Even if you identify one, working out what it does is tricky. In 2008, Visel’s team showed that an enhancer called HACNS1 has changed rapidly during human evolution, ever since our ancestors split away from those of chimps. HACNS1 seems to be active in the limbs of embryos, in areas that eventually give rise to the shoulder, wrist, and—most excitingly—the thumb. This suggests that the enhancer may have influenced the evolution of our uniquely dextrous hands. But even so, the team only had circumstantial evidence for this.
That’s why Visel turned to ZRS. It’s probably one of the best studied enhancers, and its role is clear. By controlling a gene called sonic hedgehog (it’s a long story), it governs the growth of limbs—and only the growth of limbs. Delete it from the genome of a mouse, and you get a legless rodent. Mutate it in humans, and you can get deformed limbs, including extra thumbs or fingers. What then, Visel wondered, does it do in snakes? “We wondered if it still works,” he says. “Intuitively, you wouldn’t expect it to.”
To find out, team member Evgeny Kvon scanned the genomes of six snake species, and found that five of them still contain ZRS—albeit, in a heavily altered form. Since snakes split off from the other lizards from which they arose, their versions of ZRS have accumulated mutations at a breakneck pace.
Next, Kvon loaded mouse embryos with versions of ZRS from a variety of animals, including humans, sharks, playtpuses, bats, chickens, and the five snakes. In each case, he connected ZRS to a gene that produces a blue chemical, so he could see where the enhancer was being used. And he found that in almost all cases, the embryos developed blue patches in the back part of their small budding limbs.
Only the snake enhancers broke this trend: the python and cobra versions weren’t active in the limb at all, and the rattlesnake version was just all over the place. “The activity pattern was either completely gone, or just became weird,” says Visel. In other words, the enhancer wasn’t enhancin’.
To prove that point, Kvon actually changed the ZRS of mice to match those of the other animals, using a now-famous gene-editing technique called CRISPR. Amazingly, he found that most of these changes did nothing. Even when he replaced the mouse version of ZRS with that of a coelacanth—a fish, with 400 million years of evolution separating it from a mouse—the rodent’s limbs developed normally.
That wasn’t the case when Kvon edited the mouse ZRS to match those of snakes. With the cobra version, they didn’t develop legs at all. With the python version, they developed little stubs, but not full limbs. This implies that the python ZRS still works slightly, but the cobra one absolutely doesn’t. (In an independent study, published at the same time, Francisca Leal and Martin Cohn confirm that mutations in the python ZRS do indeed curtail the activity of the snake’s sonic hedgehog gene, and so halt the growth of its hind legs.)