Husbandry of Spanish ribbed newts (Pleurodeles waltl).

Research on urodele amphibians, such as newts, is constantly contributing to our understanding of fundamental biological processes. In the present chapter, we present detailed husbandry protocols for the Spanish ribbed newt (Pleurodeles waltl ). We describe the main phases of their life cycle, with emphasis on the progressive development of sensory, motor, and integration systems, which lead to the acquisition of specific stereotyped (and conditioned) behaviors. The methods are outlined to manage housing, feeding, handling, captive breeding, health monitoring, and euthanasia in this species under laboratory conditions. With minor changes, these protocols can also be applied to other species of urodele amphibians commonly used in laboratory research.

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.

Contribution of the urodele amphibian Pleurodeles waltl to the analysis of spaceflight-associated immune system deregulation.

Immune system deregulation has been demonstrated to occur during and immediately following spaceflight. Several animal models have been used to study this phenomenon because of the limited availability of human subjects in space as well as of the need to carry out experiments requiring samples and experimental conditions that cannot be performed using humans. Here, we review major spaceflight-induced microbial and immunological modifications, some of the existing hardware developed to host amphibians in a space station and immunological information provided by space experiments performed with Pleurodeles waltl as an animal model. These data show that the urodele amphibian P. waltl fulfills many technical requirements associated with spaceflight experimentation and that this model is interesting to improve our understanding of the immunosuppressive effects of spaceflight, data required for the preparation of future deep-space missions.

Gravity changes during animal development affect IgM heavy-chain transcription and probably lymphopoiesis.

Our previous research demonstrated that spaceflight conditions affect antibody production in response to an antigenic stimulation in adult amphibians. Here, we investigated whether antibody synthesis is affected when animal development occurs onboard a space station. To answer this question, embryos of the Iberian ribbed newt, Pleurodeles waltl, were sent to the International Space Station (ISS) before the initiation of immunoglobulin heavy-chain expression. Thus, antibody synthesis began in space. On landing, we determined the effects of spaceflight on P. waltl development and IgM heavy-chain transcription. Results were compared with those obtained using embryos that developed on Earth. We find that IgM heavy-chain transcription is doubled at landing and that spaceflight does not affect P. waltl development and does not induce inflammation. We also recreated the environmental modifications encountered by the embryos during their development onboard the ISS. This strategy allowed us to demonstrate that gravity change is the factor responsible for antibody heavy-chain transcription modifications that are associated with NF-κB mRNA level variations. Taken together, and given that the larvae were not immunized, these data suggest a modification of lymphopoiesis when gravity changes occur during ontogeny.

Busulfan-mediated germ cell depletion does not alter gonad differentiation in the urodele amphibian Pleurodeles waltl.

It is not known in urodele amphibians whether germ cells (GCs) are indispensable for gonadal differentiation. In order to address this question in the newt Pleurodeles waltl, we first cloned the ortholog of VASA which is known as a GC marker in many species. Male (ZZ) and female (ZW) larvae were then exposed to the alkylating agent busulfan (25 µg/ml for 3 days) just after hatching (stage 36). In the main body of busulfan-treated larvae, PwVASA mRNA expression decreased before gonad differentiation in both sexes (at stage 50). This suggested GC depletion which was confirmed by histology, with a complete absence of GCs observed slightly earlier in females (stage 54) than in males (stage 54 + 60 days). In busulfan-treated ZW larvae, the presence of the typical central cavity and expression of a high level of aromatase mRNA confirmed the ovarian phenotype. In busulfan-treated ZZ larvae, the presence of a medulla surrounded by a thin cortex and a low level of aromatase mRNA confirmed the testis phenotype. At the juvenile stage, efferent ducts and lobules were present in the first testis lobe. Taken together, these data suggest that GC depletion does not alter gonad differentiation in P. waltl.

The sensitivity of an immature vestibular system to altered gravity.

Stimulus deprivation or stimulus augmentation can induce long-lasting modifications to sensory and motor systems. If deprivation is effective only during a limited period of life this phase is called "critical period." A critical period was described for the development of the roll-induced vestibuloocular reflex (rVOR) of Xenopus laevis using spaceflights. Spaceflight durations and basic conditions of Xenopus' development did not make it possible to answer the question whether exposure of the immature vestibular organ to weightlessness affects rVOR development. The embryonic development of Pleurodeles waltl is slow enough to solve this problem because the rVOR cannot be induced before 15 dpf. Stage 20-21 embryos (4 dpf) were exposed to microgravity during a 10-day spaceflight, or to 3g hypergravity following the same time schedule. After termination of altered gravity, the rVOR was recorded twice in most animals. The main observations were as follows: (1) after the first rVOR appearance at stage 37 (16 dpf), both rVOR gain and amplitude increased steadily up to saturation levels of 0.22 and 20°, respectively. (2) Three days after termination of microgravity, flight and ground larvae showed no rVOR; 1 day later, the rVOR could be induced only in ground larvae. Differences disappeared after 3 weeks. (3) For 10 days after 3g exposure, rVOR development was similar to that of 1g-controls but 3 weeks later, 3g-larvae showed a larger rVOR than 1g-controls. These observations indicate that the immature vestibular system is transiently sensitive to microgravity exposure and that exposure of the immature vestibular system to hypergravity leads to a slowly growing vestibular sensitization.

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.

Temporal and spatial SOX9 expression patterns in the course of gonad development of the caudate amphibian Pleurodeles waltl.

The SOX family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Namely, SOX9 expression is conserved in vertebrate sex determination or differentiation. Nevertheless, information about caudate amphibians is lacking. In this study, we provide data on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. Phylogenetic analysis of SOX9 P. waltl ortholog reveals that the deduced protein segregates from the group of anuran and could be more closely related to amniote than to anamniote. However, SOX9 lacks the PQA-rich domain present in amniotes. In larvae, SOX9 is expressed in both sexes in gonad-mesonephros complexes as soon as stage 42, before gonad differentiation. At stage 54(60d) at which testis differentiation is already in progress, analyses of isolated gonads reveal a male-enriched expression of SOX9, which was quantified by real-time PCR. At the end of metamorphosis (stage 56), SOX9 shows a nuclear localization only in the testis. In adults, SOX9 is still expressed in testes and ovaries. In the ovary, SOX9 is found in oocytes from stage I to stage VI but it is never detected in the nucleus. Our results suggest that in P. waltl, like in non mammalian vertebrates, SOX9 could play a role during the late phase of gonad differentiation rather than in sex determination. Its role in germ cells of the adult ovary has still to be elucidated.

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.

Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset.

Pleurodeles waltl is a urodele amphibian displaying a ZZ/ZW genetic mode of sex determination. Gonad differentiation can later be modulated by hormone treatment. To investigate germ cell differentiation, we analyzed the expression of the meiosis marker PwDmc1 and show that germ cells enter meiosis in late larval life in females, and 2 months after metamorphosis in males. Organotypic cultures of gonad-mesonephros complexes demonstrated that retinoic acid triggers meiosis entry in P. waltl. In vivo analyses of both PwRaldh2 and PwCyp26b1 expressions, the enzymes required for RA synthesis and degradation respectively, indicate that meiosis onset depends on PwCyp26b1 repression in the gonad during normal or steroid-induced sex-reversed development. Taken together, our results show that RA-dependent meiosis entry could be a conserved mechanism of germ cell differentiation in vertebrates and provide evidence for crosstalk between steroid and RA signaling in the course of sex differentiation. Developmental Dynamics 238:1389-1398, 2009. (c) 2009 Wiley-Liss, Inc.

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development.

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.

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.

Enameloid/enamel transition through successive tooth replacements in Pleurodeles waltl (Lissamphibia, Caudata).

Study of the evolutionary enameloid/enamel transition suffers from discontinuous data in the fossil record, although a developmental enameloid/enamel transition exists in living caudates, salamanders and newts. The timing and manner in which the enameloid/enamel transition is achieved during caudate ontogeny is of great interest, because the caudate situation could reflect events that have occurred during evolution. Using light and transmission electron microscopy, we have monitored the formation of the upper tooth region in six successive teeth of a tooth family (position I) in Pleurodeles waltl from late embryos to young adult. Enameloid has only been identified in embryonic tooth I(1) and in larval teeth I(2) and I(3). A thin layer of enamel is deposited later by ameloblasts on the enameloid surface of these teeth. From post-metamorphic juvenile onwards, teeth are covered with enamel only. The collagen-rich enameloid matrix is deposited by odontoblasts, which subsequently form dentin. Enameloid, like enamel, mineralizes and then matures but ameloblast participation in enameloid matrix deposition has not been established. From tooth I(1) to tooth I(3), the enameloid matrix becomes ever more dense and increasingly comes to resemble the dentin matrix, although it is still subjected to maturation. Our data suggest the absence of an enameloid/enamel transition and, instead, the occurrence of an enameloid/dentin transition, which seems to result from a progressive slowing down of odontoblast activity. As a consequence, the ameloblasts in post-metamorphic teeth appear to synthesize the enamel matrix earlier than in larval teeth.

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

Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan.

Effects of space environment on embryonic growth up to hatching of salamander eggs fertilized and developed during orbital flights.

In vertebrates, only few experiments have been performed in microgravity to study the embryonic development from fertilization. To date, these concern only amphibian and fish. We report here a study on the embryonic development of Pleurodeles waltl (urodele amphibian) eggs oviposited in microgravity. The experiment was performed twice on board the Mir space station and the data obtained included video recording and morphological, histological and immunocytological analyses. The data confirm that the microgravity conditions have effects during the embryonic period, particularly during cleavage and neurulation, inducing irregular segmentation and abnormal closure of the neural tube. Moreover, we observed several abnormalities hither to undescribed corresponding to cortical cytoplasm movements, a decrease of cell adhesion and a loss of cells. These abnormalities were temporary and subsequently reversible. The young larvae that hatched during the flight displayed normal morphology and swimming behavior after landing. The results obtained in the urodele Pleurodeles waltl are in accordance with those observed earlier in the anuran Xenopus laevis and in the fish Oryzias latipes.

Sequencing and expression of the CD3 gamma/delta mRNA in Pleurodeles waltl (urodele amphibian).

The CD3 complex is an essential component of the T-cell receptor (TCR) implicated in T-cell maturation and activation. This TCR has been identified in both cartilaginous and bony vertebrates. In different studies where the CD3 chains were cloned and sequenced, it appeared that the CD3 complex is composed of several chains, all susceptible to phosphorylation and able to transduce signals. Here, by an approach combining degenerative oligonucleotide primers and RACE-PCR, we report the cloning and sequencing of a CD3 cDNA from the salamander Pleurodeles waltl, highly homologous to the Xenopus and chicken CD3 gamma/delta cDNAs. Using semi-quantitative PCR and Northern blot analysis, we found the highest CD3 gamma/delta mRNA expression in the thymus; weaker expression was observed in the spleen and blood, followed by the intestine, therefore confirming the tissue and lymphoid specificities of this mRNA. The signals in the spleen, blood and intestine represented 55%, 33% and 16%, respectively, of the signal detected in the thymus. During the embryonic and larval stages of Pleurodeles waltl development, CD3 gamma/delta mRNA expression begins early at the neurula stage (stage 15, 69 h after laying), increases up to stage 33 (9 days after laying) and afterwards remains stable, at least until the larval stage 42 (28 days after laying). As the thymus primordium appears much later, the question of the formation and maturation of the first T-cell precursors outside this organ is posed.

Early development of NADPH diaphorase-expressing neurons in the brain of the urodele amphibian Pleurodeles waltl.

The distribution of nitrergic cells was studied in the brain of the urodele amphibian Pleurodeles waltl during embryonic and larval stages by means of NADPH-diaphorase histochemistry. The first positive neurons were observed at embryonic stage 30 in the ventrolateral area of the caudal rhombencephalon. Subsequently (stage 33b), weakly reactive cells appeared in the isthmic tegmentum, the mesencephalic tegmentum, the hypothalamus, and the nucleus of the solitary tract. At initial larval stages (34-38), two new groups of NADPH-diaphorase-positive cells appeared in the caudal telencephalon (the amygdaloid region) and in the middle reticular nucleus. During the beginning of the active larval life (stages 39-42), reactive cells were found in the granule cell layer of the olfactory bulb and in the telencephalic pallium. As in the adult, the nitrergic cells in the central nervous system are widely distributed during early development, pointing to important roles of nitric oxide through ontogenesis. The sequence of appearance of nitrergic cells suggests an early involvement in reticulospinal control most likely related to locomotor behavior.

[Presumed role of the gravity in the establishment of the symmetrization in amphibian embryos. Response provided by the biological experimentation in space]. / Role presume de la gravite dans la symetrisation de l'embryon des amphibiens. Reponse apportee par l'experimentation en biologie dans l'espace.

During the early embryonic development of the amphibian, several events are presumed to be dependent on the ground gravity (egg rotation of equilibrium, formation of the grey crescent, symmetrization, cleavage). Will development occur normally in the absence of the gravity? The experience FERTILE had led to obtain in weightlessness, onboard the space station Mir, the natural fertilization and the embryonic development of the salamander Pleurodeles waltl, an amphibian urodele. All the embryos developed in microgravity acquired normal bilateral symmetry. The egg rotation that occurs on earth after fertilization is clearly not necessary in urodeles to determine the position of symmetrical plan. The results obtained in P. waltl are in accordance with those observed earlier in the anuran Xenopus laevis and the fish Oryzias latipes.

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.

Design of specific hardware to obtain embryos and maintain adult urodele amphibians aboard a space station.

The study of the influence of weightlessness on fertilization and embryonic development of a vertebrate is of importance in the understanding of basic embryogenesis and in the preparation of the future exploration of space. Accordingly, specific hardware was designed to perform experiments on board the MIR space station with an amphibian vertebrate model, taking into account the biological requirements and the multiple constraints of a long-term mission. This paper describes the biological uses and presents the technological specifications of the device developed under CNES management. The hardware was adapted to and is compatible with biological requirements as confirmed by three experiments performed in space on board the orbital MIR station.