Data on chemical activation of Wnt/β-catenin during axolotl limb regeneration

Limb amputation in axolotls was performed to obtain data demonstrating that a chemical agonist of Wnt (int-related protein)/β-catenin signalling can have a role in axolotl limb regeneration (Wischin et al., 2017) [1]. The data revealed that active β-catenin protein was present during limb regeneration in some Leydig cells in the epithelium; after the chemical treatment, it was observed in more Leydig cells. In addition, the chemical agonist of Wnt generated distinct limb malformation.


a b s t r a c t
Limb amputation in axolotls was performed to obtain data demonstrating that a chemical agonist of Wnt (int-related protein)/β-catenin signalling can have a role in axolotl limb regeneration (Wischin et al., 2017) [1]. The data revealed that active βcatenin protein was present during limb regeneration in some Leydig cells in the epithelium; after the chemical treatment, it was observed in more Leydig cells. In addition, the chemical agonist of Wnt generated distinct limb malformation.
& 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Experimental factors
Limb regeneration was studied in axolotls, the effect of Wnt agonist on limb regeneration was analysed Experimental features Immunofluorescence microscope, luciferase assay, RT-PCR, skeletal staining

Data source location
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Data accessibility The data are available with this article

Value of Data
The data showed that Wnt/β-catenin signalling was active throughout the limb regeneration process.
It showed that the Wnt agonist activates Wnt/β-catenin signalling. This data can be used to determine that Wnt agonist has effects during axolotl limb regeneration. It can be used to determine that Wnt agonist is able to activate β-catenin signalling in Leydig cells. This data can be used to determine that Wnt agonist is able to induce limb malformations during limb regeneration.

Data
Data in Fig. 1A show that during the limb regeneration process, Wnt/β-catenin signalling was active in the epithelium (Fig. 1A). Active-β-catenin was observed in the nuclei of cells that apparently corresponded to Leydig gland cells but it was not observed in nuclei of other tissues (Fig. 1A).
Data in Figs. 1C and 2 show that the Wnt agonist (1 mM) treatment of amputated axolotls at 12 days post amputation (dpa) for 24 h activated β-catenin in Leydig cells nuclei, compared with controls, but not in mesenchymal cells of the blastema. Also, Wnt agonist increased expression of the target genes in the limb epithelium (Fig. 1D). Data in Fig. 3A show that Wnt agonist caused limb malformations. In animals treated 24-hours post amputation for 24 h, the anterior-most skeletal elements of the regenerated limb were missing ( Fig. 3A and B). Animals treated at 10 dpa for 24 h developed three digits and one more, which could not be clearly identified as a digit (Fig. 3C) or did not develop completely (Fig. 3D). Limb of animals treated at 12 dpa presented only three slender digits with no joints (Fig. 3E) or two small digits with no joints (Fig. 3F). Data in Fig. 3G show that only one individual could regenerate after three months after amputation, however, it only developed one digit and no carpus or ossification was observed (Fig. 3G).

Animal maintenance and treatment
All experiments were performed on 5-6.5 cm axolotls (Ambystoma mexicanum). One forelimb was amputated at mid-zeugopod after they were anesthetised with 0.05% Tricaine (Sigma-Aldrich, St. Louis, MO, USA). The animals were placed in the solution with the Wnt agonist (Calbiochem, Billerica, MA, USA) on different days after amputation. The vehicle, dimethyl sulfoxide (DMSO) was used as control. Limbs were amputated at the base of the arm and collected at the end of the experiment.
This research protocol was reviewed and approved by the Institutional Review Board for the Care and Use of Laboratory Animals of the Instituto de Investigaciones Biomédicas, UNAM. All experiments in the present study were carried out in accordance with the approved guidelines.

Tissue staining and skeletal preparation
Tissue samples were fixed in 4% paraformaldehyde at 4°C overnight and processed to be embedded in Paraplast (Sigma-Aldrich) and sectioned into 7 mm for tissue staining. They were stained with the Masson Trichrome technique. For skeleton staining, limbs were fixed in 95% ethanol and permeabilized overnight with acetone. Later, collected limbs were stained in an Alcian blue/Alizarin red solution for three days and were cleared in 1% KOH/20% glycerol and stored in 50% ethanol/50% glycerol.

RT-PCR and luciferase assay
RNA expression detected RT-PCR using RNA collected from eight 12-day post-amputated axolotl limb skins, from animals treated either with the Wnt agonist or with DMSO. We used the following primers: c-myc forward primer 5 0 -TGACCCTTCAGTGGTCTTCC-3 0 c-myc reverse primer 5 0 -CGCCTCTTGTCGTTCTCTTC-3 0  axin2 forward primer 5 0 -GAGTCTGACGCTTGGACACT-3 0 axin2 reverse primer 5 0 -AGAAACTCGGTGAGTGGCATT-3 0 The ef1α primers and PCR conditions were obtained from [3]. A luciferase assay and RKO (colon cancer cell line) cell culture were performed to determine the efficiency of the drug to activate the Wnt/β-catenin signalling as described previously [4].