2015

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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Putative Hybrid Cells in Damaged Adult Organ of Corti

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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At the fall 2014 HRP meeting, several investigators shared unpublished data and all realized that they had seen the same phenomenon—in the mature mouse inner ear, after damage that wiped our hair cells, a number of cells could be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.

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Standardized Mouse Model for Hearing Loss Studies

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Standardized mouse model for hearing loss studies
Albert Edge, Ph.D. Massachusetts Eye and Ear Infirmary, Harvard Medical School
Stefan Heller, Ph.D. Stanford University School of Medicine

Testing how the mouse inner ear responds to hair-cell damage will be greatly facilitated by the mouse model developed in this project. Using these mice, the HRP will be able to rapidly kill hair cells very selectively, determine which cells were supporting cells prior to hair cell death, and to visualize newly formed hair cells. We expect that these mice will be useful in a variety of phase I, II, and III experiments.

Bioinformatic Analysis and Integration of Existing and Pending RNA-seq and ChiP-seq Datasets

Bioinformatic analysis and integration of existing and pending RNA-seq and ChiP-seq datasets
Michael Lovett, Ph.D. Imperial College London

As with the Segil bioinformatics proposal, the Lovett group has an acute need for a bioinformaticist to analyze the data generated by the group and integrate with the other HRP genomic data. The bioinformaticist supported by this project will analyze the group’s data in more detail and determine how the mouse differs from the two regenerating species (chick and zebrafish).

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Single Cell RT-PCR-based Expression Analysis of Homeostatic and Regenerating Zebrafish Neuromasts

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Single cell RT-PCR-based expression analysis of homeostatic and regenerating zebrafish neuromasts
Tatjana Piotrowski, Ph.D. Stowers Institute for Medical Research
Stefan Heller, Ph.D. Stanford University School of Medicine

The zebrafish is one of the primary models of the HRP; hair cells in its lateral line organ show robust regeneration, the accessibility of this organ means that we can watch the whole process take place using microscopy. However, the lack of complete synchronization of this process means that when we are looking at molecular changes, they are smeared out in time. This project will use single-cell transcript analysis techniques to order supporting cell responses to damage and determine the order of expression of transcripts with high precision.

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

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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.

Methods for Delivering Adenovirus Vectors to Non-Sensory Cells in the Deaf DTR Mouse Cochleae

Methods for delivering adenovirus vectors to non-sensory cells in the deaf DTR mouse cochleae
Yehoash Raphael, Ph.D. University of Michigan

The HRP is beginning to identify molecules that could modulate hair-cell regeneration, but introducing them into the mammalian inner ear is challenging. This project will develop methods using adenoviruses to deliver biomolecules into the mature mammalian cochlea. The methods used are designed to mimic a therapeutic approach.

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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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
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.

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