Snakes used to have legs and arms

The ancestors of today's snakes sported full-fledged arms and legs, according to a new study which found that genetic mutations caused the reptiles to lose their limbs over 100 million years ago.

Researchers described a stretch of DNA involved in limb formation that is mutated in snakes. When they inserted the snake DNA into mice, the animals developed truncated limbs, suggesting that a critical stretch of DNA lost its ability to support limb growth during snake evolution.
"This is one of many components of the DNA instructions needed for making limbs in humans and, essentially, all other legged vertebrates. In snakes, it's broken," said Axel Visel, from Lawrence Berkeley National Laboratory in the US. "It's probably one of the several evolutionary steps that occurred in snakes, which, unlike most mammals and reptiles, can no longer form limbs," said Visel.

Today's serpents have undergone one of the most dramatic body plan changes in the evolution of vertebrates. To study the molecular roots of this adaptation, Visel and his colleagues started looking at published snake genomes, including the genomes from basal snakes such as boa and python, which have vestigial legs - tiny leg bones buried in their muscles - and advanced snakes, such as viper and cobra, which have lost all limb structures. Within these genomes, they focused specifically on a gene called Sonic hedgehog, or Shh, involved in many developmental processes - including limb formation.

The researchers delved further into one of the Shh gene regulators, a stretch of DNA called ZRS (the Zone of Polarising Activity Regulatory Sequence) that was present but had diverged in snakes. To determine the consequences of these mutations, the researchers used CRISPR, a genome-editing method, to insert the ZRS from various other vertebrates into mice, replacing the mouse regulator. With the ZRS of other mammals, such as humans, the mice developed normal limbs. Even when they inserted the ZRS from fish, whose fins are structurally very different from limbs, the mice developed normal limbs.
However, when the researchers replaced the mouse ZRS with the python or cobra version, the mice went on to develop severely truncated forelimbs and hindlimbs.

"Using these new genomic tools, we can begin to explore how different evolutionary versions of the same enhancer affect limb development and actually see what happens," said Visel. To identify the mutations in the snakes' ZRS that were responsible for its inactivation during snake evolution, the researchers took a closer look at the evolutionary history of individual sequence changes. By comparing the genomes of snakes and other vertebrates, they identified one particularly suspicious 17 base-pair deletion that only occurred in snakes; this deletion removed a stretch of the ZRS that has a key role in regulating the Shh gene in legged animals.