Using transcriptomics to enable a plethodontid salamander (Bolitoglossa ramosi) for limb regeneration research

Abstract

Background: Tissue regeneration is widely distributed across the tree of life. Among vertebrates, salamanders possess an exceptional ability to regenerate amputated limbs and other complex structures. Thus far, molecular insights about limb regeneration have come from a relatively limited number of species from two closely related salamander families. To gain a broader perspective on the molecular basis of limb regeneration and enhance the molecular toolkit of an emerging plethodontid salamander (Bolitoglossa ramosi), we used RNA-Seq to generate a de novo reference transcriptome and identify differentially expressed genes during limb regeneration.

Results: Using paired-end Illumina sequencing technology and Trinity assembly, a total of 433,809 transcripts were recovered and we obtained functional annotation for 142,926 non-redundant transcripts of the B. ramosi de novo reference transcriptome. Among the annotated transcripts, 602 genes were identified as differentially expressed during limb regeneration. This list was further processed to identify a core set of genes that exhibit conserved expression changes between B. ramosi and the Mexican axolotl (Ambystoma mexicanum), and presumably their common ancestor from approximately 180 million years ago.

Conclusions: We identified genes from B. ramosi that are differentially expressed during limb regeneration, including multiple conserved protein-coding genes and possible putative species-specific genes. Comparative analyses reveal a subset of genes that show similar patterns of expression with ambystomatid species, which highlights the importance of developing comparative gene expression data for studies of limb regeneration among salamanders.

Keywords: Axolotl; Bolitoglossa; Limb; Plethodontid; Regeneration; Transcriptomics; Urodele.

Fig. 1

Bolitoglossa ramosi. a Wild adult salamander of B. ramosi (7–10 cm snout to tail) where the forelimbs had completed 28 weeks of regeneration. b Tissues used during this analysis. The control limb was the intact limb of the animal; the white line indicates the point of the amputation. The regenerative tissues were the blastema of 20 days post-amputation (dpa), blastema of 40 dpa, blastema 60 dpa, and early palette of 70 dpa

Fig. 2

Flowchart of strategies used to annotate the reference trancriptome of Bolitoglossa ramosi. Different strategies were used to identify homologous genes from different vertebrate and salamander databases. The objective of these analyses was to obtain a gene list of differential expressed genes (DEG) during limb regeneration that could be compared to DEG reported for Ambystoma mexicanum [26]

Fig. 3

Heatmap of differentially expressed genes (DEGs) during limb regeneration in Bolitoglossa ramosi (Caudata: Plethodontidae). a DEGs with a ≥ 2-fold expression change and good transcriptional support (TPM ≥ 0.95) were considered (n = 602). Each column of the heatmap indicated the 2-fold changes of each sample respect the control limb, dpa: days post-amputation. Two clusters were identified that largely consisted of up-regulated (red clusters, n = 310) and down-regulated genes (green cluster, n = 292). b A group of genes exhibiting down-regulation at 20 dpa but up-regulation from 40 to 70 dpa (orange square in a). c A group of genes markedly up-regulated at 40 dpa (yellow Square in a). d ncRNAs identified as DEG, with the heatmap showing RFAM or miRBase ID

Fig. 4

Conservation of gene expression between Bolitglossa ramosi (Caudata: Plethodontidae) and Ambystoma mexicanum (Caudata: Ambystomatidae). A total of 273 significant genes were expressed similarly between the species. Representative expression profiles are shown for nine of the most highly correlated genes: Tnc, Areg, Sall4, Col6a1, Col6a2, Col6a3, Aurka, Basp1, Bub1. The log₂ expression values were derived from the expected counts as calculated by RSEM. Values on the X axis reflect time points (post-amputation) evaluated in each study

Fig. 5

RT-qPCR validation of differentially expressed genes in Bolitoglossa ramosi (Plethodontidae). Eight genes were evaluated by RT-qPCR to validate the DEG analysis in silico at 40 days post amputation (dpa) and 60 dpa against the control limb (D0). Bars represent mean ± SD of three independent measurements. The sample means (Control vs 40dpa and Control vs 60dpa) differed significantly (*) under a t-test and 0.05 p-value threshold