FGF-stimulated tendon cells embrace a chondrogenic fate with BMP7 in newt tissue culture.

Newts can regenerate functional elbow joints after amputation at the joint level. Previous studies have suggested the potential contribution of cells from residual tendon tissues to joint cartilage regeneration. A serum-free tissue culture system for tendons was established to explore cell dynamics during joint regeneration. Culturing isolated tendons in this system, stimulated by regeneration-related factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor, led to robust cell migration and proliferation. Moreover, cells proliferating in an FGF-rich environment differentiated into Sox9-positive chondrocytes upon BMP7 introduction. These findings suggest that FGF-stimulated cells from tendons may aid in joint cartilage regeneration during functional elbow joint regeneration in newts.

Step-by-step protocol for alternative injury models in newt cardiac regeneration.

Although the heart is one of the most important organs for animal survival, its regenerative capacity varies among animal species. Notably, adult mammals cannot regenerate their hearts after damage such as acute myocardial infarction. In contrast, some vertebrate animals can regenerate the heart throughout their lives. Cross-species comparative studies are important to understand the full picture of cardiac regeneration in vertebrates. Among the animal species able to regenerate the heart, some urodele amphibians, such as newts, possess a remarkable capacity for this process. Standardized methods of inducing cardiac regeneration in the newt are needed as a platform for studies comparing newts and other animal models. The procedures presented here describe amputation and cryo-injury techniques for the induction of cardiac regeneration in Pleurodeles waltl, an emerging model newt species. Both procedures consist of simplified steps that require no special equipment. We additionally show some examples of the regenerative process obtained using these procedures. This protocol has been developed for P. waltl. However, these methods are also expected to be applicable to other newt and salamander species, facilitating comparative research with other model animals.

Motilin is a regulator of gastric contraction in Japanese fire belly newts (Cynops pyrrhogaster), in vitro studies using isolated gastrointestinal strips of newts, rabbits, and chickens.

The effects of newt motilin on the contractility of the isolated gastrointestinal (GI) tract from Japanese fire belly newts (newt) were examined to clarify whether motilin regulates GI motility in urodele amphibians. In addition, contractile responsiveness to motilins from seven species of vertebrates (human, chicken, turtle, alligator, axolotol, newt and zebrafish) were compared in GI preparations from three different animals (rabbit duodenum, chicken ileum and newt stomach) to determine the species-specific action of motilin. Newt motilin (10-10 M - 10-6 M) caused a contraction of cognate gastric strips, while the upper, middle, and lower intestinal strips were insensitive. The rank order of motilins for contractile activity in newt gastric strips was newt > alligator > axolotol > chicken > turtle > human ≫ zebrafish. On the other hand, newt motilin caused a weak contraction in the rabbit duodenum (human > alligator = chicken > turtle > newt ≧ axolotol > zebrafish), and it was ineffective in the chicken ileum (chicken > turtle > alligator > human ≫ newt, axolotol and zebrafish). This study demonstrates that motilin induces contraction in the GI tract of a urodele amphibian, the newt, in a region (stomach)-specific manner and further indicates that a ligand-receptor interaction of the motilin system is a species-specific manner probably due to differences in the amino acid sequence of motilin.

Molecular Signatures Integral to Natural Reprogramming in the Pigment Epithelium Cells after Retinal Detachment in Pleurodeles waltl.

The reprogramming of retinal pigment epithelium (RPE) cells into retinal cells (transdifferentiation) lies in the bases of retinal regeneration in several Urodela. The identification of the key genes involved in this process helps with looking for approaches to the prevention and treatment of RPE-related degenerative diseases of the human retina. The purpose of our study was to examine the transcriptome changes at initial stages of RPE cell reprogramming in adult newt Pleurodeles waltl. RPE was isolated from the eye samples of day 0, 4, and 7 after experimental surgical detachment of the neural retina and was used for a de novo transcriptome assembly through the RNA-Seq method. A total of 1019 transcripts corresponding to the differently expressed genes have been revealed in silico: the 83 increased the expression at an early stage, and 168 increased the expression at a late stage of RPE reprogramming. We have identified up-regulation of classical early response genes, chaperones and co-chaperones, genes involved in the regulation of protein biosynthesis, suppressors of oncogenes, and EMT-related genes. We revealed the growth in the proportion of down-regulated ribosomal and translation-associated genes. Our findings contribute to revealing the molecular mechanism of RPE reprogramming in Urodela.

Development of the smooth newt, Lissotriton vulgaris, as observed in a garden pond during lockdown.

BACKGROUND: Lissotriton vulgaris ("smooth newt") was an important organism for experimental embryology, especially in the early 20th century. The stages of development of L. vulgaris from the appearance of the first cleavage to metamorphosis have previously been described, but only through drawings. RESULTS: Referring to Glaesner's staging and utilizing a makeshift lockdown laboratory, this study documents the development of L. vulgaris embryos in a small garden pond in Surrey, UK. This study describes varying developmental rates between individuals, identifies pigmented cells in close proximity/contributing to the heart, describes early gill development and reports a case of spontaneous, non-conjoined newt twins. CONCLUSIONS: While pigmentation in the heart has been reported in some amphibians, no pigmentation within urodele hearts has been documented to date. Furthermore, although the literature on conjoined twinning in amphibia is extensive, separate, non-conjoined twinning is largely unexplored in the literature. This study invites further research into understanding of these topics.

Model systems for regeneration: salamanders.

Salamanders have been hailed as champions of regeneration, exhibiting a remarkable ability to regrow tissues, organs and even whole body parts, e.g. their limbs. As such, salamanders have provided key insights into the mechanisms by which cells, tissues and organs sense and regenerate missing or damaged parts. In this Primer, we cover the evolutionary context in which salamanders emerged. We outline the varieties of mechanisms deployed during salamander regeneration, and discuss how these mechanisms are currently being explored and how they have advanced our understanding of animal regeneration. We also present arguments about why it is important to study closely related species in regeneration research.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Identification of motilin in Japanese fire bellied newt.

Motilin, a peptide hormone consisting of 22 amino acid residues, was identified in the duodenum of pigs in the 1970s. It is known to induce gastrointestinal contractions during the interdigestive state in mammals. Although the motilin gene has been identified in various animal species, it has not been studied in amphibians. Here, we identified the motilin gene in the Japanese fire bellied newt (Cynops pyrrhogaster), and conducted an analysis of tissue distribution, morphological observations, and physiological experiments. The deduced mature newt motilin comprises 22 amino acid residues, like in mammals and birds. The C-terminus of the newt motilin showed high homology with motilin from other species compared to the N-terminus region, which is considered the bioactive site. Motilin mRNA expression in newts was abundant in the upper small intestine, with notably high motilin mRNA expression found in the pancreas. Motilin-producing cells were found in the mucosal layer of the upper small intestine and existed as two cell types: open-and closed-type cells. Motilin-producing cells in the pancreas were also found to produce insulin but not glucagon. Newt motilin stimulated gastric contractions but not in other parts of the intestines in vitro, and motilin-induced gastric contraction was significantly inhibited by treatment with atropine, a muscarinic acetylcholine receptor antagonist. These results indicate that motilin is also present in amphibians, and that its gastrointestinal contractile effects are conserved in mammals, birds, and amphibians. Additionally, we demonstrated for the first time the existence of pancreatic motilin, suggesting that newt motilin has an additional unknown physiological role.

Identification and expression analysis of Dazl homologue in Cynops cyanurus.

The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

Immunity in salamander regeneration: Where are we standing and where are we headed?

Salamanders exhibit the most extensive regenerative repertoire among vertebrates, being able to accomplish scar-free healing and faithful regeneration of significant parts of the eye, heart, brain, spinal cord, jaws and gills, as well as entire appendages throughout life. The cellular and molecular mechanisms underlying salamander regeneration are currently under extensive examination, with the hope of identifying the key drivers in each context, understanding interspecies differences in regenerative capacity, and harnessing this knowledge in therapeutic settings. The immune system has recently emerged as a potentially critical player in regenerative responses. Components of both innate and adaptive immunity have been found at critical stages of regeneration in a range of salamander tissues. Moreover, functional studies have identified a requirement for macrophages during heart and limb regeneration. However, our knowledge of salamander immunity remains scarce, and a thorough definition of the precise roles played by its members is lacking. Here, we examine the evidence supporting roles for immunity in various salamander regeneration models. We pinpoint observations that need revisiting through modern genetic approaches, uncover knowledge gaps, and highlight insights from various model organisms that could guide future explorations toward an understanding of the functions of immunity in regeneration.

Regeneration and development. An amphibian call to arms.

BACKGROUND: Unlike axolotls, the urodele Notophthalmus viridescens completes two metamorphoses and emerges from its aquatic environment to mate as a fully terrestrial adult. Larval and adult limb regeneration are commonly treated as roughly equivalent processes and, at least in part, as a recapitulation of embryonic development. RESULTS: We compared larval limb development to regeneration of both larval and adult forelimbs and found that there are substantial differences in developmental pattern among larvae and adults. The larval pattern of preaxial dominance is absent in adult regenerates: adult regenerates instead develop digits synchronously, and they do so before proximal autopodial elements have formed discrete aggregation zones. By contrast, larval regenerates follow a pattern of sequential digit formation from anterior to posterior, like their embryonic limb buds. CONCLUSIONS: Based upon these morphological clues, we conclude that larval regenerates are unlikely to exhibit features of epimorphic regeneration seen in adults, but are more likely to represent a form of developmental regulation. Furthermore, we confirm that post-metamorphic limb regeneration is not a simple recapitulation of ontology at the morphological level. These distinctions may help to explain and interpret some experiments and observations of regeneration in neotenic or paedomorphic urodeles.

Cellular basis of brain maturation and acquisition of complex behaviors in salamanders.

The overall bauplan of the tetrapod brain is highly conserved, yet significant variations exist among species in terms of brain size, structural composition and cellular diversity. Understanding processes underlying neural and behavioral development in a wide range of species is important both from an evolutionary developmental perspective as well as for the identification of cell sources with post-developmental neurogenic potential. Here, we characterize germinal processes in the brain of Notophthalmus viridescens and Pleurodeles waltl during both development and adulthood. Using a combination of cell tracking tools, including clonal analyses in new transgenic salamander lines, we examine the origin of neural stem and progenitor cells found in the adult brain, determine regional variability in cell cycle length of progenitor cells, and show spatiotemporally orchestrated neurogenesis. We analyze how maturation of different brain regions and neuronal subpopulations are linked to the acquisition of complex behaviors, and how these behaviors are altered upon chemical ablation of dopamine neurons. Our data analyzed from an evolutionary perspective reveal both common and species-specific processes in tetrapod brain formation and function.

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 2-3 years 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.

Molecular cloning, the characterization of metallothionein and catalase, and the evaluation of testicular toxicity of Cd in the Chinese fire-bellied newt (Cynops orientalis).

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.

Multiple glacial refugia and contemporary dispersal shape the genetic structure of an endemic amphibian from the Pyrenees.

Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species' present-day genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.

Drivers of amphibian population dynamics and asynchrony at local and regional scales.

Identifying the drivers of population fluctuations in spatially distinct populations remains a significant challenge for ecologists. Whereas regional climatic factors may generate population synchrony (i.e. the Moran effect), local factors including the level of density dependence may reduce the level of synchrony. Although divergences in the scaling of population synchrony and spatial environmental variation have been observed, the regulatory factors that underlie such mismatches are poorly understood. Few previous studies have investigated how density-dependent processes and population-specific responses to weather variation influence spatial synchrony at both local and regional scales. We addressed this issue in a pond-breeding amphibian, the great crested newt Triturus cristatus. We used capture-recapture data collected through long-term surveys in five T. cristatus populations in Western Europe. In all populations-and subpopulations within metapopulations-population size, annual survival and recruitment fluctuated over time. Likewise, there was considerable variation in these demographic rates between populations and within metapopulations. These fluctuations and variations appear to be context-dependent and more related to site-specific characteristics than local or regional climatic drivers. We found a low level of demographic synchrony at both local and regional levels. Weather has weak and spatially variable effects on survival, recruitment and population growth rate. In contrast, density dependence was a common phenomenon (at least for population growth) in almost all populations and subpopulations. Our findings support the idea that the Moran effect is low in species where the population dynamics more closely depends on local factors (e.g. population density and habitat characteristics) than on large-scale environmental fluctuation (e.g. regional climatic variation). Such responses may have far-reaching consequences for the long-term viability of spatially structured populations and their ability to respond to large-scale climatic anomalies.

The geographic mosaic in parallel: Matching patterns of newt tetrodotoxin levels and snake resistance in multiple predator-prey pairs.

The Geographic Mosaic Theory of Coevolution predicts that coevolutionary arms races will vary over time and space because of the diverse ecological settings and population histories of interacting species across the landscape. Thus, understanding coevolution may require investigating broad sets of populations sampled across the range of the interaction. In addition, comparing coevolutionary dynamics between similar systems may reveal the importance of specific factors that structure coevolution. Here, we examine geographic patterns of prey traits and predator traits in the relatively unstudied interaction between the Sierra garter snake (Thamnophis couchii) and sympatric prey, the rough-skinned newt (Taricha granulosa), Sierra newt (Ta. sierrae) and California newt (Ta. torosa). This system parallels, in space and phenotypes, a classic example of coevolution between predatory common garter snakes (Th. sirtalis) and their toxic newt prey exhibiting hotspots of newt tetrodotoxin (TTX) levels and matching snake TTX resistance. We quantified prey and predator traits from hundreds of individuals across their distributions, and functional trait matching at sympatric sites. We show strong regional patterns of trait covariation across the shared ranges of Th. couchii and newt prey. Traits differ significantly among localities, with lower newt TTX levels and snake TTX resistance at the northern latitudes, and higher TTX levels and snake resistance at southern latitudes. Newts and snakes in northern populations show the highest degree of functional trait matching despite possessing the least extreme traits. Conversely, newts and snakes in southern populations show the greatest mismatch despite possessing exaggerated traits, with some snakes so resistant to TTX they would be unaffected by any sympatric newt. Nevertheless, individual variation was substantial, and appears to offer the opportunity for continued reciprocal selection in most populations. Overall, the three species of newts appear to be engaged in a TTX-mediated arms race with Th. couchii. These patterns are congruent with those seen between newts and Th. sirtalis, including the same latitudinal gradient in trait covariation, and the potential 'escape' from the arms race by snake predators. Such concordance in broad scale patterns across two distinct systems suggests common phenomena might structure geographic mosaics in similar ways.

Sperm collection and cryopreservation for threatened newt species.

The aims of this project were to transfer hormone-induced spermiation and sperm cryopreservation protocols developed in the model salamander species, Ambystoma tigrinum, to three threatened newt species. Additionally, we tested if supplementation with trehalose or thawing at different temperatures impacts post-thaw sperm parameters. Hormone stimulation protocols were applied to male Notophthalmus meridionalis (N = 10), Neurergus kaiseri (N = 5) and Tylototriton kweichowensis (N = 6) with sperm collected periodically up to 24-28 h post-spermiation dose. Samples of adequate sperm concentration (>70%) were cryopreserved in solutions of 10% Me2SO + 1% BSA with or without a 10% trehalose cryodiluent. Frozen sperm samples were thawed at either 20 °C or 40 °C and examined for post-thaw motility parameters and abnormalities in head and tail structure. The spermiation response to exogenous hormone treatment was significantly different between newt species, with a success rate of 0% for N. kaiseri, 67% for T. kweichowensis, and 100% for N. meridionalis. Sperm concentration varied with time of collection after hormone administration in both T. kweichowensis and N. meridionalis. For N. meridionalis, structural abnormalities decreased in samples collected over the 24 h period (p < 0.0001) and a thaw temperature of 40 °C resulted in higher relative total sperm motility (p < 0.0001). This is the first study to describe the cryopreservation of sperm from two newt species and demonstrates the transferability of ART developed in a salamander to two newt species.

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.

Reproductive fitness consequences of progenesis: Sex-specific pay-offs in safe and risky environments.

Progenesis is considered to have an important role in evolution because it allows the retention of both a larval body size and shape in an adult morphology. However, the cost caused by the adoption of a progenetic process in both males and females remains to be explored to explain the success of progenesis and particularly its biased prevalence across the sexes and environments. Here, through an experimental approach, we used a facultative progenetic species, the palmate newt (Lissotriton helveticus) that can either mature at a small size and retain gills or mature after metamorphosis, to test three hypotheses for sex-specific pay-offs of progenesis in safe versus risky habitats. Goldfish were used because they caused a higher decline in progenetic than metamorphic newts. We determined that progenetic newts have a lower reproductive fitness than metamorphic newts. We also found that, when compared to metamorphs, progenetic males have lower reproductive activity than progenetic females and that predatory risk affects more progenetic than metamorphic newts. By identifying ultimate causes of the female-biased sex ratios found in nature, these results support the male escape hypothesis, that is the higher metamorphosis rate of progenetic males. They also highlight that although progenesis is advantageous in advancing the age at first reproduction, it also brings an immediate fitness cost and this, particularly, in hostile predatory environments. This means that whereas some environmental constraints could favour facultative progenesis, some others, such as predation, can ultimately counter-select progenesis. Altogether, these results improve our understanding of how developmental processes can affect the sexes differently and how species invasions can impair the success of alternative developmental phenotypes.