Sometimes, a genetic tweak can make a really big difference in an animal’s appearance. That’s what likely happened when the predecessors of modern snakes lost their legs, a process that started some 150 million years ago, two separate groups of scientists have discovered. Although the teams took very different approaches to solve the mystery of how those limbs vanished, both came up with similar results: Mutations in DNA located near a gene key to limb formation keep that gene from ever turning on, they report today.

The new studies have impressed other scientists in the field. In both cases, "there is a correlation between the molecular findings on the one hand and the evolutionary trend of limb reduction and loss on the other," says Michael Richardson, a developmental biologist at Leiden University in the Netherlands. The findings show “there can be pretty minor changes in the genome that can account for very big specific changes,” adds James Hanken, an evolutionary developmental biologist at Harvard University.

Though they are reptiles, almost all snakes are completely missing the limbs typical of most land vertebrates. They didn’t start out that way: More than 100 million years ago, snakes had visible legs. And even today pythons and boas have tiny leg bones inside their bodies, suggesting they have vestiges of the molecular pathway for building these appendages.

Scientists got some of their first clues about the genes involved in the development of the serpentine body form in 1999. At the time, Martin Cohn, an evolutionary developmental biologist at the University of Florida in Gainesville, discovered snake embryos had a different pattern of activity of certain genes than other reptiles and that applying a growth factor could make those embryos start to grow limbs. But he lacked the genomic tools to look any deeper. Four years later, Hanken and his colleagues discovered that activity in one of the genes, named after the video game character Sonic the Hedgehog, played a role in leg size in lizards, hinting that it could also be important for snakes. Now, with more ways to monitor gene activity during development—and fully sequenced genomes of various snakes and other reptiles for comparison—Cohn and graduate student Francisca Leal have tracked the genetic activity in embryonic pythons to see why their legs start, but never finish, developing.

They found three DNA deletions in the genetic switch that controls the activity of the Sonic hedgehog gene. Situated in front of the gene, this switch, called an enhancer, is a docking site for proteins that control the gene’s activity. The deletions made it difficult for certain proteins to land, resulting in only a brief window of gene activity during the development of the python embryo, Cohn and Leal report today in Current Biology . In snakes with no leg bones, the enhancers have even more deletions, they report, making it likely that the gene never turns on in the first place. “It’s taking the old results to a finer level,” Hanken says.

Axel Visel, a genomicist at the Lawrence Berkeley National Laboratory in Berkeley, California, also used the python and boa to track down the cause of leglessness. He and his colleagues compared the genomes of those snakes with the genomes of snakes such as vipers and cobras, which evolved more recently and have no leg remnants. They, too, noticed that the same Sonic hedgehog enhancer had many deletions and mutations.

They then showed that a mutated Sonic hedgehog enhancer does indeed affect leg growth by testing its influence on mouse limbs. They replaced the rodent’s own version of the enhancer with the snake version using the CRISPR-Cas9 gene-editing technique. They also repeated the experiment, subbing in the same enhancer from a fish and, later, from a human. Even with the fish and human enhancers, the mouse legs developed normally, demonstrating that enhancers from other species still work in the mice. But with the snake enhancer, the legs turned into little nubs, Visel and his colleagues report today in Cell . Finally, when researchers put the missing DNA into the snake enhancer and then put the modified enhancer back into mice, their legs grew just fine.

“The Visel paper is a beautiful study; a tour de force that takes functional genomics using CRISPR-Cas9 to a new level,” Cohn says. Visel says that Cohn’s experiments with snake embryos “independently confirm what we saw in our mouse models. It's very elegant work.”

But Cohn, Visel, and Hanken caution that changes in this enhancer are not the whole story behind the evolution of the serpentine form. Recently, for example, other researchers discovered that another enhancer was responsible for snakes’ extra ribs and long backbones. And it’s possible that the Sonic hedgehog enhancer was not the first step in leg loss, Hanken says. “But it’s certainly a major player.”

The studies may also help settle a longstanding controversy about fossil snakes, some of which have legs to varying degrees. Paleontologists have long tried to squeeze the limbed fossils onto one branch of the family tree with the limbless ones sprouting off from that branch, something that would be expected had limbs been lost only once. But if it didn’t take much to lose legs, then it probably didn’t take much to re-evolve them. “It could explain the possible reappearance of limbs in some extinct snake lineages,” Richardson says. For centuries, evolutionary biologists have argued that organisms cannot re-evolve lost features. But Hanken says this work “shows that, developmentally speaking, it’s not that farfetched.”