Cells keep a memory of their tissue origin during axolotl limb regeneration

Abstract

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.

Mechanisms of urodele limb regeneration

“…Nearly half the cells of the blastema are derived from dermal fibroblasts (Muneoka, Fox, and Bryant, 1986a), but the total fibroblast contribution is probably well above 50% when the fibroblasts of the periosteum, muscle interstitial tissue, and nerve sheath are considered. Experiments in which transgenic green fluorescent protein (GFP) neurula stage axolotl tissues contributing to the limb were grafted in place of their counterparts in non‐GFP neurulae and the developed limbs amputated showed that dermal fibroblasts, Schwann cells, skeletal cells, and myogenic cells contribute to the blastema (Kragl et al., 2009; Fig. 2).…”

“…Results of experiments tracing tissues grafted from transgenic GFP axolotls to white axolotls, based on data from Kragl et al. (2009), Sandoval‐Guzman et al. (2014) and Tanaka et al.…”

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

“…Dedifferentiation, or loss of differentiation characteristics followed by the acquisition of progenitor features like proliferation, was believed to provide cells with the capacity to re-differentiate into different cell lineages [11-13]. However, the blastema was recently shown to mainly contain lineage-restricted progenitors that only give rise to the same kind of tissues from which they originated [6,8,14-16], indicating that dedifferentiation is less extensive than previously thought [13,17,18]. …”

“…One of the main tissues that contributes to the blastema is the skeletal muscle [4,8,10,14]. Skeletal muscle is mainly composed of elongated cells called muscle fibres, or myofibres, that contract upon nervous stimuli.…”

“…Further studies strongly suggested that limb and tail muscle fibres were able to fragment and that the resulting mononuclear cells proliferate and contribute to the blastema [4,21-23]. Along with muscle dedifferentiation, muscle stem cells (satellite cells) were also shown to contribute to the new muscle [7,10,14]. On the other hand, studies on Xenopus tail regeneration revealed that muscle fibres do not contribute to the blastema, indicating the absence of muscle dedifferentiation [8].…”