Fgf10 mutant newts regenerate normal hindlimbs despite severe developmental defects.

In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

Comparison of hypoxia- and hyperoxia-induced alteration of epigene expression pattern in lungs of Pleurodeles waltl and Mus musculus.

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.

CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.

Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.

Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl.

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the "agarose-embedded heat shock (AeHS) method" involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.

Cryo-injury procedure-induced cardiac regeneration shows unique gene expression profiles in the newt Pleurodeles waltl.

BACKGROUND: Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo-injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo-injury procedures are common in mouse and zebrafish. RESULTS: In the present study, we induced cryo-damage on the ventricle in Iberian ribbed newts using a liquid nitrogen-chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo-injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures. CONCLUSIONS: Notably, we established a cryo-injury procedure for the newt and confirmed that regeneration of the cryo-damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo-injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models.

Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl.

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.

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/).

Cas9 ribonucleoprotein complex allows direct and rapid analysis of coding and noncoding regions of target genes in Pleurodeles waltl development and regeneration.

Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl, a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase, pax6, tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders.

A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte.

Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis. We demonstrate that SOX9 is associated with ribonucleoprotein (RNP) transcripts of lampbrush chromosomes in an RNA-dependent manner. This association can be visualized by Super-resolution Structured Illumination Microscopy (SIM). Our results suggest that SOX9, known to bind DNA, also carries an additional function in the posttranscriptional processes. We also discuss the significance of the acquisition or loss of Sox9 expression in the oocyte during evolution at the transition between anamniotes and amniotes.

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.

Gene manipulation for regenerative studies using the Iberian ribbed newt, Pleurodeles waltl.

Newts provide a unique model animal for regenerative studies. An experimental model system for molecular genetics in newts is needed in order to clarify the mechanisms of regeneration from the perspective of gene functions. We have identified that Iberian ribbed newt (Pleurodeles waltl) is a suitable model animal for such studies. Here we describe protocols for gene manipulation using Pleurodeles waltl.

Molecular cloning and expression analysis of Pleurodeles waltl complement component C3 under normal physiological conditions and environmental stresses.

C3 is a component of the complement system that plays a central role in immunity, development and tissue regeneration. In this study, we isolated the C3 cDNA of the Iberian ribbed newt Pleurodeles waltl. This cDNA encodes a 1637 amino acid protein with an estimated molecular mass of 212.5 kDa. The deduced amino acid sequence showed that P. waltl C3 contains all the conserved domains known to be critical for C3 function. Quantitative real-time PCR (qRT-PCR) demonstrated that under normal physiological conditions, P. waltl C3 mRNA is expressed early during development because it is likely required for neurulation. Then, its expression increased as the immune system developed. In adults, the liver is the richest source of C3, though other tissues can also contribute. Further analysis of C3 expression demonstrated that C3 transcription increased when P. waltl larvae were exposed to pH or temperature stress, suggesting that environmental modifications might affect this animal's defenses against pathogens.

Modulation of Pleurodeles waltl DNA polymerase mu expression by extreme conditions encountered during spaceflight.

DNA polymerase µ is involved in DNA repair, V(D)J recombination and likely somatic hypermutation of immunoglobulin genes. Our previous studies demonstrated that spaceflight conditions affect immunoglobulin gene expression and somatic hypermutation frequency. Consequently, we questioned whether Polµ expression could also be affected. To address this question, we characterized Polµ of the Iberian ribbed newt Pleurodeles waltl and exposed embryos of that species to spaceflight conditions or to environmental modifications corresponding to those encountered in the International Space Station. We noted a robust expression of Polµ mRNA during early ontogenesis and in the testis, suggesting that Polµ is involved in genomic stability. Full-length Polµ transcripts are 8-9 times more abundant in P. waltl than in humans and mice, thereby providing an explanation for the somatic hypermutation predilection of G and C bases in amphibians. Polµ transcription decreases after 10 days of development in space and radiation seem primarily involved in this down-regulation. However, space radiation, alone or in combination with a perturbation of the circadian rhythm, did not affect Polµ protein levels and did not induce protein oxidation, showing the limited impact of radiation encountered during a 10-day stay in the International Space Station.

Molecular genetic system for regenerative studies using newts.

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture.

Subterminal lampbrush loops of one of the 12 bivalents of the oocyte karyotype of Pleurodeles waltl (Amphibian, Urodele) underwent prominent morphological changes upon in vitro culture. These loops exhibited a fine ribonucleoprotein (RNP) granular matrix, which evolved during culture into huge structures that we have named 'chaussons' (slippers). This phenomenon involved progressive accumulation of proteins in the RNP matrix without protein neosynthesis. One of these proteins, which translocated into the nucleus during the culture, was identified as a homolog of the human Ro52 E3 ubiquitin ligase. RNA polymerase III was also found to accumulate on the same loops. These results suggest that the subterminal loops of bivalent XII act as a storage site for the components of a nuclear machinery involved in the quality control of RNA synthesis and maturation in response to cellular stress. They also emphasise the considerable value of the lampbrush chromosome system for a direct visualisation of modifications in gene expression and open the question of a nuclear accumulation of Ro52 in human or animal oocytes cultured in vitro for assisted reproductive technologies (ART).

Amphibian interorder nuclear transfer embryos reveal conserved embryonic gene transcription, but deficient DNA replication or chromosome segregation.

Early interspecies nuclear transfer (iNT) experiments suggested that a foreign nucleus may become permanently damaged after a few rounds of cell division in the cytoplasm of another species. That is, in some distant species combinations, nucleocytoplasmic hybrid (cybrid) blastula nuclei can no longer support development, even if they are back-transferred into their own kind of egg cytoplasm. We monitored foreign DNA amplification and RNA production by quantitative PCR (qPCR) and RT-qPCR in interorder amphibian hybrids and cybrids formed by the transfer of newt (Pleurodeles waltl) embryonic nuclei into intact and enucleated frog (Xenopus laevis) eggs. We found a dramatic reduction in the expansion of foreign DNA and cell numbers in developing cybrid embryos that correlated with reduced gene transcription. Interestingly, expansion in cell numbers was rescued by the recipient species (Xenopus) maternal genome in iNT hybrids, but it did not improve P. waltl DNA expansion or gene transcription. Also, foreign gene transcripts, normalized to DNA copy numbers, were mostly normal in both iNT hybrids and cybrids. Thus, incomplete foreign DNA replication and/or chromosome segregation during cell division may be the major form of nuclear damage occurring as a result of nuclear replication in a foreign cytoplasmic environment. It also shows that the mechanisms of embryonic gene transcription are highly conserved across amphibians and may not be a major cause of cybrid lethality.

Decrease in antibody somatic hypermutation frequency under extreme, extended spaceflight conditions.

Somatic hypermutation diversifies antibody binding sites by introducing point mutations in the variable domains of rearranged immunoglobulin genes. In this study, we analyzed somatic hypermutation in variable heavy-chain (VH) domains of specific IgM antibodies of the urodele amphibian Pleurodeles waltl, immunized either on Earth or onboard the Mir space station. To detect somatic hypermutation, we aligned the variable domains of IgM heavy-chain transcripts with the corresponding VH gene. We also quantified NF-κB and activation-induced cytidine deaminase transcripts. Results were compared with those obtained using control animals immunized on Earth. Our data show that, as in most species of ectotherms, somatic hypermutation in P. waltl exhibits a mutational bias toward G and C bases. Furthermore, we show for the first time that somatic hypermutation occurs in space following immunization but at a lower frequency. This decrease is not due to a decrease in food intake or of the B-cell receptor/antigen interaction or to the absence of the germinal center-associated nuclear protein. It likely results from the combination of several spaceflight-associated changes, such as the severe reduction in T-cell activation, important perturbations of the cytoskeleton, and changes in the distribution of lymphocyte subpopulations and adhesion molecule expression.

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.

Molecular characterization of Pleurodeles waltl activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is involved in immunoglobulin affinity maturation, gene conversion and class switch recombination. This protein is therefore a major actor in the creation of the antibody repertoire. We have isolated, for the first time, the AID mRNA from a urodele amphibian, Pleurodeles waltl. This mRNA encodes 198 amino acids and shares 70% and 76% of similarity with Xenopus laevis and human AID sequences, respectively. All consensus motifs necessary for AID functions are present, suggesting that AID is functional in P. waltl. P. waltl AID is encoded by five exons as in other species. However, in contrast to mammalian AID, no splice variant could be detected in that species. We also noted that AID is predominantly expressed in the spleen, the major secondary lymphoid organ of P. waltl, and that the transcriptional regulation of P. waltl AID is partially different from that found in higher vertebrates. Furthermore, we showed that AID is expressed early during P. waltl embryonic development.