Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

Jeffrey Lehrberg,David M Gardiner

doi:10.1371/journal.pone.0123186

Abstract

We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

Highlights

Research on regenerating body parts has focused on identifying the signaling pathways involved in initiating and regulating this fascinating and biologically important process
Experiments using salamander tissues typically employed the technique of whole explant cultures, either using the developing limb bud or regenerating limb blastema [4,24,32,43,59,60,61]
Increased cell death, lack of reproducibility owing to the variability of sample source and preparation, and lack of access to the interior mesenchymal tissues for experimental manipulation when blastemas are cultured with the epithelium present [62]

Summary

Introduction

Research on regenerating body parts has focused on identifying the signaling pathways involved in initiating and regulating this fascinating and biologically important process. The gain-of-function assay for limb regeneration (the Accessory Limb Model, ALM) has identified a number of signals and pathways that are necessary and sufficient for induction of blastema formation and subsequent regeneration of an ectopic limb [1,2,3]. These experimental approaches have provided insights into the mechanisms of regeneration; their utility has been limited by the fact that they all involve regeneration in vivo. It is not possible to control the spatial and temporal activation or PLOS ONE | DOI:10.1371/journal.pone.0123186 April 29, 2015

Methods

Results

Conclusion