Research team determines process for regenerating neurons in the eye
Serena Zacharias | Monday, October 12, 2020
When professor of biology David Hyde and his lab first discovered that zebrafish could regenerate a part of their eye when damaged in the late 1990s, they sought to discover the mechanisms that govern this process.
Twenty-five years later, through a collaboration between researchers at Notre Dame, Johns Hopkins University, Ohio State University and the University of Florida, Hyde and his team have discovered the gene regulatory networks that control regeneration in the retina of zebrafish. Their study was recently published in Science Mag.
Understanding the process of regenerating retinal neurons can have implications for the rest of the central nervous system as well.
“If we can figure out how to make [neurons] regenerate in mouse, then we should be able to make them regenerate in other parts of the central nervous system,” Hyde said.
The hope is that this will eventually translate to identifying a mechanism to regenerate human neurons.
“The possible effects are pretty far reaching when you start thinking about spinal cord injuries, traumatic brain injury and different genetic diseases that affect the brain,” Hyde said.
The symptoms of genetic diseases like Parkinson’s and Alzheimer’s disease are caused by the death of cells in the brain, and finding a way to regenerate such cells can provide an avenue for possible treatment.
The study compared a specific cell type in the retina — Müller glial cells — of zebrafish, chicks and mice.
“Using zebrafish as an organism which can regenerate and studying what makes it regenerate is a much easier approach than using mouse which doesn’t regenerate and trying to guess what might allow it to regenerate,” Hyde said.
When the retina of a zebrafish is damaged, Müller glial cells undergo gliosis, which changes the gene expression of these cells to prevent other cells in the retina from dying. Then, zebrafish Müller glial cells progress into a progenitor state which allows regeneration to occur.
In mice and humans, Müller glial do not progress into this progenitor state. Chicks show a limited regenerative response.
“We discovered that there are gene networks in zebrafish that stimulate regeneration and gene networks in mouse that inhibit regeneration,” Hyde said.
The team worked to map the gene regulatory networks that govern the response to retinal damage in zebrafish, mice and chick by isolating Müller glial cells from each of the three organisms. They did this by performing a technique called RNA sequencing to see which genes were active in the Müller glial.
Fifth year PhD student Patrick Boyd began working on this project when he started his PhD. He spent three years beginning in 2016 gathering and interpreting data for this study.
In the beginning, Boyd worked with research assistant professor Manuela Lahne on improving protocols to isolate the Müller glial from other cells present in the retina.
“You have specific buffers and solutions to disrupt the cell and to only get the RNA, and you take away all the other components like proteins and DNA that you’re not interested in,” Lahne said regarding the isolation process.
Then, they worked to fix the cells in order to send them to other universities to sequence.
During the last year of the study, Boyd performed functional tests to see whether the genes they identified actually had an important role in the network.
To test this, Boyd said they cause a “knockdown” which decreases the levels of RNA which make specific proteins.
“We basically look and see what the Müller glial are doing after we’ve knocked down those genes, whether they still producing the same number of cells that they would normally produce,” Boyd said.
While researchers at Notre Dame worked with zebrafish, teams at Johns Hopkins University School of Medicine performed RNA sequencing for the project and worked with mice to identify gene networks. Collaborators at Ohio State University performed functional studies on chickens, and researchers at the University of Florida also worked on mapping the networks.
Seth Blackshaw, professor of neuroscience at Johns Hopkins University School of Medicine, specifically worked to identify a target set of genes in mice which resulted in regeneration when inactivated.
He thinks that this regeneration capability is repressed in mice and other mammals as an evolutionary response.
“I frankly suspect that it has a lot to do with resistance to infection,” Blackshaw said. “Cells in the Central nervous system are more likely to be infected by viruses and bacteria and other parasites and that the last thing you want to do under those circumstances is grow more cells to disperse the pathogen.”
In the future, the researchers across the universities will work to identify the key genes within each of these networks that control the process of regeneration. Hyde said they also want to compare acute versus chronic damage in the retina of zebrafish to determine whether similar gene networks are activated in the zebrafish with chronic damage as in mouse retina. His team also wants to understand how zebrafish age-dependent loss of the ability to regenerate neurons affects the gene networks they identified.
Both Hyde and Blackshaw emphasized the importance of the team effort which led to the publishing of this study.
“This has been a really enjoyable collaboration,” Blackshaw said. “It’s been really fun to work with David and the other members of the consortium.”