By tweaking a single gene, Harvard scientists engineered zebrafish that show the beginning formation of limb-like appendages. The researchers stumbled upon this mutation, which may shed light on the sea-to-land transition of vertebrates, while screening for various gene mutants and their impact on fish development. Their discovery, outlined February 4th in the journal Cell, marks a fundamental step in our understanding of fin-to-limb evolution and how surprisingly simple genetic changes can create great leaps in the development of complex structures.
"It was a little bit unbelievable that just one mutation was able to create completely new bones and joints," says M. Brent Hawkins (@Homeobox), first author of the study, who recently received his doctorate from Harvard Organismic and Evolutionary Biology. "In the 30,000 species of teleost fishes, none of them have this sort of variation, so the fact that we found a mutant like this really knocked us off our feet."
The mutation Hawkins and colleagues discovered causes a change in in the zebrafish's pectoral fin bones called "proximal radials," which attach to the fish shoulder joint, similar to how the human arm attaches to our shoulder. But unlike humans and other tetrapods, zebrafish do not have series of these skeletal elements that articulate at joints, such as the components of our arm and fingers. With this mutation, a new set of long bones called "intermediate radials" develop and are able to articulate with the existing proximal radials, making a joint similar to our elbow.
"In this one mutation, you get the new bone, you make the joint, and you make the muscle attachments all in one go," says senior author Matthew Harris (@fishyskeleton), an associate professor of Genetics at Harvard Medical School and Orthopedics at Boston Children's Hospital. "You didn't need to have a mutation in the muscle gene, a joint gene, and in the bone gene; the system is coordinated such that whatever our change is, it's able to push all these things together in unison."
Genetic analysis revealed that mutations in either of two genes, vav2 and waslb, can independently cause this developmental change. Neither gene has previously been connected to skeletal development, but the analysis revealed that both genes activate Hox programs that pattern the middle region of the limb. "With these zebrafish, we're able to actually show that these mutations are activating programs that are quintessentially thought to be only found in a limb," says Harris. "So not only do we have the phenotype of something never before seen in teleost fish, but we show we can turn on ancestral patterns that were thought to be only limb associated."
Limbs are thought to be a key evolutionary innovation, allowing vertebrates to walk on land and--in the case of birds and bats--fly. What these results show is that a group of fishes who were thought to have either lost or silenced the machinery necessary to evolve limb-like appendages actually retain an innate latency to form these structures. "With our work, we've found unexpected commonalities between fins and limbs, and I think there are even more similarities that are yet to be discovered," says Hawkins.
Despite these findings, a question still remains of whether these new bones change the functionality of the zebrafish pectoral fins. Next steps will incorporate fine-scale video microscopy to determine whether the articulation of these news bones is enough to influence how the fish move. "Normally, the structures are not present to allow for the articulation required for movement on land," says Harris. "It'd be very interesting to see, for example, if we put our mutant on a platform, whether they would have an adjusted gate."
With the discovery of these mutants, researchers open a new line of questions on how vertebrates took their first steps towards movement on land, and of the genetic and developmental mechanics necessary to make it a happen. "While it's not the whole story, what we're seeing is a window into the puzzle of how you go from a fin to the modern limb," says Hawkins.
"And for me, I'm left with what can these mutants tells us about development and the ability to form complex structures. It's great reminder that not all monsters are scary. If you look closely, sometimes they can actually tell you a lot about yourself," says Harris.
This study was supported by the National Science Foundation and the Children's Orthopedic Research Foundation.
Cell, Hawkins et al.: "Latent developmental potential to form limb-like skeletal structures in zebrafish" https://www.cell.com/cell/fulltext/S0092-8674(21)00003-9
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