On a Data-Driven Mission

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

The annual meeting of Hearing Health Foundation’s (HHF) Hearing Restoration Project was held in Seattle November 11-12, 2018. We used this meeting to update one another on recent progress on HHF-funded projects, discuss in detail the implications of new data, evaluate the directions of ongoing projects, and plan for the next funding period.

As you may recall, in November 2016 the Hearing Restoration Project (HRP) made a deliberate turn toward funding only the highest-impact science that our group leads the world in researching—we have termed this the “Seattle Plan.” We therefore devoted a substantial portion of our efforts to cross-species comparisons that contrast molecular responses to inner ear sensory hair cell damage in species that regenerate their hair cells, especially chickens and fish, with responses in mice, which like other mammals do not regenerate their hair cells. We also have been examining the “epigenetic” structure of key genes in the mouse, as one hypothesis is that epigenetic modifications of the DNA—that is, the inactivation of genes through chemical changes to the DNA—causes mouse (and human) cells of the cochlea to no longer respond to hair cell damage by regenerating hair cells.

Avian and mammal supporting cell subtypes differ, but Stefan Heller, Ph.D., and team are investigating if an evolutionary homogenous equivalent exists in the organ of Corti, and if this knowledge could be used for hair cell regeneration. Credit: Chris Gralapp / Otolaryngology Head and Neck Surgery (OHNS) - Stanford University School of Medicine

I am happy to report that progress over the past two years on these two major projects has been outstanding. For the cross-species comparisons, Stefan Heller, Ph.D., and Tatjana Piotrowski, Ph.D., reported on single cell analysis of, respectively, chick and fish hair cell organs responding to damage. Using single cell analysis—isolating hundreds to thousands of individual cells and quantifying all of the protein-assembly messages they express—we can determine the molecular pathways by which hair cells are formed during development and regeneration. This approach has always been promising, but this year we have begun to reap the expected benefits, as those projects have given us an unprecedented view of hair cell formation.

The epigenetics project overseen by Neil Segil, Ph.D., has now reached maturity, and using the voluminous data acquired over the past several years his lab has shown how supporting cells (from which we intend to regenerate hair cells) change the epigenetic modification of their DNA so they no longer are able to switch on key genes used for turning them into hair cells. A topic of great interest at the meeting was that of genetic reprogramming: Can we use genes (like transcription factors, proteins that control the transfer of genetic information) or small molecules (which often can be taken orally and still reach their targets) to overcome the epigenetic modification and push supporting cells to turn into hair cells? Preliminary results from Segil’s lab and from others in the field make us optimistic that the reprogramming approach will eventually be part of a regeneration strategy.

We also heard from Seth Ament, Ph.D., a bioinformatics expert we recently recruited to the HRP to explicitly compare our various datasets and find the common threads between them. Ament has used gene expression data from the chick, fish, and mouse, as well as the epigenetic data from the mouse, to hypothesize which genes may be important for hair cell regeneration. As a systems biology specialist, Ament brings a fresh eye to the field of auditory science and has not only identified some of the genes we expected to be important, but new ones as well. His success nicely justifies our cross-species approach, and the bioinformatics comparisons that he has been able to achieve in his initial HRP project have been impressive.

Finally, two other Seattle Plan projects have gone well, including our data-sharing platform called the gEAR (gene Expression Analysis Resource), developed by Ronna Hertzano, M.D., Ph.D., which allows us to analyze our data privately but also to efficiently share data with the public. In addition, John Brigande, Ph.D., reported on his project developing mouse models for testing interesting new genes; his group will be adding several powerful models in the year to come.

The excitement at the meeting extended to our future plans. We agreed that the Seattle Plan was the still the proper course, and we eagerly anticipate more data and results to come from our consortium of researchers. We are truly getting a clearer picture of hair cell regeneration due to the HRP’s efforts. That said, there is a long way to go; our efforts show us how surprisingly intricate biology is, despite knowing from the start that systems like the inner ear are remarkably complex. Nature always has surprises for us, by turns dashing treasured hypotheses while revealing unexpected mechanisms. The HRP is most definitely on track for success, and all of us in the HRP sincerely thank you for your continued support.

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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