2018

Comparison of Three Reprogramming Cocktails

Comparison of three reprogramming cocktails
Andy Groves, Ph.D., Baylor College of Medicine

Each cell type in the human body is defined by its activation of a unique combination of genes that endow each cell type with specific properties. The activation of these genes is achieved by special proteins known as transcription factors. These “switches” are responsible for turning on appropriate genes in one cell type and preventing inappropriate genes from being activated. In recent years, investigators have identified a number of these transcription factors that lead to the formation of hair cells in the inner ear. The goal of this project is to rigorously test the extent to which a cocktail of transcription factors is able to reprogram supporting cells of the inner ear to turn into hair cells.

Transcriptome Changes in Single Chick Cells

Transcriptome changes in single chick cells
Stefan Heller, Ph.D., Stanford University

Chicks regenerate hair cells in auditory and vestibular organs after damage, making them a valuable animal model to study the signals controlling hair cell regeneration. This project aims to identify changes in gene expression after hair cell loss in the chick cochlea and vestibular system. The collected data will be comprehensive because it will cover all detectable expressed genes. Subsequent data analysis will focus on establishing a sequence of gene expression changes that we hypothesize will correlate with important steps of the hair cell regeneration process. These steps include the signals that initiate, execute, sustain, and ultimately terminate the regenerative process. Comparison among the different organs and across species through collaborations with other HRP investigators will allow us to draw conclusions about species-specific specialized mechanisms as well as more general processes that control hair cell regeneration.

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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.

Signaling Molecules Controlling Avian Hair Cell Regeneration

Signaling molecules controlling avian hair cell regeneration
Jennifer Stone, Ph.D., University of Washington

HRP members have spent the past three years gathering information about genes that are turned on or off after inner ear hair cell damage in the chick, fish, and mouse. Some of these genes may encode therapeutic agents that can be applied to stimulate hair cell regeneration in humans. Our HRP studies and others have found that five signaling pathways (Wnt, VEGF, BMP4, Notch, and FGF) are important regulators of hair cell regeneration in the chick cochlea (the basilar papilla). The expression and activity of these pathways change significantly after hair cell damage, and the experimental manipulation of activity in each pathway either boosts or dampens hair cell regeneration. Furthermore, each pathway shows distinct regional expression patterns in the basilar papilla, which implicates it in either mitotic regeneration or non-mitotic regeneration—two distinct ways in which hair cells are replaced after damage. Studies in other growing tissues demonstrate that these five pathways regulate one another in temporally and spatially restricted patterns, in order to coordinate cell growth, differentiation, and patterning. Thus, it is likely that any therapy leading to safe and stable hair cell regeneration will require coordinated manipulation of more than one gene or pathway in the cochlea. In this study, we propose to begin to determine how these five powerful pathways interact to enable and control hair cell regeneration in the chick basilar papilla after hair cell damage.

Integrated Systems Biology of Hearing Restoration

Integrated systems biology of hearing restoration
Seth Ament, Ph.D., University of Maryland School of Medicine

The goal of this proposal is to support the HRP through data integration and systems biology. We propose two related goals for 2018, based on our preliminary network modeling results and discussions with other HRP investigators. We will (a) predict regulatory genes driving cell fate decisions in the developing mouse cochlea using refined transcriptional regulatory networks, gene co-expression networks, protein-protein interaction networks, and related methods. We will (b) extend these analyses to the zebrafish and chick models by projecting networks from the mouse cochlea onto data from these other species. Our goal is to generate testable predictions about driver genes and perturbations (deviations) that could influence hearing restoration.

Implementing the gEAR for Data Sharing within the HRP

Implementing the gEAR for data sharing within the HRP
Ronna Hertzano, M.D., Ph.D. University of Maryland

The HRP takes a multi-investigator, multi-species, multi-omic (methods of tracking gene expression, or instructions), and cell type‐specific approach to define the underpinnings of differences among hair cell regeneration in the chick, fish, and mouse with the aim of identifying keys for hair cell regeneration in mammals. Consequently, the consortium generates large amounts of data that are difficult to visualize, conceptualize, and analyze. The gEAR portal (gene Expression Analysis Resource, umgear.org) allows for simple visualization of multi-omic, multi-species datasets in the public or private domain—without the need for advanced informatics skills. In the first two years of funding from the HRP, we focused primarily on developing tools for multi-omic, multi-species data upload and visualization. Numerous features were added, and all available HRP datasets were uploaded for sharing within the consortium. In parallel, all tools and features developed for the consortium were made available in the public domain—leading the gEAR to be a primary portal for multi-omic data sharing and visualization within the field. With the next two years of funding committed (years three and four), the vast majority of our efforts will be focused on (a) the continued upload of HRP and public datasets, and (b) the development and integration of analysis tools.

Mouse Functional Testing

Mouse functional testing
John Brigande, Ph.D. Oregon Health & Science University

The conceptual framework of this project wrestles with a persistent challenge facing the HRP consortium: We must verify that the candidate genes we advance as regenerative genes actually perform as advertised. Is our altering of the gene expression of a candidate gene truly the trigger that turns supporting cells into hair cells? Our solution is to devise a mammalian model system that meets several definitive criteria. First, we need deafened adult mammalian inner ears to detect the production of new hair cells; we achieve this genetically by specifically killing hair cells that are uniquely sensitive to a bacterial toxin. Second, we need a way to turn on or turn off the candidate gene after the hair cells are dead; we achieve this by chemically activating a gene that in turn unmasks the expression of the proposed candidate. Third, we need a way to detect newly produced hair cells in the cochlea; we achieve this by using a tissue clearing and staining procedure developed with HRP funding that allows us to detect hair cells produced from supporting cells.

This entire approach is called a model system for validating candidate genes for hair cell regeneration. But one size does not fit all, and we need to continually adapt the core model system to achieve full functionality. In this proposal, we aim to test our model system in healthy ears to see if tweaking our candidate genes can produce hair cells from supporting cells in the absence of widespread hair cell death. The idea here is to make sure that the bacterial toxin–mediated destruction of hair cells is not interfering with our candidate gene activity and new hair cell production. Our second goal is to test a new virus delivery system that will allow us to evaluate larger candidate genes. Presently, we can only express very small genes with the virus we are using, restricting candidate gene verification. Our final goal is to evaluate a modified viral vector that is encased in lipid membranes to learn if it can express candidate genes more efficiently in supporting cells. The benefit of this approach is that viral production is quick, inexpensive, and requires no special training or expertise. Successful completion of this proposal will establish a comprehensive, cost-effective approach to aggressively validate candidate genes for hair cell regeneration.

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