Limbs can be incredibly useful. Whether it’s the wing of a bat, the elongated leg of a hopping frog or our own grasping arms, limbs have been adapted to all sorts of ecosystems and functions through the course of evolutionary time.

The earliest limbs date back to over 375 million years ago. The fossil record has beautifully documented how the fleshy fins of ancient fish became more and more limb-like and allowed our amphibious ancestors to come ashore. These creatures, like us, are known as tetrapods—or “four limbs.” Now a study on a modern fish familiar to aquarium enthusiasts has provided new insight into the genetic underpinnings of this transcendent change. Boston Children’s Hospital biologist M. Brent Hawkins and colleagues published a study today in Cell that demonstrates mutations to either of two zebrafish genes can create a very limb-like fin in these fish. By using gene-editing techniques to replay the mutation in the lab, the researchers were able to pinpoint how some zebrafish grow fins that have more of a resemblance to our arms.

Finding the relevant genes started with looking for fish with particular mutations. The Harris Lab, of which Hawkins is a part, screened over 10,000 mutated animals for particular skeletal defects. Among those that stuck out were zebrafish that had extra bones in their fins. Much like lab mice and fruit flies, zebrafish are classic study animals for understanding genetics and development. They’re classified as teleosts—bony fish that support their fins on pointed rays. Only, some of the mutant zebrafish had fins that had extra bones. Not only that, but the new bones were attached to muscles and even formed joints, just like a limb. “Finding a fish with extra fin bones that should never be there was quite the ‘Eureka!’ moment,” Hawkins says.

Most striking of all was that the new bones required other changes to the fish’s anatomy. “Because development is an integrated process, this one mutation creates a new bone, but also creates a joint and brings along changes in musculature,” Hawkins says. With a single mutation, fins became something much more like arms. And so Hawkins and colleagues set about finding what could have been responsible for such a change.

Starting with zebrafish that had obvious mutations, Hawkins and colleagues used a process called a forward genetic screen to find the genes responsible for the limb-like fins. The researchers identified two—known as vav2 and waslb —that influenced the mutation. To confirm the connection, the researchers used CRISPR gene editing techniques to make zebrafish with limb-like fins in the lab, confirming the connection between the genes and anatomy.

“Prior to our discovery, we had no idea that these genes were involved in making the skeleton,” Hawkins says. Both of the genes were thought to have roles in cell maintenance and no one suspected that they might have a larger role to play in how skeletons are organized. In broad strokes, either of these two genes can somehow influence what are known as regulatory genes that lay out the pattern of the fins in the fish.

Zebrafish With Limbs
This image shows how mutant zebrafish transform their limbs to be close to human limbs.

(M. Brent Hawkins)

The study is “ground breaking,” says McGill University development expert Ehab Abouheif, who was not involved with the new paper. When a single gene takes on a new role, an entirely new and complex structure can suddenly appear and then be further molded by natural selection. This is the main driver of evolution—each individual has variations that affect their ability to survive and reproduce, and variations that lead to more offspring get passed on only to be modified further until organisms dramatically change. “The latent potential to produce new elements in the fish fin that resemble tetrapod limbs is mind-blowing,” Abouheif says.

Naturally, Hawkins and colleagues are looking at modern-day mutations in fish that belong to a different group than our distant forebears, which were more like lungfish. While zebrafish typically have fins supported by spine-like rays, our distant ancestors had fins supported by thick branches of skeletal parts that were the anatomical equivalents of our arm and leg bones. What’s important, Hawkins notes, is that the genes involved and the biological interaction that allows them to influence body patterns are very, very ancient. The fact that fruit flies have vav2 and waslb, too, means that these genes originated in early animals and were later inherited by prehistoric fish.

Understanding these developmental pathways may be the key to revealing what happened millions and millions of years ago. “These exact mutations, even if they are not observed in tetrapods, do provide new insights into how the early tetrapod limb evolved,” Abouheif says.

Ancient fish had the potential to make limbs long before the actual event happened, with luck likely allowing some of the ancient mutants to start pioneering a new way of living that brought them ever further ashore. “What our mutants reveal is that the latent ability to make limb-like things was already present in the bony fish ancestor and was not just a tetrapod-specific innovation,” Hawkins says. And such changes are not limited to fish. Looked at one way, humans are just a highly-modified form of fish and our bodies have been greatly influenced by just these kinds of developmental tweaks. “If a fish can make a limb,” Hawkins asks, “what are humans able to do?”