With its rapid incubation and long list of genetic features, the humble zebrafish is assisting Kellogg researchers in groundbreaking basic science to regenerate the eye.

Zebrafish labThe zebrafish has become the focus of research in several laboratories at Kellogg Eye Center.

What makes it the perfect animal model for ophthalmology? Unlike mice or other mammals, zebrafish embryos are transparent and develop outside of the mother’s body. Moreover, zebrafish progress from single-cell fertilized eggs to a developed, hatched organism in just five days. Such features allow investigators to observe zebrafish development and manipulate their genetic makeup in real time.

Zebrafish also provide an ideal platform to study cells with specific generative properties relevant to eye development, including neural crest, muscle, and Müller glia cells.

Studying the anterior segment

Dr. Brenda Bohnsack and lab

Brenda Bohnsack, M.D., Ph.D., assistant professor of Ophthalmology (left), with her laboratory group

To assistant professor Brenda Bohnsack, M.D., Ph.D., congenital diseases of the eye’s anterior segment hold the key to understanding how the eye develops. Dr. Bohnsack uses zebra-fish to study neural crest cells, early embryonic cells from which many of the structures in the anterior segment are derived. She investigates the genes that regulate ocular neural crest migration, proliferation and differentiation, along with mutations in those genes known to give rise to problems with eye formation.

One example is the gene CYP1B1, which, when mutated, is known to cause primary infantile-onset glaucoma. “Despite all we know about CYP1B1,” Dr. Bohnsack explains, “no one has yet described its role in eye development. That’s one area where we’re breaking new ground here at Kellogg.”

Dr. Bohnsack is applying the same approach in her study of mutations to PITX2 and PAX6, the former known to lead to Axenfeld-Rieger syndrome and the latter linked to aniridia.

In addition to singling out genetic mutations, her lab is looking at the role collagens play in conditions like Stickler syndrome, a hereditary multisystem disease that involves the eye, and osteogenesis imperfecta (“brittle bone disease”) to understand how collagens influence eye development.

“Our hope,” Dr. Bohnsack says, “is that this work will eventually lead to breakthroughs in genetic testing and new therapeutic approaches to prevent blindness in children affected by these congenital diseases.”

Building on her understanding of the genetic origins of eye development, Dr. Bohnsack also wants to know whether the same genes may play a role in the structure and function of the adult eye. For her work in this emerging area, Bohnsack was awarded a 2016 Alcon Research Institute Young Investigator Grant.

Studying the retina

Peter Hitchcock Lab

Peter Hitchcock, Ph.D., professor of Ophthalmology (left), with his laboratory group

Another advantage of studying zebrafish is that the retina in all vertebrates, whether fish or human, is quite similar. Professor Peter Hitchcock, Ph.D. is using zebrafish to answer two questions: How is the retina formed? Can essential cells in the retina be regenerated?

To study retinal formation, Dr. Hitchcock was an early adopter of technologies like CRISPR-Cas, a genome editingtool that allows researchers to introduce changes directly into an organism’s genome. “In zebrafish, we use a CRISPR-Cas system to disable select genes, creating different mutations,” he explains. “It’s a revolutionary tool for analyzing the molecular mechanisms governing the retina’s development.”

Dr. Hitchcock’s lab is also looking for ways to translate particularly advantageous characteristics of zebrafish into humans. Within the retina of the zebrafish are intrinsic stem cells called Müller glia, which have the ability to regenerate new photoreceptors after injury. Human retinas have Müller glia cells as well, but have no such regenerative capacity.

“One way to achieve regeneration is to transplant exogenous stem cells fated to make photoreceptor cells into an injured retina,” Dr. Hitchcock says. “Another is to understand how zebrafish Müller glia behave like stem cells, then induce human Müller glia to do the same.”

Dr. Hitchcock is also developing a similar model to study the regenerative potential of retinal ganglion cells destroyed in glaucoma. “While all of these studies are pre-clinical,” he says, “stem cell therapy is clearly on the horizon in ophthalmology.”

Studying the orbit

Dr. Alon Kahana and lab

Alon Kahana, M.D., Ph.D., associate professor of Ophthalmology (fifth from left), with his laboratory group

Associate professor Alon Kahana, M.D., Ph.D., an oculoplastic surgeon who specializes in orbital disease, is using zebrafish to better understand the biology of orbital disease, with a focus on stem cells, tissue regeneration, and cancer.

A pioneer in the use of zebrafish to study the orbit and periocular structures, Dr. Kahana’s research takes advantage of the unique characteristics of adult zebrafish to study the biology of strabismus and repair of the muscles that move the eyes following injury or disease. Using an innovative model developed in his lab, Dr. Kahana is able to use genomic and epigenetic analyses to study how to coax damaged tissues to regenerate and recover function.

“Eye movement in both humans and zebrafish is controlled by the same six pairs of extraocular muscles,” he explains. “Disorders in these muscles result in amblyopia in children and double vision in adults. We have identified a number of pathways through which zebrafish can regenerate fully functional muscles even after up to 80 percent of the muscle is lost.”

Dr. Kahana believes that translating findings from zebrafish to humans will result in improved treatments of disorders that affect eye alignment, in effect allowing the body to heal itself.

The biology of zebrafish regeneration appears to share many commonalities with cancer. “The genetic and epigenetic changes that occur in zebrafish stem cells mimic, to a large extent, the biology of cancer stem cells,” he explains. “It’s a truly remarkable finding that has led to an important collaboration with top cancer researchers at Michigan, including Dr. Max Wicha.”

Dr. Kahana is also actively translating his findings in zebra-fish to human eye disorders. “Using zebrafish, we discovered that thyroid eye disease (also known as Graves eye disease) may be caused by a unique biological interaction between thyroid hormone and neural crest-derived cells in the orbit, resulting in ‘hijacking’ of the immune system’s inflammatory response.”

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