Shh protein in axolotl limb regeneration

Understanding how a protein that looks like everyone’s favorite videogame hedgehog, plays an important role in axolotl limb regeneration.

Apurva Joshi
3 min readDec 18, 2020
  • *IMPORTANT DISCLAIMER: This is a summary review of the paper: “Cyclopamine Induces Digit Loss in Regenerating Axolotl Limbs.” I have no affiliation with the authors of this paper, only that this experiment fascinates me, and I, a limb regeneration enthusiast, am writing this article to better my understanding of their work. Cheers!**

Ambystoma mexicanum, better known as the mexican axolotl, is perhaps best known for its amazing regeneration capabilities. It’s one of the amphibians, let alone one of the only animals in the world, that can regrow its own limbs!

Naturally, it’s a lot more complex than just having an amputated limb. There are many biological factors, like various genes and proteins, involved in regrowing an axolotl’s limb. One of the key genes in the limb regeneration process is the Sonic hedgehog protein (shh).

Yup, you read that right. The Sonic hedgehog protein. It’s named that way because a mutation in this protein makes it look like Sonic the hedgehog.

Shh is a developmental gene that’s found across vertebrates and invertabrates, and plays a key role in segmentation of a limb, specifically for the anterior-posterior (can be thought of top-down) axis in both development and regeneration. In axolotls, thought, it’s still poorly misunderstood. Here’s insight as to why that’s the case:

The start of the axolotl limb regeneration process is a bud-shaped clump of cells called a blastema. Expression of shh comes towards the bottom half of the regrowing limb from the blastema.

Image Credits: Nature

Contrast that with bhh, another protein just like shh, which is present throughout the blastema, and therefore throughout regeneration of the limb. A common hypothesis was that bhh precedes the signaling of shh. If that’s the case, then what role does shh really play in the regeneration of a limb?

All isn’t lost, though. Previously conducted studies have shown that wherever scientists try to grow extra digits on the axolotl limb, they observed the presence of shh. Clearly, shh does play a role in axolotl limb patterning….but the question remains: What exactly does it do?

In 2000, Doctors Roy and Gardiner set out to better answer that question.

The Big Idea💡

The researchers wanted to use a compound called cyclopamine, a compound derived from the corn lily that acts as an antagonist for the shh/hh protein families, to induce loss of function of shh during regeneration.

Cyclopamine blocks the anterior-posterior patterning of developing zebrafish fin buds and that of mice and chick limb buds as well. A clear pattern across different vertebrates for sure! They figured, “why don’t we see how cyclopamine affects shh?”

The Results 📊

  • Cyclopamine treatment caused a reduction in the number of digits in all the regenerating limbs treated with the compound. This was the same for both types of limbs, forelimbs AND hindlimbs!
  • Even more interesting is that it didn’t just happen to a few of the axolotls in this study: every salamander treated with the same concentration of cyclopamine showed the same phenotype (ex. all the axolotls treated with 1microg of cyclopamine only had 3 fingers on every individual axolotl limb, when that concentration of cyclopamine was doubled, only 1 digit formed on those limbs, etc)
  • On a macro-level, cyclopamine disrupts shh, and therefore segmentation of an axolotl limb on the anterior-posterior axis.

Key Takeaway:

We now better understand the role of shh in limb regeneration — it’s essential in digit formation of developing limbs. Plus, its function is the same for both development and regeneration of limbs!

Another piece of the limb regeneration puzzle 🧩 unlocked. We’re one step closer to understanding how limb regeneration works at the genetic level — someday soon, we can figure out how this could work in humans too!

Thanks for reading this review paper! My name’s Apurva Joshi, and I’m a 17-year-old working a project called Likagen, focused on understanding the fundamental biomechanisms of limb regeneration to develop limb regeneration methods for humans. If you have any questions or want to help me in making our vision into a reality, I’d love to get in touch to discuss more!

You can contact me on Linkedin , or at my personal email:



Apurva Joshi

Currently conducting independent research in iPSC derivation. Outside of that: 2nd-yr bchm & neuro @ brandeis, alum @ TKS, writer of medium articles