A Simple Genetic Change Adds Limb-Like Bones to Zebrafish Fins
A Simple Genetic Change Adds Limb-Like Bones to Zebrafish Fins

A Simple Genetic Change Adds Limb-Like Bones to Zebrafish Fins

A gain-of-function mutation in a single gene reveals ancient limb-forming capacity that has been preserved for hundreds of millions of years.

Abby Olena
Abby Olena
Feb 5, 2021

ABOVE: An image of dissected zebrafish pectoral fin bones demonstrates that fish with a gain-of-function mutation in waslb have two more long bones (right, arrowheads) than wildtype fish (left).

While the differences in form and function between the fins of fish and the jointed forearms of those of us with four limbs are quite obvious, there’s a common link: both descend from the same appendage borne by our shared ancestor, a bony fish. In a study published today in Cell, researchers identify a gain-of-function mutant in the zebrafish (Danio rerio) that grows an extra set of bones at the ends of two long bones in the fin. The genetic program that makes the extra bones is similar to the instructions for forearm bones in tetrapods. 

“There is this latent potential to build morphological structures in lineages where you think these structures have long been gone,” says Ingo Braasch, an evolutionary developmental biologist at Michigan State University who did not participate in the work. Because fish fins have looked similar for millions of years, scientists did not know that the genomes that build them could still make limb-like bones as their ancestors did. “With relatively simple genetic manipulations, you can re-evoke ancestral genetic programs to build structures that are seen in very distant evolutionary lineages within vertebrates.”

The approximately 30,000 species of fish that are part of the teleost lineage all grow pectoral fins in a similar way: Closest to the body, they have four long bones side-by-side, two of which stay connected to the shoulder in adulthood. These long bones are in contact with small radial bones, which support the bony rays that extend through the fin.

As we study more and more, we’re understanding that fish are not as different as we thought from humans or land animals.

—Gage Crump, University of Southern California

In 2017, a team from Matthew Harris’s lab at Harvard Medical School and Boston Children’s Hospital published a genetic screen, the goal of which was to find dominant mutations that affected adult zebrafish. They exposed zebrafish males to a mutagen, then crossed those males with wildtype females, and then crossed the mutant offspring with wildtype fish again. This strategy allowed them to identify 72 dominant mutants that had skeletal defects, pigmentation abnormalities, or both and lived into adulthood. One of these had striking anatomical changes that caught the eyes of Katrin Henke, who led the screen and now runs a lab at Emory University, along with Harris and graduate-student-turned-postdoc Brent Hawkins.

“Once we took a closer look at the skeleton . . . we noticed that the pectoral fin had these extra bones that should never be there, and this really kind of knocked us off our feet,” says Hawkins.

The researchers examined the skeletons of heterozygous mutant fish over the course of development and found that the extra bones appeared around three to five weeks after fertilization, with an initial lengthening and subsequent joint formation separating two of the existing long bones into four segments. “We analyzed that segmentation process more carefully and saw a few similarities to the way that tetrapod limbs do the segmentation in their bones,” Hawkins tells The Scientist. Not only do these fish make bones that aren’t present in wildtype animals, but the joint space that separates the new bone from the old bone looks a lot like the synovial joints that are present in limbs, both in terms of how they’re shaped and the patterns of joint-specific proteins, he adds.

Next, the researchers mapped the mutation to a gene called wiskott-aldrich syndrome-like b (waslb). Its protein product is involved in forming filamentous actin, which has multiple roles, including in transcription and cell migration. They confirmed that their fish carried a gain-of-function mutation by knocking out the mutant allele, which restored a normal appearance in the animals. When the researchers knocked out the gene in the tissue of mouse embryos that usually becomes the limbs, the mice ended up with fusions of their bones and lost elements of their digits.

What’s happening, Hawkins says, is the more waslb function there is, the more bones and joints grow in number. With reduction, there are fewer bones.

Then, the research team investigated how waslb ties in with known limb development regulators, such as the homeobox (hox) genes. It’s been shown in mice before, for instance, that hox11 is required for making the forearm. The team found that waslb needs the function of hox11 to make extra bones, and it interacts with other hox genes to pattern the pectoral fins in fish. This interaction with hox11 indicates that the extra bones in the mutant fish are homologous to forearm bones in tetrapods, meaning that the fish’s genome still carries the information necessary to make a limblike bone, despite not doing so for millions of years.

“The big black box now is . . . the molecular process that’s linking waslb to control of the hox genes,” Hawkins explains. Actin functions in a wide variety of cellular contexts, he adds. “And for each one, you could imagine a way that it eventually integrates with the hox program.”

“As we study more and more, we’re understanding that fish are not as different as we thought from humans or land animals. They have a lot of the same patterning information, but they use it in different ways,” says Gage Crump, a stem cell biologist at the University of Southern California who did not participate in the study. One open question, he says, is, “if this information is present in fish, why is a limb not forming?”

It’s intriguing to consider “why teleosts don’t seem to have made use of this latent potential,” agrees Braasch, who hypothesizes that understanding the connection between waslb and the hox genes could provide clues. “There must be extreme pleiotropic effects there that could explain why evolution hasn’t tinkered with that pathway.”

M.B. Hawkins et al., “Latent developmental potential to form limb-like skeletal structures in zebrafish,” Cell, doi:10.1016/j.cell.2021.01.003, 2021.