Adventures in limb regeneration

As you may have heard previously, three out of four limbs were eaten off my axolotl by his sibling before I got him. (Yes, he's a male. Axolotl testes become apparent, one might even say obtrusive, with maturity.) Some owners might see fit to crow to our mailing list about how great it is to own a cannibal. I prefer to observe and then show off the chief amazing feature of the mighty axolotl: limb regeneration.

Here is Hex's right front foot from the time it started to be more than a stub. The first two photos are awful — I then installed a light on the tank for a dramatic improvement in imaging performance! — but the weird little elf-boot curl the stub took on at first is discernible. Tetrapod limb development is supposed to require apoptosis to separate the fingers, so I was very interested in seeing whether Hex grew a paddle and then dissolved parts away to make digits. At the macro level, though, it looked a whole lot like little toes popping out of a foot and getting longer. This is why we need science.

Luckily, there's no shortage of axolotl-foot biology. Urodele (newts and salamanders) limb regeneration is such a fertile topic for developmental biologists that at the SDB conference over the summer I had the only poster for the entire plant kingdom, compared to a whole row of axolotls. There are obvious applications to medicine if we can thoroughly understand what lets these organs grow back; newts can even regenerate optical lenses from pigmented epithelial cells [1], which is possible for bird and mammal cells only in culture so far [2]. (Unsurprisingly, some of the miRNAs found in regenerating lenses/dedifferentiating PECs matched genes expressed in tumors, so there's an equally evident danger for organisms that are too regeneration-happy. How the newts manage to avoid cancer is another very interesting question.)

I wondered about the developmental signals regulating axolotl limb regeneration, and it turns out that Wnt signaling is critical: if you block it (by using a virus to express a natural antagonist there), the limbs can fail to regenerate completely, or develop a single, Tim-Burtonesque, pointed toe. [3]

Wnt signaling in development is best known from the "canonical Wnt pathway" in which Wnt and β-catenin control dorsalization, and thus the patterning of the neural tube, in animal embryos. But what does dorsality have to do with limbs? It's not completely clear how just yet, but we know that Wnts also affect stem cells and cell differentiation, maybe with different molecular partners. For regeneration, it seems that Wnt function is required very early in the process, when the dedifferentiated cell mass called a blastema is forming [4].

If Wnt signaling is only necessary to make a bunch of undifferentiated cells, what causes those cells to become a new foot? It's another massively important signaling molecule from embryogenesis, BMP. BMP is required for both cell division in the growing limb bud and appropriate apoptosis, as shown by overexpressing BMP and its antagonist Noggin in different limb buds [5]. The images below are in situ hybridizations, which show in blue where axolotl BMP-2 is expressed in progressive stages of development: first all over, maybe regulating cell division, then where cells are being pared away to create toes — just like Hex's, which now look perfect. - KMP

1. Maki N, Martinson J, Nishimura O, Tarui H, Meller J, Tsonis PA, Agata K. Expression profiles during dedifferentiation in newt lens regeneration revealed by expressed sequence tags. Molecular Vision 2010; 16:72-78.

2. Eguchi G, Okada TS. Differentiation of lens tissue from the progeny of chick retinal pigment cells cultured in vitro: a demonstration of a switch of cell types in clonal cell culture. Proc Natl Acad Sci USA. 1973; 70:1495-9.

3. Kawakami Y, Rodriguez Esteban C, Raya M, Kawakami H, Martí M, Dubova I, Izpisúa Belmonte JC. Wnt/β-catenin signaling regulates vertebrate limb regeneration. Genes Dev 2006; 20:3232-3237.

4. Yokoyama H, Ogino H, Stoick-Cooper CL, Grainger RM, Moon RT. Wnt/β-catenin signaling has an essential role in the initiation of limb regeneration. Dev Biol 2007; 306(1):170–178.

5. Guimond JC, Lévesque M, Michaud PL, Berdugo J, Finnson K, Philip A, Roy S. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs. BMC Dev Biol 2010; 10:15.


  1. Replies
    1. If I'd known its ultimate fate I would have been more sensitive!


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