Comparative transcriptomics of limb regeneration: Identification of conserved expression changes among three species of Ambystoma

Abstract

Transcriptome studies are revealing the complex gene expression basis of limb regeneration in the primary salamander model - Ambystoma mexicanum (axolotl). To better understand this complexity, there is need to extend analyses to additional salamander species. Using microarray and RNA-Seq, we performed a comparative transcriptomic study using A. mexicanum and two other ambystomatid salamanders: A. andersoni, and A. maculatum. Salamanders were administered forelimb amputations and RNA was isolated and analyzed to identify 405 non-redundant genes that were commonly, differentially expressed 24 h post amputation. Many of the upregulated genes are predicted to function in wound healing and developmental processes, while many of the downregulated genes are typically expressed in muscle. The conserved transcriptional changes identified in this study provide a high-confidence dataset for identifying factors that simultaneous orchestrate wound healing and regeneration processes in response to injury, and more generally for identifying genes that are essential for salamander limb regeneration.

Keywords: Ambystoma; Limb regeneration; Mexican axolotl; Transcriptomics.

Fig. 1.

(A) Phylogenetic relationships among A. mexicanum, A. andersoni, and A. maculatum. Numbers at nodes of the topology are estimated divergence times in millions of years. (B) The analytical pipeline used to identify 1) 2360 A. mexicanum transcripts that were identified as differentially expressed by microarray analysis and RNA-Seq, and 2) 474 genes that were commonly, differentially expressed among all three species.

Fig. 2.

Comparison of log2 fold change values of 2360 commonly, differentially expressed transcripts identified by microarray analysis and RNA-Seq. Red dots represent upregulated transcripts and blue dots represent downregulated transcripts. Inset plots show highly, differentially expressed transcripts (log2FC > 4 or log2FC < −2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

Pairwise comparison of RNA-Seq log2 fold change of A. andersoni and A. maculatum, to A. mexicanum. Transcripts exhibiting high DE between species are shown.

Fig. 4.

Expression difference between muscle-specific cox7a1 and the more ubiquitously expressed cox4a1. Data are from Voss et al. [27].

Fig. 5.

Differentially expressed metabolic (orange background) and muscle genes (green background) among three ambystomatid salamander species. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)