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      Transcriptomic Analysis of Mouse Cochlear Supporting Cell Maturation Reveals Large-Scale Changes in Notch Responsiveness Prior to the Onset of Hearing

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          Abstract

          Neonatal mouse cochlear supporting cells have a limited ability to divide and trans-differentiate into hair cells, but this ability declines rapidly in the two weeks after birth. This decline is concomitant with the morphological and functional maturation of the organ of Corti prior to the onset of hearing. However, despite this association between maturation and loss of regenerative potential, little is known of the molecular changes that underlie these events. To identify these changes, we used RNA-seq to generate transcriptional profiles of purified cochlear supporting cells from 1- and 6-day-old mice. We found many significant changes in gene expression during this period, many of which were related to regulation of proliferation, differentiation of inner ear components and the maturation of the organ of Corti prior to the onset of hearing. One example of a change in regenerative potential of supporting cells is their robust production of hair cells in response to a blockade of the Notch signaling pathway at the time of birth, but a complete lack of response to such blockade just a few days later. By comparing our supporting cell transcriptomes to those of supporting cells cultured in the presence of Notch pathway inhibitors, we show that the transcriptional response to Notch blockade disappears almost completely in the first postnatal week. Our results offer some of the first molecular insights into the failure of hair cell regeneration in the mammalian cochlea.

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          Proteomic characterization of the human centrosome by protein correlation profiling.

          The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.
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            Notch inhibition induces cochlear hair cell regeneration and recovery of hearing after acoustic trauma.

            Hearing loss due to damage to auditory hair cells is normally irreversible because mammalian hair cells do not regenerate. Here, we show that new hair cells can be induced and can cause partial recovery of hearing in ears damaged by noise trauma, when Notch signaling is inhibited by a γ-secretase inhibitor selected for potency in stimulating hair cell differentiation from inner ear stem cells in vitro. Hair cell generation resulted from an increase in the level of bHLH transcription factor Atoh1 in response to inhibition of Notch signaling. In vivo prospective labeling of Sox2-expressing cells with a Cre-lox system unambiguously demonstrated that hair cell generation resulted from transdifferentiation of supporting cells. Manipulating cell fate of cochlear sensory cells in vivo by pharmacological inhibition of Notch signaling is thus a potential therapeutic approach to the treatment of deafness. Copyright © 2013 Elsevier Inc. All rights reserved.
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              Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo.

              Loss of cochlear hair cells in mammals is currently believed to be permanent, resulting in hearing impairment that affects more than 10% of the population. Here, we developed two genetic strategies to ablate neonatal mouse cochlear hair cells in vivo. Both Pou4f3(DTR/+) and Atoh1-CreER™; ROSA26(DTA/+) alleles allowed selective and inducible hair cell ablation. After hair cell loss was induced at birth, we observed spontaneous regeneration of hair cells. Fate-mapping experiments demonstrated that neighboring supporting cells acquired a hair cell fate, which increased in a basal to apical gradient, averaging over 120 regenerated hair cells per cochlea. The normally mitotically quiescent supporting cells proliferated after hair cell ablation. Concurrent fate mapping and labeling with mitotic tracers showed that regenerated hair cells were derived by both mitotic regeneration and direct transdifferentiation. Over time, regenerated hair cells followed a similar pattern of maturation to normal hair cell development, including the expression of prestin, a terminal differentiation marker of outer hair cells, although many new hair cells eventually died. Hair cell regeneration did not occur when ablation was induced at one week of age. Our findings demonstrate that the neonatal mouse cochlea is capable of spontaneous hair cell regeneration after damage in vivo. Thus, future studies on the neonatal cochlea might shed light on the competence of supporting cells to regenerate hair cells and on the factors that promote the survival of newly regenerated hair cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                5 December 2016
                2016
                : 11
                : 12
                : e0167286
                Affiliations
                [1 ]Department of Neuroscience, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, United States of America
                [2 ]Program in Developmental Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, United States of America
                [3 ]Department of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, United States of America
                [4 ]Department of Pediatrics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, United States of America
                [5 ]The Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, United States of America
                [6 ]Department of Otolaryngology, Hospital Clínico Universidad de Chile and Interdisciplinary Program of Physiology and Biophysics ICBM Universidad de Chile, Santiago, Chile
                [7 ]Department of Otolaryngology, Clínica Alemana de Santiago, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo, Santiago, Chile
                University of Washington, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: JCM AKG.

                • Formal analysis: YWW ZL.

                • Funding acquisition: JCM AKG.

                • Investigation: JCM RG JSTA TC HIJ SCC RKE HZ.

                • Methodology: JCM RG JSTA AKG.

                • Project administration: JCM AKG.

                • Supervision: AKG JCM.

                • Validation: RKE HIJ HZ TC.

                • Visualization: JCM RG HIJ TC AKG.

                • Writing – original draft: JCM AKG.

                • Writing – review & editing: JCM AKG.

                [¤a]

                Current address: Sound Pharmaceuticals, Seattle, WA, United States of America

                [¤b]

                Current address: Thermo Fisher Scientific, Stem Cell Research Unit, Waltham, MA, United States of America

                Article
                PONE-D-16-37442
                10.1371/journal.pone.0167286
                5137903
                27918591
                7e12b2f8-4a8c-4696-ab91-edb0b89db431
                © 2016 Groves et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 September 2016
                : 13 November 2016
                Page count
                Figures: 3, Tables: 12, Pages: 26
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000055, National Institute on Deafness and Other Communication Disorders;
                Award ID: DC006185
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000055, National Institute on Deafness and Other Communication Disorders;
                Award ID: DC011657
                Award Recipient :
                Funded by: CONIYCT
                Award ID: 11130247
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000005, U.S. Department of Defense;
                Award ID: W81XWH-11-2-004
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100002046, Hearing Health Foundation;
                Award ID: HRP Consortium Grant
                Award Recipient :
                This project was supported in part by (1) The Genomic and RNA Profiling Core and the RNA In Situ Core at Baylor College of Medicine with the expert assistance of Dr. Lisa D. White, Ph.D., Cecilia Ljunberg, Ph.D., and funding from the NIH grant P30HD024064 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development; (2) The Cytometry and Cell Sorting Core at Baylor College of Medicine with the expert assistance of Joel M. Sederstrom and funding from the NIH grants P30 AI036211, P30 CA125123, and S10 RR024574; (3) NIH Grants DC006185 and DC011657 to A.K.G.; (4) Department of Defense Grant DOD W81XWH-11-2-004 to A.K.G.; (5) A Hearing Restoration Project consortium grant from the Hearing Health Foundation to A.K.G.; and (6) An Initiation into Research grant FONDECYT 11130247 from CONICYT (Chile) to J.C.M.
                Categories
                Research Article
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Anatomy
                Head
                Ears
                Inner Ear
                Cochlea
                Medicine and Health Sciences
                Anatomy
                Head
                Ears
                Inner Ear
                Cochlea
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Notch Signaling
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Chemical synthesis
                Biosynthetic techniques
                Nucleic acid synthesis
                RNA synthesis
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA synthesis
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Transcriptome Analysis
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Transcriptome Analysis
                Custom metadata
                Fastq files of paired end reads for all RNA-seq data have been deposited in the NCBI GEO database, Accession No. GSE83357.

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                Uncategorized

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