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

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/).

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

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.

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

Sexual development of the urodele amphibian Pleurodeles waltl.

Pleurodeles waltl is a urodele amphibian that displays a ZZ/ZW genetic mode of sex determination involving a putative W-borne dominant determinant. This determining pathway can be environmentally inhibited since heat treated ZW larvae undergo a functional female to male sex reversal. Moreover, both genetic sexes can be reversed by treatment of larvae with steroid hormones suggesting they are the major players in the differentiation process. Indeed we demonstrated that i) aromatase expression and activity increase just before ovarian differentiation, ii) aromatase inhibitors induce a female to male sex reversal, iii) estrogens induce male to female sex reversal whereas the opposite is obtained with non-aromatizable androgens, iv) steroidogenic factor 1 and estrogen receptor alpha both display a female-enriched expression following the increase in aromatase activity. The role of endogenous hormones was investigated in a parabiosis model. Surprisingly, in ZW/ZZ associations, the ZW gonad could not differentiate suggesting that the ZZ parabiont produces an inhibiting factor, prior to ovarian differentiation. The role of AMH in this process is discussed, keeping in mind that Mullerian ducts are maintained in males. The development of antibodies and new molecular tools in the near future should help us to better understand the sexual development of this vertebrate.

Comparative evaluation of the toxicity and genotoxicity of cadmium in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test.

The toxic and genotoxic potential of Cadmium (CdCl(2)) were evaluated by the micronucleus test (MNT) and comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that Cd is toxic to Xenopus larvae exposed from 2 to 50 mg/L and to Pleurodeles from 5 to 50 mg/L, depending on the nature of the water (reconstituted water containing mineral salts or mineral water MW (Volvic)). The MNT results obtained in MW showed that Cd (2 mg/L) is genotoxic to Xenopus, whereas it was not genotoxic to Pleurodeles at all concentrations tested. The CA established that the genotoxicity of Cd to Xenopus and Pleurodeles larvae depends on the concentration, the exposure times, and the comet parameters (Tail DNA, ETM, OTM, and TL). The CA and MNT results were compared for their ability to detect genotoxic effects, considering the concentrations of Cd applied and the exposure time. The CA showed Cd to be genotoxic from the first day of exposure. In amphibians, the CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by Cd.

In vitro effect of metal ions on the activity of two amphibian glyceraldehyde-3-phosphate dehydrogenases: potential metal binding sites.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified from two amphibian species, Xenopus laevis and Pleurodeles waltl. Comparative studies revealed that the two proteins differ by their subunit molecular masses, pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes. X. laevis and P. waltl GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions. Different half-lives and differential sensitivity to various metals was observed between the two enzymes with P. waltl GAPDH being remarkably tolerant to cadmium ions compared to the X. laevis enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both X. laevis and P. waltl GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length P. waltl GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the X. laevis or the P. waltl GAPDH sequences.

NUDC expression during amphibian development.

To identify gene products important for gastrulation in the amphibian Pleurodeles waltl, a screen for regional differences in new protein expression at the early gastrula stage was performed. A 45 kDa protein whose synthesis was specific for progenitor endodermal cells was identified. Microsequencing and cDNA cloning showed that P45 is highly homologous to rat NUDC, a protein suggested to play a role in nuclear migration. Although PNUDC can be detected in all regions of the embryo, its de novo synthesis is tightly regulated spatially and temporally throughout oogenesis and embryonic development. New PNUDC synthesis in the progenitor endodermal cells depends on induction by the mesodermal cells in the gastrula. During development, PNUDC is localized in the egg cortical cytoplasm, at the cleavage furrow during the first embryonic division, around the nuclei and cortical regions of bottle cells in the gastrula, and at the basal region of polarized tissues in the developing embryo. These results show for the first time the expression and compartmentalization of PNUDC at distinct stages during amphibian development.

Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl.

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop-associated proteins (RNA/protein interactions). North-western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.

The genotoxicity of iron and chromium in electroplating effluents.

Electroplating effluents were tested for their genotoxicity with the micronucleus test on newt larvae. The metallic content of the tested samples was responsible for the induction of micronuclei in red blood cells (RBC). Then, iron (Fe3+), chromium (Cr3+, Cr6+) and zinc (Zn2+) which were identified in these samples, were tested either separately or combined, at their concentrations in the electroplating effluents. Fe3+ induced a high level of micronuclei at 12.5 and 25 mg/l (nominal concentrations). Both soluble and non-soluble forms of iron were responsible for these genotoxic effects. At lower concentrations (0.6 and 4.5 mg/l) Fe3+ was not systematically genotoxic. Zinc could not be considered genotoxic on newt. Cr3+ gave negative responses, but exposure to Cr6+ (1 mg/l) could result in a significant number of micronucleated RBC in some cases. The most dramatic genotoxic effects were registered when Fe3+ and Cr6+ were combined. This study demonstrates that interactions between pollutants and the effects of non-soluble chemicals on aquatic vertebrates and invertebrates can no longer be neglected.

A new approach of urodele amphibian limb regeneration: study of myosin isoforms and their control by thyroid hormone.

In P. waltlii, an urodele amphibian species which undergoes spontaneous metamorphosis, study of native myosin in pyrophosphate gels at various stages of normal development demonstrates a complete larval to fast myosin isoforms transition, which occurs more precociously in forelimb muscles than in the dorsal and ventral muscles. In the neotenic species A. mexicanum, forelimb muscles development also presents a complete myosin isoforms transition which is in contrast with the partial myosin isoforms transition observed in the dorsal muscle. In metamorphosed or neotenic animals of both species aged 1 year, forelimb regeneration is characterized by a complete transition from larval to fast myosin isoforms, that occurs earlier and more rapidly than in normal forelimb development. When forelimb regeneration is studied in P. waltlii aged 4 years, the adult fast and slow isomyosins are expressed very early in the regeneration process. In experimental hypothyroidian P. waltlii, the larval to fast isoforms transition in regenerating forelimb muscles is slightly delayed. Experimental hyperthyroidism accelerates the disappearance of larval isomyosins in regenerating forelimb muscles, both in P. waltlii and A. mexicanum aged 1 year. This work demonstrates that changes in myosin isoform pattern during forelimb regeneration in adult urodele amphibians are different from changes occurring in the normal forelimb development. They take place without any thyroid hormone influence, as opposed to normal development, and appear to be age-dependent.

Isolation and characterization of a cDNA clone encoding a Pleurodeles lectin.

A cDNA encoding a lectin secreted by the oviduct of Pleurodeles waltl has been isolated and sequenced. The cDNA was identified by comparing the N-terminal amino-acid sequence of the purified P. waltl lectin polypeptides with the amino-acid sequence deduced from the cDNA. The two chains of the mature protein can be encoded within a unique mRNA. Two mRNA were also found in the oviduct extracts. However, they probably result from differential polyadenylation events. The mRNA are strictly localized in the anterior part of the oviduct and increase after estradiol stimulation, two characteristics which have been previously demonstrated for the protein. P. waltl is known to possess a very high DNA content (approximately 2 x 10(10) bp) but the aforementioned results and Southern-blot experiments suggest a unique or at least a very low gene-copy number for this protein. The amino-acid sequence of the P. waltl lectin deduced from the cDNA sequence shows similarities with the C-type carbohydrate-recognition domains of animal lectins as defined by Drickamer [Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560]. Although it is regulated by estradiol, the P. waltl lectin amino-acid sequence shows a higher similarity with animal lectins involved in the defence of the organism than with those involved in reproduction and development.

Cloning and sequencing of mRNAs coding for two adult alpha globin chains of the salamander Pleurodeles waltlii.

A cDNA library of erythrocyte mRNAs was established from immature red blood cells of the adult amphibian, Pleurodeles waltlii (urodele; salamander). The cDNA clones corresponding to the four adult globin chains were first identified and characterized by positive selection and the cDNAs derived from the two (major and minor) alpha-globin chains sequenced. The sequences presented contain both the complete 3'-noncoding region and the coding region of both chains, with the exception of the first nine codons of the minor alpha-chain, and a portion of the 5'-noncoding region of the major chain. The amino acid sequences of the encoded alpha-globin polypeptides have been deduced and compared with those of Xenopus laevis and of man. These comparative studies suggest that the alpha-globins of Pleurodeles waltlii and Xenopus laevis may have diverged from a common ancestral gene at the time when mammalian and amphibian lines diverged, and that they then evolved separately. Duplication of the alpha-gene, which is responsible for the polypeptide heterogeneity, appears to have occurred earlier in Pleurodeles waltlii than in Xenopus laevis.

Comparative plasma levels of androgens and 17 beta-estradiol in the diploid and triploid newt, Pleurodeles waltl.

Seasonal changes in the plasma levels of androgens (testosterone plus dihydrotestosterone) and 17 beta-estradiol in diploid and triploid adult newts, Pleurodeles waltl were studied. In both male and female diploid individuals, large variations were reported with highest levels being found during breeding periods. In triploid newts seasonal variations were also found, similar to the diploid ones, but the plasma concentrations of the 17 beta-estradiol and androgens in triploid females and androgens in triploid males were lower throughout the year than those reported for the diploids. This difference is discussed in relation to the genetic sexual constitution.