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Limb regeneration involves re-establishing a limb development program from cells within adult tissues. Identifying molecular handles that provide insight into the relationship between cell differentiation status and cell lineage is an important step to study limb blastema cell formation. Here, using single cell PCR, focusing on newly isolated Twist1 sequences, we molecularly profile axolotl limb blastema cells using several progenitor cell markers. We link their molecular expression profile to their embryonic lineage via cell tracking experiments. We use in situ hybridization to determine the spatial localization and extent of overlap of different markers and cell types. Finally, we show by single cell PCR that the mature axolotl limb harbors a small but significant population of Twist1(+) cells.

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

INTRODUCTIONA remarkable property of axolotls (Ambystoma mexicanum) is their amenability to grafting at any stage of the life cycle. In addition to the ease of transplantation in embryonic axolotls, the graftability of tissues extends to larval, juvenile, and adult tissues. Amazingly, there seems to be no rejection of tissue grafts from individual to individual. Grafting techniques have been important for studying tissue contributions to the blastema and other cellular aspects of blastema formation, growth, and patterning. This protocol describes grafting of axolotl limb skin and cartilage from green fluorescent protein (GFP)+ donors to normal hosts.

INTRODUCTIONAppendage regeneration is a well-known and well-studied trait in salamanders including the axolotl (Ambystoma mexicanum). Depending on the size of the animal, functional regeneration of the limb or tail can occur within 3 wk (in 2-cm-long animals) to several months (in >10-cm-long animals). After appendage amputation, epithelial wound healing is followed by formation of a progenitor cell zone called the blastema that first undergoes expansion and then morphogenesis and differentiation to replace the appendage. To obtain reproducible, morphologically perfect regeneration, it is important to cut the appendage so that the wound site is evenly open over its surface. This allows cells from the basal layer of the surrounding epidermis to crawl over the cut end. This protocol describes limb amputation in 3- to 5-cm and 5- to 8-cm axolotls, as well as tail amputation in 3- to 8-cm axolotls.