Stéphanie Ventéo

Low-Threshold Mechanoreceptor Subtypes Selectively Express MafA and Are Specified by Ret Signaling

Low-threshold mechanoreceptor neurons (LTMs) of the dorsal root ganglia (DRG) are essential for touch sensation. They form highly specialized terminations in the skin and display stereotyped projections in the spinal cord. Functionally defined LTMs depend on neurotrophin signaling for their postnatal survival and functioning, but how these neurons arise during development is unknown. Here, we show that specific types of LTMs can be identified shortly after DRG genesis by unique expression of the MafA transcription factor, the Ret receptor and coreceptor GFRalpha2, and find that their specification is Ngn2 dependent. In mice lacking Ret, these LTMs display early differentiation defects, as revealed by reduced MafA expression, and at later stages their central and peripheral projections are compromised. Moreover, in MafA mutants, a discrete subset of LTMs display altered expression of neurotrophic factor receptors. Our results provide evidence that genetic interactions involving Ret and MafA progressively promote the differentiation and diversification of LTMs.

Tmprss3, a transmembrane serine protease deficient in human DFNB8/10 deafness, is critical for cochlear hair cell survival at the onset of hearing

Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3Y260X homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3Y260X mice by using rotating rod and vestibular behavioral tests. Tmprss3Y260X mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.

Development of the rat efferent vestibular system on the ground and in microgravity

We investigated whether plastic changes occurred in the organization of the vestibular efferent network in the rat utricle during a 17-day episode of microgravity, from postnatal (PN) day 8 to PN23, and on return to earth on PN25. We also determined the normal pattern of efferent development from birth to PN25. Immunofluorescence experiments were performed with a specific biochemical marker of the efferent system, the calcitonin gene-related peptide (CGRP), and vibratome sections of the utricles were analyzed by laser scanning confocal microscopy. At birth, a few efferent fibers were detected beneath the sensory epithelium. These then massively invaded the epithelium between PN2 and PN4. At the time of launch, PN8, most fiber paths in the utricular epithelium, after following transient courses (towards the epithelial surface for example) returned to the base and were stabilized in the lower part of the epithelium, in which they established synaptic contacts with sensory cells, except at a few immature locations. The main difference between this stage (on PN8) and subsequent more mature stages was the lower density of fibers and synapses in the utricle. The maturation of the vestibular efferent system was similar in microgravity and on the ground. Thus, maturation of the efferent system between PN8 and PN23 was not sensitive to a change in gravitational environment. These results suggest that periods of microgravity at earlier stages are required to identify critical periods in peripheral vestibular system development.

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

Efferent function of vestibular afferent endings? Similar localization of N-type calcium channels, synaptic vesicle and synaptic membrane-associated proteins

We investigated the distribution of N-type voltage-dependent calcium channels that mediate Ca(2+) entry initiating transmitter release in the rat vestibular sensory epithelium. We used confocal microscopy to assess the in vitro labeling by fluorescent specific ligand binding, omega-conotoxin-GVIA and also the immunolabeling of presynaptic soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE) proteins, syntaxin, 25,000 mol. wt synaptosome-associated protein and synaptotagmin: components of the neurotransmitter exocytosis machinery. We found that there was a close anatomical association between the voltage-gated calcium channels, the synaptic vesicle and synaptic membrane-associated proteins on the afferent nerve calyces and probably afferent boutons, which are postsynaptic compartments. Our data suggest that these peripheral afferent endings possess the presynaptic Ca(2+) channels and the components of the presynaptic SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. They provide additional evidence for a secretory function and efferent role of these endings in hair cell neurotransmission.

A SAGE-based screen for genes expressed in sub-populations of neurons in the mouse dorsal root ganglion

BackgroundThe different sensory modalities temperature, pain, touch and muscle proprioception are carried by somatosensory neurons of the dorsal root ganglia. Study of this system is hampered by the lack of molecular markers for many of these neuronal sub-types. In order to detect genes expressed in sub-populations of somatosensory neurons, gene profiling was carried out on wild-type and TrkA mutant neonatal dorsal root ganglia (DRG) using SAGE (serial analysis of gene expression) methodology. Thermo-nociceptors constitute up to 80 % of the neurons in the DRG. In TrkA mutant DRGs, the nociceptor sub-class of sensory neurons is lost due to absence of nerve growth factor survival signaling through its receptor TrkA. Thus, comparison of wild-type and TrkA mutants allows the identification of transcripts preferentially expressed in the nociceptor or mechano-proprioceptor subclasses, respectively.ResultsOur comparison revealed 240 genes differentially expressed between the two tissues (P < 0.01). Some of these genes, CGRP, Scn10a are known markers of sensory neuron sub-types. Several potential markers of sub-populations, Dok4, Crip2 and Grik1/GluR5 were further analyzed by quantitative RT-PCR and double labeling with TrkA,-B,-C, c-ret, parvalbumin and isolectin B4, known markers of DRG neuron sub-types. Expression of Grik1/GluR5 was restricted to the isolectin B4+ nociceptive population, while Dok4 and Crip2 had broader expression profiles. Crip2 expression was however excluded from the proprioceptor sub-population.ConclusionWe have identified and characterized the detailed expression patterns of three genes in the developing DRG, placing them in the context of the known major neuronal sub-types defined by molecular markers. Further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities.

Regulation of the Na,K-ATPase gamma-subunit FXYD2 by Runx1 and Ret signaling in normal and injured non-peptidergic nociceptive sensory neurons.

Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons which relay nociceptive, thermoceptive, mechanoceptive and proprioceptive information from peripheral tissues toward the central nervous system. These neurons establish constant communication with their targets which insures correct maturation and functioning of the somato-sensory nervous system. Interfering with this two-way communication leads to cellular, electrophysiological and molecular modifications that can eventually cause neuropathic conditions. In this study we reveal that FXYD2, which encodes the gamma-subunit of the Na,K-ATPase reported so far to be mainly expressed in the kidney, is induced in the mouse DRGs at postnatal stages where it is restricted specifically to the TrkB-expressing mechanoceptive and Ret-positive/IB4-binding non-peptidergic nociceptive neurons. In non-peptidergic nociceptors, we show that the transcription factor Runx1 controls FXYD2 expression during the maturation of the somato-sensory system, partly through regulation of the tyrosine kinase receptor Ret. Moreover, Ret signaling maintains FXYD2 expression in adults as demonstrated by the axotomy-induced down-regulation of the gene that can be reverted by in vivo delivery of GDNF family ligands. Altogether, these results establish FXYD2 as a specific marker of defined sensory neuron subtypes and a new target of the Ret signaling pathway during normal maturation of the non-peptidergic nociceptive neurons and after sciatic nerve injury.

Zfh1 promotes survival of a peripheral glia subtype by antagonizing a Jun N-terminal kinase-dependent apoptotic pathway.

In Drosophila subperineurial glia (SPG) ensheath and insulate the nerve. SPG is under strict cell cycle and survival control because cell division or death of such a cell type would compromise the integrity of the blood–nerve barrier. The mechanisms underlying the survival of SPG remain unknown. Here, we show that the embryonic peripheral glia expresses the Zfh1 transcription factor, and in zfh1 mutants a particular SPG subtype, ePG10, undergoes apoptosis. Our findings show that in ePG10, Zfh1 represses the pro‐apoptotic RHG‐motif gene reaper in a cell‐autonomous manner. Zfh1 also blocks the activation of the Jun N‐terminal kinase (JNK) pathway, and reducing or enhancing JNK signalling in zfh1 mutants prevents or promotes ePG10 apoptosis. Our study shows a novel function for Zfh1 as an anti‐apoptotic molecule and uncovers a cryptic JNK‐dependent apoptotic programme in ePG10, which is normally blocked by Zfh1. We propose that, in cells such as SPG that do not undergo self‐renewal and survive long periods, transcriptional control of RHG‐motif gene expression together with fine tuning of JNK signalling is crucial for cell survival.

Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification.

Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species. To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce. Here, based on specific combinations of expression patterns of the Col1a1, Col1a2, and Col2a1 fibrillar collagen genes, we compare the molecular footprint of endoskeletal elements from the chondrichthyan Scyliorhinus canicula and the tetrapod Xenopus tropicalis. We find that, depending on the anatomical location, Scyliorhinus skeletal calcification is associated to cell types expressing different subsets of fibrillar collagen genes, such as high levels of Col1a1 and Col1a2 in the neural arches, high levels of Col2a1 in the tesserae, or associated to a drastic Col2a1 downregulation in the centrum. We detect low Col2a1 levels in Xenopus osteoblasts, thereby revealing that the osteoblastic expression of this gene was significantly reduced in the tetrapod lineage. Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

Fibroblast growth factor homologous factor 1 (FHF1) is expressed in a subpopulation of calcitonin gene-related peptide-positive nociceptive neurons in the murine dorsal root ganglia

Dorsal root ganglia (DRG) neurons exhibit a wide molecular heterogeneity in relation to the various sensory modalities (mechanoception, thermoception, nociception) that they subserve. Finding markers of subpopulations is an important step in understanding how these neurons convey specific information. We identified fibroblast growth factor homologous factor 1 (FHF1) in a search for markers of subpopulations of DRG neurons. FHFs constitute a family of four factors that share some structural properties with fibroblast growth factors (FGFs) but are functionally distinct. They are expressed in specific subsets of neurons and are involved in the modulation of sodium channel activity. The pattern of expression of FHF1 in the DRG was determined during development, in the adult and after axotomy. We show that in the adult, FHF1 is expressed in two populations, one composed of nociceptors and another in which no neurotrophic factor receptors were detected (panTrk−/c‐Ret−). Interestingly, in the nociceptors, FHF1 expression was restricted to a subset of TrkA+/calcitonin gene‐related peptide (CGRP)‐positive neurons. Neurofilament 200 (NF‐200) and peripherin labeling indicates that 70% of the FHF1‐expressing neurons contribute to A‐fibers and 30% to C‐fibers. FHF1 interacts with the Nav1.9 sodium channel isoform, which is strongly expressed in cRet+/isolectin‐B4 binding neurons, but we show that FHF1 is not expressed in the cRet+/IB4+ subclass and that it does not colocalize with Nav1.9. Our results argue strongly against the possibility that FHF1 has a modulatory effect on this channel in cRet+/IB4+ neurons, but FHF1 could play a role in a distinct subset of TrkA+/CGRP+ nociceptors. J. Comp. Neurol. 507:1588–1601, 2008.