Headlines in Hearing Restoration

By Yishane Lee

The cornerstone of Hearing Health Foundation for six decades has been funding early-career hearing and balance researchers through its Emerging Research Grants (ERG) program. Many ERG scientists have gone on to obtain prestigious National Institutes of Health (NIH) funding to continue their HHF-funded research; since 1958, each dollar awarded to ERG scientists by HHF has been matched by NIH investments of more than $90. Within the scientific community, ERG is a competitive grant awarded to the most promising investigators, and we’re always especially pleased when our ERG alumni who are now also members of or affiliated with our Hearing Restoration Project consortium make headlines in the mainstream news for their scientific breakthroughs.

Hair cells in the mouse cochlea courtesy of the lab of Hearing Restoration Project (HRP) member Andy Groves, Ph.D., Baylor College of Medicine.

Ronna Hertzano, M.D., Ph.D. (2009–10): Hearing Restoration Project consortium member Hertzano, an associate professor at the University of Maryland School of Medicine, and colleagues identified a gene, Ikzf2, that acts as a key regulator for outer hair cells whose loss is a major cause of age-related hearing loss. The Ikzf2 gene encodes helios, a transcription factor (a protein that controls the expression of other genes). The mutation of the gene in mice impairs the activity of helios in the mice, leading to an outer hair cell deficit.

Reporting in the Nov. 21, 2018, issue of Nature, the team tested whether the opposite effect could be created—if an abundance of helios could boost the population of outer hair cells. They introduced a virus engineered to overexpress helios into the inner ear hair cells of newborn mice, and found that some mature inner hair cells became more like outer hair cells by exhibiting electromotility, a property limited to outer hair cells. The finding that helios can drive inner hair cells to adopt critical outer hair cell characteristics holds promise for future treatments of age-related hearing loss.

Patricia White, Ph.D. (2009, 2011), with Hearing Restoration Project member Albert Edge, Ph.D.: White, a research associate professor at the University of Rochester Medical Center, Edge, a professor of otolaryngology at Massachusetts Eye and Ear and Harvard Medical School, and team have been able to regrow the sensory hair cells found in the mouse cochlea. The study, published in the European Journal of Neuroscience on Sep. 30, 2018, builds on White’s prior research that identified a family of receptors called epidermal growth factor (EGF) that is responsible for activating supporting cells in the auditory organs of birds. When triggered, these cells proliferate and foster the generation of new sensory hair cells. In mice, EGF receptors are expressed but do not drive regeneration of hair cells, so it could be that as mammals evolved, the signaling pathway was altered.

The new study aimed to unblock the regeneration of hair cells and also integrate them with nerve cells, so they are functional, by switching the EGF signaling pathway to act as it does in birds. The team focused on a specific receptor called ERBB2, found in supporting cells. They used a number of methods to activate the EGF signaling pathway: a virus targeting ERBB2 receptors; mice genetically altered to overexpress activated ERBB2; and two drugs developed to stimulate stem cell activity in the eye and pancreas that are already known to activate ERBB2 signaling. The researchers found that activating the ERBB2 pathway triggered a cascading series of cellular events: Supporting cells began to proliferate and started the process of activating other neighboring stem cells to lead to “apparent supernumerary hair cell formation,” and these hair cells’ integration with the network of neurons was also supported.

This was prepared using press materials from the University of Maryland and the University of Rochester. For more, see hhf.org/hrp.

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The Hearing Restoration Project: Update on the Seattle Plan and More

By Peter G. Barr-Gillespie, Ph.D.

Hearing Health Foundation launched the Hearing Restoration Project (HRP) to understand how to regenerate inner ear sensory cells in humans to restore hearing. These sensory hair cells detect and turn sound waves into electrical impulses that are sent to the brain for decoding. Once hair cells are damaged or die, hearing is impaired, but in most species, such as birds and fish, hair cells spontaneously regrow and hearing is restored.

The overarching principle of the HRP consortium is cross-discipline collaboration: open sharing of data and ideas. By having almost immediate access to one another’s data, HRP scientists are able to perform follow-up experiments much faster, rather than having to wait years until data is published.

Regenerated hair cells from chicken auditory organs, with the cell body, nucleus and hair bundle labeled with various colored markers. Image courtesy of Jennifer Stone, Ph.D.

You may remember that two years ago, we changed how we develop our projects. We decided together on a group of four projects—the “Seattle Plan”—that are the most fundamental to the consortium’s progress. These projects, which grew out of previous HRP projects, have now been funded for two years, and considerable progress has been made. We have also funded several other projects that have bubbled up out of new observations and capabilities, and they have added considerably to our knowledge base. With this in mind, I am pleased to share with you the latest updates for our 2018–19 projects.

SEATTLE PLAN PROJECTS

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

• Found that all “tall” hair cells are exclusively regenerated mitotically in this animal model.

• Compiled evidence for different supporting cell subtypes.

• Obtained good quality single cell RNA sequencing (scRNA-seq) data and are in the process of evolving an analysis strategy for the baseline cell types (control group). Identified about 50 novel marker genes for hair cells, supporting cells, and homogene cells, including subgroups.

• Developed a strategy to finish all scRNA-seq using a novel peeling technique and latest generation library construction methods.

•  Established two methods for multi-color in situ hybridization (PLISH, proximity ligation in situ hybridization) and SGA (sequential genomic analysis) for spatial and temporal mRNA expression validation.
  
  

Epigenetics of the mouse inner ear
Michael Lovett, Ph.D., David Raible, Ph.D., Neil Segil, Ph,D., Jennifer Stone, Ph.D.

• Completed epigenetic, chromatin structure, and RNA-seq datasets for FACS-purified cochlear hair cells and supporting cells from postnatal day 1 and postnatal day 6 mice, and provision of these data sets to the gEAR (gene Expression Analysis Resource portal) for mounting on their webpage through EpiViz for access by the HRP consortium.

• Established a webpage (EarCode) so that HRP consortium members can access the current data directly through a University of California, Santa Cruz, genome browser.

• Discovered maintenance of the transcriptionally silent state of the hair cell gene regulatory network in perinatal supporting cells is dependent on a combination of H3K27me3 and active H3k27-deacetylation, and that during transdifferentiation, these epigenetic marks are modified to an active state.

Mouse functional testing
John Brigande, Ph.D.

• Defined in vitro and in vivo model systems to interrogate genome editing efficacy using CRISPR/Cas9.
  
  

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

• Added scRNA-seq workbench for easy sharing and viewing of scRNA-seq data. Such data, which are now driving the field forward, have been particularly difficult to share

• Created additional public datasets to improve data sharing.

• Completely rewrote the gEAR backbone to be updated to the latest technologies, allowing the portal to now to handle a much larger number of datasets and users.

• Performed hands-on gEAR workshops at the Association for Research in Otolaryngology and the Gordon Research Conference, increasing the number of users with accounts to greater than 300.

Single Cell RNA-seq of homeostatic neuromasts
Tatjana Piotrowski, Ph.D.

• Optimized protocols for fluorescent-activated cell sorting and scRNA-seq; obtained high quality scRNA-seq transcriptome results from 1,400 neuromast cells; clustered all cells into seven groups; and performed analyses to align the cells along developmental time, providing a temporal readout of gene expressions during hair cell development.
  
  

OTHER PROJECTS

Integrated systems biology of hearing restoration
Seth Ament, Ph.D.

• Discovered 29 novel risk loci for age-related hearing difficulty through new analyses of genome-wide association studies of multiple hearing-related traits in the U.K. Biobank (comprising 330,000 people), and predicted the causal genes and variants at these loci through integration with transcriptomics and epigenomics data from HRP consortium members.

• Generated scRNA-seq of 9,472 cells in the neonatal mouse cochlea and utricle (postnatal days 2 and 7).

• Conducted systems biology analyses that integrate multiple HRP datasets to characterize gene regulatory networks and predict driver genes associated with the development and regeneration of hair cells. These analyses utilize scRNA-seq of sensory epithelial cells in mouse, chicken, and zebrafish hearing and vestibular organs, as well as epigenomic data (ATAC-seq) from hair cells, support cells, and non-epithelial cells in the mouse cochlea.

Comparison of three reprogramming cocktails
Andy Groves, Ph.D.

• Created and validated transgenic mouse lines expressing three different combinations of reprogramming transcription factors.

• Demonstrated these lines can produce new hair cell–like cells in the undamaged and damaged cochlea of the immature mouse.

• Compiled preliminary data showing Atoh1 and Gfi1 genes can create ectopic hair cells in the adult mouse cochlea.

Signaling molecules controlling avian auditory hair cell regeneration
Jennifer Stone, Ph.D.

• Identified four molecular pathways (FGF, BMP, VEGF, and Wnt) that control hair cell regeneration in the bird auditory organ. These pathways were identified in Phase I (gene discovery) as being transcriptionally dynamic in birds, fish, and mice during regeneration, which indicated they may be universal regulators of hair cell regeneration.

• Determined that the Notch signaling pathway (a powerful inhibitor of stem cells) also blocks supporting cell division in the chicken auditory organ after damage. This discovery shows that Notch is a negative regulator of regeneration, conserved in birds, fish, and mice.

• Identified signaling molecules in birds that are correlated with either mitotic or non-mitotic modes of hair cell regeneration, and are now exploring how these signaling molecules interact to determine which mode of regeneration occurs. Since mammals only exhibit non-mitotic regeneration, we are particularly interested in determining how this mode is controlled.
  
  

UP NEXT

We look forward to our annual meeting, which will be held in Seattle in November. There we will discuss and integrate these data to develop our plans for our 2019–20 projects.

As always we are very grateful for the donations we receive to fund this groundbreaking research to find better treatments for hearing loss and related conditions. Every dollar counts, and we sincerely thank our supporters.

HRP scientific director Peter G. Barr-Gillespie, Ph.D., is a professor of otolaryngology at the Oregon Hearing Research Center, a senior scientist at the Vollum Institute, and the interim senior vice president for research, all at Oregon Health & Science University. For more, see hhf.org/hrp.

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A Powerful New Bioinformatics Tool

By Stefan Heller, Ph.D.

Our paper describing a new bioinformatics tool—and to showcase the software, a very detailed investigation as to how inner ear hair cells assemble their hair bundles—appeared in Cell Reports on June 5, 2018.

The creation of the CellTrails tool was supported in part by Hearing Health Foundation’s Hearing Restoration Project (HRP) but moreover, it is the product of recognizing existing limitations of data analysis, going back to the drawing board multiple times, and finally getting to a “product” that is going to be the workhorse to analyze a good part of the bioinformatics data that the HRP has been accumulating for years.

An image taken at 40x magnification using a confocal microscope in the Stefan Heller lab shows a 7-day-old chicken cochlea. Credit: Amanda Janesick, Ph.D.

The ideas came from conversations between HRP scientific director Peter Barr-Gillespie, Ph.D., and me and our getting stuck with trying to make sense of all the data—so the tool is the direct product of interactions through the HRP.  It follows on our work utilizing single-cell gene expression analysis to examine the genetic instructions allowing individual cells to differentiate (change) into other types of cells, such as inner ear supporting cells that turn into hair cells in species other than mammals, and thereby restoring hearing.

The tool helps us pinpoint where specific single cells are located in an organ, and their trajectories as they undergo transformations, information that was lost or fuzzy before. With it we can create a more robust, visually rendered gene expression landscape. Two postdoctoral fellows in my lab were instrumental in CellTrails: bioinformatics researcher Daniel Ellwanger, Ph.D., the tool’s primary developer, and Mirko Scheibinger, Ph.D., who validated its predictions.

I hope many researchers make use of CellTrails, accessible online, to analyze their own mountains of data. As I told Stanford’s SCOPE Blog, “Single cell transcriptome analysis and reconstruction of spatial and temporal relationships among cells is an exploding new technology. A lot of labs are faced with the challenge of analyzing the data from single cells. This study is a rather extensive study that goes beyond the inner ear field because it provides a new way to analyze single cell transcriptomic data.”

I truly feel that the seeds that were planted years ago are now growing into sizable plants—we have a massive "chick regeneration inner ear plant” that is starting to thrive!

Find the tool at hellerlab.stanford.edu/celltrails.

Stanford University’s Stefan Heller, Ph.D., is a member of HHF’s Hearing Restoration Project, where Oregon Health & Science University’s Peter Barr-Gillespie, Ph.D., is the scientific director.

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