Comparative RNA-seq analysis in the unsequenced axolotl: the oncogene burst highlights early gene expression in the blastema.

Ron Stewart,Srikumar Sengupta,Michael Volkmer,Colin N Dewey,Mitchell D Probasco,Jeff Nie,Shulan Tian,Elly M Tanaka,Hamisha Ardalani,Bianca Habermann,Li-Fang Chu,James A Thomson,Jennifer M Bolin,Ning Leng,Ryan J Wagner,Chris Barry,Cynthia Alexander Rascón

doi:10.1371/journal.pcbi.1002936

Abstract

The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.

Highlights

The salamander’s capability to regenerate various body parts, including limbs, has been the focus of study for almost 200 years [1]
Quantification of transcript abundances with respect to the human gene set Full details of our comparative RNA sequencing (RNA-seq) analysis approach are provided in Materials and Methods
These contigs were matched to human transcripts to allow for differential expression and functional analyses using the human gene set

Summary

Introduction

The salamander’s capability to regenerate various body parts, including limbs, has been the focus of study for almost 200 years [1]. A study focusing on the nerve dependence of limb generation utilized microarrays and 454 sequencing to identify genes expressed in the blastema with and without the presence of nerves [3]. A more recent study utilized a microarray with 20,000 probe sets to identify genes expressed during the regenerative process [11]. There are limitations to this approach, as microarrays can only identify the expression of genes for which probes are designed, and the shallow depth of 454 sequencing in the first study limits the ability to detect low abundance transcripts. Another study in Xenopus identified several genes likely to be important to hind limb regeneration [12], and a proteomic analysis of the blastema has been published [2]. In addition to the above axolotl studies, Expressed Sequence Tag (EST) and other sequencing efforts have made inroads in defining the transcriptome of the newt [14,15,16]

Methods

Results

Discussion

Conclusion