Ribbed Newt Pleurodeles Waltl Research Articles

Photoperiod-independent testicular development in the model newt Pleurodeles waltl.

Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P.waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P.waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P.waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P.waltl proceeds irrespective of day length. Our results show that P.waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P.waltl bioresources.

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 Pleurodeleswaltl 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.

Effective to census population size ratios in two Near Threatened Mediterranean amphibians: Pleurodeles waltl and Pelobates cultripes

Efforts to mitigate amphibian declines are hindered by a lack of information about basic aspects of their biology and demography. The effective to census population size ratio (Ne/Nc) is one of the most important parameters for the management of wildlife populations because it combines information on population abundance and genetic diversity and helps predict population viability in the long term. Few studies have calculated this ratio in amphibians, which sometimes show low ratios, associated with a higher extinction risk. Here we integrate field-based (capture-mark-recapture studies, egg string counts) and molecular approaches (estimation of the effective number of breeders (Nb) and the effective population size (Ne) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the Ne/Nc and Nb/Nc ratios in two amphibians, the Iberian ribbed newt Pleurodeles waltl and the western spadefoot Pelobates cultripes. Additionally, we investigate sex-biased dispersal in both species based on direct (field observations) and indirect (genetic) evidence. Both species showed similar ratios, slightly lower in Pleurodeles (0.21–0.24) than in Pelobates (0.25–0.30). Observed displacement rates were low in both species (P. waltl = 0.51%; P. cultripes = 1.23%). We found no evidence for sex-biased dispersal in P. cultripes, but both direct and indirect evidences suggest a tendency for female-biased dispersal in P. waltl. We discuss differences in the genetic estimates of Ne and Nb provided by three inference methods and the implications of our findings for the management of these species, characteristic of Mediterranean wetlands in the Iberian Peninsula and listed as Near Threatened.

Male Courtship Pheromones Induce Cloacal Gaping in Female Newts (Salamandridae).

Pheromones are an important component of sexual communication in courting salamanders, but the number of species in which their use has been demonstrated with behavioral evidence remains limited. Here we developed a behavioral assay for demonstrating courtship pheromone use in the aquatically courting Iberian ribbed newt Pleurodeles waltl. By performing an in-depth study of the courtship behavior, we show that females invariably open their cloaca (cloacal gaping) before engaging in pinwheel behavior, the circling movement that is the prelude to spermatophore uptake. In contrast, cloacal gaping was not observed in failed courtships, where females escaped or displayed thanatosis. Since gaping mainly occurred during male amplexus and cloacal imposition, which is the obvious period of pheromone transfer, we next investigated whether male courtship water (i.e., water holding courtship pheromones) alone was able to induce this reaction in females. These tests showed that courtship water induced cloacal gaping significantly more than water, even in the absence of a male. Cloacal gaping thus provides a simple and robust test for demonstrating courtship pheromone use in the Iberian ribbed newt. Since opening the cloaca is an essential prerequisite for spermatophore pick-up in all internally fertilizing salamanders, we hypothesize that variations on this assay will also be useful in several other species.

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 212.5kDa. 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.

Determination of mRNA-transcripts and heat shock proteins HSP70 and HSP90 in the retina of the adult Spanish Ribbed Newt Pleurodeles waltl

Expression of genes and heat shock proteins in normal intact retina of the Spanish Ribbed Newt Pleurodeles waltl was studied using polymerase chain reaction, Western blot hybridization, and immunohistochemistry. It was shown that the proteins HSP70 and HSP90, as well as their encoding transcripts of relevant genes, are constitutively expressed in eye tissues. These proteins were distributed differentially, and they were characterized by expression of different levels in the retina: HSP70 dominated in the external retina, while HSP90 dominated in the internal one, in particular, in Muller glial cells and the optic nerve. Transcripts and heat shock proteins HSP70 and HSP90 were also found in the retinal pigment epithelium and eye growth zone.

Dynamics of body mass and oxygen consumption in the ribbed newt Pleurodeles waltl ontogenesis: 1. Endotrophic development

The correlation between parameters of growth and energy metabolism in the example of embryonic and larval development of the Iberian ribbed newt Pleurodeles waltl has been studied. The wet body mass increases five times during this period due to water absorption by developing tissues and the yolk, and the dry mass decreases 1.18 times. The highest specific growth rate of developing tissues was noticed at the 33rd stage of embryogenesis (13th-14th day of development). These indexes decreased after the hatching, but increased after larvae switched to external nutrition. Comparison of the studied parameters has identified a similar features in alteration of the specific growth rate, intensity of oxygen consumption, and watering of developing tissues in early development of the Iberian ribbed newt.

Hurt yourself to hurt your enemy: new insights on the function of the bizarre antipredator mechanism in the salamandridPleurodeles waltl

The Spanish ribbed newt Pleurodeles waltl shows a bizzare defensive mechanism against predators. X-ray analysis before and after a simulated threat shows that this newt rotates its ribs anteriorly. The maximum measured angle to which the ribs moved was 65°. This forward movement causes the sharply pointed rib tips to lacerate the body wall and project freely from the sides of the trunk as spines. Light microscopy shows the microanatomy, and computed tomography shows the 3D morphology of these unusual weapons. They are ‘spear-shaped’ and hollow proximally, massive distally and are connected to the corresponding vertebra by a well-developed, two-headed joint. The skin in the penetration areas lacks permanent pores through which the ribs could be projected and is pierced de novo by every antipredator posturing. This investigation provides new insight into the functionality of a highly complex, integrated and unusual defensive strategy.

Lifelong testicular differentiation in Pleurodeles waltl (Amphibia, Caudata).

BackgroundIn numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented.MethodsTestes were obtained from Pleurodeles waltl of different ages reared in our laboratory. Testis evolution was studied by several approaches: morphology, histology, immunohistochemistry and RT-PCR. Surgery was also employed to study testis regeneration.ResultsIn this species, the testis is linked to the lung. This association consists of connective tissue derived from the mesorchium and the coelomic epithelium surrounding the lung and takes place at the end of larval life. This tissue contains lobules including primordial germ cells with a typical large and polylobular nucleus. The anterior part of the testis remains thin and undifferentiated while the posterior part differentiates in a large first testis lobe where spermatogenesis occurs during the first year of life. The undifferentiated status of the anterior part is attested by the lack of expression of the testis marker Dmrt1 and the meiosis entry marker Dmc1. Three-year-old Pleurodeles waltl possess multiple testes made up of two lobes. The second lobe appears at the caudal extremity of the first one from residual primordial germ cells located near or even inside efferent ducts in the glandular tissue that usually appears following spermatozoa extrusion. Surprisingly, in the case of surgical elimination of the anterior part of the testis, de novo spermatogenesis is stopped in the first lobe which becomes restricted to the glandular tissue. Following first testis lobe removal, the anterior part of the testis regenerates a new testis lobe, a process stimulated in the presence of DHT.ConclusionPleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood.

IgX antibodies in the urodele amphibian Ambystoma mexicanum

Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.

Oxygen Consumption during Spontaneous Amphibian Metamorphosis

Previous articleNext article No AccessOxygen Consumption during Spontaneous Amphibian MetamorphosisAnne Funkhouser and K. S. MillsAnne Funkhouser Search for more articles by this author and K. S. Mills Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Volume 42, Number 1Jan., 1969 Article DOIhttps://doi.org/10.1086/physzool.42.1.30152461 Views: 5Total views on this site Citations: 12Citations are reported from Crossref Journal History This article was published in Physiological Zoology (1928-1998), which is continued by Physiological and Biochemical Zoology (1999-present). Copyright 1969 The University of ChicagoPDF download Crossref reports the following articles citing this article:Lucas J. Kirschman, Marshall D. McCue, Justin G. Boyles, Robin W. Warne Exogenous stress hormones alter energetic and nutrient costs of development and metamorphosis, Journal of Experimental Biology 220, no.1818 (Sep 2017): 3391–3397.https://doi.org/10.1242/jeb.164830I. G. Vladimirova, S. Yu. Kleimenov, T. A. Alekseeva Dynamics of body mass and oxygen consumption in the ontogeny of the Spanish ribbed newt (Pleurodeles waltl): 2. Larval stage, Biology Bulletin 39, no.11 (Jan 2012): 10–14.https://doi.org/10.1134/S106235901201013XI. G. Vladimirova, S. Yu. Kleimenov, T. A. Alekseeva Dynamics of body mass and oxygen consumption in the ribbed newt Pleurodeles waltl ontogenesis: 1. Endotrophic development, Biology Bulletin 37, no.66 (Dec 2010): 558–564.https://doi.org/10.1134/S1062359010060026Tobias Landberg, Emanuel Azizi Ontogeny of escape swimming performance in the spotted salamander, Functional Ecology 24, no.33 (Nov 2009): 576–587.https://doi.org/10.1111/j.1365-2435.2009.01662.xSarah A. Orlofske, William A. Hopkins Energetics of metamorphic climax in the pickerel frog (Lithobates palustris), Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 154, no.22 (Oct 2009): 191–196.https://doi.org/10.1016/j.cbpa.2009.06.001Carolyn L. Fordham, John D. Tessari, Howard S. Ramsdell, Thomas J. Keefe Effects of malathion on survival, growth, development, and equilibrium posture of bullfrog tadpoles ( Rana catesbeiana ), Environmental Toxicology and Chemistry 20, no.11 (Jan 2001): 179–184.https://doi.org/10.1002/etc.5620200120William C. Crowder, Minghua Nie, Gordon R. Ultsch Oxygen uptake in bullfrog tadpoles (Rana catesbeiana), The Journal of Experimental Zoology 280, no.22 (Feb 1998): 121–134.https://doi.org/10.1002/(SICI)1097-010X(19980201)280:2<121::AID-JEZ3>3.0.CO;2-QGad Degani, Effrat Nevo Osmotic stress and osmoregulation of tadpoles and juveniles of Pelobates syriacus, Comparative Biochemistry and Physiology Part A: Physiology 83, no.22 (Jan 1986): 365–370.https://doi.org/10.1016/0300-9629(86)90591-8Martin E. Feder Effect of developmental stage and body size on oxygen consumption of anuran larvae: A reappraisal, Journal of Experimental Zoology 220, no.11 (Mar 1982): 33–42.https://doi.org/10.1002/jez.1402200105Martin E. Feder Effect of body size, trophic state, time of day, and experimental stress on oxygen consumption of anuran larvae: An experimental assessment and evaluation of the literature, Comparative Biochemistry and Physiology Part A: Physiology 70, no.44 (Jan 1981): 497–508.https://doi.org/10.1016/0300-9629(81)92561-5M.H.I. Dodd, J.M. Dodd THE BIOLOGY OF METAMORPHOSIS, (Jan 1976): 467–599.https://doi.org/10.1016/B978-0-12-455403-0.50015-3 Anne Funkhouser , and K. S. Mills Oxygen Consumption during Induced Amphibian Metamorphosis, Physiological Zoology 42, no.11 (Sep 2015): 22–28.https://doi.org/10.1086/physzool.42.1.30152462