Jennifer Stone Ph.D.

, , , , , ,

Integrative Analysis

, , , , , ,

Integrative Analysis
Seth Ament, Ph.D. (co-chair), University of Maryland
Ronna Hertzano, M.D., Ph.D. (co-chair), National Institute on Deafness and Other Communication Disorders
Albert Edge, Ph.D., Mass Eye & Ear
Stefan Heller, Ph.D., Stanford University
David Raible, Ph.D., University of Washington
Jennifer Stone, Ph.D., University of Washington

This group will take the lead on data curation and analysis. A dedicated full-time HRP analyst is working across groups to help collect and process data, thereby facilitating a broader analysis of cell states and trajectories across species. The group will start by annotating hair cell types from all species so that anyone in the field can assess what kind of hair cell their regeneration approaches may produce, while also easing identification of common hair cell genes, which will help the Cross-Species Epigenetics group. Analysis of the hair cells produced in mouse organoids will be performed as an example. The Ament lab will leverage their expertise in bioinformatics, while the Hertzano lab will continue to oversee upkeep of gEAR, with a goal of making it even easier for HRP members to post their new data and for others in the community to analyze those data. The Edge lab will take the lead on the development of organoids as a screening platform for the future. The Heller, Hertzano, Raible, and Stone labs will validate markers by in situ hybridization across species, and all working group members will help direct the analysis.

, , ,

Epigenetics of the Mouse Inner Ear

, , ,

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.

,

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

,

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.

, , ,

Epigenetics of Mouse Inner Ear Maturation and Transdifferentiation

, , ,

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.

, , , ,

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

, , , ,

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.

, ,

An Epigenetic Framework for Regulating HC Regeneration

, ,

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.

, ,

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

, ,

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.

,

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

,

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.

, , ,

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

, , ,

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.

, , ,

Putative Hybrid Cells in Damaged Adult Organ of Corti

, , ,

Putative hybrid cells in damaged adult organ of Corti
Albert Edge, Ph.D. Massachusetts Eye and Ear Infirmary, Harvard Medical School
Jennifer S. Stone, Ph.D. University of Washington
Mark Warchol, Ph.D. Washington University School of Medicine
Liz Oesterle, Ph.D. University of Washington
Edwin Rubel, Ph.D. University of Washington

At the fall 2014 HRP meeting, several investigators shared unpublished data and all realized that they had seen the same phenomenon—in the mature mouse inner ear, after damage that wiped our hair cells, a number of cells could be detected that had molecular signatures of hair cells yet still maintained a morphology similar to supporting cells. These hybrid cells could be supporting cells that have partially differentiated towards hair cells, and the group will characterize them in more depth. The project is exciting as it shows that there is some supporting cell response to damage and because we will be able to determine more precisely where the molecular block to full hair cell regeneration occurs.

, ,

A Cross-Species Approach to Functional Testing of Hair Cell Regeneration Pathways

, ,

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

This group has developed a multi-modal, cross-species analysis platform that will allow the HRP to test the role of molecules identified in our previous and ongoing experiments. Coupling gene knockout in zebrafish using the newly emerging CRISPR technology, which allows rapid testing, with pathway manipulation and gene knockouts in chicks and mice, we will be able to rapidly interrogate the many molecules the group wishes to examine. This project exemplifies the consortium’s shift towards phase II of the project.

, ,

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

, ,

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.

, , ,

Single Cell Transcriptional Profiling of Chicken Utricle and Basilar Papilla Sensory Epithelium Cells After Aminoglycoside-Induced Hair Cell Loss

, , ,

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
Mark Warchol, Ph.D. Washington University School of Medicine
Jennifer S. Stone, Ph.D. University of Washington
Michael Lovett, Ph.D. Imperial College London

While the HRP has excellent datasets showing the response of chicken auditory and vestibular cells to damage, the experiments examine all cells (including both hair cells and supporting cells) and are not temporally precise. In these experiments, the investigators will damage hair cells, then examine the molecular responses of hundreds of individual cells. Bioinformatics techniques will allow them to order those cells along a timeline that will reveal the molecular changes that unfold during hair cell regeneration.

Sign up to receive groundbreaking hearing and balance research updates.