Cell-type profiling in salamanders identifies innovations in vertebrate forebrain evolution.

The evolution of advanced cognition in vertebrates is associated with two independent innovations in the forebrain: the six-layered neocortex in mammals and the dorsal ventricular ridge (DVR) in sauropsids (reptiles and birds). How these innovations arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we built a cell-type atlas of the telencephalon of the salamander Pleurodeles waltl. Our molecular, developmental, and connectivity data indicate that parts of the sauropsid DVR trace back to tetrapod ancestors. By contrast, the salamander dorsal pallium is devoid of cellular and molecular characteristics of the mammalian neocortex yet shares similarities with the entorhinal cortex and subiculum. Our findings chart the series of innovations that resulted in the emergence of the mammalian six-layered neocortex and the sauropsid DVR.

Unique high sensitivity to heat of axolotl TRPV1 revealed by the heterologous expression system.

The thermosensation mechanism plays critical roles in various animals living in different thermal environment. We focused on an axolotl, which is a tailed amphibian originally from Lake Xochimilco area in the Vally of Mexico, and examined its behavior response to heat stimulation. Mild heat at 33 °C induced noxious locomotive activity to axolotls, but the noxious response of another tailed amphibian, Iberian ribbed newt, was not observed at 33 °C. To explore the mechanism for the temperature sensitivity of axolotls, we isolated a cDNA of TRPV1. Using the degenerate primer PCR method, we identified the DNA fragment encoding axolotl TRPV1 (axTRPV1), and then cloned a full-length cDNA. We studied the chemical and thermal sensitivities of axTRPV1 by two-electrode voltage clamp method using Xenopus oocyte expression system. Capsaicin, acid, and 2-aminoethoxydiphenylborane apparently activated axTRPV1 channels in a dose-dependent manner. The analysis of thermal sensitivity showed that axTRPV1 was significantly activated by heat but not by cold. The average temperature threshold for heat-activation was 30.95 ±â€¯0.12 °C. This thermal activation threshold of axTRPV1 is unique and significantly low, when compared with the known thresholds of TRPV1s from various animals. Further, this threshold of axTRPV1 is well consistent with the observation of heat-induced behavior of axolotls at 33 °C, demonstrating that axolotl shows noxious response to mild heat mediated through axTRPV1.

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.

Static osteogenesis does not precede dynamic osteogenesis in periosteal ossification of Pleurodeles (Caudata, Amphibia) and Pogona (Squamata, Lepidosauria).

Two successive mechanisms have been described in perichondral ossification: (1) in static osteogenesis, mesenchymal cells differentiate into stationary osteoblasts oriented randomly, which differentiate into osteocytes in the same site; (2) in dynamic osteogenesis, mesenchymal cells differentiate into osteoblasts that are all oriented in the same direction and move back as they secrete collagen fibers. This study is aimed at testing the hypothesis that the ontogenetic sequence static then dynamic osteogenesis observed in the chicken and in the rabbit is homologous and was acquired by the last common ancestor of amniotes or at a more inclusive node. For this we analyze the developmental patterns of Pleurodeles (Caudata, Amphibia) and those of the lizard Pogona (Squamata, Lepidosauria). We processed Pleurodeles larvae and Pogona embryos, prepared thin and ultrathin sections of appendicular bones, and analyzed them using light and transmission electron microscopy. We show that static osteogenesis does not precede dynamic osteogenesis in periosteal ossification of Pleurodeles and Pogona. Therefore, the null hypothesis is rejected and according to the parsimony method the ontogenetic sequence observed in the chicken and in the rabbit are convergent. In Pleurodeles and Pogona dynamic osteogenesis occur without a previous rigid mineralized framework, whereas in the chicken and in the rabbit dynamic osteogenesis seems to take place over a mineralized support whether bone (in perichondral ossification) or calcified cartilage (in endochondral ossification). Interestingly, in typical dynamic osteogenesis, osteoblasts show an axis (basal nucleus-distal endoplasmic reticulum) perpendicular to the front of secreted unmineralized bone matrix, whereas in Pleurodeles and Pogona this axis is parallel to the bone matrix.

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.

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.

Müllerian inhibiting substance in the caudate amphibian Pleurodeles waltl.

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-ß family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.

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.

Segmental oscillators in axial motor circuits of the salamander: distribution and bursting mechanisms.

The rhythmic and coordinated activation of axial muscles that underlie trunk movements during locomotion are generated by specialized networks in the spinal cord. The operation of these networks has been extensively investigated in limbless swimming vertebrates. But little is known about the architecture and functioning of the axial locomotor networks in limbed vertebrates. We investigated the rhythm-generating capacity of the axial segmental networks in the salamander (Pleurodeles waltlii). We recorded ventral root activity from hemisegments and segments that were surgically isolated from the mid-trunk cord and chemically activated with bath-applied N-methyl-d-aspartate (NMDA). We provide evidence that the rhythmogenic capacity of the axial network is distributed along the mid-trunk spinal cord without an excitability gradient. We demonstrate that the burst generation in a hemisegment depends on glutamatergic excitatory interactions. Reciprocal glycinergic inhibition between opposite hemisegments ensures left-right alternation and lowers the rhythm frequency in segments. Our results further suggest that persistent sodium current contributes to the rhythmic regenerating process both in hemisegments and segments. Burst termination in hemisegments is not achieved through the activation of apamine-sensitive Ca(2+)-activated K(+) channels and burst termination in segments relies on crossed glycinergic inhibition. Together our results indicate that the basic design of the salamander axial network is similar to most of axial networks investigated in other vertebrates, albeit with some significant differences in the cellular mechanism that underlies segmental bursting. This finding supports the view of a phylogenetic conservation of basic building blocks of the axial locomotor network among the vertebrates.

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.

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

BACKGROUND: In 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. METHODS: Testes 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. RESULTS: In 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. CONCLUSION: Pleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood.

Three types of cutaneous glands in the skin of the salamandrid Pleurodeles waltl. A histological and ultrastructural study.

Histological and ultrastructural investigations revealed three different multicellular skin gland types in the salamandrid Pleurodeles waltl. The mucous glands are small, with one layer of secretory cells surrounding a central lumen; they produce the viscous and slippery mucus film that has various functions in amphibians. The serous glands can be divided based on their histological and ultrastructural characters into the granular gland Type I (GGI) and the granular gland Type II (GGII). The first type (GGI) is moderately sized and distributed throughout the body surface, with higher concentrations in the parotoid and back regions. In contrast, the second type (GGII) is very large (for Pleurodeles) and was found only in the tail, with highest concentration in the tail dorsum. Both granular gland types contain mainly proteinaceous materials but differ in their morphological features including size, shape, cellular organization and vesicle distribution, vesicle size and vesicle shape. Both GGI and GGII are especially concentrated in body parts that are presented to an attacking predator and are hypothesized to produce repellent to poisonous substances to thwart potential aggressors.

Muscarinic control of the excitability of hindlimb motoneurons in chronic spinal-transected salamanders.

The excitability of spinal motoneurons (MNs) is regulated by acetylcholine via the activation of muscarinic receptors. The objective of the present study was to determine whether this cholinergic modulation of MN excitability is altered following a chronic spinal cord transection. Juvenile salamanders (Pleurodeles waltlii) were spinally transected at the mid-trunk level, and patch-clamp recordings from hindlimb MNs in spinal cord slices were performed 9-30 days after transection, with and without bath application of muscarine (20 mum). Our results showed that the input-output relationship was larger in MNs recorded 2 weeks after spinal transection than in MNs recorded 3-4 weeks after spinal transection. They further revealed that muscarine increased both the gain of MNs and the proportion of MNs that could exhibit plateau potentials and afterdischarges, whereas it decreased the amplitude of the medium afterhypolarizing potential. Moreover, muscarine had no effect on the hyperpolarization-activated cation current (I(h)), whereas it increased the inward rectifying K(+) current (I(Kir)) in MNs recorded > or = 2 weeks after spinal transection. We conclude that following chronic spinal cord injury, the muscarinic modulation of some intrinsic properties of MNs previously reported in acute spinal-transected animals [S. Chevallier et al. (2006)The Journal of Physiology, 570, 525-540] was preserved, whereas that of other intrinsic properties of MNs was suppressed, either transiently (I(Kir)) or definitively (I(h)). These alterations in muscarinic modulation of MN excitability may contribute to the spontaneous recovery of locomotion displayed in long-term chronic spinal-transected salamanders.

Calbindin-D28k and calretinin expression in the forebrain of anuran and urodele amphibians: further support for newly identified subdivisions.

A general pattern of organization of the forebrain shared by amphibians, mainly anurans, and amniotes has been proposed considering the relative topography of the territories, their connectivity, and their neurochemistry. These criteria were needed because the amphibians possess limited cell migration from the ventricle that precludes a parcellation into circumscribed nuclei. In the present study we have tested the identity of most newly described forebrain territories in anurans and urodeles with regard to their content in calbindin-D28k (CB) and calretinin (CR). By means of immunohistochemistry, these proteins were demonstrated to be particularly abundant and specifically distributed in the amphibian forebrain and were extremely useful markers for delineating nuclear boundaries otherwise indistinguishable. In the telencephalon, labeled cells in the pallium allowed the identification of particular regions with marked differences between anurans and urodeles, whereas the subpallium showed more conservative patterns of distribution. In particular, the components of the amygdaloid complex and the basal ganglia were distinctly labeled. The distribution in the nonevaginated secondary prosencephalon and diencephalon showed abundant common features between anurans and urodeles, highlighted using the prosomeric model for the comparison. In the pretectum, thalamus, and prethalamus of urodeles, the CB and CR staining was particularly suitable for the identification of diverse structures within the simple periventricular gray layer. However, the analysis across species also revealed a considerable degree of heterogeneity, even within comparatively well-defined neuronal populations. Therefore, the content of a particular calcium binding protein in a neuronal group is not a fully reliable criterion for considering homologies.

Acrosome biosynthesis in spermatocytes and spermatids revealed by HPA lectin cytochemistry.

The origin of the acrosome is controversial, because of both its lysosomal nature and at the moment of its appearance, which seems to be species-specific. Considering the amazing organization shown by the acrosome of some urodele amphibians, HPA-colloidal gold cytochemistry was used to analyze the biogenesis of the acrosome in the urodele Pleurodeles waltl at electron microscopy level. The results showed that HPA-labeling is useful to label the acrosome and its precursor vesicles and, consequently, HPA-histochemistry could be used as a marker of acrosomal content. Labeling of the Golgi apparatus and precursor vesicles was seen in primary spermatocytes and round (stage I) spermatids, thus contributing solid evidence for the beginning of acrosome biogenesis before meiosis. In both primary spermatocytes and round spermatids, an enigmatic vesicle, probably related to the biosynthesis of the neck piece or the tail, was also labeled. Labeling in elongating spermatids (stage II-IV), showed a homogeneous distribution of colloidal gold particles in the acrosomal cap, but the perforatorium was not positive to the lectin. However, in mature (stage V-VI) spermatids, a regional distribution of labeling in the acrosome was seen, with the apical knob showing a stronger labeling than the lateral barb, and the lateral barb showing a stronger labeling than the principal piece of the acrosomal cap. This regional distribution of the labeling suggests that the acrosome develops several domains with different glycoconjugate compositions.

Sexual development of the urodele amphibian Pleurodeles waltl.

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

Female-enriched expression of ERalpha during gonad differentiation of the urodele amphibian Pleurodeles waltl.

In the amphibian Pleurodeles waltl, estradiol treatment of genetically male larvae (ZZ) induces male-to-female sex reversal whereas heat treatment of genetically female larvae (ZW) inhibits estradiol synthesis and leads to female-to-male sex reversal. No data are available on estrogen receptors in this species. In the present study, we have isolated a unique full-length pwERalpha cDNA and its 5'-flanking region whose promoter activity was confirmed by transfection assays. RT-PCR studies performed in adult animals using ERalpha-specific primers, revealed that pwERalpha mRNA was present mainly in reproductive tissues: gonads, fat body and oviduct. PwERalpha transcript was also detected in liver, suggesting its implication in vitellogenesis control as in numerous oviparous species. The level of pwERalpha transcripts was also studied during gonad differentiation by quantitative real-time PCR. At stage 54(30d) pwERalpha expression in gonads of ZW larvae was significantly higher than in ZZ ones. This sex-specific discrimination was confirmed when gonad-mesonephros-interrenal complexes (GMI), taken at the same stage, were subjected to whole mount in situ hybridization. In comparison, the female-enriched expression of P450 aromatase, which was studied as a control of ovary differentiation, was observed earlier (stage 54). In ZW larvae reared at 32 degrees C, a condition leading to sex reversal, pwERalpha mRNA level at stage 54(30d) was lower than in control females. Taken together, these results showing a female-enriched and thermosensitive expression of pwERalpha suggest an important role for this receptor in gonad differentiation of the urodele amphibian Pleurodeles waltl.

From swimming to walking with a salamander robot driven by a spinal cord model.

The transition from aquatic to terrestrial locomotion was a key development in vertebrate evolution. We present a spinal cord model and its implementation in an amphibious salamander robot that demonstrates how a primitive neural circuit for swimming can be extended by phylogenetically more recent limb oscillatory centers to explain the ability of salamanders to switch between swimming and walking. The model suggests neural mechanisms for modulation of velocity, direction, and type of gait that are relevant for all tetrapods. It predicts that limb oscillatory centers have lower intrinsic frequencies than body oscillatory centers, and we present biological data supporting this.

Female-enriched and thermosensitive expression of steroidogenic factor-1 during gonadal differentiation in Pleurodeles waltl.

In the urodele amphibian Pleurodeles waltl, sex differentiation is genetically controlled, that is, ZZ male vs ZW female, but may be influenced by temperature, which induces a female-to-male sex reversal. We investigated whether steroidogenic factor 1 (SF-1) could be involved in Pleurodeles sex differentiation or in temperature-dependent sex reversal by cloning a Pleurodeles SF-1 cDNA and examining its developmental expression. The 468-amino-acid deduced protein is highly conserved in comparison with other species. In ZZ and ZW control larvae, SF-1 mRNA is detected at the first stage of the thermosensitive period (TSP) in the gonad-mesonephros-interrenal complex (GMI). By the end of TSP at stage 55, SF-1 is expressed in the gonad (Gd) and in the mesonephros-interrenal (MI) both in ZZ and ZW larvae. During this stage, a transient, ZW-specific increase of SF-1 transcription occurs not only in Gd but also in MI, this increase starting earlier in Gd than in MI. Therefore, in P. waltl, an SF-1 upregulation occurs after the onset of the ovarian-specific increase of aromatase mRNA expression. At the end of metamorphosis, the SF-1 transcription level in Gd and MI is nearly the same in both ZZ and ZW larvae. Besides, after long-term heat treatment leading to sex reversal, SF-1 mRNA upregulation is not observed in ZW larvae, in either Gd or MI. However, SF-1 expression is not decreased after a 48-h heat shock applied at the end of the TSP, suggesting that temperature has no inhibitory effect by itself in long-term heat treatment. Estradiol benzoate treatments show that, at the end of the TSP, SF-1 gene transcription could be controlled by the estrogen level. This is in accordance with the female-enriched SF-1 expression and the decreased SF-1 expression following long-term, sex-reversing heat treatment, which is known to decrease aromatase expression and activity. Thus, it is unlikely that SF-1 is directly involved in Pleurodeles temperature-dependent sex reversal.