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Background Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool. Methods We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3). Results Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel. Conclusions Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.

Because of its high prevalence and social impact, hearing impairment is a major public health problem. Whatever the cause--heredity, acoustic trauma, aminoglycoside antibiotics, noise exposure or aging--the hearing impairment is often caused by an irreversible loss of sensory hair cells. So far, hearing aids and cochlear implants are the only possibility to "treat" profound deafness. With the advent of regenerative medicine, extensive studies aimed to repair, regenerate or replace lost hair cells have been initiated. Recently, Stefan Heller and colleagues described a guidance protocol to induce mouse embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) to differentiate into mechanosensitive hair cells. The resulting hair cells hold promise as a tool for hair cell molecular physiology and physiopathology, drug discovery, and possibly also hair cell replacement. The next challenges, alternative strategies, their limitations and prospects are also discussed.

The sensitivity and frequency selectivity of the mammalian cochlea involves a mechanical amplification process called electromotility, which requires prestin-dependent length changes of the outer hair cell (OHC) lateral wall in response to changes in membrane electric potential. The cortical lattice, the highly organized cytoskeleton underlying the OHC lateral plasma membrane, is made up of F-actin and spectrin. Here, we show that alphaII and two of the five beta-spectrin subunits, betaII and betaV, are present in OHCs. betaII spectrin is restricted to the cuticular plate, a dense apical network of actin filaments, whereas betaV spectrin is concentrated at the cortical lattice. Moreover, we show that alphaII-betaV spectrin directly interacts with F-actin and band 4.1, two components of the OHC cortical lattice. betaV spectrin is progressively recruited into the cortical lattice between postnatal day 2 (P2) and P10 in the mouse, in parallel with prestin membrane insertion, which itself parallels the maturation of cell electromotility. Although betaV spectrin does not directly interact with prestin, we found that addition of lysates derived from mature auditory organs, but not from the brain or liver, enables betaV spectrin-prestin interaction. Using this assay, betaV spectrin, via its PH domain, indirectly interacts with the C-terminal cytodomain of prestin. We conclude that the cortical network involved in the sound-induced electromotility of OHCs contains alphaII-betaV spectrin, and not the conventional alphaII-betaII spectrin.

International audience; Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.

International audience; Usher syndrome type IIa (USH2A) combines moderate to severe congenital hearing impairment and retinitis pigmentosa. It is the most common genetic form of USH. USH2A encodes usherin, which was previously defined as a basement membrane protein. A much larger USH2A transcript predicted to encode a transmembrane (TM) isoform was recently reported. Here, we address the role of TM usherin in the inner ear. Analysis of the usherin alternative transcripts in the murine inner ear revealed the existence of several predicted TM usherin isoforms with modular ectodomains of different lengths. In addition, we identified in the usherin cytoplasmic region a predicted 24 amino acid peptide, derived from a newly defined exon that is predominantly expressed in the inner ear but not in the retina. In mouse and rat inner ears, we show that TM usherin is present at the base of the differentiating stereocilia, which make up the mechanosensitive hair bundles receptive to sound. The usherin immunolabeling is transient in the hair bundles of cochlear hair cells (HCs), but persists in mature hair bundles of vestibular HCs. Several lines of evidence support the involvement of TM usherin in the composition of the ankle links, a subset of filamentous lateral links connecting stereocilia at the base. By co-immunoprecipitation and in vitro binding assays, we establish that the usherin cytodomain can bind to whirlin and harmonin, two PDZ domain-containing proteins that are defective in genetic forms of isolated deafness and USH type I, respectively. These PDZ proteins are suitable to provide the anchoring of interstereocilia lateral links to the F-actin core of stereocilia. Our results strongly suggest that congenital deafness in USH type I and type II shares similar pathogenic mechanisms, i.e. the disruption of hair bundle links-mediated adhesion forces that are essential for the proper organization of growing hair bundles.

Une description phénoménologique précise de la différenciation de la touffe ciliaire des cellules sensorielles de l’oreille interne a été réalisée il y a environ 20 ans chez le poulet. Pourtant, les mécanismes cellulaires concourant à la formation de cette structure remarquablement organisée, qui assure la transformation d’un son ou d’une accélération en un signal électrique (transduction mécano-électrique), demeurent très mal connus. Récemment, par des approches génétiques conjointes chez l’homme et la souris, cinq des protéines directement impliquées dans le syndrome de Usher de type I, un double déficit sensoriel qui concerne à la fois l’audition et la vision, ont pu être caractérisées. Ces découvertes ont d’ores et déjà permis d’identifier les liens interstéréociliaires comme des acteurs essentiels de la différenciation des touffes ciliaires., Defects in myosin VIIa, the PDZ-domain-containing protein harmonin, cadherin 23, protocadherin 15, and the putative scaffolding protein sans, underlie five genetic forms of Usher syndrome type I (USH1), the most frequent cause of hereditary deafness-blindness in humans. Mice mutants defective for any of these proteins have a severe hearing impairment and display similar inner ear phenotypes characterized by the abnormal spreading of the sensory cells’ stereocilia. These are highly specialized mechanoreceptive organelles derived from microvilli, that normally form a well-structured hair bundle at the apex of inner ear sensory cells. All the USH1 proteins, except sans, have been detected in the growing stereocilia. Moreover, biochemical studies have started to unravel the multiple direct molecular interactions between USH1 proteins. In particular, harmonin can bind to the other four USH1 proteins and to F-actin. Finally, cell biology studies have provided the first insights into the functions of these proteins, and revealed that cadherin 23, and probably protocadherin 15 also, are associated with transient lateral links that interconnect growing stereocilia. These connectors play a critical role in the differentiating hair bundle.

International audience; By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.

To investigate the commitment potentialities of the adenohypophyseal tissue, its presumptive territory, localized in the anterior ridge of the neural plate in the chick embryo, was surgically removed from 2- to 4-somite stages of development. At later stages, operated embryos were progressively collected to study the extent (if any) of the adenohypophyseal tissue. The results revealed that Rathke's pouch and later the adenohypophysis were completely absent in 33 out of the 45 studied embryos. The surrounding tissues in the operated embryos developed in a comparable manner to those in normal embryos. The head morphogenesis was not affected by the surgical ablation. Strikingly, no regeneration of adenohypophyseal tissue was observed in the operated embryos. At younger stages, a very small invagination was observed close to the diencephalon of 9 out of the 45 studied embryos. Its extent was reduced to less than 20% when compared to normal embryos at the same stages. This epithelial invagination is believed therefore to be a residuum of the stomodeal ectoderm and/or the stomodeo-adenohypophyseal channel which normally regresses at later stages of development. Therefore, the anterior ridge of the neural plate seems irrevocably destined to form adenohypophyseal tissue even as early as 2- to 4-somite stages. This suggests that the adenohypophysis presumptive territory is already committed prior to neural tube closure and also seems to confirm its origin from the neural primordium in avian embryos. Since close topological relationships link the presumptive adenohypophysis territory with both those of the hypothalamus and ectoderm of nasal cavity, possible developmental influences through such intimate contacts cannot be totally excluded.

International audience; Listeria monocytogenes is a bacterial pathogen with the capacity to invade non-phagocytic cells. This dynamic process involves coordinated membrane remodelling and actin cytoskeleton rearrangements. Although some of the molecular factors promoting these events have been identified, the driving force allowing internalization is unknown. One of the receptors for L. monocytogenes on epithelial cells is E-cadherin, a transmembrane protein normally involved in homophilic interactions that allow cell-cell contacts at the adherens junctions. E-cadherin has to be connected to the actin cytoskeleton to mediate strong cell-cell adhesion and to trigger Listeria entry; alpha- and beta-catenins play key roles in these processes. We have recently identified an unconventional myosin, myosin VIIa and its ligand vezatin, at the adherens junctions of polarized epithelial cells. Here, we demonstrate by pharmacological and genetic approaches that both myosin VIIa and vezatin are crucial for Listeria internalization. These results provide the first evidence for the role of an unconventional myosin in bacterial internalization and a novel example of the exploitation of mammalian proteins, by a pathogen, to establish a successful infection.

International audience; The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

International audience; The whirler mouse mutant (wi) does not respond to sound stimuli, and detailed ultrastructural analysis of sensory hair cells in the organ of Corti of the inner ear indicates that the whirler gene encodes a protein involved in the elongation and maintenance of stereocilia in both inner hair cells (IHCs) and outer hair cells (OHCs). BAC-mediated transgene correction of the mouse phenotype and mutation analysis identified the causative gene as encoding a novel PDZ protein called whirlin. The gene encoding whirlin also underlies the human autosomal recessive deafness locus DFNB31. In the mouse cochlea, whirlin is expressed in the sensory IHC and OHC stereocilia. Our findings suggest that this novel PDZ domain–containing molecule acts as an organizer of submembranous molecular complexes that control the coordinated actin polymerization and membrane growth of stereocilia.

Nherf1, a PDZ-domain-containing protein, was identified in the hair bundle in differentiating outer hair cells (OHCs). Nherf1-/- mice showed apparently mild hearing-threshold elevations at mid/high sound frequencies, associated to OHC hair-bundle shape anomalies, prominent in the basal cochlea. This mild impact on hearing sensitivity was discordant with the finding of almost non-responding OHCs in the basal cochlea as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Unlike normal mice, responses of Nherf1-/- mice to high-frequency test tones were not masked by tones of neighboring frequencies. Efficient masker tones displayed unusual characteristics: maximal efficiency at lower frequencies (up to two octaves lower than the test tone), and at low levels (up to 25 dB below test-tone level). This, and the relative growth of the masker and test tones, suggests that mid-high frequency tones of moderate intensity are detected off-frequency, in the functionally unaffected apical cochlear region. Our results establish that Nherf1 is critical for hair bundle morphogenesis and reveal a novel mode of off-frequency detection, probably involving the persistent contact between OHCs and the tectorial membrane. These findings suggest how to circumvent major pitfalls in hearing assessment of some patients, by avoiding misleading interpretations of hearing thresholds.

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, an autosomal dominant form of progressive hearing loss in humans. In the mouse cochlea, the transcript has been found exclusively in the outer hair cells. By using specific antibodies, we now show that KCNQ4 is situated at the basal membrane of these sensory cells. In the vestibular organs, KCNQ4 is restricted to the type I hair cells and the afferent calyx-like nerve endings ensheathing these sensory cells. Several lines of evidence suggest that KCNQ4 underlies the I K,n and g K,L currents that have been described in the outer and type I hair cells, respectively, and that are already open at resting potentials. KCNQ4 is also expressed in neurons of many, but not all, nuclei of the central auditory pathway, and is absent from most other brain regions. It is present, e.g., in the cochlear nuclei, the nuclei of the lateral lemniscus, and the inferior colliculus. This is the first ion channel shown to be specifically expressed in a sensory pathway. Moreover, the expression pattern of KCNQ4 in the mouse auditory system raises the possibility of a central component in the DFNA2 hearing loss.

Branchio-Oto-Renal (BOR) syndrome is an autosomal dominant, early developmental defect characterised by varying combinations of branchial (fistulas, sinuses, and cysts), outer, middle and inner ear, and renal anomalies. The gene underlying this syndrome, EYA1, is homologous to the Drosophila developmental gene eyes absent which encodes a transcriptional co-activator required for eye specification. We report here the temporal and spatial pattern of expression of the murine homologue, Eya1, throughout ear and kidney development in relation to the anomalies of BOR syndrome. The expression of Eya1 in the branchial arch apparatus (namely in the 2nd, 3rd, and 4th branchial clefts and pharyngeal pouches) at embryonic day (E)10.5, can be correlated with the branchial fistulas, sinuses, and cysts but not with the outer and middle ear anomalies. In contrast, Eya1 is expressed during the slightly more advanced stage of outer and middle ear morphogenesis at E13.5, in the mesenchyme adjacent to the first branchial cleft (the cleft will give rise to the external auditory canal and the surrounding mesenchyme to the auricular hillocks) and surrounding the primordia of the middle ear ossicles, and in the epithelium of the tubotympanic recess (the future tympanic cavity). During early inner ear development, Eya1 is expressed in the ventromedial wall of the otic vesicle (the site of the future sensory epithelia), in the statoacoustic ganglion, and in the periotic mesenchyme, consistent with the cochlear anomalies and sensorineural hearing loss of BOR syndrome. Subsequently, Eya1 expression is observed in the differentiating hair and supporting cells of the sensory epithelia, as well as in the associated ganglia, and persists after differentiation has taken place. This suggests that, in addition to a role in the morphogenetic process, Eya1 could also be implicated in the differentiation and/or survival of these inner ear cell populations. Finally, Eya1 expression in the condensing mesenchymal cells of the kidney is consistent with the excretory and collecting system anomalies of BOR syndrome. From the comparison of the Eya1 and Pax2 expression patterns during ear and kidney development, a contribution of these two genes to the same regulatory pathway can only be suggested in the mesenchymal-epithelial transition directing renal tubule formation.

Efforts to identify the specific components of the mammalian inner ear have been hampered by the small number of neuroepithelial cells and the variety of supporting cells. To circumvent these difficulties, we used a PCR-based subtractive method on cDNA from 2-day-old mouse cochlea. A cDNA encoding a predicted 2910-amino acid protein related to mucin has been isolated. Several lines of evidence indicate, however, that this protein does not undergo the O- glycosylation characteristic to mucins. As confirmed by immunocytochemistry and biochemical experiments, this protein is specific to the inner ear. Immunohistofluorescence labeling showed that this protein is a component of all the acellular membranes of the inner ear: i.e., the tectorial membrane of the cochlea, the otoconial and accessory membranes of the utricule and saccule, the cupula of the semicircular canals, and a previously undescribed acellular material covering the otoconia of the saccule. The protein has been named otogelin with reference to its localization. A variety of nonsensory cells located underneath these membranes could be identified as synthesizing otogelin. Finally, this study revealed a maturation process of the tectorial membrane, as evidenced by the progressive organization of otogelin labeling into thick and spaced radial fiber-like structures.

International audience; The gene encoding myosin VIIA is responsible for the mouse shaker-1 phenotype, which consists of deafness and balance deficiency related to cochlear and vestibular neuroepithelial defects. In humans, a defective myosin VIIA gene is responsible for Usher syndrome type IB, which associates congenital deafness, vestibular dysfunction and retinitis pigmentosa. In an attempt to progress in the understanding of the function(s) of myosin VIIA, we studied the expression of the myosin VIIA gene during mouse embryonic development. Embryos from day 9 (E9) to E18 were analyzed by in situ hybridization and immunohistofluorescence. The myosin VIIA mRNA and protein were consistently detected in the same embryonic tissues throughout development. Myosin VIIA was first observed in the otic vesicle at E9, and later in a variety of tissues. The olfactory epithelium and the liver express it as early as E10. In the retinal pigment epithelium, choroid plexus, adrenal gland and tongue, expression begins at E12 and in the testis and the adenohypophysis at E13. In the small intestine, kidney and hair follicles of the vibrissae, expression of myosin VIIA starts only at E15. Myosin VIIA expression was observed only in epithelial cell types, most of which possess microvilli or cilia. Interestingly, myosin VIIA expression seems to be concomitant with the appearance of these structures in the epithelial cells, suggesting a role for this myosin in their morphogenesis. The cellular location of myosin VIIA within sensory hair cells and olfactory receptor neurons also argues for a role of this protein in the synaptic vesicle trafficking.

International audience; Tyrosine hydroxylase (TH) immunoreactive (IR) central catecholaminergic neurons have been observed in human CNS from 4.5 gestational weeks (g.w.) on [Verney, C., Zecevic, N. and Puelles, L., Eur. J. Neurosci., Suppl. 8 (1995) 7044; Zecevic, N. and Verney, C., J. Comp. Neurol., 351 (1995) 509–535]. We describe here a discrete TH-IR cell population localized in the rostral nasal region during embryonic development. Tyrosine hydroxylase-IR cells spread from the olfactory placode towards the basal and medial telencephalon. They follow the same migration path as the gonadotropin-releasing hormone (GnRH)-IR hypothalamic neurons. Tyrosine hydroxylase-IR neurons are first detected at 4.5 g.w., while GnRH-IR cells are visualized later at 5.5 g.w. Double immunocytochemical labeling reveals the presence of three neuronal populations comigrating along the developing vomeronasal-nervus terminalis complex. These populations express either one or both TH and GnRH phenotypes depending on their position in the migration route. At 6 g.w., most of the neurons express TH immunoreactivity as they leave the vomeronasal organ whereas most of the GnRH-IR neurons are detected closer to the CNS and in the CNS itself. These results emphasize the early phenotypic heterogeneity of the different migrating neuronal populations generated in the olfactory placode in humans. At later stages, very few TH-IR neurons are detected in the anterior forebrain suggesting a transient expression of TH immunoreactivity within these neuronal populations.

Usher syndrome type I (USH1) associates severecongenital deafness, vestibular dysfunction andprogressive retinitis pigmentosa leading to blindness.The gene encoding myosin VIIA is responsible forUSH1B. Mutations in the murine orthologous gene leadto the shaker-1 phenotype, which manifests cochlearand vestibular dysfunction, without any retinal defect. Toaddress this phenotypic discrepancy, the expression ofmyosin VIIA in retinal cells was analyzed in human andmouse during embryonic development and adult life. Inthe human embryo, myosin VIIA was present first in thepigment epithelium cells, and later in these cells as wellas in the photoreceptor cells. In the adult human retina,myosin VIIA was present in both cell types. In contrast,in mouse, only pigment epithelium cells expressed theprotein throughout development and adult life. MyosinVIIA was also found to be absent in the photoreceptorcells of other rodents (rat and guinea-pig), whereas thesecells expressed the protein in amphibians, avians andprimates. These observations suggest that retinitispigmentosa of USH1B results from a primary rod andcone defect. The USH1B/shaker-1 paradigm illustrates aspecies-specific cell pattern of gene expression as apossible cause for the discrepancy between phenotypesinvolving defective orthologous genes in man andmouse. Interestingly, in the photoreceptor cells, myosinVIIA is mainly localized in the inner and base of outersegments as well as in the synaptic ending region whereit is co-localized with the synaptic vesicles. Therefore,we suggest that myosin VIIA might play a role in thetrafficking of ribbon-synaptic vesicle complexes andthe renewal processes of the outer photoreceptor disks.INTRODUCTIONUsher syndrome (USH) is an autosomal recessive defect whichaffects both the inner ear and the retina (1). It is regarded as themost frequent cause of deaf-blindness in humans and accounts for3 to 6% of deaf children ( 2,3). Three clinical subtypes have beendescribed, based on the severity of the hearing loss, the presenceor absence of balance problems and the age of onset of the retinitispigmentosa. The most severe form, USH type I (USH1), ischaracterized by profound congenital sensorineural hearing loss,constant vestibular dysfunction and prepubertal onset of retinitispigmentosa leading to blindness. At least four loci have beenassigned to USH1, three of which, USH1A, 1B and 1C, havealready been mapped to chromosomes 14q32, 11q13.5 and 11p15respectively (4–7). We have identified the myosin VIIA gene asresponsible for USH1B (which accounts for about one half of allUSH cases) (8). Mutations in the murine orthologous gene leadto the shaker-1 phenotype (9), characterized by hearingimpairment and balance problems due to damaged cochlear andvestibular neuroepithelia (10,11). However, none of the sixdifferent shaker-1 mutants seems to display any retinal defect(10,11). To explain the discrepancy between the human andmouse retinal phenotypes, several hypotheses can be considered:(i) a difference in the nature of the mutations; (ii) a dissimilaritybetween mouse and human retinal development (12,13); (iii) adifferent influence of modifier genes; (iv) the expression of a genewith redundant function in the mouse retina; (v) an accumulationprocess at the origin of the retinal phenotype, too slow to bedeleterious during the mouse lifetime.The shaker-1 mutations are unlikely to account for the retinalphenotype discrepancy, since at least two of the three mutations

International audience; The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

For some 40 years, the development of the pituitary gland has been the subject of numerous studies. Several aspects of the origin, differentiation, and commitment of the pituitary cells, however, are still in doubt. The pituitary gland of several species apparently originates from the anterior ridge of the neural plate. Glandular pituitary cells can be committed very soon in embryonic life, as early as at the open neural stages. Numbers of differentiation and transcription factors may then control the expression of a given phenotype from the committed cells. Many questions remain unanswered about factors underlying such cellular commitment. These issues call for further studies to elucidate the molecular mechanisms of known and unknown factors controlling the embryology of the pituitary gland.

In chick embryo, the adenohypophysis shows close morphological relationships with hypothalamic and nasal presumptive territories. However, we do not know how long the adenohypophysis depends on its surrounding tissues for its development and differentiation nor do we know anything about factors and mechanisms invovled. This study was undertaken to investigate whether any interactions between these neighbouring tissues influence adenohypophyseal cell growth and differentiation. The ablation of the presumptive hypothalamus and neurohypophysis results in the failure of hypothalamic and infundibular process development. However, the adenohypophysis was present, although it was drastically modified. Moreover, gonadotrophs and corticotrophs can be detected in the developing adenohypophyseal tissue. After the ablation of nasal presumptive territory, from where GnRH neurons originate, the adenohypophyseal length and the number of gonadatrophs and corticotrophs are not significantly altered when compared to control embryos. These results suggest that the presumptive hypothalamus and neurohypophysis are committed during open neural stage. At the following stages, these territories may act to promote the future adenohypophysis development and morphogenesis. However, it seems that pituitary cells are committed from the very early embryonic stages, but interactions between the presumptive adenohypophysis and adjacent territories before the open neural stage cannot be ruled out.

The origin and the migration of gonadotropin-releasing hormone (GnRH)-producing neurons were studied using the indirect immunoperoxidase method in normal and surgically operated chick embryos. In normal embryos, during early embryonic development, GnRH neurons were located only in the respiratory and the olfactory epithelia. Then, these neurons followed the nearest nerve bundle and occupied, thereafter, the dorsal, medial or ventral part of the olfactory nerve according to the time and area of the olfactory epithelium they emerged from. At the junction with the forebrain, the majority of GnRH neurons passed ventromedially round the olfactory bulb. Therefore, they penetrated through the interhemispheric space and coursed obliquely toward caudal and dorsal telencephalon from where they will be later distributed to reach their adult-like position. In view of the large distribution of these neurons in the nasal region, unilateral surgical ablation either of the whole or of each presumptive territory of nasal structures was performed from 2 to 4 somite stages. As expected, when both olfactory placode and ectoderm of nasal cavity presumptive territories were unilaterally removed, olfactory nerve, nasal structures and GnRH neurons failed to develop in the operated side. After the unilateral removal of the olfactory placode anlage, the distribution pattern of GnRH neurons was not disturbed in the operated as well as in the control side although ipsilateral olfactory structures were greatly reduced. In contrast, when the presumptive ectoderm of nasal cavity was unilaterally removed, GnRH neurons were detected only in the control side where this territory was left intact. Therefore, from early neurogenesis, GnRH neurons seem to be already committed, and they originate from the ectoderm of nasal cavity presumptive territory.

Cell–cell adhesion maintains the structural and functional integrity of multicellular organisms. It ensures cell recognition, sorting, and signaling in various tissues and organs. In line with the diversity of the structural and physiological properties of the cell–cell junctions (tight junctions, adherens junctions, desmosomes, and gap junctions), related disorders span a highly heterogeneous group of diseases. Abnormal or loss of cell–cell adhesion and/or associated signaling are hallmarks of tumor growth, malignant transformation, and metastases (see Berx and van Roy 2009). In addition, nonneoplastic diseases caused by defects in genes encoding proteins of the tight junctions, adherens junctions, desmosomes, and gap junctions have been identified (Table 1) (see also Lai-Cheong et al. 2007; Awad et al. 2008; Org et al. 2009). In this article, we summarize the current understanding of human inherited disorders because of defects in members of the cadherin superfamily, i.e., classical cadherins, desmosomal cadherins, protocadherins, and atypical cadherins. Table 1. Integral membrane proteins of cell–cell junctions involved in human diseases. Cadherins are characterized by the presence of 1–34 extracellular cadherin (EC) domains (comprised of ∼110 amino acids). Variations in their cytoplasmic domains impart functional specificity by conferring to each molecule the ability to interact with different ligands (see Shapiro and Weis 2009). These cadherins initiate and build up two types of specialized cell–cell contacts: (1) adherens junctions, and (2) desmosomes, where they tether the plasma membrane to actin microfilaments and intermediate filaments, respectively. Clinical interest in cadherins stems from the discovery, about 25 years ago, of autoantibodies directed against desmosomal cadherins, desmoglein 1 and desmoglein 3, in the autoimmune blistering skin diseases pemphigus foliaceous and pemphigus vulgaris, respectively (Waschke 2008). Pemphigus-affected patients display a loss of intercellular adhesion between keratinocytes (acantholysis) caused by the binding of autoantibodies to desmosomal cadherins (Waschke 2008). Later on, when a dominantly inherited skin disease, striate palmoplantar keratoderma (SPPK; OMIM 148700), was mapped at chromosome 18q12.1, near the cluster of desmosomal cadherin genes (Hennies et al. 1995), the corresponding genes were considered as candidate causative genes for this disorder (Allen et al. 1996). However, it was not until 1999 that the first mutation—an in-frame deletion—was detected in the desmoglein 1 gene in SPPK patients (Rickman et al. 1999). Genome sequence analysis has revealed an impressive diversity of the cadherin superfamily, with 113 cadherins identified in humans to date (Hulpiau and van Roy 2009). Despite functional redundancy among cell–cell junction proteins, several genetic defects affecting the skin, heart, nervous system, ear, or eye have been reported in mice and humans carrying mutations in some cadherin genes (Table 1). So far, mutations in genes encoding 11 cadherins (two classical, four desmosomal, three protocadherins, and two atypical) have been associated with hereditary human diseases (Table 1). These have highlighted a particular need for dynamic and positional cues for axon guidance during the establishment of neural circuits, or for high adhesive strength in tissues that experience mechanical stress, such as the epidermis and hairs, myocardium, and inner ear sensory epithelia.

PURPOSE. Human Usher syndrome is the most common form of combined deafness and blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction, and prepubertal onset retinitis pigmentosa. Previous studies have shown that the USH1-proteins myosin VIIa, harmonin, and cadherin 23 interact and form a functional network during hair cell differentiation in the inner ear. The purpose of the present study was to analyze the molecular and cellular functions of these USH1 proteins in the mammalian retina. METHODS. Antibodies to USH1 proteins were generated and used in Western blot analysis of subcellular photoreceptor fractions and immunofluorescence and electron microscopy of the retina. RESULTS. Splice variants of harmonin were differentially expressed in the photoreceptor cell compartments. Whereas harmonin b isoforms were restricted to the light-sensitive outer segment, the harmonin a and c isoforms were more ubiquitously distributed in the photoreceptors. At the synaptic terminal of photoreceptor cells, harmonin a and c colocalized with myosin VIIa and cadherin 23. CONCLUSIONS. USH1 molecules can assemble to a supramolecular complex at photoreceptor synapses. Such a complex may contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions, which are thought to play a fundamental role in the organization of synaptic junctions. Dysfunction of any of the USH1 complex partners may lead to synaptic dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of patients with USH1. Furthermore, in photoreceptor outer segments, harmonin may also contribute to the clustering of outer segment proteins into supramolecular complexes.

International audience; Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identi®ed by a yeast two-hybrid screen. Vezatin is a ubiquitous protein of adherens cell±cell junctions, where it interacts with both myosin VIIA and the cadherin±catenins complex. Its recruitment to adherens junctions implicates the C-terminal region of a-catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin±catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell±cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane±membrane interaction site, namely at the ®brillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA-defective mutants, inactivity of the vezatin±myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.

Usher Syndrome (USH) is the first cause of deafness blindness in humans. 3 USH clinical types (USH1-3) are defined. Type III clinical form patients hearing loss is not congenital, but progressive, usually occurring during or after adolescence, and the presence of vestibular defects and age of onset of retinitis pigmentosa is variable. I studied the role of clarin-1, causing USH3A. CLRN1 gene encodes clarin-1. The characterization of Clrn1 mutant mice revealed that clarin-1 is essential for the structural organization and function of the presynaptic channels Cav1.3 Ca2+ at the inner hair cell ribbon synapse and for the distribution of postsynaptic AMPA receptors. The viral-mediated transfer of the intact Clrn1 into the clarin-1 mutant mice in cochlea durably prevented synaptic defects and occurrence of the hearing loss. I also explored the role of clarin-2 another member of the clarin family, the absence of which leads to hearing loss. The clarin-2 mutant mice have a progressive, early-onset hearing loss. Our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance. Finally, I studied the compensatory mechanisms involving the two clarins which might conceal important functions in the inner ear. The inactivation of both Clrn1 and Clrn2 impairs prematurely the vestibular function, total loss of mechano-electrical transduction & extreme disruptions of the hair bundle stereocilia. Further elucidation of the mechanisms through which the two clarins interact, and the importance of such interactions in the vestibular and cochlear systems is underway.; Le syndrome d'Usher (USH) est la première cause de surdité et de cécité chez l'homme. 3 types cliniques USH (USH1-3) sont définis. La perte auditive des patients de forme clinique de type III n'est pas congénitale, mais progressive, survenant généralement pendant ou après l'adolescence, et la présence de défauts vestibulaires et l'âge d'apparition de la rétinite pigmentaire sont variables. J'ai étudié le rôle de la clarine-1, à l'origine de l'USH3A. Le gène CLRN1 code pour la clarine-1. La caractérisation des souris mutantes Clrn1 a révélé que la clarine-1 est essentielle pour l'organisation structurelle et la fonction des canaux présynaptiques Cav1.3 Ca2+ au niveau de la synapse du ruban des cellules ciliées internes et pour la distribution des récepteurs AMPA postsynaptiques. Le transfert à médiation virale de la Clrn1 intacte dans les souris mutantes clarin-1 dans la cochlée a empêché durablement les défauts synaptiques et l'apparition de la perte auditive. J'ai également exploré le rôle de clarine-2, dont l'absence entraîne une perte auditive. Les souris mutantes clarin-2 présentent une perte auditive progressive et précoce. Nos résultats démontrent un rôle clé pour la clarin-2 dans l'audition des mammifères, fournissant des informations sur l'interaction entre la transduction mécano-électrique et le maintien des stéréocils. Enfin, j'ai étudié les mécanismes compensatoires impliquant les deux clarines qui pourraient cacher des fonctions importantes dans l'oreille interne. L'inactivation de Clrn1 et Clrn2 altère prématurément la fonction vestibulaire, la perte totale de transduction mécano-électrique et les perturbations extrêmes des stéréocils du faisceau de cheveux. Une élucidation supplémentaire des mécanismes par lesquels les deux clarines interagissent, et l'importance de ces interactions dans les systèmes vestibulaire et cochléaire est en cours.

Usher Syndrome (USH) is the first cause of deafness blindness in humans. 3 USH clinical types (USH1-3) are defined. Type III clinical form patients hearing loss is not congenital, but progressive, usually occurring during or after adolescence, and the presence of vestibular defects and age of onset of retinitis pigmentosa is variable. I studied the role of clarin-1, causing USH3A. CLRN1 gene encodes clarin-1. The characterization of Clrn1 mutant mice revealed that clarin-1 is essential for the structural organization and function of the presynaptic channels Cav1.3 Ca2+ at the inner hair cell ribbon synapse and for the distribution of postsynaptic AMPA receptors. The viral-mediated transfer of the intact Clrn1 into the clarin-1 mutant mice in cochlea durably prevented synaptic defects and occurrence of the hearing loss. I also explored the role of clarin-2 another member of the clarin family, the absence of which leads to hearing loss. The clarin-2 mutant mice have a progressive, early-onset hearing loss. Our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance. Finally, I studied the compensatory mechanisms involving the two clarins which might conceal important functions in the inner ear. The inactivation of both Clrn1 and Clrn2 impairs prematurely the vestibular function, total loss of mechano-electrical transduction & extreme disruptions of the hair bundle stereocilia. Further elucidation of the mechanisms through which the two clarins interact, and the importance of such interactions in the vestibular and cochlear systems is underway., Le syndrome d'Usher (USH) est la première cause de surdité et de cécité chez l'homme. 3 types cliniques USH (USH1-3) sont définis. La perte auditive des patients de forme clinique de type III n'est pas congénitale, mais progressive, survenant généralement pendant ou après l'adolescence, et la présence de défauts vestibulaires et l'âge d'apparition de la rétinite pigmentaire sont variables. J'ai étudié le rôle de la clarine-1, à l'origine de l'USH3A. Le gène CLRN1 code pour la clarine-1. La caractérisation des souris mutantes Clrn1 a révélé que la clarine-1 est essentielle pour l'organisation structurelle et la fonction des canaux présynaptiques Cav1.3 Ca2+ au niveau de la synapse du ruban des cellules ciliées internes et pour la distribution des récepteurs AMPA postsynaptiques. Le transfert à médiation virale de la Clrn1 intacte dans les souris mutantes clarin-1 dans la cochlée a empêché durablement les défauts synaptiques et l'apparition de la perte auditive. J'ai également exploré le rôle de clarine-2, dont l'absence entraîne une perte auditive. Les souris mutantes clarin-2 présentent une perte auditive progressive et précoce. Nos résultats démontrent un rôle clé pour la clarin-2 dans l'audition des mammifères, fournissant des informations sur l'interaction entre la transduction mécano-électrique et le maintien des stéréocils. Enfin, j'ai étudié les mécanismes compensatoires impliquant les deux clarines qui pourraient cacher des fonctions importantes dans l'oreille interne. L'inactivation de Clrn1 et Clrn2 altère prématurément la fonction vestibulaire, la perte totale de transduction mécano-électrique et les perturbations extrêmes des stéréocils du faisceau de cheveux. Une élucidation supplémentaire des mécanismes par lesquels les deux clarines interagissent, et l'importance de ces interactions dans les systèmes vestibulaire et cochléaire est en cours.

Usher syndrome (USH) causes a combined deafness-blindness in humans. At least nine causative genes are known. While the analysis of USH knockout mice has shed light on the origin of the auditory deficit, the causes of vision loss are still unclear. Nevertheless, USH1B protein, myosin VIIa, appears to contribute to intracellular traffic in photoreceptor cells. To better understand the role of this myosin in the retina, I studied the functions of its interacting partner, spectrin βV. We found that spectrin V, along with USH1 proteins, participates in intracellular transport by coupling motor proteins (myosin VIIa, kinesin II, dynein/dynactin complex) to the cargoes en route towards the outer segment of photoreceptor cells. Evidence from comparative studies in frog and mouse inner ear, biochemical assays and phylogenetic analyses point to cargo trafficking to and from the apical cell region, as the likely ancestral function of this spectrin. Our analyses also suggest that evolutionary pressures in the mammalian lineage drove the recruitment of spectrin βV to the lateral wall of auditory outer hair cells, probably to support a new function: electromotility. Finally, I explored the origin of hearing loss in Usher syndrome of type III (USH3). So far, the only causal gene known is CLRN1, which codes for clarin-1. The comparative characterization of two Clrn1 mouse mutants revealed that clarin-1 is required for the maturation and maintenance of the hair bundle in the hair cells. Moreover, our results indicate that clarin-1 is also essential to cluster the voltage-gated Ca2+ channels in close proximity to the exocytotic machinery of the ribbon synapse of inner hair cells.; Le syndrome d’Usher (USH) cause une surdité-cécité chez l’homme. Au moins neuf gènes responsables ont été identifiés. L’origine de l’atteinte auditive a été dévoilée par l’analyse de souris mutantes, mais les causes de la cécité sont encore obscures. Néanmoins, unes des protéines de Usher, la myosine VIIa, a été impliquée dans le transport intracellulaire dans les photorécepteurs. Pour mieux comprendre le rôle dans la rétine, j’ai étudié l’un de ses partenaires, la spectrine βV. Nous avons conclu que cette spectrine, en collaboration avec les protéines USH1, est impliquée dans le trafic cellulaire: elle couple les moteurs (myosine VIIA, kinesine II et le complexe dynéine/dynactine) à leurs cargos en route vers le segment externe des photorécepteurs. La combinaison d’études comparatives dans les cellules ciliées (CC) de l'oreille interne de grenouille et de souris, de tests biochimiques et d’analyses phylogénétiques indique que le transport vers et depuis la surface apicale des cellules est la fonction ancestrale de cette spectrine. Chez les mammifères, une pression évolutive a engendré le recrutement de la spectrine βV à la paroi latérale des CC externes auditives, probablement pour participer à une nouvelle fonction: l’électromotilité. Enfin, j’ai étudié l’origine de la surdité dans le syndrome d’Usher III. Le seul gène causal connu est CLRN1, qui code pour la clarine-1. Nous avons conclu que la clarine-1 est nécessaire à la maturation et le maintien des touffes ciliaires des CC. De plus, la clarine-1 est essentielle pour le regroupement des canaux calciques voltage-dépendants au voisinage immédiat de la machinerie exocytotique de la synapse à ruban des CC internes.