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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