Project description:We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions using integrated miRNA, transcriptome and proteome profiles with advanced in silico analysis. By looking at both the transcript and protein levels of expression, a thorough coverage of miRNA regulation was obtained. Microdissected auditory and vestibular sensory epithelia were used as the model system, thus being the first time such a comparison was carried out in a neuroepithelial system. Moreover, this is one of only a few studies employing proteome screening for the identification of miRNA targets. Notably, this approach can be employed for the study of other tissues and organs. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. By searching for enrichment and depletion of miRNA targets in the transcript and protein datasets with a reciprocal or similar expression, respectively, as the regulatory miRNA, we identified functionally important miRNAs. Finally, the interaction between miR-135b and PSIP1-P75, a transcriptional coacitvator previously unknown in the inner ear, was identified and validated experimentally. We suggest that miR-135b may serve as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells. We investigated the mRNA expression profile of the cochlear and vestibular sensory epithelia from inner ears of postnatal day 2 mice using the Affymetrix GeneChip® 430 2Mouse Genome array. Cochlear and vestibular sensory epithelia were dissected from wild type C3H mice and collected separately. The vestibular epithelia consisted of the saccule, utricle and the lateral and anterior cristae. Both the cochlear and vestibular sensory epithelia were dissected with their underlying mesenchyma. Altogether three pools, three biological replicates, of each tissue type were collected consisting of cochlear or vestibular sensory epithelia dissected from 10 to 12 inner ears.
Project description:The cochlear duct is tonotopically organized, such that the basal cochlea responds more sensitively to high frequency sounds and the apical cochlea to low frequency sounds. In effort to understand how the tonotopic organization is established in mammals, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Eighty temporal bones were dissected from C57BL/6 mice at P0 and P8. The cochlear tissues were divided into three equal pieces representing the basal, middle and apical turns, and pooled separately. Six total RNA from the pooled samples were applied to 6 GeneChips.
Project description:Inner ear auditory and vestibular tissues differ in their responses to mechanical stimuli. Chick cochlea and utricle sensory epithelia were microdissected at E20-E21. RNA was extracted and cRNA hybridized to Affymetrix microarrays.
Project description:This study demonstrates the baseline data of gradient gene expression in the cochlea. Especially for genes whose mutations cause autosomal dominant non syndromic hearing loss (Pou4f3, Slc17a8, Tmc1, and Crym) as well as genes important for cochlear function (Emilin-2 and Tectb), gradual expression changes help to explain the various pathological conditions. Four C57BL/6 mice aged 6 weeks cochlea samples including the lateral wall, stria vascularis, spiral ligament, spiral prominence, and the organ of corti were dissected and separated into the apical, middle and basal turns to compare gene expression profiles of each cochlea turn.
Project description:The vomeronasal organ (VNO) of mice contains two main types of vomeronasal sensory neurons (VSNs)- Apical and Basal. Apical VSNs express vomeronasal receptors (VRs) of the V1R family and project to the anterior accessory olfactory bulb (AOB) and VSNs in the basal portions of the epithelium express receptors of the V2R family and project to the posterior portion of the AOB. In the vomeronasal epithelium of mice we found active BMP signaling. By generating Smad4 conditional mutants we disrupted canonical TGF-b/BMP signaling in either maturing basal VSNs or in mature apical and basal VSNs.
Project description:Cre recombinase-mediated conditional knockout of floxed Dicer1 alleles causes depletion of small RNAs including microRNAs, which function to repress target mRNA expression by inhibiting translation and/or stimulating mRNA degradation. We used microarrays to examine gene expression in apical versus basal organ of Corti from the cochleae of control and mutant mice in which Dicer1 was deleted and microRNAs were depleted specifically in sensory hair cells by Atoh1 promoter-driven Cre recombinase expression. Each biological replicate represents the combined apical or combined basal segments of organ of Corti from both cochleae of a single mouse. Two biological replicates for apical and basal organ of Corti from Dicer1 conditonal knockout and littermate controls were collected for RNA extraction and microarray analysis.
Project description:The cochlear duct is tonotopically organized, such that the basal cochlea responds more sensitively to high frequency sounds and the apical cochlea to low frequency sounds. In effort to understand how the tonotopic organization is established in mammals, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development.
Project description:The inner ear utilizes sensory hair cells as mechano-electric transducers for sensing sound and balance. In mammals, these sensory hair cells lack the capacity for regeneration and if damaged lead to hearing or balance disorders. However, non-mammalian vertebrates such as birds maintain their regenerative abilities throughout their life. In a previous study we conducted a gene expression profiling time course of regenerating sensory epithelia (SE) in avian cochlea and utricle on a custom transcription factor microarray following damage by both laser and chemical ablation. We identified several known signaling cascades such as The Pax-Eya-Six-Dach pathway, Ap-1 pathway, the Tgf-β pathway and sonic hedgehog signaling that are differentially expressed during SE regeneration. In this study we selected 27 of these genes for knockdown by siRNA or small molecule inhibition to determine their requirement for SE regeneration and identify downstream targets. We assessed phenotypes using a 96 well proliferation assay and expression profiled each knockdown on a custom transcription factor microarray. Using these techniques we have determined several genes that are required for SE proliferation and identified novel epistatic relationships between many of these genes. Pure sensory epithelia was isolated from avian utricles. Sensory epithelia was physically dissociated and grown in 96 well cultures for 3 days. Prior to confluency, dissociated sensory epithelia were transfected with siRNAs (12 pmol/well) or small molecule inhibitor in 0.1% DMSO. RNA was isolated 24 hrs post transfection and assayed on a custom oligonucleotide transcription factor microarray compared to controls (GFP siRNA or 0.1% DMSO only). siRNA: CEBPG, JunD, BTAF1, LRP5, PAX2, PAX5, PAX7, Wnt4, BCL11A, CBX3, CBX4, CTNNB1, CUTL1, MYT1L, RARA, TIMELESS, TRIP15, PAX3, EZH2, HES1, ID1, CDKN1B, and PPARGC1 small molecule inhibition: IGF, MAPK, SHH, and JNK