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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Identification of Candidate Regulators of Hair Cell Regeneration in the Chick Cochlea and Utricle

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Identification of candidate regulators of hair cell regeneration in the chick cochlea and utricle
Jennifer Stone, Ph.D. University of Washington
Mark Warchol, Ph.D. Washington University in St. Louis

This project has two components. In the first, the investigators will focus on validation of miRNA-seq data recently acquired by the HRP consortium; these new experiments will determine whether the genomics experiments accurately reflected miRNA fluctuations in the tissue, and may suggest candidate miRNA modulators of hair cell regeneration. In the second component, this project will test which specific signaling pathways are important for the proliferation of supporting cells and the regeneration of hair cells using pharmacological approaches.

Fish Epigenomics and Enhancer Screening

Fish epigenomics and enhancer screening
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research

As explained in Project #2, many genes are turned off by chemical modifications (epigenetic marks) that silence genes and prevent their activation. This inactivation often occurs at enhancers, which are regions of DNA that control the activation of genes. In this project, Piotrowski will use the ATAC-seq and H3K27ac ChIP-seq methods, which were successful for the mouse inner ear, to find enhancers that are active during hair cell regeneration in the fish. Identification of regeneration enhancers will enable the HRP to examine epigenetic marks comparatively—to determine whether regenerating species, such as the zebrafish and chick, utilize different enhancers than non-regenerating species like the mouse, or whether these enhancers are inactive in mammals.

Fish CRISPR/Cas9 Screen, Enhancer screen

Fish CRISPR/Cas9 screen, enhancer screen
David Raible, Ph.D. University of Washington

The functional testing of candidate genes is essential for us to be able to wade through the dozens or hundreds of candidates that have been put forward from the Lovett-Warchol RNA-seq experiments. Raible has successfully developed a zebrafish CRISPR screen approach that allows for the rapid testing of candidate genes for their role in hair cell development and regeneration. Raible will continue in his characterization of genes that affect hair cell regeneration. In addition, the project includes a second aim that proposes to test candidate enhancers in the zebrafish.

Integrative Systems Biology of Hearing Restoration

Integrative systems biology of hearing restoration
Ronna Hertzano, M.D., Ph.D. University of Maryland

The HRP consortium aims to identify factors that can either block or promote regeneration and has generated multiple genomics datasets from inner ears of regenerating and non-regenerating model organisms. This proposal aims to predict these causal factors by a detailed analysis of those datasets. The project’s two investigators—Hertzano, who is well versed in inner ear development and genomics, and Seth Ament, Ph.D., an expert in systems biology and neurobiology—will develop a quantitative model for the gene regulatory networks in hair cells and hair cell precursors that will allow them to predict key genes (e.g., master regulator transcription factors) that are associated with the ability to regenerate hair cells. They will use cutting-edge network biology approaches that integrate information about the preservation and divergence of gene co-expression across species and conditions, with information about the targets of hundreds of transcription factors.

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Establishing the Human Utricle from Surgical Patients as a Translational in Vitro Model for Hair Cell Regeneration

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Establishing the human utricle from surgical patients as a translational in vitro model for hair cell regeneration
Alain Dabdoub, Ph.D. University of Toronto
Stefan Heller, Ph.D. Stanford University
Michael Lovett, Ph.D. Imperial College London

These investigators will work with human utricles harvested during surgery to examine whether the response of human inner ear tissue to damage is similar to that of our mammalian model, the mouse. They will culture human utricles for extended lengths of time following damage with aminoglycoside drugs and determine whether the utricles show any proliferation of their supporting cells (making new supporting cells) or regeneration of their hair cells (making new hair cells). Mouse utricles have a limited ability to show proliferation and regeneration, and it is important to determine whether they are a good model for humans. The investigators also intend to examine gene expression in these human utricles to determine the molecular similarity of the cells of interest to their human counterpart cells.

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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.

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

Here the gEAR (gene expression for auditory research) portal will be further developed for a second year to perform key gene comparison tasks for the HRP. The gEAR allows for the graphic visualization of gene expression data in an intuitive way: Multiple datasets can be displayed on 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.

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Transcriptomics-based Analysis of Hair Cell Regeneration in Non-Mammals

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Transcriptomics-based analysis of hair cell regeneration in non-mammals
Stefan Heller, Ph.D. Stanford University
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research
Jennifer Stone, Ph.D. University of Washington
Mark Warchol, Ph.D. Washington University in St. Louis
Michael Lovett, Ph.D. Imperial College London

In this third-year project, we will disrupt hair cells, then analyze how gene expression changes in single cells from species that show robust hair cell regeneration. Heller’s lab will examine the consequences of aminoglycoside damage at the single-cell level in the chick utricle, while the Piotrowski lab will examine fish lateral-line cells. In another component, the project will add two time points to the chick cochlea and utricle bulk RNA-seq datasets that were generated by Lovett and Warchol and which are extremely valuable datasets for the HRP consortium.

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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.

An In Vivo Model System to Interrogate Gene and Pathway Candidates Involved in Sensory Epithelial Regeneration of the Mammalian Inner Ear

An in vivo model system to interrogate gene and pathway candidates involved in sensory epithelial regeneration of the mammalian inner ear
John Brigande, Ph.D. Oregon Health & Science University

An example of the consortium moving into phase II, this project will deliver into the embryonic inner ear of mice genes that could regulate hair cell regeneration; these genes will be in an inactive state. Delivery of an activating molecule in the mature inner ear will then allow that gene to be expressed an appropriate time relative to hair cell damage. While this delivery method is not appropriate for any eventual therapy, it will allow us to examine to determine whether any specific molecules are capable of triggering hair cell regeneration in the mouse.

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