Rethinking the evolution of limbs and digits

Developmental biologists have unexpectedly found that the genetics underpinning the formation of limbs and digits in vertebrates is distinctly different than what scientists have believed for nearly 30 years.

The long-held theory has been that a gene called Sonic Hedgehog (Shh) plays the lead role in directing the formation of limb skeletal elements and determining the specific characteristics, or identity, of each digit. Earlier studies showed that when Shh is missing, animals end up with stubby limbs and only one digit.

In the Aug. 29 issue of Nature, however, researchers at the UW–Madison Medical School and Vanderbilt University report that, to their great surprise, limbs and digits will form in mice lacking Shh and Gli3, a gene previously suspected of counteracting Shh activity.

Furthermore, the researchers found that mice missing both genes develop up to 11 digits that lack normal digit identities and look essentially the same.

Examining the interactions between the two genes, the research team found that Gli3 plays a much more important role than was expected. In fact, the scientists found that Shh has no effect on limb patterning without Gli3. By sending signals through Gli3, Shh restricts the number of digits formed to no more than five, and it dictates digit identity.

“These are fundamental new observations about limb development,” says John Fallon, UW Medical School’s Harland Winfield Mossman Professor of Anatomy, co-senior author on the paper. Fallon has studied limb development and digit patterning for nearly 40 years.

UW–Madison graduate student Randall Dahn was the co-lead author on the Nature paper with Ying Litingtung, a post-doctoral associate of Chin Chiang, an associate professor of cell and developmental biology at Vanderbilt.

The researchers propose that Shh and Gli3 work together to create the pattern in which five fingers and toes will appear. Depending on the amount of Shh present, Gli3 becomes either an activator or repressor, influencing other genes involved in digit patterning by turning them on or off. Higher levels of Gli3 repressor produce the thumb, while higher levels of Gli3 activator produce the pinkie.

The new findings push Fallon and Dahn to rethink how the two genes may figure in the evolutionary shift that took place 340 million years ago, when some genetic mechanism first limited to the number of digits in animals to five per limb.

In the study, the limbs of animals lacking Shh and Gli3 resembled the multiple-digit limbs of animals that lived prior to that point in evolutionary history, notes Dahn. Biologists call the modern appearance of structures lost during evolution an “atavism,” which means ancestral form.

“Shh and Gli3 constrain the potential all tetrapods, or four-legged animals, have to produce more than five digits, a condition called polydactyly,” says Fallon. “Digit number is probably constrained because the syndromes that accompany mutations causing polydactyly are not compatible with overall fitness of an animal.”

Polydactyly is among the most common birth defects in humans, affecting as much as 1 percent of the world population. Syndromes involving other body systems are almost always associated with these genetic disorders.

The new findings also are relevant to cancer research, says the researchers, in that disruptions in the Shh and Gli3 signaling pathway can produce the unchecked cell proliferation that leads to basal cell carcinoma, the most common of all human cancers.

Shh and Gli3 have also been implicated in regulating growth and patterning during embryonic and fetal development of the brain, spinal cord, lungs, gut, genitalia and several other tissues.

“Further work is needed to assess whether the mechanisms we describe for limb development may also apply to the development of these organs and tissues,” Fallon says.