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Illuminating Research Reveals Genes Responsible for Healthy Gut Development in Zebrafish

green finger smudges in black light

Like a string of bonfires at night, orange, red and yellow lights connected by delicate, neon-green filaments illuminated the front cover of a recent issue of Developmental Biology. The photo captures a moment in the neuronal development of two zebrafish embryos.

“What makes it really nice is that you can see the individual neurons and the connections that those neurons make as they travel along the zebrafish gut,” said Julia Ganz, assistant professor in the Department of Integrative Biology in MSU’s College of Natural Science. “It looks like an abstract adaptation of an image, but that is how it actually looked under the microscope.”

For Ganz and her research team, the zebrafish that live and reproduce in a steamy room of MSU’s Giltner Hall are much more than just a pretty picture.

The tiny fish native to India are ideal models for investigating the largest part of the peripheral nervous system — the Enteric Nervous System, or ENS — that regulates the extraordinarily complex and interconnected functions of the gut.

Immunofluorescent images of zebrafish larva

Immunofluorescent images of zebrafish larva show the wildtype intestines (top) and the Uhrf1 mutant intestines (bottom). The mutant has fewer positive enteric neurons (green) stained with the pan-neuronal marker (red) than its wildtype sibling, demonstrating epigenetic factor Uhrf1’s required role in normal development of enteric neurons. Images constructed by J. Ganz, MSU, and E. Melancon, University of Oregon.

“The ENS sits on top of the gut and is closely connected to all the other parts of this fascinating organ,” Ganz explained. “It is sometimes called the ‘brain in the gut’ because it can actually work independently of the Central Nervous System, which is pretty cool,” Ganz said.

The photograph, and the information it provides about specific genes involved in ENS development, took hours of painstaking genetic mapping, technically difficult experiments and a little luck.

A few years ago, Ganz was presented with an interesting zebrafish phenotype identified through a forward genetic screen, the technique of mutagenizing a male zebrafish, crossing it with a wildtype fish and then in-crossing the offspring to find a homozygous carrier of an unknown mutation.

The result was a zebrafish with fewer neurons in the ENS.

But, like explorers looking for harbor on a coastline with no map, Ganz had a potentially interesting phenotype with no idea of the location for the gene responsible.

“The joy and pain of a forward genetic screen is that you know that you have a good phenotype, but you don’t know what gene you are going to land on when you map this mutation — it’s always a surprise,” said Ganz, who also conducted postdoctoral work at the University of Oregon, the birthplace of zebrafish research and this particular phenotype.

“We did not expect it to be a gene that played a role in epigenetic modifications, but it was. Then we were curious — what does this gene do, and how does it lead to this phenotype?” Ganz said.

Ganz and her co-workers were able to sift through candidate genes and find the mutation that made sense.

They zeroed in on Uhrf1, a gene that, in tandem with its DNA methylation partner Dnmt1, plays a role in epigenetic modifications.

“Once we located the gene, we did a challenging experiment where we took enteric progenitor cells out of a labeled donor embryo and transplanted them into a host embryo,” Ganz explained. “Because zebrafish lay eggs externally and have transparent embryos, we can take out cells that will give rise to the ENS and transplant them back into the same place of a host fish and literally watch as they contribute to the ENS.”

In order to find out if Uhrf1 played a role solely in the ENS or also contributed to surrounding tissues such as the smooth muscle cells, the researchers set up two experiments. In the first, they transplanted wildtype cells into a mutant embryo. In the second, they transplanted mutant cells into a wildtype embryo.

Ganz and her coworkers watched as the transplanted cells made their way along the zebrafish gut, using antibody staining techniques to track the creation of neurons, or bonfires, and their connections as they journeyed through the embryo.

What they discovered was as much a surprise as the gene itself. The mutant environment never fully allowed the wildtype enteric progenitor cells to reach the most posterior part of the gut.

Conversely, the mutant enteric progenitor cells were unable to expand and populate the gut of a wildtype host.

“Our results indicate that Uhrf1 has a role within the ENS as well as the surrounding tissue,” Ganz said. “You have both a cell-autonomous role within the ENS cells and a cell-nonautonomous role, meaning a mutation in the surrounding tissue also does not allow normal ENS development.”

These two genes are now potential Hirschsprung disease candidate genes, a congenital disease developed in utero that results in lack of neurons in the gut. The disease affects 1 in 5,000 live births and requires surgery to remediate.

“We are intrigued with these genes because of their potential role in Hirschsprung disease and because ENS development and the factors that control it are so poorly understood,” Ganz said. “Using this research and the fantastic zebrafish, we plan to focus our next research steps on regulators of neuron differentiation within the ENS.”

Val Osowski Via MSU Today

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