Molecular basis for positional memory and its reprogrammability in limb regeneration

Upon limb amputation in salamanders, anterior and posterior connective tissue cells form distinct signalling centres that together fuel successful regeneration. The molecular properties that distinguish anterior and posterior cells prior to injury, which enable them to initiate different signalling centres after amputation, are not known. These anterior and posterior identities, crucial for regeneration, were thought to be established during development and to persist through successive regeneration cycles as positional memory. However, the molecular nature of these memory states and whether these identities can be engineered have remained outstanding questions. Here, we identify a positive feedback mechanism encoding posterior identity in the axolotl limb, which can be used to newly encode positional memory in regenerative cells. Posterior cells express residual levels of the bHLH transcription factorHand2from development and this is a priming molecule necessary and sufficient to establish aShhsignalling centre after limb amputation. During regeneration,Shhfeeds back and reinforcesHand2expression in nearby cells.Hand2is sustained following regeneration, safeguarding posterior memory, whileShhis shut off. As a consequence of thisHand2-Shhsystem, anterior and posterior identities are differentially susceptible to alteration. Posterior cells are stabilised against anteriorisation as their expression ofHand2poises them to trigger theHand2-Shhloop. In contrast, anterior cells can be reprogrammed: a transient exposure toShhduring regeneration causes anterior cells to gainHand2expression and a lasting competence to expressShh. In this way, regeneration is an opportunity and entry point to re-write positional memory. Our results implicate positive feedback in the stability of positional memory and explain why positional memory is more easily altered in one direction (anterior to posterior) than the other. Because modifying positional memory changes signalling outputs from regenerative cells, our findings have wider implications for tissue engineering.