Professor Sarah Kucenas and recently minted doctor of neuroscience Ashtyn Wiltbank take a moment to reflect on Wiltbank’s journey working in the lab over several years. (Photo by Dan Addison, University Communications)
In their immaculately kept tanks at the University of Virginia, the approximately 12,000 striped zebrafish, otherwise transparent, all look the same at first. But these zebrafish are both special and specialized.
Turn out the lights, and you’ll see: they absolutely glow.
Yet from tank to identical 2.8-liter tank, the fish glow differently, depending on what aspect of neuroscience graduate students in biology professor Sarah Kucenas’ lab hope to illuminate. The effect is a product of gene manipulation and selective breeding, all with the intent of creating developmental deviations that can literally be highlighted for study.
What’s revealed in the Kucenas Fish Facility may have implications for diseases such as multiple sclerosis and muscular dystrophy, forms of epilepsy, bipolar disorder and schizophrenia, and even later-stage diseases such as Alzheimer’s.
Zebrafish, aka Danio rerio, have brains and spines like we do. In fact, they have about 80% of the same genome. But their see-through bodies develop outside of their mothers, which is convenient for researchers who can more easily observe the evolution of pathologies.
Kucenas’ lab is specifically interested in glia – cells in the nervous system that don’t conduct electrical impulses. They perform vital roles, though, often involving the maintenance and repair of the nerves. Humans and zebrafish have all the same glial cells.
When the lights are off, the genetically engineered zebrafish glow. (Photo by Dan Addison, University Communications, and courtesy of Kucenas Lab)
These days, the lab is learning that glia may have a more important role in nervous system development than anyone ever realized.
Even with such findings, Kucenas will tell you her real mission is to be a helpful guide to students as they pursue their scholarly bliss.
“My own training has been in environments where people just inherently trusted me the first day,” said the scientist, who has directed the lab for 13 years and heads UVA’s Program in Fundamental Neuroscience. “My mentors saw that I was creative and engaged and passionate, while knowing I needed mentorship along the way; I wasn’t a professional scientist yet. So when I started my own lab, it would have been unnatural to do it any other way.”
For her students, just as with the zebrafish, Kucenas creates the conditions for life to unfold in a certain way. In doing so, she’s spawning discovery.
Fishing for New Scientific Knowledge
Kucenas’ office is a celebration of her chosen path. Facing each other on opposing walls are two joyful, swirling abstract paintings of fish. On another wall are snapshots of her former students, intermixed with a few family photos. Her protégés share equal space along a strand of yarn with the moment she introduced her wrinkly newborn daughter, Madelyn, to the world.
Last month, Ashtyn Wiltbank sat beside the array of photos and discussed how she followed her own thread and landed on some important scientific findings. She was a little tired when she spoke to UVA Today, but exceedingly happy. It was the day after she successfully defended her doctoral dissertation.
In 2018, within weeks of joining Kucenas’ lab, Wiltbank had scanned a spreadsheet of tens of thousands of genes – a “snapshot” the lab took of a developing zebrafish using a method called RNA sequencing – and found a gene that seemed to be a worthy candidate for her dissertation study. Having previously worked in labs focused on immunology, she homed in on CD59.
“I stumbled on this, and I said, ‘I think this is super-important,’” Wiltbank said of her unfolding discoveries. (Photo by Dan Addison, University Communications)
“I saw these genes, traditionally known as immune genes, that were highly expressed in glia,” she recalled.
“… that I had been ignoring,” Kucenas said, equal parts humor and irony in her voice.
“Because you were, ‘Eh, that’s just some immune gene – not interesting.’ And I was like, ‘This is interesting! Like, no one has looked at this!’”
CD59 (sometimes written by scientists as “cd59” in reference to zebrafish) regulates inflammation and protects the integrity of cell walls and membranes from rupture. The gene is useful throughout life for inhibiting cell degradation – from viruses, for example. But little had been known about CD59’s role in the neurological development process.
Looking at the data, Wiltbank could see the gene’s fingerprints were all over myelin – the fatty tissue that insulates nerve fiber in order to conduct electrical impulses efficiently and accurately.
“This was striking because most other cells do not express CD59 during development, whereas during adulthood, most cells in the body do express CD59,” she said.
Her comparisons to other RNA-sequencing databases of developing zebrafish, mice and humans found the same thing: CD59 was highly expressed in the brain and peripheral nerves, and to a lesser extent in two other organs, but otherwise not present in all other cells until adulthood.
So she checked the scientific literature to see what was known.
“There was actually a paper in the 1980s looking at CD59 expression in the brain, and they’re like, ‘It’s really highly expressed in these myelinating cells, and we think it’s important for development.’ But it was one sentence, and nobody has looked at it since. And then I stumbled on this, and I said, ‘I think this is super-important.’
“I suspect a lot of scientists noticed that myelinating glia expressed CD59 during development and just assumed that other cells do as well.”
Kucenas and Wiltbank take a closer look at some of the fish from her studies. (Photo by Dan Addison, University Communications)
Kucenas wanted to emphasize the significance of her student’s find. Trusting Wiltbank to pursue what she deemed interesting, based on her own background, resulted in a pulling a needle from a virtual haystack of information.
“She picked ‘the’ gene out of the list, right? She joined the lab, and I’m not a neuroimmunologist – I’m a developmental biologist, a glial biologist. She took this gene list that had 26,000 genes in it, and she came to me and said, ‘Here’s the gene, and here’s why we have to look at it.’”
Congenital disorders were one reason. In a rare disease now known as CD59 deficiency, it first seemed as though the immune system was attacking healthy cells. But despite therapies to suppress immune activation, people born with the deficiency still have nervous system dysfunction throughout their lives.
“That was another indicator that CD59 may play an additional role in nervous system development,” Wiltbank said.
CRISPR Leads to Surprising Findings
Zebrafishes’ selective bioluminescence is genetically engineered using lab-based transgenesis – a gene transfer from one organism to another. The process involves inserting into the fish the same protein that makes jellyfish glow.
In the case of Wiltbank’s study, the fish glowed specifically for CD59.
After the fish started to breed, Wiltbank inserted specific instructions into one of the embryos using CRISPR gene-editing technology. Pronounced “crisper,” the tech allows for precision alterations within living organisms.
“I basically gave the embryo instructions to cut the CD59 gene and tools to help the embryo make the cut,” she said. “The embryo then repaired the gene incorrectly, which lead to a mutation in the gene. This mutation stopped the gene from producing a functioning CD59 protein. Ultimately, this allowed me to look at what happens during Schwann cell development when these cells do not have a functional CD59 gene and protein.”
Schwann cells support a healthy nervous system, while actively myelinating nerves throughout the periphery of the body. Wiltbank and colleagues found that the mutant zebrafish had an excess in Schwann cells due to unregulated inflammation from the missing CD59. These fish also had impaired development of myelin and nodes of Ranvier, electrically active gaps in the myelin sheaths.
But when the team suppressed inflammation in fish with normally functioning CD59 using the anti-inflammatory drug dexamethasone, that too created disrupted neurological development, including reduced Schwann cell count.
“The inflammation part was very surprising,” Wiltbank said. “When we inhibited inflammation in our normal fish, that also impaired the developmental processes, which meant that inflammation is actually there to help nerve development. But CD59 is there to make sure it doesn’t go overboard.”
The team made another exciting finding when it suppressed inflammation in the mutant fish. Sodium channel clusters that help conduct electrical activity and myelin volume were restored almost to levels found in wild zebrafish. The results may have implications for future interventions and therapeutics.
Kucenas, for her part, largely stood out of the way during the research. Just as in any other lab, she expects her students to persevere, and she has found that encouraged students do so. Still, anyone can get stuck. That’s where the more experienced scientist is happy to assist.
Wiltbank captured nerve myelination, left, and a Schwann cell creating myelination, right, during her research. (Photos courtesy of Ashtyn Wiltbank, Kucenas Lab)
“Ashtyn would come to me with me a problem and say something is not working, and I would say, ‘Have you looked at this?’ or ‘Have you read this paper?’” Kucenas said. “I like to set up an environment where we feel like equal partners in the science. I don’t have all the answers. I just try to give them enough runway so they can take off. I see my job as kicking the propellers and making sure the plane is working.”
That includes making sure all milestones are surpassed and all deadlines are met en route to the graduation requirement of having a scholarly paper published. As lead author, Wiltbank – along with Emma R. Steinson, Stacey J. Criswell, Melanie Piller and Kucenas – premiered the findings in the journal eLife in June.
Grants from the National Institutes of Health and the Owens Family Foundation supported the research. The discoveries are in keeping with UVA’s Grand Challenge Research Initiatives in neuroscience, which are looking to better understand specifics of how the brain and nervous system work.
What Wiltbank observed will now give neurobiologists much to study and discuss.
“I learned that the immune system and the nervous system are actually working together during development, and you need a certain amount of inflammation to make that happen,” she said. “This gene that normally interfaces with the immune system is actually making sure our glia are forming the right number of copies of themselves, and in turn making sure myelin and nodes of Ranvier form properly during development.
“Traditionally we think of inflammation as a bad thing, but we’re finding more and more that inflammation can actually be constructive and helpful.”
The Lab’s Benefits Are Transparent
Wiltbank said working in Kucenas’ lab provides numerous benefits, for the fish and the humans. For example, zebrafish often can be anesthetized for viewing under a microscope, then safely returned to the tank to wake up.
Also, “I can cross my fish, and I can have embryos the next day,” Wiltbank said. “You can’t do that with a mouse. And zebrafish live as long as a mouse. We can apply drugs and apply stains to the living fish.”
The students thrive, too. Another recently graduated doctoral student, Kimberly Arena, studied the role of perineurial glia in motor neuron regeneration and was able to determine the mechanisms that drive repair. The work could have implications for people suffering from serious physical injuries.
“We know the cells regenerate, but how do we help patients do that faster?” Kucenas said of the significance of Arena's research, published in the journal Gliain May.
Yunlu Zhu, a 2019 UVA doctoral graduate, studied neural crest cells in Kucenas’ lab and learned they act like phagocytes – cells that clean up dead cells – before the body has an immune system to perform that function.
In Kucenas’ lab, the benefits are transparent for both the fish and the human researchers. (Photo by Dan Addison, University Communications)
After graduation, Wiltbank will work at the National Institutes of Health to help science understand the role of glial cells on the fine hairs that help us hear. She will be one of the relatively few practitioners in the growing field of neuroimmunology.
The Westminster College graduate became an expert in developmental neuroscience while taking courses from multiple instructors at UVA, but Wiltbank credits Kucenas not only with fostering her hands-on skills in genetic engineering, but also with her becoming a better writer and presenter. (The student even picked up some of Kucenas’ mannerisms from watching how she delivers talks.)
All of this mentoring resulted in Wiltbank becoming more self-assured.
“That’s the great thing about the mentor that is Sarah. She is your biggest champion and cheerleader, and she really teaches you how to own what you know and be proud of that. In science, there is criticism and skepticism, and to a certain level that is healthy and necessary, but sometimes it creates unhealthy interactions. And so Sarah did a fantastic job with making you be OK being yourself and being your best scientist.”
“I’m going to get teary,” Kucenas said.
“I might start crying,” Wiltbank said. “I was so proud of myself yesterday in my private defense because I feel like I did it confidently – such a big change since I started in this lab.”
Seated at Kucenas’ desk, the two women mirrored each other.
“Yep, that’s why I do this job,” the lab director affirmed. “The reason I’m in academic science is discovery. But my real motivation is watching other people discover and seeing them get to that point where they’re standing up in an academic defense – and I’m just sitting there grinning like a goofy person because I had something to do with that.
“That happened with me, and that makes me really proud.”
