How much do you know about axolotl anatomy? - Part 3, regeneration

Written By LibertyLand Axolotl Rescue

Hi everyone! As many are probably aware, axolotls are capable of regeneration of limbs and organ tissues. In this post, we’ll dive into just how this occurs, as well as some limitations and expectations. This will be one of our longer posts, so buckle up for some fascinating information!

Let’s start from the beginning: wound healing. If an amputation of, let’s say a leg, occurs then the process starts by sealing the wound. The wound bleeds minimally, and within 24 hours of amputation the wound is encapsulated in epithelial cells that travel up and around the stump. This covering develops into an apical epithelial cap (AEC). The AEC provides molecular signals critical for the potential to regenerate. Without this AEC and specialized wound healing, regeneration will not occur.

Diagram showing the steps in limb regeneration (credit: Whited and Tabin, Journal of Biology 2009)

Axolotl limb regeneration begins with a blastema. This is a bundle of diverse proliferating cells that forms the new limb. Physically, it appears as a clear almost cone shaped appendage on the distal portion of the amputated limb. It is uncertain the origin of blastema cells. They are undifferentiated cells, but the controversy is over which type of cell differentiates to create the blastema.

These cells have a quality called positional memory, which allows the cells to know what types of cells used to be there, and the cells differentiate to remake the limb. Differentiating is the quality of cells to change and specialize into the cell types needed. Stem cells are the most undifferentiated form of cell, they are basically a clean slate with the capability to turn into any cell type (nerve cells, bone cells, tissue cells, etc.).

The blastema contains cells that used to be specialized, but undifferentiated and use this positional memory to reassign themselves (so a previous tissue cell may turn into a bone cell because that is what is needed in the new limb).

After the blastema has formed, regeneration is broken down into two phases: the “early tiny limb” phase and the “late tiny limb” phase.

After the blastema has formed, regeneration is broken down into two phases: the “early tiny limb” phase and the “late tiny limb” phase. During the early tiny limb phase the blastema forms the structure and shape of the new limb, while the late tiny limb phase is the growth of this limb to scale. The early phase contains more rapid cell and tissue growth, while it slows down in the late phase.

The growth rate is increased due to decreased cell death and increased cellular growth (both in number and size). The limb will typically grow proportionately and in necessary areas. The catch with the blastema is that it requires innervation within a critical period of time in order to regenerate a new limb. In other words, without a viable (active) nerve, the amputated limb will not regenerate. In fact, if the nerve is severed at any point, regeneration will stop.

Regeneration can sometimes go too far. An extra limb, hand, finger, or fimbriae may develop, as with the . This is a result of the cellular positional memory becoming skewed and the wound believes it is actually a different position on the body. Another common sign of over-regeneration is thrombosed gill fimbriae. These are swollen blood vessels, and are pretty harmless. They do not tend to bother the axolotl, but if snagged they can bleed quite a bit. 

Over-regeneration: sometimes, axolotl regeneration goes too far, as with the axolotl above, who has 5 legs

Regeneration is a fascinating, wildly complex process that involves a multitude of genetics, environmental factors, and molecules to replicate an amputated body part. Axolotls have this capability to regrow limbs and organ tissues up to several times. However, it is not always a guarantee that a limb will regenerate properly. Lack of innervation, inability of the AEC to develop and wound heal, lack of necessary chemical messengers, and incorrect positional memory can all hinder an axolotls ability to regenerate correctly. 

Metamorphosis leads to regenerative defects in axolotls. (A), (B) Representative images of a paedomorph and metamorph at the end of the study. (C), (D) Representative images of a paedomorphic limb (C) and a metamorphic limb (D) at the time of amputation. (E), (F) Histological sections of a paedomorphic limb (E) and metamorphic limb (F) at the amputation plane. Green arrowheads indicate the fasciculi of the anconeus muscle. (G)−(J) Gross morphology of the morphogenesis phase of limb regeneration in paedomorphs (G) and metamorphs (H), and images of the limbs at the end of the experiment (I), (J).

If a limb is lost, and it does not appear to be growing back, it is possible that it will not regenerate. There is also a difference in time frames for different body parts (gills being one of the slowest parts to regenerate, taking the longest time). Good quality nutrients, and fresh clean water will be the best for creating a supportive environment for a regenerating axolotl (remember: always monitor for infections).

Questions?

Reach out to the LLA team on Facebook and ask how you can join our private Axolotl Help group. Amongst us, the LLA team has nearly 20 years of experience working hands-on with axolotls, and we’re happy to offer you the benefit of our experience!


Additional academic/scholarly resources on axolotl regeneration:

LibertyLand Axolotl Rescue https://www.libertylandaxolotlrescue.org
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How much do you know about axolotl anatomy? - Part 2, the respiratory system