Living in a murky lake around Mexico City, surrounded by aggressive and cannibalistic neighbors, the axolotl lives at constant risk of losing a limb to a neighbor's nibble. Fortunately, lost limbs regrow and are functional in as few as eight weeks. To achieve this feat, the regrowing body parts must "know" their position within the axolotl body to regenerate the right structure for a specific location. A long-sought code that tells the cells where they are located and thereby gives body parts their identity has now been cracked by Elly Tanaka and her group at IMBA, the Institute of Molecular Biotechnology of the Austrian Academy of Sciences. The study, published in the journal Nature on 21 May, shows how cells "remember" their position and, upon injury, switch on a signal that is broadcast across the one side of the limb and instructs cells to regenerate structures that match with their location.
Once regeneration starts, stem cells on the anterior (thumb) side express the signaling factor FGF8, while posterior (pinky) side stem cells express Shh. These two signals reinforce each other to tell cells to grow and pattern the regenerating arm - a previous discovery from the Tanaka lab. "What we did not know was which cues ensure that FGF8 and Shh are turned on at the two sides of the limb during regeneration, the master mechanism that underlies positional information", explains Leo Otsuki, first author of the study. Axolotls have very large and complex genomes, and genetic tools readily available for other model organisms often do not exist for axolotls. Only recent advances with these molecular tools allowed the scientists to systematically search for signaling cues at the root of regeneration.
Many cues to encode positional memory
To their surprise, the researchers found hundreds of factors that differed in the anterior (thumb-side) versus the posterior (pinky-side) half of the limb even before an injury. But one gene stuck out: The appropriately named Hand2 is only expressed on the posterior side, and not at all in the anterior half. "Hand2 caught our attention because it is expressed in the right place to act as a positional cue", says Otsuki. Experiments on developing and regenerating limbs confirmed an important role of Hand2 in switching on Shh after injury – demonstrating its role as a central player in providing positional information.
The scientists arrived at a new model for limb regeneration, resembling a radio broadcast: In the fully developed limb, only cells on the posterior side express Hand2 at low levels. This Hand2 expression maintains the cells' stable memory of "being in the pinky zone". Upon injury, these cells dial up Hand2 expression to a higher level, which switches on the Shh signal in a subset of Hand2-expressing cells. Shh cells near the Shh source then regenerate in the form cells with posterior-part identity; cells far away from the Shh signal regenerate as anterior-part-cells. Once the limb is fully regenerated, cells again express Hand2 at a low level, ensuring a stable memory of their position is ready for the next cycle of injury and regeneration. These findings can for the first time explain how a preexisting positional memory signal re-activates upon injury to repeatedly induce correct patterning.
Promising possibilities for tissue and organoid engineering
The discovery, made possible by genetic manipulation and cell tracing tools developed in the Tanaka lab, is a major breakthrough for the entire regeneration field. "We uncovered a more flexible model of regeneration than we had expected, and this is really exciting. Our model predicted that we should be able to switch cells from an anterior identity to a posterior identity by taking advantage of the Shh broadcast", Otsuki explains.
Indeed, when the researchers placed cells from the arm's thumb side into the pinky side, these thumb cells regenerated and behaved like pinky cells as they fell into the range of the Shh 'broadcast'. "We were able to reprogram cells from the anterior and change their identity."
The ability to alter cell identities holds immense potential for tissue engineering and regenerative therapies. This concept—essentially "reprogramming" cells to change their function—could enable scientists to transform cells in different parts of the body.
"Being able to convert cells remaining after an injury and change their function is critically important for applications in regenerative therapies", Otsuki points out. "It also enhances our ability to work with organoids and engineer tissues: We now know signals that can transform cell identity and change their regenerative outputs. Harnessing such signals might allow us to push cells beyond their normal biological limits." This could allow them to take on entirely new roles—an exciting prospect for medical innovation.
The discovery that the axolotl relies on the Hand2-Shh signaling circuit for limb regeneration is particularly promising. "These same genes are also present in humans, and the fact that the axolotl reuses this circuit during adult life to regenerate a limb is exciting. It suggests that, if similar memory exists in human limbs, scientists may one day be able to target them to unlock new regenerative capabilities", Elly Tanaka says.
Moreover, by expressing this gene in areas where it is not typically active, such as the anterior half of the limb, it could direct cells to initiate limb formation from scratch. "This finding fuels optimism that, by using Hand2 expression along with other insights from the axolotl model, we may eventually be able to regrow limbs in mammals", Tanaka adds. "Such advances hold promise for the field of regenerative medicine."
