During salamander limb regeneration, a blastema – a mass of progenitor cells – forms at the site of amputation. The blastema proliferates, differentiates and is patterned to form the regenerated tissues (McCusker et al., 2015) in a process that has often been compared to the initial development of the limb. Indeed, there are many important molecular parallels between the blastema and the developmental limb bud; however, regeneration presents the blastema with unique challenges that are not present during development. During development, the limb bud follows a consistent progression of steps, emerging from a predefined position at a specific time in the animal's development. Regeneration, by contrast, must restore missing structures, the composition and size of which varies greatly from injury to injury and as the animal increases in size through growth (McCusker et al., 2015). Crucially, the blastema must correctly establish its position along the proximodistal axis for precise replacement of missing components. Further, orientation coordinates, such as dorsoventral and anterior-posterior axes, must be properly specified to regenerate the appropriate structures correctly, which is largely achieved through the redeployment of developmental fibroblast growth factor (FGF), sonic hedgehog (SHH) and bone morphogenetic protein (BMP) signaling gradients, but increasing evidence supports the hypothesis that a specific population of cells in the mature limb retains positional memory through their migration into the blastema and instructs the patterning of the regenerated limb (McCusker et al., 2015; Otsuki and Tanaka, 2021). Until recently, the molecular identity and mechanisms of such a cell population remained mysterious.

In their preprint, Otsuki and colleagues make a compelling case that a group of Hand2-expressing dermal fibroblasts in the posterior limb may represent a seat of this memory in the axolotl salamander (Ambystoma mexicanum) (Otsuki et al., 2023 preprint). Hand2 is a transcription factor that activates Shh expression in the zone of posteriorizing activity (ZPA) during limb development and Shh signaling then initiates the regulatory cascade that directs the anterior-posterior polarity of the developing limb (Charité et al., 2000). Through transcriptomics and the development of several sophisticated reporter animals, Otsuki and colleagues demonstrate that, whereas Shh gene expression fades shortly after development, a population of posterior cells maintains Hand2 expression in the mature limb. The study uses inducible Cre reporters to trace the lineage of cells that expressed Shh during limb development, and real-time reporters for mature cells that express Hand2 through regeneration. The authors find that cells descended from Shh-expressing cells in the limb bud ZPA do not, as a whole, make a major contribution to the regenerating limb. In contrast, they see evidence that cells descended from those which express Hand2 in the limb bud are later enriched in the regenerative ZPA.

Having found that Hand2-expressing cells in the mature limb give rise to cells within regenerative ZPA, Otsuki and colleagues sought to examine whether Hand2 is functionally required for this role, using CRISPR mutagenesis. The authors found that 45% of F0Hand2 CRISPants displayed defects during limb development, which were not seen in the controls. These defects were amplified during regeneration with 75% of CRISPants showing regenerative defects. In Shh reporter animals, mosaic Hand2 knockout reduces Shh expression and the degree of Shh disruption correlates with the severity of both developmental and regenerative phenotypes. The authors also tested the posteriorizing ability of Hand2 CRISPant tissue with the accessory limb model (ALM). In the ALM, skin from the posterior limb is grafted to an innervated wound in the anterior part of the limb; wild-type posterior tissue induces an ectopic limb at the graft site (Carlson, 1974; Endo et al., 2004). Hand2 CRISPant posterior tissue does not induce an ectopic limb, suggesting that Hand2 is required for this posteriorizing activity. Otsuki and colleagues also overexpressed Hand2 using Prrx1-promoter driven Hand2; Prrx1 is expressed throughout the limb bud mesenchyme. In F0 mosaic animals, they saw a range of phenotypes from polydactyly to the formation of ectopic limbs, indicating that Hand2 overexpression is sufficient to induce ectopic Shh expression. Anterior tissue from animals with strong ectopic Hand2 expression also induced limbs when transplanted within the ALM, whereas anterior tissue from animals with weak ectopic Hand2 expression did not. Together, these results suggest that Hand2 expression is necessary for posteriorizing activity in both axolotl limb development and regeneration.

With results suggesting that Hand2 expression likely carries posterior positional memory, Otsuki and colleagues tested whether they could reprogram this memory during regeneration. They created animals with both an Alx4-mCherry reporter to label anterior limb tissue and a Hand2-GFP reporter to label posterior tissue. They then transplanted Alx4-expressing anterior tissue to the posterior part of a wild-type limb, and Hand2-expressing posterior tissue to the anterior part of the limb during regeneration. When anterior, Alx4-expressing cells were transplanted to the posterior limb, they stopped expressing Alx4 and began to express Hand2 and Shh, suggesting that anterior limb tissue can be reprogrammed to a posterior identity during regeneration. Conversely, Hand2-expressing posterior cells continued to express Hand2 and Shh when transplanted to anterior locations, suggesting that, once programmed, posterior identity becomes fixed. They performed similar experiments in the presence of Shh inhibition, Shh overexpression and Shh agonists, finding evidence that Shh signaling initiates Hand2 expression, potentially enforcing a fixed posterior identity during regeneration.

Considering these results, Otsuki and colleagues speculate that developmental Shh signaling specifies posterior cells in the limb bud, initiating long-lasting Hand2 expression in a small subpopulation of these cells. In their model, these cells retain this expression throughout the axolotl's life, allowing it to recall posterior identity during regenerative events. Recent transcriptional and lineage-tracing studies suggest that many cells that transition through the blastema arguably retain some memory of their previous identities: although most blastema cells are of fibroblast origin, several other mature and progenitor cells contribute to the blastema. These populations retain distinct transcriptional identities throughout the entire regenerative process, often giving rise to the same cell populations from which they derived (Kragl et al., 2009; Currie et al., 2016; Choi et al., 2017; Flowers et al., 2017; Gerber et al., 2018; Leigh et al., 2018). When combined with the recent findings of Otsuki and colleagues, a picture emerges in which blastema cells have mechanisms to retain discrete aspects of their identities, despite undergoing drastic changes in behavior, morphology and gene expression. Inevitably, however, some of these identities must be reprogrammed, because there is likely no way to restore fingers from cells that stubbornly retain elbow memories, and, arguably, the most profound findings in this preprint pertain to reprogramming. Otsuki and colleagues demonstrate that some cellular identities, such as Hand2 posterior identity, are more ‘stubborn’ than others, such as Alx4, expressing anterior identity. They also beautifully illustrate (in figure 5d), that competence to reprogram is limited to cells that transition through the blastema state. An obvious future direction is to study the circuitry that retains Hand2 expression in the mature limb, and perhaps more interesting is the regenerative circuitry that massively upregulates Hand2 in response to injury, and the circuitry that allows for anterior-posterior reprogramming within the blastema. Another recent preprint from our group shows that amputation triggers a body-wide proliferation response in axolotls, priming tissues for blastema creation (Payzin-Dogru et al., 2023). As Hand2 cells are arguably already primed for a regenerative role, it would be interesting to see whether they also respond to systemic signals provoked by amputation. Ultimately, the field will benefit greatly as we elucidate the mechanisms by which cells of different origins and discrete descendent populations heed the common call to enter the blastema.

Funding

The authors acknowledge funding from the National Science Foundation (IOS-2145925 to J.L.W.) and the National Institute of Child Health and Human Development (R01HD095494 to J.L.W.).

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Competing interests

The authors declare no competing or financial interests.