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Protocol for patterning competency assay in regenerative axolotl limb cells using the competency accessory limb

Michael J Raymond¹, Catherine D McCusker²

¹Brigham and Women's Hospital, Boston, MA 02115, USA.

|June 28, 2025

Summary

We developed the Competency Accessory Limb Model (CALM) to test axolotl limb cell responsiveness to regeneration signals. This assay helps understand how cells gain the ability to respond to specific patterning cues during limb regrowth.

Keywords:

Cell Biology Developmental biology Model Organisms Molecular Biology

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Area of Science:

Developmental Biology
Regenerative Medicine
Amphibian Research

Background:

Understanding limb regeneration in axolotls is crucial for advancing regenerative medicine.
A key challenge is determining how limb cells acquire competence to respond to regeneration signals.

Purpose of the Study:

To introduce the Competency Accessory Limb Model (CALM) as a novel assay.
To assess the competency of axolotl limb cells to respond to specific patterning signals.

Main Methods:

The CALM assay involves precise wound positioning on the limb.
Nerve deviation into the wound site and retinoic acid injection are key procedural steps.
Detailed protocols for anterior and posterior CALM preparation and analysis are provided.

Main Results:

The CALM assay effectively evaluates limb cell responsiveness to patterning signals.
This model provides a framework for studying the acquisition of regenerative competency.
The study details the practical execution of the CALM protocol.

Conclusions:

The Competency Accessory Limb Model (CALM) is a valuable tool for studying limb regeneration.
This assay facilitates research into the cellular mechanisms underlying axolotl limb development and repair.
Further research can utilize CALM to explore factors influencing regenerative signal response.