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.

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.

<h2>Summary</h2> The avian utricle, a vestibular organ of the inner ear, displays turnover of sensory hair cells throughout life. This is in sharp contrast to the mammalian utricle, which shows limited regenerative capacity. Here, we use single-cell RNA sequencing to identify distinct marker genes for the different sensory hair cell subtypes of the chicken utricle, which we validated <i>in situ</i>. We provide markers for spatially distinct supporting cell populations and identify two transitional cell populations of dedifferentiating supporting cells and developing hair cells. Trajectory reconstruction resulted in an inventory of gene expression dynamics of natural hair cell generation in the avian utricle.

Hearing impairment arises from the loss of either type of cochlear sensory hair cells. Inner hair cells act as primary sound transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 hours, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained intact until three days post-treatment, after which deterioration in structure and number was observed, culminating in cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.

Understanding the complex pathologies associated with hearing loss is a significant motivation for conducting inner ear research. Lifelong exposure to loud noise, ototoxic drugs, genetic diversity, sex, and aging collectively contribute to human hearing loss. Replicating this pathology in research animals is challenging because hearing impairment has varied causes and different manifestations. A central aspect, however, is the loss of sensory hair cells and the inability of the mammalian cochlea to replace them. Researching therapeutic strategies to rekindle regenerative cochlear capacity, therefore, requires the generation of animal models in which cochlear hair cells are eliminated. This review discusses different approaches to ablate cochlear hair cells in adult mice. We inventoried the cochlear cyto- and histo-pathology caused by acoustic overstimulation, systemic and locally applied drugs, and various genetic tools. The focus is not to prescribe a perfect damage model but to highlight the limitations and advantages of existing approaches and identify areas for further refinement of damage models for use in regenerative studies.

Human skin fibroblasts labeled with [5,6,8,9,11,12,-14,15-3H]arachidonic acid produce a radioactive metabolite that has a shorter retention time on reverse-phase high-performance liquid chromatography than arachidonic acid. This product is not retained in the cells; it is released entirely into the extracellular fluid in a time-dependent manner. The metabolite does not cochromatograph with any of the eicosanoid standards, and its formation is not prevented by the addition of cyclooxygenase, lipoxygenase, or cytochrome P-450 inhibitors. The compound is not produced by fibroblasts labeled with [1-14C]arachidonic acid, suggesting that it is formed through an oxidative process. Chemical analyses indicated that the metabolite is 4,7,10-hexadecatrienoic acid (16:3). Peroxisome-deficient human skin fibroblasts did not produce 16:3, indicating that it probably is formed through peroxisomal beta-oxidation. Human umbilical vein endothelial cells and porcine pulmonary artery smooth muscle cells also release radioactive 16:3 following labeling with [3H]arachidonic acid. Therefore, the production of this metabolite is not limited only to fibroblasts. The fact that 16:3 is released into the extra-cellular fluid suggests that it may be a new type of lipid mediator derived from arachidonic acid, formed through a peroxisome-dependent oxidative process.

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.

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.

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.

The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels.Background: Physiological function(s) and activation mechanism(s) of TRPML2 and TRPML3 channels are largely unknown.Results: TRPML2 and TRPML3 channels are activated by different small chemical compounds and low extracellular sodium. Mutations in the first and second extracellular loops render TRPML3 constitutively active in high extracellular sodium.Conclusion: TRPML2 and TRPML3 display similar activation mechanisms.Significance: Novel insights into TRPML2 and TRPML3 activation are provided. The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels. Background: Physiological function(s) and activation mechanism(s) of TRPML2 and TRPML3 channels are largely unknown. Results: TRPML2 and TRPML3 channels are activated by different small chemical compounds and low extracellular sodium. Mutations in the first and second extracellular loops render TRPML3 constitutively active in high extracellular sodium. Conclusion: TRPML2 and TRPML3 display similar activation mechanisms. Significance: Novel insights into TRPML2 and TRPML3 activation are provided.

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.

The avian inner ear can regenerate sensory hair cells after damage and has served as a model for the study of hearing regeneration for more than 30 years. Here we present a detailed surgical protocol to induce rapid apoptosis of all hair cells in the chicken cochlea and utricle with a single, local infusion of the aminoglycoside sisomicin. S-phase entry of supporting cells engaged in proliferative regeneration peaks at 48 h and newly regenerated hair cells emerge as early as 4–5 days post-sisomicin. We provide reliable read-outs for hair cell loss, such as overt manifestations of vestibular deficiencies, and quick validation of regeneration using reliable markers that can be detected with commercial antibodies. Titrating down the dose of sisomicin reveals differential susceptibilities of hair cell subtypes: cochlea versus utricle, cochlear tall versus cochlear short hair cells, vestibular type I versus type II hair cells, and proximal versus distal location along the cochlea. We provide a method to quantitate cells within the sensory epithelium in 3D, leveraging vibratome sectioning and imaging methods that are presented in a companion chapter. Finally, we present the technique of cold-peeling the cochlear sensory epithelium for the purposes of RNA or protein extraction, and single-cell dissociation in preparation for RNA-seq.

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.

While the mouse has been a productive model for inner ear studies, a lack of highly specific genes and tools has presented challenges. The absence of definitive otic lineage markers and tools is limiting in vitro studies of otic development, where innate cellular heterogeneity and disorganization increase the reliance on lineage-specific markers. To address this challenge in mice and embryonic stem (ES) cells, we targeted the lineage-specific otic gene Fbxo2 with a multicistronic reporter cassette (Venus/Hygro/CreER = VHC). In otic organoids derived from ES cells, Fbxo2VHC specifically delineates otic progenitors and inner ear sensory epithelia. In mice, Venus expression and CreER activity reveal a cochlear developmental gradient, label the prosensory lineage, show enrichment in a subset of type I vestibular hair cells, and expose strong expression in adult cerebellar granule cells. We provide a toolbox of multiple spectrally distinct reporter combinations for studies that require use of fluorescent reporters, hygromycin selection, and conditional Cre-mediated recombination.

This chapter describes the application of immunohistochemistry and in situ mRNA detection to vibratome sections derived from chicken and mouse inner ears. The protocols portray simple strategies to investigate the cellular organization of vestibular and cochlear sensory epithelia. All inner cell types are confidently identified due to the excellent three-dimensional preservation of the inner ear tissue in vibratome sections. Shown are examples for detecting proteins that label cell subtypes as well as subcellular structures such as synapses. Vibratome sections are suitable for conventional colorimetric in situ hybridization as well as fluorescence-based hybridization chain reaction.

Journal of NeurobiologyVolume 37, Issue 4 p. 672-683 A transient role for ciliary neurotrophic factor in chick photoreceptor development Sabine Fuhrmann, Sabine Fuhrmann Institute of Anatomy I, University of Freiburg, P.O. Box 111, D-79001 Freiburg, GermanySearch for more papers by this authorStefan Heller, Stefan Heller Max-Planck-Institut für Hirnforschung, Deutschordenstr. 46, D-60528 Frankfurt, GermanySearch for more papers by this authorHermann Rohrer, Hermann Rohrer Max-Planck-Institut für Hirnforschung, Deutschordenstr. 46, D-60528 Frankfurt, GermanySearch for more papers by this authorHans-Dieter Hofmann, Corresponding Author Hans-Dieter Hofmann Institute of Anatomy I, University of Freiburg, P.O. Box 111, D-79001 Freiburg, GermanyInstitute of Anatomy I, University of Freiburg, P.O. Box 111, D-79001 Freiburg, GermanySearch for more papers by this author Sabine Fuhrmann, Sabine Fuhrmann Institute of Anatomy I, University of Freiburg, P.O. Box 111, D-79001 Freiburg, GermanySearch for more papers by this authorStefan Heller, Stefan Heller Max-Planck-Institut für Hirnforschung, Deutschordenstr. 46, D-60528 Frankfurt, GermanySearch for more papers by this authorHermann Rohrer, Hermann Rohrer Max-Planck-Institut für Hirnforschung, Deutschordenstr. 46, D-60528 Frankfurt, GermanySearch for more papers by this authorHans-Dieter Hofmann, Corresponding Author Hans-Dieter Hofmann Institute of Anatomy I, University of Freiburg, P.O. Box 111, D-79001 Freiburg, GermanyInstitute of Anatomy I, University of Freiburg, P.O. Box 111, D-79001 Freiburg, GermanySearch for more papers by this author First published: 12 December 1998 https://doi.org/10.1002/(SICI)1097-4695(199812)37:4<672::AID-NEU14>3.0.CO;2-1Citations: 21AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract 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 (CNTFRα). As shown immunocytochemically, CNTFRα 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. 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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.