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

Abstract

A key question in limb regeneration is how axolotl limb cells become competent to specific types of regeneration signals. Here, we present the competency accessory limb model (CALM), a regenerative assay that assesses whether limb cells have achieved the ability to respond to patterning signals. We describe the steps for positioning a wound, deviating the nerve into a limb wound site, and injecting retinoic acid. We also detail the harvest and analyses in both anterior and posterior CALMs. For complete details on the use and execution of this protocol, please refer to Raymond et al.¹.

Keywords: Cell Biology; Developmental biology; Model Organisms; Molecular Biology.

Graphical abstract

Figure 1

Positioning of wounds for CALM nerve deviations To determine the position of anterior or posterior, begin by pulling the limb out from the body and aligning the A/P axis of the autopod with the A/P body axis. The wound sites should be positioned in the mid-stylopod. (A) The CALM-A position will be on the side of the arm closest to the head of the animal. The Brachial Artery runs along the dorsal-anterior region of the limb and serves as a good landmark for the position of the wound site. (B) The CALM-P wound position is on the side of the limb, facing towards the tail of the animal. The white-toned nerve bundle and Auxiliary Artery can be used as visual landmarks because they enter the limb from the trunk on the posterior side of the limb. The Blue dotted line indicates the dorsal apex.

Figure 2

Surgical setup (A) Animal is submerged in a container filled with Tricaine solution. Also pictured is a timer and an example surgical tray. (B) Once the animal is anesthetized, it is laid on its side on a surgery tray covered with moistened kimwipes, then covered with moistened kimwipes and kept moist by frequently misting with Working Holfreter’s Solution. (C) The animal is placed under the stereomicroscope with gooseneck lamps for surgery. The required surgical tools for nerve-deviation surgery – 2 #5 watchmaker forceps and a pair of micro-dissecting (Noyes) scissors – are also pictured. (D) The animal is placed on and covered with ice after surgery. The targeted limb and wound site should remain exposed to prevent disruption of the surgery site and frequently misted with Working Holfreter’s Solution.

Figure 3

Surgical procedure for the CALM-A and CALM-P (A) For the CALM-A nerve deviation, position the limb to access the anterior surface. Use the branchial artery, which marks the dorsal end of where the wound should be positioned, as a visual landmark run through this region. (A’) For the CALM-P nerve deviation, blood vessels and nerve bundles that enter the limb on this site are visible through the flank skin. (B-B’) Make incisions through the full thickness of the skin without damaging the underlying mesenchyme to generate a small window. (C-C’) Cut away the flap of skin to create an even wound site. (D) Make an incision through the skin along the posterior-ventral side of the limb, extending from the base of the limb to the elbow. (E) Carefully peel back the skin and dissect the brachial nerve out from the surrounding connective tissue. (F) Sever the nerve at the elbow joint and carefully dissect the nerve out to the base of the limb, handling only the distal tip. (G-G’) Create a tunnel from the posterior-ventral incision to the wound site in the shortest direction possible. (H-H’) Pull the nerve through the tunnel into the wound handling only the distal tip. (I-I’) Position the nerve in the center of the wound site and trim away the excess distal tip that was handled during surgery. CALM-A-specific steps are colored in red, CALM-P-specific steps are colored in blue, and general steps are colored in black.

Figure 4

Blastema harvest and expressional analysis for CALM-P (A) Position the limb so that the posterior-located blastema is accessible. (B) Using forceps, pinch along the contours of the original wound site to help separate the blastema from the surrounding mature tissue. (C) At the blastema’s base, pinch using forceps to separate the blastemal mesenchyme from the underlying corrective tissue. (D) Complete blastema dissection by pulling the tissue away from the limb, severing any remaining connections. (E) Process blastema samples for qRT-PCR (using primers for Ef1α and Shh) or alternate expressional analysis technique. (F) Example bar graphs of Shh qRT-PCR data represent treatment outcomes on non-competent (left) vs patterning competent blastemas (right).

Figure 5

CALM-A sample processing and expected outcomes (A) After DMSO or RA injection, CALM-A blastemas are imaged weekly in housing water to monitor for ectopic pattern formation. Once patterning is completed after 6-10 weeks, the limbs are harvested, and whole-mount skeletal staining is employed to score the ectopic skeletal patterns for patterning competency. (B) Expected outcomes: if the CALM-A fails to achieve competency, then a simple nodule or no pattern will form. If the CALM-A achieves patterning competency, then a hypomorph limb phenotype or a fully patterned single or 2+ limb phenotype will form.