Neil Segil Ph.D.

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Reprogramming and Gene Delivery

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Reprogramming and Gene Delivery
Andy Groves, Ph.D. (chair), Baylor College of Medicine
John Brigande, Ph.D., Oregon Health & Science University
Yehoash Raphael, Ph.D., University of Michigan
Segil Lab, University of Southern California

This group will take the lead on transitioning to Phase II, testing candidate genes. They will study the effects of current transcription factor reprogramming cocktails on supporting cell behavior, including the collection of additional transcriptomic and epigenetic data that will be shared with the CSE group. These experiments will be performed in a variety of systems, including a flattened epithelium guinea pig model that shares features with chronic human deafness. The Groves lab will use transgenic mice to detail the effects of the reprogramming cocktails in the organ of Corti. The Brigande lab will continue its work on creating efficient mouse model systems to interrogate candidate genes for sensory hair cell regeneration, as pioneered by testing whether viral delivery of these same reprogramming factors is also efficacious. The Raphael lab will take a similar approach in the guinea pig model, in this case using a virus designed and generated by the Groves and Segil labs. These experiments are part of the group’s broader efforts to develop new methods to deliver molecules and/or genes, for example by endogenous activation of reprogramming factors via CRISPR/Cas-9.

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Cross-Species Epigenetics

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Cross-Species Epigenetics
Tatjana Piotrowski, Ph.D. (chair), Stowers Institute for Medical Research
Alain Dabdoub, Ph.D., Sunnybrook Research Institute
Andy Groves, Ph.D., Baylor College of Medicine
Stefan Heller, Ph.D., Stanford University
The Lab of Neil Segil, Ph.D., University of Southern California
Litao Tao, Ph.D., Creighton University

This group will complete the collection of transcriptomic and epigenetic data from systems that regenerate (neonatal mouse, zebrafish, chick) and those that do not (mature mouse and human). In addition, they will begin to perform cross-species comparisons of the behavior of a shared set of hair cell loci across species. Collection of chick data is spearheaded by the Heller lab; the Groves, Segil, and Tao labs are responsible for mouse data; the Piotrowski lab leads work on zebrafish; and the Dabdoub lab will add data from humans. High-quality single-cell RNA-seq, ATAC-seq, and SHARE-seq data from multiple timepoints and conditions will be generated by all member labs.

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Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle

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Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle
Neil Segil, Ph.D., University of Southern California
Andy Groves, Ph.D., Baylor College of Medicine

The broad premise of the HRP is to identify molecules that could control hair cell regeneration. To do this, we are studying cell types and regenerative processes in multiple contexts and species, then integrating these data together to identify mechanisms that could potentially be turned on in the mouse cochlea to drive transdifferentiation (activating the correct set of hair cell–promoting genes in supporting cells). The role of this systems biology project is to provide the necessary data integration “glue,” binding together the results from the data generation projects. We will combine much of the data that is being generated by the HRP to advance our knowledge of hair cells, supporting cells, conversion of one cell type to another, and the potential for regeneration. By modeling all of the available HRP data, we will identify regulatory molecules that may contribute to regeneration.

Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle (2019)

Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle
Neil Segil, Ph.D., University of Southern California

Although hair cell regeneration does not occur in mammals, newborn mice harbor a latent capacity for some regenerative responses. However, this capability disappears within the first few weeks of life. This observation provides an experimental window that this proposal exploits to address fundamental questions about the failure of hair cell regeneration in mammals. Specifically, we propose experiments to identify those changes in the genetic material, the chromatin, that are responsible for orchestrating the differentiation of new hair cells within the newborn organ of Corti in the inner ear, and to investigate the changes in the chromatin that lead to the failure of regeneration in the adult mammalian inner ear.

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Epigenetics of the Mouse Inner Ear

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Epigenetics of the mouse inner ear
Michael Lovett, Ph.D., Imperial College London
David Raible, Ph.D., University of Washington
Neil Segil, Ph.D., University of Southern California
Jennifer Stone, Ph.D., University of Washington

Inner ear supporting cells from newborn mice harbor a latent capacity for some regenerative responses, but these disappear within the first few weeks of life. This observation provides an experimental window this proposal will exploit to address fundamental questions about the failure of hair cell regeneration in mammals. Specifically, we propose experiments to identify those changes in the genetic material, the chromatin, that are responsible for orchestrating the differentiation of new hair cells within the perinatal organ of Corti; and investigating the changes in the chromatin, the epigenome, that lead to the failure of regeneration in the adult inner ear.

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Epigenetics of Mouse Inner Ear Maturation and Transdifferentiation

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Epigenetics of mouse inner ear maturation and transdifferentiation
Neil Segil, Ph.D. University of Southern California
Jennifer Stone, Ph.D. University of Washington
Michael Lovett, Ph.D. Imperial College London
David Raible, Ph.D. University of Washington

Many genes are turned off by chemical modifications (epigenetic marks) that prevent activation of the gene. One hypothesis is that mammals cannot activate a hair cell regeneration program after the first few postnatal days because the responsible genes have been epigenetically silenced. This second-year project looks at these epigenetic marks in the ear for every gene, during both early and late development. The investigators will use this analysis to find candidate promoter regions, which control gene activity. Preliminary data support the idea that supporting cells turn off expression of key hair cell genes (e.g., Atoh1), and so a plausible approach to triggering regeneration in the mammalian ear is to reverse such changes.

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An Epigenetic Framework for Regulating HC Regeneration

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An epigenetic framework for regulating HC regeneration
David Raible, Ph.D. University of Washington
Neil Segil, Ph.D. University of Southern California
Jennifer S. Stone, Ph.D. University of Washington

Many genes are turned off by chemical modifications (epigenetic marks) that prevent activation of the gene. One hypothesis is that mammals cannot activate a hair cell regeneration program after the first few postnatal days because the responsible genes have been epigenetically silenced. This project uses HRP data that looks at these epigenetic marks in the ear for every gene, both during early and late development. The investigators will use this analysis to find candidate promoter regions, which control gene activity. Candidate genetic sequences from the mouse experiments will then be tested in zebrafish. These experiments will allow us to better understand how key hair cell regeneration genes are controlled.

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Growing Supporting Cells in Culture: Toward High-Throughput Screens

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Growing supporting cells in culture: toward high-throughput screens
Alain Dabdoub, Ph.D. University of Toronto
Andy Groves, Ph.D. Baylor College of Medicine
Neil Segil, Ph.D. University of Southern California

There is now a need for experimental systems to quickly test the candidates identified in Phase I experiments before moving into animal models and ultimately clinical trials. These researchers will develop a supporting cell culture system that can be used to quickly screen for factors that promote supporting cell growth or that promote supporting cells to generate hair cells. They will then test if this system can be used to grow cells at low density (allowing fewer cells to be used), to grow cells from older animals (necessary for a real drug), and to grow cells from the adult mouse utricle (which already shows limited regeneration). This project may give us the assay system needed to identify small molecule or gene therapeutics.

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RNA-seq Analysis of Vestibular Supporting Cells During Hair Cell Regeneration in Adult Mouse Vestibular Organs

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RNA-seq analysis of vestibular supporting cells during hair cell regeneration in adult mouse vestibular organs
Neil Segil, Ph.D. University of Southern California
Jennifer S. Stone, Ph.D. University of Washington

Because the adult mouse utricle, a vestibular organ, shows limited hair cell regeneration, the investigators will examine which genes are active in supporting cells that allow this activity. They will first complete RNA-seq analysis for adult mouse utricular supporting cells, to learn how gene expression is altered in these cells after hair cell damage. Second, they will perform ATAC-seq to locate regions in mouse chromatin that are altered after utricle damage, and correlate these data with RNA-seq results. Finally, they will define genes that are only expressed in type I or type II vestibular hair cells, which will help them determine strategies to promote regeneration of both cell types after damage in adult mammals. The data they generate from these three sets of experiments will be highly useful for defining potential therapeutic strategies for hair cell regeneration in humans.

Bioinformatic Support for Transcriptional and Epigenetic Profiling Projects of the Inner Ear

Bioinformatic support for transcriptional and epigenetic profiling projects of the inner ear
Neil Segil, Ph.D. University of Southern California

Like the Lovett group, the Segil group has generated and is continuing to generate large amounts of genomic data, and faces a substantial need for expertise to analyze those data and compare them to other HRP consortium datasets. Using bioinformatics tools, which allow computational analysis of the data, the group will investigate all of the HRP data to find key molecules that block hair cell regeneration in mammals.

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Comparative MiRNA Profiling of the Postnatal Mouse Cochlea and Regenerating Avian Basal Papilla

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Comparative miRNA profiling of the postnatal mouse cochlea and regenerating avian basal papilla
Michael Lovett, Ph.D. Imperial College London
Neil Segil, Ph.D. University of Southern California
Mark Warchol, Ph.D. Washington University School of Medicine

Micro RNAs (miRNAs) are small molecules that are transcribed from genomic DNA but not translated into protein. They provide regulation to many processes, uniquely affecting many genes simultaneously. This feature makes miRNA regulation of hair-cell regeneration a particularly interesting target for pharmacological intervention. This project will examine miRNA expression in the inner ears of mice and chicks, and determine the differences in response to hair-cell damage between organs that regenerate their hair cells (chick basilar papilla) and those that do not (mouse cochlea).

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RNAseq Analysis of Mouse Auditory and Vestibular Supporting Cells Following Hair Cell Killing In Vitro in DTR Mice

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RNAseq analysis of mouse auditory and vestibular supporting cells following hair cell killing in vitro in DTR mice
Neil Segil, Ph.D. University of Southern California
Jennifer S. Stone, Ph.D. University of Washington
Andy Groves, Ph.D. Baylor College of Medicine

At the fall 2014 meeting, the HRP consortium realized that we lacked an important genomics dataset, which would characterize at a molecular level how supporting cells respond to immediate death of hair cells. Using a mouse model developed by Ed Rubel, an HRP investigator, this project will examine how RNA expression changes in mice whose hair cells are killed with an application of diphtheria toxin. These “DTR mice” respond to the diphtheria toxin because all of their hair cells are uniquely sensitive to the toxin because of expression of a specific receptor. We will be able to compare the responses of supporting cells to the toxin with responses to hair cell death from aminoglycosides, already carried out by this group.

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