2016

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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.

Implementing the gEAR for Discretionary Data Sharing within the HRP (2016)

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

Here the gEAR portal (gene Expression for Auditory Research portal) will be adapted to perform key gene comparison tasks for the HRP. The gEAR allows for graphic visualization of gene expression data in an intuitive way: multiple datasets can be displayed in a single page, all represented by cartoons and dynamically colored based on levels of gene expression. Additional features will be added through dedicated developer time to specifically serve the needs of the consortium, including additional dataset-specific graphics, tools for cross-dataset and cross-species comparisons, and intuitive integration of DNA structure and gene expression. Once the gEAR has been adapted for the HRP, all HRP members will be able to browse and interrogate the consortium data on a regular basis, allowing for computational identification of targets that lead to hair cell regeneration.

Pou4f3/DTR Mouse Colony Maintenance

Pou4f3/DTR mouse colony maintenance
Edwin Rubel, Ph.D. University of Washington

The Pou4f3/DTR mouse has become an important mammalian model in which to study inner ear hair cell regeneration. Most HRP consortium labs and many other labs that study potential genes and drugs to induce or facilitate hair cell regeneration rely on this model to behave in a systematic fashion. Unfortunately, on occasion variations of this model have occurred in the colony such that hair cells are preserved after a treatment intended to remove all hair cells. To prevent this from recurring in the HRP’s colony and to replace mice when this occurs elsewhere, the HRP has established a single location where we can be assured of having consistent behavior of the model.

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A Cross-Species Approach Toward Functional Testing for Hair Cell Regeneration

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A cross-species approach toward functional testing for hair cell regeneration
Andy Groves, Ph.D. Baylor College of Medicine
David Raible, Ph.D. University of Washington
Jennifer S. Stone, Ph.D. University of Washington

This multi-modal, cross-species analysis project allows the HRP to test the role of molecules identified in our previous and ongoing experiments. Experience gained in the first year has led to first screening pathway inhibitors in chicks, then validating these initial hits by generating gene knockouts in zebrafish using CRISPR technology. Targets that pass both screens will then be assessed in the mouse. This project exemplifies the consortium’s shift toward Phase II of the HRP’s strategic research plan.

A Mouse Model System to Interrogate Candidate Genes for Sensory Hair Cell Regeneration

A mouse model system to interrogate candidate genes for sensory hair cell regeneration
John Brigande, Ph.D. Oregon Health & Science University

This Phase II project involves the delivery of genes and reagents that could regulate hair cell regeneration into the embryonic inner ear of mice. Two approaches will be taken. In the first, transcription-factor genes that activate pathways that may be involved in regeneration will be delivered, and their ability to trigger hair cell regeneration after those cells are killed will be assessed. In the second, CRISPR/Cas9 reagents will be used to turn off genes that might be preventing hair cell regeneration. While these are experimental and not therapeutic approaches, the project will allow us to determine whether any specific molecules are capable of triggering hair cell regeneration in the mouse.

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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.

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Single-cell RNA-seq Expression Analysis of Homeostatic Zebrafish Neuromasts

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Single-cell RNA-seq expression analysis of homeostatic zebrafish neuromasts
Stefan Heller, Ph.D. Stanford University School of Medicine
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research

The zebrafish is one of the primary models of the HRP, as hair cells in its lateral line organ show robust regeneration. Because of its superficial location in the skin, we can watch the regeneration process at the single-cell level using microscopy. We discovered that even within a regenerating cell population, individual cells are not synchronized in their behavior and gene expression. Therefore, conventional
gene expression analyses of the entire cell population provide only averages of gene expression, masking the cell heterogeneity.This project will use single-cell expression analysis techniques to determine how many supporting cell types exist and in future experiments how they respond to damage with high precision.

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Single-cell transcriptional profiling of chicken utricle and Basilar Papilla Sensory Epithelium Cells After Aminoglycoside-induced Hair Cell loss

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Single-cell transcriptional profiling of chicken utricle and basilar papilla sensory epithelium cells after aminoglycoside-induced hair cell loss
Stefan Heller, Ph.D. Stanford University School of Medicine
Jennifer S. Stone, Ph.D. University of Washington
Michael Lovett, Ph.D. Imperial College London
Mark Warchol, Ph.D. Washington University School 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.

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