Project description:Mechanosensory outer hair cells (OHC) play an essential role in the amplification of sound-induced vibrations in the cochlea because of their ability to contract or elongate following changes in the intracellular potential. To study the critical roles of OHC, we generated a novel mouse model, OHC-TRECK, for hearing impairment by introducing the human diphtheria toxin (DT) receptor gene under the control of the OHC-specific promoter of the mouse prestin gene. DT administration to OHC-TRECK mice resulted in severe hearing impairment with a decrease in the amplitude of distortion product otoacoustic emissions and elevated auditory brainstem responses. We performed a differential expression analysis between DT-administered OHC-TRECK and wild-type mice using high-throughput microarray analysis to screen for novel, specifically expressed and functional genes involved in the development and maintenance of OHC.
Project description:In order to determine the regulators of outer hair cell postnatal maturation, we utilized the RiboTag mouse model to perform a detailed transcriptomic analysis of outer hair cells at five postnatal developmental time points: P8, P14, P28, 6 weeks (6wk) and 10 weeks (10wk). This analysis resulted in consistent enrichment of outer hair cell expressed genes in the immunoprecipitated RNA compared to whole cochlear input RNA from each time point. Using transcription factor binding motif prediction on a set of defined outer hair cell enriched genes, we further use this dataset to identify the helios transcription factor as a regulator of the postnatal outer hair cell transcriptome.
Project description:To further understand the biological properties of hair cells of the mammalian cochlea, we examined the transcriptome of adult inner and outer hair cells. Morphologically distinct inner and outer hair cells were isolated from the organ of Corti from adult CBA/J mice. One thousand inner and outer hair cells were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of two biological replicates of IHCs and three biological replicates of OHCs, each with two technical repeats, was performed. The resulting sequenced reads were mapped. Comparisons between inner and outer hair cells allow identification of enriched genes, as well as differentially expressed genes that result in cellular specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult mouse inner and outer hair cells.
Project description:The transcriptome is the complete set of all RNA transcripts produced by the genome in a cell and reflects the genes that are being actively expressed. Transcriptome analysis is essential for understanding the genetic mechanism controlling the phenotype of a cell. Using DNA microarray technique we examined transcriptomes of 2,000 individually collected inner (IHCs) and outer hair cells (OHCs), two types of auditory sensory cells critical for hearing. Among approximately 16,645 and 17,711 transcripts considered to be expressed, 1,296 and 256 genes showed significant differential expression in IHCs and OHCs, respectively. The top ten differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, Map4k4 in IHCs, and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, Ankrd22 in OHCs. Many unknown sequences and non-coding RNAs were also expressed in hair cells. The differentially expressed genes underlie the genetic mechanism for unique functions of IHCs and OHCs. The total RNA was extracted from the collected pools of single outer hair cell (OHC) and inner hair cell (IHC). Their whole-genome transcriptome expression was detected using GeneChip microarray analysis. The analysis and comparison between OHC and IHC allow us to determine what genes are expressed and what genes are uniquely or differential expressed in each population.
Project description:The transcriptome is the complete set of all RNA transcripts produced by the genome in a cell and reflects the genes that are being actively expressed. Transcriptome analysis is essential for understanding the genetic mechanism controlling the phenotype of a cell. Using DNA microarray technique we examined transcriptomes of 2,000 individually collected inner (IHCs) and outer hair cells (OHCs), two types of auditory sensory cells critical for hearing. Among approximately 16,645 and 17,711 transcripts considered to be expressed, 1,296 and 256 genes showed significant differential expression in IHCs and OHCs, respectively. The top ten differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, Map4k4 in IHCs, and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, Ankrd22 in OHCs. Many unknown sequences and non-coding RNAs were also expressed in hair cells. The differentially expressed genes underlie the genetic mechanism for unique functions of IHCs and OHCs.
Project description:The mouse auditory organ cochlea contains two types of sound receptors: inner hair cells (IHCs) and outer hair cells (OHCs). Tbx2 is expressed in IHCs but repressed in OHCs, and neonatal OHCs that misexpress Tbx2 transdifferentiate into IHC-like cells. However, the extent of this switch from OHCs to IHC-like cells and the underlying molecular mechanism remain poorly understood. Furthermore, whether Tbx2 can transform fully mature adult OHCs into IHC-like cells is unknown. Here, our single-cell transcriptomic analysis revealed that in neonatal OHCs misexpressing Tbx2, 85.6% of IHC genes, including Slc17a8, are upregulated, but only 38.6% of OHC genes, including Ikzf2 and Slc26a5, are downregulated. This suggests that Tbx2 cannot fully reprogram neonatal OHCs into IHCs. Moreover, Tbx2 also failed to completely reprogram cochlear progenitors into IHCs. Lastly, restoring Ikzf2 expression alleviated the abnormalities detected in Tbx2+ OHCs, which supports the notion that Ikzf2 repression by Tbx2 contributes to the transdifferentiation of OHCs into IHC-like cells. Our study evaluates the effects of ectopic Tbx2 expression on OHC lineage development at distinct stages of either male or female mice and provides molecular insights into how Tbx2 disrupts the gene-expression profile of OHCs. This research also lays the groundwork for future studies on OHC regeneration.Significance Statement Elucidation of the molecular and genetic mechanisms governing the determination and stability of cochlear inner hair cells (IHCs) and outer hair cells (OHCs) should provide valuable insights into the regeneration of damaged IHCs and OHCs. Here, we conditionally overexpress Tbx2 in vivo in cochlear sensory progenitors, neonatal OHCs, or adult OHCs. Our results show that Tbx2 overexpression alone can partially destabilize the OHC fate but cannot fully convert OHCs into IHCs. Specifically, we demonstrate that Ikzf2 repression due to Tbx2 overexpression is one of the key pathways disrupting the OHC fate.
Project description:Next Generation Sequencing (NGS) based mRNA sequencing (RNA-seq) has provided high-throughput measurements to analyze the transcriptome of cells. We report the comprehensive transcriptome profiling of primarily isolated human mesenchymal stem cells from hair follicle outer root sheath (MSCORS) in series cultivation passages using NGS-based RNA-seq method. Human anagen hair follicles were non-invasively plucked from scalp of healthy donors, cultivated on a Transwell membrane, from which the MSCORS were isolated and subcultured onto normal culture flask. Attached and proliferating MSCORS were cultivated and characterized as mesenchymal stem cells (MSC) according to the minimum criteria for defining multipotent MSC from the International Society for Cellular Therapy (ISCT). mRNA profiles of MSCORS in p0, p1 and p2 were quality controlled, and generated by deep sequencing, in triplicate, using Illumina PE150. Paired-end raw data were pre-processed using a published protocol. HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts) was used to align reference genome. HTSeq was used to calculate Reads Count, and gene abundence was determined by analyzing FPKM (fragments per kilobase of exon model per million reads mapped).