Mutant Fish Eyes

On the second floor of the biology building, massive confocal microscopes and tanks of sleek, stripy zebrafish inhabit the developmental biology lab. This is the domain of biology professor Kara Cerveny and her student researchers, who work to tease apart one of biology’s most stunning and complex phenomena: the transformation of a single fertilized egg into a fully functioning, multicellular creature. “Developmental biology is the context in which we study all of biology,” Cerveny said. “Ecology, molecular biology, cell signaling, they’re all crucial in development. It’s a really wide umbrella; it encompasses all sorts of things.”

Cerveny wasn’t always a developmental biologist. She started out working in cell and molecular biology, researching mitochondrial inheritance in yeast. One day she came across an intriguing gene involved in mitochondrial splitting that had an equivalent gene expressed in eyes. Mutations in this eye gene, Opa1, cause the optic nerve to atrophy early in development and are linked to glaucoma. The more Cerveny learned about Opa1 and how it contrasted with what she had seen in mitochondria, the more she became intrigued by the process of embryonic cells growing and differentiating into complete, functioning organs such as eyes.

Cerveny’s research focuses on eyebdevelopment in zebrafish, a common model organism for studying many developmental processes andbthe genes that govern them. “Zebrafish are especially cool because they continue to grow throughout their lives,” Cerveny explained. “Because they grow forever, until the day they die, we can ask a lot of questions about how those growing cells are specialized and maintained.” The developmental biology lab’s experiments focus on identifying the genes and chemical signals that coordinate the formation of eyes in zebrafish, and how cells in the visual system are coordinated to maintain this unique continuous growth.

Every year biology students have the opportunity to join the lab and undertake zebrafish experiments of their own. Alicia Uchida, ‘17, took Cerveny’s class her sophomore year and was so intrigued that she decided to work in the development lab over the summer. “There are so many
signaling systems that work within our bodies, and it was so cool to learn how they intertwine and somehow develop into a full-functioning body.

The fact that a small defect in a signaling system could have such detrimental defects was both captivating and terrifying to me,” Uchida said.

For Cerveny, both teaching and working with students in the lab are essential to being a scientist, and she emphasized the benefits of working closely with undergraduate students at a small institution. “My job is three things: teaching, research, and service to the college. My favorite is combining teaching and research, when they really synergize with each other. It’s hard to get lab experience at bigger institutions. Here there are a lot of opportunities right away. You get to learn science by doing science.”

Over the summer, Uchida’s lab work focused on conducting fate-mapping studies, a type of experiment where cells are labeled with fluorescent molecules in the early stages of the embryo’s development and are then tracked to see which type of cells they become later on. “I focused on the ciliary marginal zone of zebrafish, and how it arises. The CMZ is a stem-cell niche within the periphery of the zebrafish eye, which is really cool!” Uchida explained.

In addition to growing continuously throughout their lives, zebrafish are an excellent species to study in developmental biology because their embryos are transparent and easy to manipulate. As if they didn’t already have enough going for them as experimental organisms, zebrafish also have stem cell niches, clusters of tissue made up of unspecialized cells that are set aside in different parts of the zebrafish body and are able to differentiate into new cells. Zebrafish have over 17 different stem cell niches in just their central nervous system, and Cerveny’s lab
pays special attention to the niches that supply the eyes with new nerve cells, known as the ciliary marginal zone. The CMZ is found in the very back of each retina and contributes stem cells that become new nerve cells, allowing the eye to keep growing throughout the zebrafish’s lifetime. By studying CMZ cells, the lab hopes to understand how chemical signaling in the stem cell niches of the retina control the way cells divide and when they transform into specialized, mature nerve cells.

“Zebrafish are really, really cool fish,” says Uchida. “They continuously grow even in adulthood, and the fact that they have a stem-cell niche in their eye (the CMZ) is mind blowing to me. Their embryos are also very cute. I love that the embryos are transparent so you can literally see the little zebrafish moving around within their chorions.”

An important part of working in the developmental biology lab is taking pictures of the zebrafish embryos as they develop under different conditions. “My favorite part of working in Kara's lab this summer was learning how to use the confocal microscope,” Uchida said. “It's a powerful imaging tool, and I've always been very intimidated by it since it’s so huge and very expensive. The images I was able to capture and see were really beautiful, and I'm looking forward to hopefully getting some more images for thesis.”

In addition to capturing confocal microscope images, research students must learn to perform embryological manipulations, such as transplanting cells from one embryo to another and delivering chemical treatments via microinjections. According to Uchida, acquiring these skills made for some of the most frustrating moments she encountered during the summer. “The [microinjection] setup and needle [are] very finicky, and it requires steady hands. I definitely learned to reduce my caffeine intake.” Zebrafish embryos spend the first couple days of their development wrapped in tiny clear envelopes called chorions, which measure about 2 mm in diameter. Working with such small creatures and delivering chemical treatments precisely and efficiently is notoriously difficult.

Cerveny’s lab takes in 3-5 thesising seniors every year, as well as other research students over the summer. “Students really own it,” said Cerveny. “Many thesis and summer students were in a paper we published last April [2016].” The paper, titled “Antagonism between Gdf6a and retinoic acid pathways controls timing of retinal neurogenesis and growth of the eye in zebrafish,” compiles experimental work from 18 researchers in Cerveny’s lab, many of them students. The paper presents evidence gathered from two different mutant zebrafish strains with damage in a gene called Gdf6a; these mutants develop into embryos with reduced CMZ activity, less growth in the retina, and abnormally small eyes.

By analyzing these mutant embryos and performing molecular tests, the results of several experiments in Cerveny’s lab added up to the conclusion that retinoic acid, another signaling molecule, regulates the delicate timing of when CMZ cells stop dividing and start becoming specialized nerve cells.

When asked what exciting things are up and coming in the field of developmental biology, Cerveny said: “Regenerative medicine, stem cells, all sorts of things. Everything in dev bio is cutting edge these days.”

Uchida urged students to check out what the developmental biology lab has to offer, even if researching mutant fish eyes isn’t your cup of tea: “Anyone who hasn't seen the fish room in Kara's lab should definitely come visit—it’s really amazing how many fish we have!”