A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology

Abstract

Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).

Keywords: Iberian ribbed newt; NGS; model organism; transcriptome.

Figure 1

Organs and embryos used for RNA preparation. Panel (A) provides a picture of a whole adult female. (B–N) Examples of organs, tissues, and embryos used for RNA extraction. dpa, days post amputation. Scale bars: 1 mm.

Figure 2

Venn diagram of shared and unique orthogroups in five vertebrates. Orthogroups were identified by clustering of orthologous groups using OrthoFinder.

Figure 3

MDS plot for RNA-Seq gene expression of P. waltl tissues, organs, and embryogenesis samples. Multi-dimensional scaling (MDS) plot showing relatedness between transcript expression profiles of organs, tissues, and embryos of P. waltl at different developmental stages. Red dots represent the expression profiles of adult tissues/organs and pink dots represent those of juveniles (3 or 7 months). The labels indicating the tissues and sources are defined in Table 1. Yellow dots represent the expression profiles in samples undergoing regeneration after amputation, where the labels Lb0, Lb3, and Lb19 indicate limb or limb blastema expression profiles at 0, 3, and 19 dpa, respectively; HtR and HtN indicate expression profiles of the hearts regenerating after amputation and in unamputated controls, respectively.

Figure 4

Gene co-expression analysis of the P. waltl transcriptome. Hierarchical cluster tree of the P. waltl genes showing co-expression modules identified using WGCNA. Modules correspond to branches are labelled by colours as indicated by the colour band underneath the tree.

Figure 5

Co-expression gene modules. The co-expression gene modules identified using WGCNA are shown. Each grey dot represents the value of the respective module’s Eigengene. The number at the top left in each panel indicates the number of genes belonging to that module that exhibit unique expression. The modules are classified into four categories based on the expression pattern: modules associated with (A) specific tissues/organs, (B) embryogenesis, (C) regeneration processes, and (D) others. The sample abbreviations indicated by labels at the bottom of each panel are defined in Fig. 3 legend.

Figure 6

Phylogenetic tree of bmp2/4/16 genes among vertebrates. This phylogenetic tree was reconstructed using 34 vertebrate orthologs, including 12 bmp4, 16 bmp2, and 7 bmp16 genes; 2 ascidian bmp2/4 genes were used as the outgroup. The number at each node represents the bootstrap probability.

Figure 7

Expression profile of hox genes during oogenesis and embryogenesis. A total of 37 hox genes are listed from the assembly data of PLEWA04. The sample abbreviations indicated by the labels at the top of each panel are defined in Table 1. Ovaries were sampled at three and seven months after metamorphosis (Ov3 and Ov7, respectively). Note that P. waltl hoxc1, and hoxd12 orthologs were not identified from our transcriptome data. A hoxb13 ortholog was not confidently identified; the candidate contig (M1173232_PLEWA04) showed low homology compared with other vertebrates (Supplementary Data S5 and S6). Most of the hox genes were zygotically activated; only the hoxd1 mRNA was synthesized during oogenesis and stored at the one-cell stage. RPKM values of each gene are indicated as a colour gradient on a log₂ scale, ranging from red (maximum) to white (minimum).

Figure 8

Expression profile of regenerating limb-enriched genes. (A) Expression (during regeneration) of transcription factor-encoding genes involved in limb development. The hoxa13, hoxc13, hoxd13, msx1 and 2, prrx1 and 2, tbx5, and hand2 genes were significantly up-regulated in the forelimb at 19 dpa. RPKM values of each gene were determined from the assembly data of PLEWA04. (B) Details of co-expressed genes in regenerating limb at 19 dpa. A total of 274 genes in this WGCNA module (indicated by lightcyan symbol in Fig. 5) were identified. Notably, genes encoding proteins of the large and small ribosomal subunits accounted for 25% (69 out of 274) of the genes in this module.