Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells

PLOS ONE, Dec 2019

Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.

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Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells

July Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells Michael Ebeid 1 2 3 Prashanth Sripal 1 2 3 Jason Pecka 1 2 3 Kirk W. Beisel 1 2 3 Kelvin Kwan 0 1 3 Garrett A. Soukup 1 2 3 0 Department of Cell Biology and Neuroscience, W. M. Keck Center for Collaborative Neuroscience, Rutgers University , Piscataway, New Jersey , United States of America 1 the Bellucci DePaoli Family Foundation (GAS), Nebraska Stem Cell Research Act LB606 (GAS and KWB), Nebraska Health Care Funding Act LB692 (GAS and KWB), National Institute of General Medical Sciences P20GM103471 (GAS), and National Institute on Deafness and Other , USA 2 Department of Biomedical Sciences, Creighton University , Omaha, Nebraska , United States of America 3 Editor: Bruce B. Riley, Texas A&M University , UNITED STATES Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance. - Data Availability Statement: The data presented in this study have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE81667. Introduction Cochlear hair cells (HCs) within the inner ear are the mechanoreceptor epithelial cells of the auditory system. These cells are vulnerable to damage by different factors including noise, drugs or aging. The inability of mammalian HCs to regenerate after ototoxic damaged leads to sensorineural hearing loss [ 1 ]. Hearing loss is a major health concern that affects over 5% of the world's population, (approximately 360 million people) [2]. Currently, the only clinical Communication Disorders R01DC009025 (GAS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. treatment for a severe-to-profound sensorineural hearing loss is cochlear implantation, which provides variable outcomes [ 3 ]. Current research hopes to provide a basis for HC regeneration or replacement therapies that will restore natural hearing. Such efforts require better understanding of the molecular mechanisms orchestrating HC development and maintenance. Development of the inner ear requires a precisely timed cascade of molecular events leading to a sequence of cell fate determinations. The molecular mechanisms guiding this process are not fully elucidated, yet some genes are shown to be crucial for the process. One of the key events is the expression of a proneural basic helix-loop-helix (bHLH) transcription factor Atoh1, which is necessary for HC development and is thought to be the earliest determinant of HC fate [ 4,5 ]. Furthermore, overexpression of Atoh1 is shown to be contextually sufficient to drive ectopic HC generation [ 6,7 ]. Since Atoh1 is crucial for differentiation of other neuronal cell types such as cerebellar granule cells [ 8 ] and spinal cord interneurons [ 9 ], as well as nonneuronal cell types such as intestinal secretory cells [ 10 ], it is believed that the cellular context is an important determinant of Atoh1 function. Previous studies have described some Atoh1 target genes in the cerebellum [ 11 ] and the developing spinal cord [ 12 ] utilizing chromatin immunoprecipitation and sequencing. Yet only a handful of Atoh1 target genes have been validated for HCs [ 13 ]. To date, the molecular mechanisms underlying the role of Atoh1 in HC differentiation and the contextual prerequisite for such a role are not well understood. Another level of gene regulation during development is achieved by small no (...truncated)


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Michael Ebeid, Prashanth Sripal, Jason Pecka, Kirk W. Beisel, Kelvin Kwan, Garrett A. Soukup. Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells, PLOS ONE, 2017, Volume 12, Issue 7, DOI: 10.1371/journal.pone.0180855