Gene Expression

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.

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.

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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.

 

Empower the Hearing Restoration Project's life-changing research. If you are able, please make a contribution today.

 
 
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NIH Researchers Show Protein in Inner Ear Is Key to How Cells That Help With Hearing and Balance Are Positioned

By the National Institute on Deafness and Other Communication Disorders (NIDCD)

Line of polarity reversal (LPR) and location of Emx2 within two inner ear structures. Arrows indicate hair bundle orientation. Source: eLife

Line of polarity reversal (LPR) and location of Emx2 within two inner ear structures. Arrows indicate hair bundle orientation. Source: eLife

Using animal models, scientists have demonstrated that a protein called Emx2 is critical to how specialized cells that are important for maintaining hearing and balance are positioned in the inner ear. Emx2 is a transcription factor, a type of protein that plays a role in how genes are regulated. Conducted by scientists at the National Institute on Deafness and Other Communication Disorders (NIDCD), part of the National Institutes of Health (NIH), the research offers new insight into how specialized sensory hair cells develop and function, providing opportunities for scientists to explore novel ways to treat hearing loss, balance disorders, and deafness. The results are published March 7, 2017, in eLife.

Our ability to hear and maintain balance relies on thousands of sensory hair cells in various parts of the inner ear. On top of these hair cells are clusters of tiny hair-like extensions called hair bundles. When triggered by sound, head movements, or other input, the hair bundles bend, opening channels that turn on the hair cells and create electrical signals to send information to the brain. These signals carry, for example, sound vibrations so the brain can tell us what we’ve heard or information about how our head is positioned or how it is moving, which the brain uses to help us maintain balance.

NIDCD researchers Doris Wu, Ph.D., chief of the Section on Sensory Cell Regeneration and Development and member of HHF’s Scientific Advisory Board, which provides oversight and guidance to our Hearing Restoration Project (HRP) consortium; Katie Kindt, Ph.D., acting chief of the Section on Sensory Cell Development and Function; and Tao Jiang, a doctoral student at the University of Maryland College Park, sought to describe how the hair cells and hair bundles in the inner ear are formed by exploring the role of Emx2, a protein known to be essential for the development of inner ear structures. They turned first to mice, which have been critical to helping scientists understand how intricate parts of the inner ear function in people.

Each hair bundle in the inner ear bends in only one direction to turn on the hair cell; when the bundle bends in the opposite direction, it is deactivated, or turned off, and the channels that sense vibrations close. Hair bundles in various sensory organs of the inner ear are oriented in a precise pattern. Scientists are just beginning to understand how the hair cells determine in which direction to point their hair bundles so that they perform their jobs.

In the parts of the inner ear where hair cells and their hair bundles convert sound vibrations into signals to the brain, the hair bundles are oriented in the same direction. The same is true for hair bundles involved in some aspects of balance, known as angular acceleration. But for hair cells involved in linear acceleration—or how the head senses the direction of forward and backward movement—the hair bundles divide into two regions that are oriented in opposite directions, which scientists call reversed polarity. The hair bundles face either toward or away from each other, depending on whether they are in the utricle or the saccule, two of the inner ear structures involved in balance. In mammals, the dividing line at which the hair bundles are oriented in opposite directions is called the line of polarity reversal (LPR).

Using gene expression analysis and loss- and gain-of-function analyses in mice that either lacked Emx2 or possessed extra amounts of the protein, the scientists found that Emx2 is expressed on only one side of the LPR. In addition, they discovered that Emx2 reversed hair bundle polarity by 180 degrees, thereby orienting hair bundles in the Emx2 region in opposite directions from hair bundles on the other side of the LPR. When the Emx2 was missing, the hair bundles in the same location were positioned to face the same direction.

Looking to other animals to see if Emx2 played the same role, they found that Emx2 reversed hair bundle orientation in the zebrafish neuromast, the organ where hair cells with reversed polarity that are sensitive to water movement reside.

These results suggest that Emx2 plays a key role in establishing the structural basis of hair bundle polarity and establishing the LPR. If Emx2 is found to function similarly in humans, as expected, the findings could help advance therapies for hearing loss and balance disorders. They could also advance research into understanding the mechanisms underlying sensory hair cell development within organs other than the inner ear.

This work was supported within the intramural laboratories of the NIDCD (ZIA DC000021 and ZIA DC000085).

Doris Wu Ph.D. is member of HHF’s Scientific Advisory Board, which provides oversight and guidance to our Hearing Restoration Project (HRP) consortium This article was repurpsed with permission from the National Institute on Deafness and Other Communication Disorders. 

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Our 2016 Annual Report Is Now Available!

By Frankie Huang

Hearing Health Foundation (HHF) is happy to announce that its 2016 annual report is now available. The report is an in-depth review of our activities, events, and achievements for fiscal year 2016. We are very proud of top marks from top charity-rating agencies and even more proud our audited financial statements.

We are fortunate to have such generous supporters who raised funds to further HHF’s mission of prevention, education, and research. Check out our supporters' creative and unique fundraisers; if you feel inspired and would like to organize an event of your own, please contact us at development@hhf.org.

In 2016, the Hearing Restoration Project (HRP) has made significant strides, bringing us closer to finding a cure for hearing loss and tinnitus. Here are just two of the HRP’s discoveries:

  • Successfully disrupted gene expression in the adult mouse cochlea, including capturing high-quality images—necessary for testing genes in regeneration.
     

  • Confirmed that the “DTR mouse” is an excellent platform for studying ways to stimulate hair cell regeneration in the mammalian inner ear.

HHF awarded nine Emerging Research Grants (ERGs) to early-career scientists who are pursuing projects in the areas of Central Auditory Processing Disorder (CAPD), Hyperacusis, Ménière's disease, Stria, and Tinnitus. Through ERG, we hope to uncover better treatment options and deeper understanding of these disorders.

Last but certainly not least, we want to express our gratitude and appreciation for our many donors; because of their support, we were able to continue with our important work. To see your name on our next donors’ list, we gladly welcome and appreciate your gift in any amount made by Sept. 30, 2017.

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