To escape predators beneath the waves, a flying fish can shoot out of the water and glide long distances because its paired pectoral and pelvic fins, longer and more rigid than those of other fish, act as airfoils. In a quirky triumph of evolution, creatures that were once strictly aquatic transformed into temporarily airborne ones through a few modifications in body shape.
Recently, a group of researchers led by Matthew Harris of Harvard Medical School and Boston Children’s Hospital reported the genetic basis for the evolution of those unusual fins: Through an innovative combination of techniques, they discovered that changes in just two genes were sufficient to create the distinctive body shape of flying fishes. When those mutations occurred in a species of common aquarium fish, its proportions began to shift in similar ways.
“When we started out in evo-devo, there was no thinking that we would be able to make these large jumps in form with such simple rules,” Harris said. The study appeared in the November 22 issue of Current Biology.
The findings are noteworthy in part because they hint that bioelectric signals within developing tissues, not just “morphogenetic” chemical ones, can regulate the growth and shape of developing fins and possibly other structures. This study and earlier work also clearly illustrate how small genetic changes can sometimes produce big morphological changes that have important evolutionary consequences.
Much of the unfathomable diversity of animal forms in nature arises from natural selection’s tinkering with the genetic programs that control development. Tweaks to the timing and speed of tissue growth can stretch or shrink structures, or even insert and delete bones, to create novel adaptations that open new niches for species. Evo-devo, the biological study of this process, has a long history, but only relatively recently have researchers been able to start probing for the genes responsible for specific changes.
To search for the genetic basis of the flying fish’s body shape, researchers in the Harris lab began by sequencing and comparing the genomes of 35 species of flying fish and their close relatives. By looking for regions of DNA that had changed unusually quickly between species, they identified genes that seemed to have evolved under selection pressure.
This comparative analysis allowed researchers to look for the main factors driving the formation of the new body type, explained Joost Woltering, an evolutionary biologist at the University of Konstanz in Germany who works on the evolution and development of fin and limb diversity. “But how are you going to find out if the gene really is the smoking gun that makes a difference? You cannot modify it in the flying fish,” he said. “You have to turn to something where you can actually do that.”
Harris’ team accordingly turned to zebra fish (Danio rerio), freshwater minnows widely kept as aquarium pets but also as research animals. His team used chemicals and gamma rays to create random mutations in more than 10,000 zebra fish embryos. They searched those that survived to adulthood for interesting adult traits or phenotypes. This approach was unusual because genetic studies of zebra fish typically focus on the embryonic development of the animals.
Jacob Daane, a postdoc in the Harris lab at the time, and his colleagues also screened a collection of previously known zebra fish mutants with long fins to refine their search for gene variants that might be regulating the growth of the flying fishes’ fins. They homed in on two: kcnh2a, a mutation that makes cells overexpress potassium channels on their outer membrane, and lat4a, a loss-of-function mutation that disables cells’ ability to transport the amino acid leucine.
The researchers showed that in zebra fish, loss-of-function mutations in the leucine transporter cause all fins to be short, while the overexpression mutation of the potassium channels causes all fins to be long. Either of those mutations by itself produces a clumsy fish. But when the two mutations are combined, the resulting zebra fish has long paired pectoral fins and shorter median fins, exactly the form of the flying fish.
“A single point mutation in some cases can give you really, really big fins,” said Daane, who recently launched his own lab at the University of Houston. “I don’t know of many other systems where there’s that level of simplicity in terms of major scale changes to an organ’s size like that.”
The flying fish body plan evolved independently several times in various lineages, and it always relied on the same types of mutations in the leucine transporter and the potassium channel. The leucine transporter mutations in the different lineages are not identical, but they cause the same amino acid change — a clue that the lineages independently hit on the same genetic trick to evolve this shape. “Nature has targeted the same specific gene in a couple of different contexts,” said Sarah McMenamin, a fish evolutionary developmental biologist at Boston College.
How the potassium channel mutation causes the extra growth in the fin is still a mystery. “It’s not like a receptor-ligand interaction where things are binding to the receptor on the inside of the cell, and they travel down and trigger transcription of something,” Harris said. Rather, overexpression of the potassium channel changes the resting membrane potential and the pH of the cytoplasm, which makes the cell more active and responsive. As a result, the fin cells start to exhibit signaling traits usually associated with neurons and stem cells. It’s possible that the changes in cell signaling might alter how the fin grows, Harris said, but that’s still speculative. “All of this is new, fertile ground, and people don’t really understand much about it,” he said.
When the researchers prevented potassium ions from passing between the fin cells — in effect, negating the potassium channel mutation — they found that it blocked the growth of the fins. They hypothesize that during some stages of development, the cells of the fin operate like a syncytium, a single cytoplasmic mass with many nuclei floating in it. If so, the potassium ions could be setting up an electric field that extends across the entire fin, creating “more potential for long-range signaling coordination than your typical morphogen or secreted factor,” Harris said. (Other researchers have also seen evidence that electric fields may play an underappreciated role in guiding the form of developing tissues.)
The new work builds on an earlier exciting discovery about the evolution of appendages that came from the Harris lab in February 2021. A study by Brent Hawkins, Katrin Henke and Harris in Cell showed that a single mutation can awaken a latent potential for limb patterning in zebra fish fins, even though the ancestors of zebra fish diverged about 450 million years ago from the lineages that later gave rise to tetrapods. Two studies from other laboratories published online with this one in Cell looked at the genomes of early branching ray-finned fishes and African lungfish and suggested that a capacity to build limbs was present in the common ancestor of all bony fish.
The pectoral fin of a zebra fish normally connects to the body with just one layer of bony elements, the proximal radials, which articulate directly with the fish’s “shoulder.” But in the mutant zebra fish that the Harris team uncovered in their genetic screen, two new “intermediate radial” bones formed a joint with the proximal radials. The researchers even found muscles attached to the new bones.
“So with only one mutation, we’re not just making this new piece of bony tissue, we’re making a brand new structure that’s well integrated, well patterned,” Hawkins said. This kind of throwback, or atavism, reaching back over hundreds of millions of years of evolution, reveals how ancient and shared the genetic “grammar” for making fins and limbs is.
“What the Harris lab has shown with this work is really that the genetic potential to make these elaborations in the endoskeleton are still retained in modern ray-finned fish, and they have the developmental potential to build more elaborated structures,” McMenamin said.
New studies continue to roll in from other labs, further reinforcing the point that fins and limbs develop under the genetic influence of highly conserved mechanisms. In November, a paper in the Proceedings of the National Academy of Sciences identified a gene that regulates both the formation of digits in tetrapod limbs and the structure of the outer edges of fins. That same month, a study in Current Biology revealed that the elongated hind feet of jerboas — tiny bipedal rodents that can hop, skip and run at extraordinary speeds — result from a gene that causes disproportionate bone growth in their limbs, not unlike the allometric growth seen in the fins of flying fish.
To Marcus Davis, an evolutionary developmental biologist at James Madison University, the accumulation of evidence that limbs and fins have a shared developmental genetic basis “really leads to the most interesting question, which is where the hell did the original developmental program come from?” The developmental program for fins and limbs was likely modified from an even more ancient developmental program for other parts of the body. “It had to come from somewhere, and it didn’t simply build overnight,” he said. “So what other part of building the body was modified over time to build that appendage program?”
Tetsuya Nakamura, a developmental biologist at Rutgers University who studies the fish-tetrapod transition, suspects that the genetic program for building paired fins and limbs is derived from the development of dorsal and anal fins, which are even more ancient than paired fins. Lampreys, the group of jawless fish that first evolved about half a billion years ago, have dorsal and anal fins but no paired fins.
But even though disparate appendages and body shapes have roots in the same ancient genetic networks, Woltering notes that the shifts between those forms were major transitions. “The tetrapod limb is an evolutionary novelty — I definitely believe that,” he said. There is consequently much left to learn about what enabled those changes to evolve.
The unconventional approach used in the Harris laboratory, which Davis praised as “atypical and modern” and McMenamin hailed as “creative” and “a tour de force,” points to one way that evo-devo researchers might find answers. In the hunt for genes regulating developmental programs, studies often look to certain usual suspects, such as insulin signaling for allometry and Hox genes for limb and fin patterning. But the Harris team took a more agnostic approach, using comparative genomics and large-scale genetic screens to identify fish with interesting, relevant phenotypes. “They had to be totally willing to follow where that phenotype led them,” McMenamin said.
“When we start asking the right questions of the organisms, unexpected things come out — things that we wouldn’t expect from doing classic population-level studies,” Harris said.