Molecular basis for positional memory and its reprogrammability in limb regeneration

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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 factor Hand2 from development and this is a priming molecule necessary and sufficient to establish a Shh signalling centre after limb amputation. During regeneration, Shh feeds back and reinforces Hand2 expression in nearby cells. Hand2 is sustained following regeneration, safeguarding posterior memory, while Shh is shut off. As a consequence of this Hand2-Shh system, anterior and posterior identities are differentially susceptible to alteration. Posterior cells are stabilised against anteriorisation as their expression of Hand2 poises them to trigger the Hand2-Shh loop. In contrast, anterior cells can be reprogrammed: a transient exposure to Shh during regeneration causes anterior cells to gain Hand2 expression and a lasting competence to express Shh . 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.

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  1. In this manuscript, the authors use RNA-sequencing, lineage tracing, and pharmacological treatments to identify the molecular basis of anterior-posterior positional memory in salamanders.

    The experiments performed in this manuscript and the presentation of the results are strikingly elegant, both in visual and narrative clarity. I found Fig. 5, in which the authors visually demonstrate reprogramming of cell identity by recruitment to the blastema, to be particularly beautiful in terms of the simplicity and clarity of the results.

    The results of these experiments – indicating that axolotl limbs use Hand2 as a memory marker for positional identity – would be interesting to explore in other regenerative, partially-regenerative, and non-regenerative organisms. Given that the axolotl limb seems to have somewhat different contribution of cell types to the limb compared to other well-studied vertebrates (for example, the proportion of cells descended from the Shh population), it would also be interesting to know whether this particularity of axolotl limb development plays any role in their capacity for limb regeneration.

  2. Taken together, these results indicate that anterior blastema cells readily acquire a posterior memory, while posterior blastema cells retained their posterior memory in this transplantation assay.

    Are Hand2 or its downstream targets known to result in changes in chromatin state? It would be interesting to determine what aspects of Hand2 regulation block posterior -> anterior switches.

  3. but consistent with the axolotl’s reported ability to generate accessory limbs at sites of anterior-posterior discontinuity

    This is a very nice demonstration of the consistency of the model and experimental results – effectively, due to the mosaicism of the insertion, the authors generated an ALM transplantation experiment in situ.

    I wonder if decreasing the amount of the construct used for injection or using electroporation later in development (or as a juvenile), rather than injection, would result in a higher rate of accessory limbs due to a greater chance of discontinuity in identity? It seems surprising to me that this phenotype was only observed in cases of high Hand2 misexpression.