How Did the Snake Lose Its Legs? Genome Sequencing May Have Found an Answer

Researchers have identified a DNA sequence responsible for the evolutionary loss of limbs in snakes. They found that a section of genome necessary for normal limb development in many vertebrate classes has mutated in snakes. Splicing this specific snake DNA sequence into the mouse genome also caused mice to have abnormally shortened legs. The study was published in the October 20, 2016, edition of Cell.

“The molecular basis of loss of limbs in snakes as they evolved from their limbed ancestor has been the subject of extensive speculation,” write the authors. They examined a DNA sequence called the zone of polarizing activity regulatory sequence (ZRS), a limb-specific enhancer of the sonic hedgehog gene. The ZRS enhancer is active in embryonic limb development. ZRS is present in many vertebrates, even fish, and ZRS mutations have been shown to cause limb malformations.

The investigators compared the genomes of six snake species: two basal snakes (Burmese python and boa constrictor), which have a vestigial pelvic girdle and hind limbs, and four advanced snakes (king cobra, speckled rattlesnake, viper, and corn snake), which have no skeletal limb elements. They found the ZRS enhancer in all of the snakes except the corn snake. In the basal snakes, ZRS was about 80% similar to the ZRS of limbed lizards and had about the same rate of mutations as other enhancer sequences. In the advanced snakes, however, the mutation rate in ZRS was substantially higher than in other enhancer sequences. The authors conclude that a rapid evolutionary change in ZRS coincided with the complete loss of limb structures in advanced snakes.

Further testing using a transgenic mouse reporter assay showed that the ZRS of many vertebrates—but not snakes—displayed activity patterns identical to those of mouse ZRS in embryonic mouse limb buds. This result, say the authors, shows that ZRS function is conserved across vertebrates with appendages but has been lost in snakes.

The researchers also demonstrated that snake ZRS causes limb loss or malformation in vivo. They used CRISPR genome editing technology to replace the ZRS enhancer sequence in mice with coelacanth, human, python, or cobra ZRS. Mice with coelacanth or human ZRS developed normal limbs, even though coelacanth fins are morphologically very different from mouse legs. Mice with snake ZRS had severely truncated limbs. The effect was most pronounced with ZRS from cobras, the advanced snake species.

Finally, the investigators edited snake ZRS by inserting a small nucleotide section that is missing in snake genomes but present in the genomes of limbed vertebrates. Using CRISPR, they replaced mouse ZRS with the edited snake sequence. In this case, the mice developed normal limbs.

“Our genomic enhancer replacement experiments in mice conclusively demonstrate that the loss of function in a single enhancer observed in snakes is sufficient to cause severe limb reduction in mice, raising the possibility that ZRS deactivation contributed to the loss of limbs in the snake lineage,” conclude the authors. They point out, however, that changes in other gene sequences probably also contributed to the absence of limbs in snakes. “…We do not expect that the reintroduction of a fully functional ZRS into a snake genome alone would be sufficient to induce the formation of fully or even partially developed limbs in snakes.”

The study was funded by the National Institutes of Health, the Olga Mayenfisch Foundation, and the University of Basel. Fellowships from the Helen Hay Whitney Foundation and the Swiss National Science Foundation supported two of the authors.

Dr. Laurie Anne Walden received her doctorate in veterinary medicine from North Carolina State University. After an internship in small animal medicine and surgery at Auburn University, she returned to North Carolina, where she has practiced small animal primary care medicine for over 20 years. Dr. Walden is also a board-certified editor in the life sciences and owner of Walden Medical Writing, LLC.