A cutting-edge tool reveals the secrets of a salamander with superpowers

Scientists at Yale use gene-editing technology  to understand the remarkable regenerative abilities of an adorable amphibian.

In 1864, a small shipment arrived in Paris from French colonialists in Mexico. It consisted of six fairly unremarkable animals — three female deer and three small dogs — and thirty-four monumentally strange animals that were like nothing to have set foot in France before. These aquatic organisms had buggy eyes and bizarre, lacy gills, and carried with them a strange name from the New World: axolotl.  

This was the first introduction of the axolotl to the European scientific community. Over the next 150 years, the study of this eccentric amphibian contributed to fundamental changes in Western biological thought, including the emergence of embryology and molecular genetics. Today, they are used to study a fascinating biological phenomenon: the regeneration of complex body parts. Axolotls are particularly fantastic subjects for this research because they can regenerate entire limbs.

The development of a simple clump of cells into a fully functional, complex animal is a massively complicated task. Cells must divide over and over again to build larger and larger body parts. They must also differentiate: activate sets of genes that turn them into specialized cells that will perform different jobs in the body – for example, neurons that form the brain, or skin cells that serve as a protective barrier to the outside world. All this growing and differentiation occurs exactly once: over the course of embryonic development (for humans, that’s in the womb). Most organisms gradually lose the ability to “build” organs and tissues as they reach adulthood. Interestingly, human children (before the age of 7 or 8), can regenerate the tippy tops of their fingers. Don’t try this at home — like most animals, we can’t regenerate after childhood.

AxolotlBE
An axolotl (as known as the Mexican salamander, Ambystoma mexicanum) in captivity. As adults, these amphibians can regenerate whole limbs. By th1098 – Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=30918973

However, there are a few lucky beasts that retain the power of self-construction well into adulthood. The axolotl is one of them. This salamander-like creature can regenerate entire limbs as an adult. It’s a complicated, gorgeous process, one that biologists have yearned to understand for decades. Central to the regeneration of structures like the axolotl limb are a set of mysterious, rapidly-reproducing cells. Last month, scientists at Yale University came one step closer to understanding them, thanks to a cutting-edge tool that the embryologists of 19th-century France would have killed for: genome-editing technology.

Upon amputation, the axolotl squeezes the muscles around its wound site extremely tightly, creating an in-house tourniquet. Skin cells migrate over the wound, forming a thin protective layer between the exposed flesh and the outside world. Then, somehow — as if by magic — seemingly random cells scattered deep within the axolotl limb start to crawl towards the site, as if sensing an emergency. On their trip towards the wound site, they start to divide, shedding their differentiated characteristics and taking on a much more naive, youthful profile: cells in an adult that resemble what you’d find in the growing limb of a fetus.

In unison, these youthful cells wiggle out towards the wound, multiplying along the way, and settle in right below the thin layer of protective skin. This group of cells is called a blastema, Greek for “offspring” or “offshoot”.

Screen Shot 2017-10-27 at 12.38.10 PM
After amputation, the wound is covered by a thin layer of skin. Blastema cells congregate at the wound site, begin to divide, and re-build the limb.
Diagram credit: Whited and Tabin, Journal of Biology 2009.

These newly active cells communicate madly to each other, and to the skin cells above them, broadcasting chemicals that induce unique cellular behaviors. Over time, the blastema rebuilds the missing limb by multiplying and differentiating: a process almost indistinguishable from the one that constructed the limb during embryonic development. Cells at the center of the blastema start to manufacture thick collagen, the material that makes up the stiff, flexible stuff in your ear and nose. While speedily churning out this collagen network, the cells spew chemicals that induce their neighbors to do other limb-building tasks. The nearby cartilage calcifies into bone, and the cells assemble the new limb around it. Fundamentally, it is the mysteriously youthful blastema cells that reconstruct the limb of the axolotl.

Axolotl limbs are made of the same basic stuff as our own: muscle, sinew, skin, and bone. If axolotls can, as adults, resurrect the processes required to grow a limb, could we do it too? For now, the answer is no. But that question — why only some animals can regenerate, how they pull it off, and how we might someday do it too — is at the heart of regenerative biology research.

A central problem in the field is to understand the de-differentiation process that those blastema cells undergo so gracefully, transforming themselves into a newly youthful state. It’s not easy for cells to revert back to an embryonic state. Just as the average middle-aged person might turn down an invitation to a fraternity party, adult cells do not like to go back to their youthful, naïve ways. Neurons stay neurons, skin stays skin. But, if we’d like to, someday, grow organs for transplant in a dish, or initiate limb regeneration in amputees, we need to convince a source of cells to mimic the axolotl blastema.

This is why the axolotl has stayed such a fixture of biological inquiry after landing with a splash in that Paris harbor. Since the days of horse-drawn carriages and chamber-pots, researchers have focused with dogged endurance on understanding the blastema cells: identifying where they come from and what allows them to engage in such unusual regenerative behaviors. Despite more than a century of research, there’s still considerable mystery surrounding these cells. Some scientists posit that there’s an as-yet unidentified “blastema” population, sprinkled through the body of the axolotl, whose members lie dormant, then kick into gear upon injury. Others counter that there is no such hidden group, and instead each cell in the axolotl has the ability to revert back to an embryonic state if it’s required: volunteers from every tissue are then sent to contribute to the rebuilding of the limb.

If the first theory turns out to be right, the hunt will be on for the human blastema-population counterpart. If the second theory is right, perhaps all cells can be called on to help rebuild tissues, and by understanding this process in axolotls we’ll gain some clues as to how to coax human cells to regenerate. It’s also possible that both scenarios are taking place at once – some special blastema-ready populations exist, but when limb regeneration occurs they’re accompanied in the reconstruction effort by members of lots of other tissues.

To pin down the origins of the mysterious blastema cells, Craig Crews and his team at Yale University needed a way to mark and keep track of the cells in the body of the axolotl. For this, they used CRISPR, a powerful gene-editing tool that allows scientists to remove or insert DNA into almost) any spot in a genome. The group inserted unique DNA sequences — biochemical barcodes — into the genomes of particular cells, such that each cell type carried a specific barcode. This produced an axolotl with many uniquely identifiable cell populations. They then amputated this special animal’s limb (don’t worry — it was asleep). When its limb regenerated, the researchers compared the distribution of barcodes in the new limb to that of the original. Amazingly, the profile of barcodes was almost identical between the original and the regenerated limb.

According to this study, cells that make up the blastema don’t seem to be derived from a unique population; in fact, most tissue types in the axolotl limb — muscle, cartilage, bone — each contribute cells that go on to form the blastema. The newly regenerated limb is actually even more similar to the original than previously thought – virtually every single flavor of cell that was present in the original limb is reproduced and integrated into the regenerated limb, making a carbon copy with mind-boggling fidelity.

Screen Shot 2017-10-27 at 12.38.48 PM
An overview of Crews’ experiments. Each colored dot represents a cell type with a unique DNA barcode. Depending on where the barcodes end up in the newly formed limb, the researchers could distinguish between two possibilities: that all cell types can produce the youthful blastema cells, or, instead, that the majority of the blastema cells come from one predesignated population.
Diagram credit: Crews et al., eLife 2017.

 

If there was a specific blastema population from which the cells originated, its cellular descendants would build the new limb, and its DNA barcode would dominate the proportion of cells. This is not what Crews and his team observed. Their findings hint that every one of the axolotl’s cells retains the remarkable ability to backtrack into youthful naïveté, undergoing dramatic changes that allow it to re-enact the choreography of organ-building it performed as an embryo.

If this is the case, it’s important to identify exactly what signal kickstarts the cells to undergo the transition to youthfulness. If we understand the biology of the axolotl blastema, we may uncover ways to nudge human cells towards their deeply buried regenerative capabilities. We would be another step closer to the stuff of science fiction: a world where losing a limb, just like healing a paper cut, is only a temporary ailment.

 

Featured image courtesy of Timothy Hsu. 

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