Network based transcription factor analysis of regenerating axolotl limbs

Deepali Jhamb,Fengyu Song,David L Stocum,Mathew J Palakal,Derek J Milner,Nandini Rao,Jo Ann Cameron

doi:10.1186/1471-2105-12-80

Abstract

BackgroundStudies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs.ResultsWe used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-β1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1.ConclusionsOur study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration.

Highlights

Studies on amphibian limb regeneration began in the early 1700’s but we still do not completely understand the cellular and molecular events of this unique process
We focus on the networks and pathways regulated by the transcription factors (TFs) c-Myc and SP1, which we found to be connected to 36.2% of the proteins expressed during axolotl limb regeneration blastema formation
The 82 proteins common to all time points were enriched in biological processes related to general development, cell structure and motility, muscle carbohydrate metabolism, cell cycle, and mRNA splicing

Summary

Introduction

Studies on amphibian limb regeneration began in the early 1700’s but we still do not completely understand the cellular and molecular events of this unique process. We followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs. Beyond this is another goal: the chemical induction of regeneration directly at the site of tissue damage [2]. Achievement of this goal will require a deep understanding of the molecular components, networks and pathways that characterize regenerative competence. Urodele amphibians (axolotls, salamanders and newts), which regenerate amputated limbs perfectly throughout larval and adult life, provide a research model that lends itself well to furthering our understanding of this process. Within a few days after amputation, these cells accumulate under the apical epidermal cap (AEC), where they proliferate and are patterned into the missing limb parts

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