Breakthrough findings in an ancient animal

As much as we’ve learned about the Hox genes, the origin and evolution of this crucial gene family has remained an unsolved mystery. Where did Hox genes come from, and how did their crucial roles in body patterning arise during ancient evolution? This past fall, more interesting discoveries regarding Hox genes emerged from the Stowers Institute—this time from studies on the sea anemone Nematostella vectensis in the lab of Investigator Matt Gibson, PhD.

The role of Hox genes in body patterning had been well-defined in bilaterally symmetric animals like flies, mice, and humans. Surprisingly, these genes are also found in our distant relatives, including simple animals known as Cnidarians (jellyfish, sea anemones and corals). Cnidarians feature radial patterning and lack a head-to-tail axis, raising the question of what Hox genes could be doing. By developing new methods to study developmental gene function in Nematostella, the Gibson Lab found that Hox genes also play an unexpected role in controlling the radially symmetric body plan of Nematostella.

The breakthrough findings, published in Science, were remarkable because Gibson didn’t start his lab at Stowers to investigate Hox genes in sea anemones. He has spent most of his career studying epithelia—highly organized layers of cells that line the internal and external surfaces of the body, such as the epidermis, the outermost layer of the skin. That work takes advantage of the fruit fly, the genetic powerhouse where many Hox genes were initially discovered.

Gibson was drawn to Nematostella because of its large and complex genome, and because it’s flush with epithelial cells. But dedicating part of his research to a much less studied organism was a decidedly risky career move, so Gibson asked for Krumlauf’s input first.

“There was a moment where I saw great potential in Nematostella, but I wasn’t absolutely sure it was a good idea. I asked, ‘Should I really jump into a completely new area?’ And he said, ‘Yes, definitely be bold if that’s what you want to explore.’”

Encouraged by Krumlauf’s enthusiastic support, Gibson ramped up his lab’s focus on the starlet sea anemone. He hired Postdoc Aissam Ikmi, PhD, from the University of Paris, who was willing to take a chance on the new model organism.

“From my perspective, studying different models is like studying different books,” Ikmi says. “Why should we restrict ourselves to studying a few books if we have access to an open library from which we can learn how living organisms solve many problems that matter to our health, as well as other aspects of our lives?”

Ikmi went on to play an essential role in establishing the enormous starlet sea anemone colony needed for research, with support from the Stowers Reptile & Aquatics Facility. He also developed tools, based on cutting-edge gene-editing technologies, to manipulate genes in the sea anemone in collaboration with the Stowers Molecular Biology Facility.

A risk worth taking

Over the course of a decade, the initial risk Gibson took in studying the starlet sea anemone has paid off, yielding several scientific papers and important discoveries, and laying the foundation for the Hox research recently published in Science.

The study’s findings were a major step forward in Hox gene research, because understanding the function of Hox genes in the sea anemone helps us understand their roles in our most ancient common ancestor, some 600 million years in the past.

Using gene knockdown technology, researchers in the Gibson Lab disrupted the function of the genes involved in body patterning of the starlet sea anemone through treatment with short hairpin RNAs, which silence gene function. They also used the CRISPR-Cas9 gene editing approach to remove these Hox genes from the genome.

The results were striking, says Gibson.

“Early in development, Nematostella have a larval stage where the animals look a bit like a little lemon. There’s an outer layer of cells (the rind) covering an inner layer of cells (the fruit). And just like a lemon, when you slice through, Nematostella larvae are divided up into eight segments. When we removed the Hox genes, specific segment boundaries were completely lost. It was a really obvious effect and told us right away that Hox genes control radial segmentation in this animal,” Gibson says. “Furthermore, these segmentation defects led to even more dramatic disruptions of the tentacles that develop right after the larval state.”

In sum, the work shows a key role for Hox genes in radial patterning. The findings were surprising because in bilaterally symmetric animals, Hox genes control the identity of segments along the head-to-tail axis—for example, which part of your spine makes a rib versus a vertebra. In contrast, Hox genes in the sea anemone are responsible for both making and patterning segments.

These functions may have separated over hundreds of millions of years such that Hox genes in present-day bilaterians have lost the role in segment formation and just control segment identity, Gibson says. The finding builds on Krumlauf’s earlier discoveries.

“In the mouse, we showed that a couple of Hox genes work also as segmentation genes, making segments, but their primary role was in segmental identity. People thought that (the finding) was just an odd one-off. But what’s cool about what Matt and his lab have shown is that it may be an ancient feature of Hox genes in making these integrated segmental structures and also giving them an identity. It’s really exciting. It makes everyone reevaluate models on the ancient role of how these systems work,” Krumlauf says.

Training the next generation of Hox researchers

Stowers research trainees have played important roles in advancing Hox gene research.

Ikmi now leads his own lab at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, where he continues to study Nematostella, and how the interaction between genetic factors and the environment controls developmental processes.

“One key environmental stress is injury, and we focus on understanding the spectacular ability of sea anemones to regenerate—if you cut them in half, each half will grow into a new sea anemone. They’re particularly wellsuited for trying to find out why some animals are better at regeneration than others,” he says.

Working at Stowers gave him the opportunity to develop new technical skills and mature his research plan to help push his scientific knowledge forward, he adds.

Ikmi’s work paved the way for Shuonan He, a predoctoral researcher at the Graduate School of the Stowers Institute for Medical Research and first author of the 2018 Science paper. He joined the Graduate School in 2014, after graduating from Peking University with a bachelor’s degree in biological science. He says his love of marine biology led him to join Gibson’s lab.

“As a mentor, Matt is very open-minded and allows us to develop our own ideas and projects. The freedom to explore is what I enjoy most,” He says. “When I first started in the lab, we already had a well-established, daily-spawning Nematostella colony, thanks to Aissam and the Reptile and Aquatics team, and the CRISPR-Cas gene editing approach had just been demonstrated in Nematostella. I do think it was the perfect opportunity to start my own project on Nematostella.”

His work has centered on generating mutants for different starlet sea anemone Hox genes. The first two years into his project, he had minimal success despite numerous trials. But after a year of exploration and optimization, he was the first researcher to successfully apply short hairpin RNA, or shRNA, to silence gene expression in the starlet sea anemone. The lab now has a much more efficient shRNA protocol that they have shared with many other Cnidarian labs, as well as a more efficient CRISPR-Cas system they used to generate the Hox mutants in the Science paper.

Post publication, He is continuing to focus on the regulatory properties and downstream targets of Nematostella Hox genes. He plans to stay in academia after graduation and continue research in the evolutionary developmental biology field.

What's next?

Scientists at Stowers will continue to dig deeper into Hox gene research, aided by the Institute’s unique research environment which provides access to modern laboratory space, advanced equipment, and scientific support services staffed by expert researchers.

But in addition to equipment and resources, Gibson credits the philosophy of the Stowers Institute, which encourages researchers to take substantial risks to pursue the most interesting ideas. He also points to the support and guiding expertise of Krumlauf, a “phenomenal mentor and a phenomenal human being,” for spurring him to consider taking a different path—one that coincidentally led to breakthrough discoveries about Hox genes that Krumlauf had studied for most of his career.

“When I first came to Stowers, I had no idea that a big part of my lab would wind up working on Nematostella,” Gibson says. “Traditionally, if you set up a fruit fly lab, that’s what you do for 40 years. The support and environment at Stowers really make it possible for scientists to go in new research directions, and that’s a big part of what makes this institution so unique on the world stage.”