Dynamic expression of two thrombospondins during axolotl limb regeneration

Abstract

The molecular processes underlying regeneration remain largely unknown. Several potential factors have been elucidated by focusing on the regenerative function of genes originally identified in a developmental context. A complementary approach is to consider the roles of factors involved in wound healing. Here we focus on the Thrombospondins, a family of secreted extracellular matrix proteins that have been implicated in skin wound healing in mammals. We show that a subset of Thrombospondins are expressed at distinct times and in particular cell types during axolotl limb regeneration. Our studies have revealed the axolotl orthologs of thrombospondin-1 (tsp-1) and thrombospondin-4 (tsp-4) are highly upregulated during limb regeneration in patterns both distinct and similar to larval limb development. Our data suggest that thrombospondins may be key regulators of limb regeneration in axolotl, while their activation appears to be relegated solely to wound healing in vertebrates that have lost the ability to regenerate limbs.

A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors

“…Depending upon orientation, T7 or Sp6 polymerase was used to transcribe the probe. In situ hybridization was performed as in (Whited et al, 2011). …”

Inducible genetic system for the axolotl

“…This expression intensifies as the blastema cells proliferate over the next 2 wk. However, once differentiation commences within the regenerate, tsp-4 expression becomes restricted to the perichondrium around the developing cartilage, consistent with, for instance, a role in the branching of the skeletal elements (32). We cloned the axolotl tsp-4 ORF downstream of our CA mini -O1O2O3 promoter/ operator sequence (termed pO-Tsp4) and coinjected newly fertilized axolotl eggs with pO-Tsp4 and pCAG-LacI to generate mosaic F 0 cotransgenic axolotls.…”

“…Previously, we had found that two genes encoding axolotl thrombospondins, thrombospondin-1 (tsp-1) and thrombospondin-4 (tsp-4), displayed dynamic expression patterns in discrete cell types during the course of limb regeneration (32). Although thrombospondins have been implicated in numerous aspects of mammalian biology (33), including some that may be pertinent to regeneration such as wound healing and angiogenesis, nothing is known about their potential role in appendage regeneration.…”

Pseudotyped retroviruses for infecting axolotl in vivo and in vitro

“…Limbs were processed as described previously (Whited et al, 2011). Cryosections were rehydrated in PBS, blocked in PBS/2% BSA/0.1% Triton for 30 minutes, incubated in primary antibody (chicken anti-GFP, 1:1000, Abcam), washed three times in PBS for 5 minutes and incubated in secondary antibody (donkey anti-chick or goat anti-chick, AlexaFluor 488-conjugated, 1:250, Jackson Labs).…”

“…Animals were bred in-house or obtained from the Ambystoma Genetic Stock Center (AGSC) (Lexington, KY) and were between 6 and 12 cm long and size-matched within experiments, except where noted. Amputations were performed as described previously (Whited et al, 2011).…”