Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration

Abstract

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.

Fig. 1

The Iberian ribbed newt P. waltl is a prime model for adult regeneration. Compared to other research animal models, P. waltl is the most regenerative adult vertebrate amenable to laboratory breeding. Phylogenetic tree from TimeTree. Regeneration capacities based on^{, , –}. Sex chromosomes from refs. ^–. Genome sizes from https://www.ncbi.nlm.nih.gov/genome/ (summed from Size (Mb) column)

Fig. 2

Genomic expansion and the expression of Harbinger DNA transposable elements pluripotency miRNAs and salamander-specific methyltransferases during adult limb regeneration in P. waltl. a Portion of Gypsy and Harbinger repeats (>1 kb) retaining a detectable protein domain. b Phylogeny of Gypsy and Harbinger elements. While the tree-based on Gypsy elements indicates a uniform expansion, the tree-based on Harbinger elements shows three branches (brown) resulting from a second distinct burst. The second Harbinger burst also results in a bimodal pairwise genomic distance distribution, compared to a unimodal distribution for the Gypsy elements. c Phylogenic tree of 361 mature P. waltl miRNAs with miR-427 and miR-93b expansion highlighted (top). Sequence logo based on multiple alignment of 155 miR-427 sequences and 66 miR-93b sequences (bottom). Note the embryonic stem cell-specific cell cycle regulating seed AAGUGC within the first eight nucleotides. d Split violin plot showing the tau score distribution of P. waltl genes that have non-salamander vertebrate orthologs (blue) and genes that only have salamander orthologs (beige). e RNAseq quantification of five Harbinger orthology groups encoding Myb or Harbi domain-bearing proteins (top), four Gypsy orthology groups encoding reverse transcriptase domain-bearing proteins (middle) and mir-427 and mir-93b primary transcripts (bottom). In parentheses are the number of transcripts and gene models belonging to each group. The groups chosen were significantly upregulated in the regenerating limb 3 days post amputation (dpa). Differential expression calculated by DESeq2 (Supplementary Table 9). TPM transcripts per million. f Eleven orthology groups of salamander methyltransferase are expressed specifically in appendages of the adult animal. In parentheses are the number of transcripts and gene models belonging to each group

Fig. 3

Newt limb muscle development and regeneration is independent of Pax7. a Pax7 ^−/− F₁ P. waltl have normal limb skeletal muscle. Note the similar myosin heavy chain (MHC) and laminin (Lam) staining in Pax7 ^−/− and WT animals. Scale bars = 200 µm in the overview and 20 µm in the insert. b Pax3-CRISPR mosaic animals have muscle-less limbs. Immunofluorescent staining for MHC in transverse sections of Pax3-CRISPR and control-CRISPR limbs. Scale bar = 100 µm. c Limb regeneration is not impaired in post-metamorphic Pax7 ^−/− F₁ animals. Scale bar = 1 mm. d Pax7 ^−/− F₁ animals regenerate normal skeletal muscle in the new limb. Note the indistinguishable myosin heavy chain (MHC) and laminin staining in Pax7 ^−/− and WT animals. Scale bar = 100 µm. e muscle-less limbs in Pax3-CRISPR mosaic animals (stage 45) regenerate muscle-less limbs. Immunofluorescent staining for MHC and the neuronal marker βIII-tubulin in transverse sections. Scale bar = 1 mm in the left panels showing animals and 100 µm in the right panels showing sections. Red dotted lines indicate amputation plane. White lines indicate section plane