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

Esther Kim
By Martin Kragl, Dunja Knapp, Eugen Nacu, Shahryar Khattak, Malcolm Maden, Hans Henning Epperlein & Elly M. Tanaka
Lake Forest College
Lake Forest, Illinois 60045

A group of researchers associated with the Max Planck Institute of Molecular Cell Biology and Genetics and the Center for Regenerative Therapies at the University of Technology Dresden conducted a study on limb regeneration in axolotls. The researchers were able to find that the axolotl tissue does not completely de-differentiate into pluripotent cells. An emerging property and area of research is the subject of the regeneration in animals and its application in humans using stem cells and glial cells. One key link to regeneration is neoteny – the retention of juvenile features. Neoteny can be seen in the axolotl, Ambystoma mexicanum, an aquatic salamander. In the experiment, researchers studied the limb-regeneration in axolotls and their amazing ability to recover post-injury with a fully functional limb.


Kim Fig 1

The tissues observed in the limb were full-thickness skin samples, tissue that included the epidermis, dermis, muscle, nerves, blood vessels, Schwann cells and other parts of the skeletal structure. The term “blastema” refers to the progenitor cell zone, undifferentiated progenitors, that allows for the regeneration of the missing tissues in the axolotl limb; the researchers were interested in the specialization of the cells. The dermal layer of skin was especially necessary for the creation of the blastema. In the research article, researchers focused on the upper arm, lower arm, and hand. The blastema is a particularly intriguing area of research as it is still unknown whether blastema cells are multipotent or pluripotent stem cells.


In order to determine limb tissue de-differentiation and pluripotent cells, the researchers used a variety of methods to observe the regeneration. First, they used an integrated GFP transgene. The GFP was used to label each major limb tissue through a grafting procedure. The procedure consisted of the following two options: first, GFP transgenic donor embryonic limb tissue was implanted into GFP- host embryos or second, there was a direct grafting of GFP+ limb tissue to unlabeled hosts. The GFP was used to observe the major tissues during regeneration and the production of progenitor cells with restricted potential. Additionally, the axolotl tissue was also immunostained for muscle-specific-myosin heavy chain (MHCl), anti-MHCl, and PAX7+. MHCl  and PAX7+ were used for muscle markers. Cells, mostly GFP+ blastema cells, were also tested for Myf5 expression via a single-cell polymerase chain reaction protocol.

Kim Fig 2

After closely inspecting the restricted potential of the blastema, the researchers deduced the positional identity and cell-type-specific property of blastema cells. The researchers found that the dermis cells did not create muscle cells but did replace cartilage and tendons. Furthermore, the cartilage cells did not make muscle cells. The GFP+ blastema cells demonstrated no Myf5 expression in dermis-derived blastema cells, however, 31% of skeletal muscle-derived blastema cells did produce Myf5. The researchers concluded that the positional identity of the axolotl cells is tissue specific (Nature 62). Essentially, the tissue the regenerated cells were derived from did affect regeneration. Moreover, the researchers expanded on their results by surmising, “[t]hese tracking experiments have established that the blastema is a heterogeneous pool of progenitor cells from the outset of regeneration. This result has important implications for another central problem in limb regeneration—the control of positional identity along the proximal/distal limb axis” (Nature 63). The proximal positional identity was also affected by the cell type the regenerated cells were derived from – Schwann cell-derived blastema cells did not have this property while cartilage-derived cells did. Schwann cells were tracked also using GFP+. The researchers utilized molecular markers with the MEIS 1 + 2 protein and HoxA13 messenger RNA as they are linked with proximo-distal properties.


The questions the researchers were left with address the limb-regeneration in different cell types and life-stages of the axolotls and more avenues of research left. It is exciting what this research could mean for both the future of axolotls and humans alike. If axolotls and their limb-regenerative properties and cell-differentiation were used for new applications of organ donations and skin grafts, the possibilities are endless.


Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.


From the article:                                                          

Figure 1 | Dermis does not make muscle but makes cartilage and tendons. a, Schematic of experiment. b, Representative time course. Inset shows cross-section at dashed line immunostained for MHCI (see Supplementary Fig. 4a, b). c, Longitudinal section of 12-day blastema. GFP1 (arrowhead) and PAX71 signals did not overlap. d, e, Cross-sections through regenerated limbs. GFP1 cells (arrowheads) were negative for the indicated muscle markers (red). f–h, Longitudinal sections immunostained for anti-MHCI. Fluorescent cells contributed to connective tissue (f), tendons (g) and cartilage (h, arrowheads). Blue shows DAPI in merge panels (d–h). Scale bars: b, 0.5 mm; c–h, 50 mm.                                                                  

Figure 3 | Muscle does not make cartilage or epidermis. a, Schematic of labelling. b, Time course. Inset shows cross-section at dashed line (see Supplementary Fig. 4e). c, Section of 12-day blastema. GFP1 cells (arrowheads) were positive for PAX7. d, Single-cell PCR showed that GFP1 muscle-derived blastema cells expressed Myf5 (see also Supplementary Fig. 9a) but GFP1 cells from other tissues did not. RP4 acted as quality control. Numbers of cells/blastemas/animals/experiments analysed were as follows for each tissue. Skeleton: 152/8/8/4, Schwann cells: 402/6/6/6, dermis: 230/12/12/6, muscle: 184/6/6/3. e, Longitudinal section through regenerated limb. No GFP1 cells were found in cartilage or epidermis (above dotted line). Scale bars: b, 0.5 mm; c, 50 mm; e, 100 mm.


Works Cited

Kragl, Martin, et al. “Cells Keep a Memory of Their Tissue Origin during Axolotl Limb Regeneration.”

Nature, vol. 460, 2 July 2009, doi:doi:10.1038/nature08152.


Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.

Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.