The gene that gave us our limbs has finally crawled out… of salamanders

Have you ever looked in the mirror and seen a salamander staring back at you? Probably not, but in a way, it would if that was a genetic mirror.

You are a tetrapod (four-legged vertebrate). Never mind that humans don’t actually walk on four legs. Nearly all tetrapods develop their limbs postaxially, meaning posterior bones start to grow from the outside edge in. When you were in utero, your pinky finger formed before your thumb, but in salamanders, also known as uredole amphibians, the opposite happens. Embryos develop their limbs preaxially or from the inside edge out.

The “but why?” and “but how?” about this had been giving scientists headaches for nearly a hundred years. While preaxial development is unique to salamanders now, this is because they are the only tetrapods, and the only vertebrates, that have somehow held on to it over millions of years. It is an ancestral state. Vertebrates that would eventually evolve into everything from salamanders to humans used to develop limbs preaxially.

Why this happened remains a mystery, but how has finally been revealed by researchers Randal Voss, Susan Mackem and their colleagues, who recently published a study in Current Biology. Turns out a protein coding gene known as Gli3 is involved.

“Limb development is complexly regulated by several interacting proteins,” Voss told SYFY WIRE. “Where and when these genes are expressed regulates the limb patterning process and determines the overall axis of polarity—preaxial or post-axial.”

Gli3 interacts with other genes in order to determine limb development. These include a signaling gene, SHH, that zaps instructions to the brain to make a protein appropriately named Sonic Hedgehog, which then sends signals for how an embryo will form, including its limbs. Sonic is expressed posteriorly more than it is anteriorly. What is interesting about SHH is that it has also been found to be one of the genes that, when activated in the right order and at the right time, is behind the regeneration of limbs in salamanders and other vertebrates.

Also part of limb formation are Hoxd11-13 proteins, which determine how active Gli3 ends up being. High Gli3 activity leads to preaxial development while low activity means the limbs will develop postaxially. Voss and his team tried turning Gli3 on and off in mice to see whether they would revert to the ancestral state if it was switched on. Sure enough, mouse embryos with high Gli3 activity levels developed preaxially. They also tried switching it off in salamanders to see if the opposite will happen. Those salamander embryos began to develop postaxially.

“The activity level of Gli3R is affected by the presence of Hoxd11-13 proteins through direct binding interactions,” Voss said. “Deleting Hoxd11-13 increases Gli3 activity and this yields the salamander pattern of limb development.”

Expression of the same genes that control how limbs form are is also thought to have been part of the transition from fins to limbs. When what were once fish morphed into the first tetrapods, permanently leaving water for land, they had somehow evolved to survive on all fours, with some (like us) later standing up on two legs. This had to have meant a genetic shift. While the exact changes in DNA that caused it have not been proven, there are theories on how it could have happened.

Then there is the question of how Gli3 factors into not just the generation, but regeneration of limbs in salamanders. Humans are thought to be incapable of this because the right genes don’t do what they have to at the right time. It starts to get weirder when you realize that we share many of the same limb tissues with lungfish, which are capable of regenerating fins. Voss believes that future studies with axolotls that have had the Gli3 gene switched off may offer more insight into how they are capable of what, at least to us, seems like a superpower.

“We tend to think that limb regeneration redeploys mechanisms of limb development, however much of the data supporting this idea are correlative,” he said. "We still need to learn whether the Gli3 associated mechanism of limb development recapitulated during regeneration or a different mechanism is used.”

We don’t need to rewind ourselves back to an ancestral state, but would it be possible to genetically engineer humans to regenerate limbs like salamanders? That is going to have to live in the realm of science fiction for a while.