Skull development in the Iberian newt, Pleurodeles waltl (Salamandridae: Caudata: Amphibia): timing, sequence, variations, and thyroid hormone mediation of bone appearance.

The cranial ossification sequence in Pleurodeles waltl is widely used in phylogenetic analyses of amphibian origin and evolution. However, the patterns published to date are far from completely resolved and contain certain discrepancies. Based on a large sample of P. waltl specimens ranging from early post-hatching larvae to post-metamorphic newts, we determined the most common cranial ossification sequence and revealed its intraspecific variations. Since thyroid hormones (THs) are involved in the mediation of skull development in salamanders, we studied the role of THs in the cranial development of P. waltl. The normal sequence and timing of bone appearance were compared with those in larvae reared under conditions of high (in 1 and 2 ng mL-1 triiodothyronine) and low [in 0.02% thiourea (TU), which inhibits thyroid gland activity] TH levels. Metamorphosis was greatly accelerated in the TH-treated larvae and was arrested in the TU-treated larvae, which retained the larval pattern of the palate and rudimentary external gills even after 2 years of the experiment. Early-appearing bones (the coronoid, vomer, palatine, dentary, squamosal, premaxilla, parasphenoid, pterygoid, prearticular, vomer, frontal, parietal, exoccipital, in this order) arise at the same stages and ages, and follow the same ossification sequence under different TH levels. The timing of the appearance of bones normally arising in the late larval and metamorphic periods (the quadratojugal, orbitosphenoid, prootic, maxilla, nasal, os thyroideum, prefrontal, quadrate, in this order) changes depending on the TH level. The maxilla and nasal display the most pronounced reaction to changes in the TH level: they appear precociously in TH-treated animals, while their appearance is postponed and they remain rudimentary in TU-treated animals. Because of different responses to THs, the order in which late-arising bones appear changes depending on the TH level. Although bones appearing early in larval ontogeny (e.g. the premaxilla, vomer, squamosal, palatine) display no TH-induced reaction when they start to develop, their further differentiation shows dependence on THs, and these bones become TH-inducible closer to metamorphosis. These findings indicate that TH involvement in the mediation of cranial development changes from minimal (if at all) in its early stages to maximal during metamorphosis. It is likely that the appearance of bones early in development is mediated by factors other than THs. Their further development is accompanied by changes in the mechanisms mediating their morphological differentiation. That is, likely non-hormonal mediation becomes replaced or/and complemented by hormonal mediation. The constituent parts of the same bone may exhibit differences in their reactions to changes in TH levels. Although in normal development, the overall cranial ossification sequence is constant, there was variation in the order in which late-appearing bones was recorded. These observations suggest that this variation results from individual variability in the internal TH level. Comparison with other salamanders suggests that (a) the pattern of TH mediation described in P. waltl is common for cranial development of metamorphosing urodeles and (b) the same bone may differ in its TH dependence in different salamanders, e.g. there are interspecific variations in the degree of TH dependence of individual cranial bones.

Identification and characterization of POU class V family genes in Japanese red bellied newt, Cynops pyrrhogaster.

Mammalian Pou5f1 encodes the POU family class V (POU-V) transcription factor which is essential for the pluripotency of embryonic cells and germ cells. In vertebrates, various POU-V family genes have been identified and classified into the POU5F1 family or its paralogous POU5F3 family. In this study, we cloned two cDNAs named CpPou5f1 and CpPou5f3, which encode POU-V family proteins of the Japanese red bellied newt Cynops pyrrhogaster. In the predicted amino acid sequence encoded by CpPou5f1, the typical MAGH sequence at the N-terminus and deletion of arginine at the fifth position of POU-homeodomain were recognized, but not in the sequence encoded by CpPou5f3. Phylogenetic analysis using Clustal Omega software indicated that CpPou5f1 and CpPou5f3 are classified into the clade of the POU5F1 and POU5F3 families, respectively. In a real-time polymerase chain reaction (RT-PCR) analysis, the marked gene expression of CpPou5f1 was observed during oogenesis and early development up to the tail-bud stage, whereas weak gene expression of CpPou5f3 was detected only in the early stages of oogenesis and gastrula. In adult organs, CpPou5f1 was expressed only in the ovary, while gene expression of CpPou5f3 was recognized in various organs. A regeneration experiment using larval forelimb revealed that transient gene expression of CpPou5f1 occurred at the time of wound healing, followed by gene activation of CpPou5f3 during the period of blastema formation. These results suggest that CpPou5f1 and CpPou5f3 might play different roles in embryogenesis and limb regeneration.

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones.

BACKGROUND: Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be regulated to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~ 2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to the synthesis of rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history: following gene duplication, the protein coding sequence diversified from positive sexual selection while the untranslated regions have been conserved by purifying selection. The molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and conservation of the untranslated regions remain to be determined. RESULTS: Using Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3' UTR. CIRBP, along with other proteins associated with stress response, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis. CONCLUSIONS: The P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of coding and noncoding segments in rapid gene evolution, and necessitate the study of co-evolution between pheromone gene products and their transcriptional/translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated tissue proliferation and subsequent resorption within the dermis and share molecular links to skin cancer biology.

Influence of temperature on female, embryonic and hatchling traits in syntopic newts, Ichthyosaura alpestris and Lissotriton vulgaris.

Amphibian populations have been declining globally for the last several decades, and climate change is often regarded as one of the most important factors driving these declines. Amphibians are particularly sensitive to climatic changes due to their physiological, ecological and behavioral characteristics. Here we performed a laboratory experiment to investigate how temperature affects ovipositing females, eggs and hatchlings in two syntopic populations of alpine newts, Ichthyosaura alpestris, and smooth newts, Lissotriton vulgaris. Female newts were assigned to two different oviposition temperatures (11°C and 14°C) for the duration of their oviposition period. Deposited eggs were equally divided and assigned to three different incubation temperatures (11°C, 14°C and 17°C). We hypothesized that oviposition will be affected by temperature, that the combination of different oviposition and incubation temperatures may have an effect on embryonic and hatchling traits (embryonic mortality, days to hatch and hatchling length), and that these effects might differ between the two newt species. Temperature affected the number of deposited eggs in smooth newts, but not in alpine newts. Larval hatching success was not affected by oviposition or incubation temperature. Temperature effects on hatching time and hatchling length differed between the two species. These results suggest that temperature changes may have disparate effects on amphibian reproduction, even in syntopic taxa.

Effects of temperature on embryonic and early larval growth and development in the rough-skinned newt (Taricha granulosa).

We investigated the effects of temperature on the growth and development of embryonic and early larval stages of a western North American amphibian, the rough-skinned newt (Taricha granulosa). We assigned newt eggs to different temperatures (7, 14, or 21°C); after hatching, we re-assigned the newt larvae into the three different temperatures. Over the course of three to four weeks, we measured total length and developmental stage of the larvae. Our results indicated a strong positive relationship over time between temperature and both length and developmental stage. Importantly, individuals assigned to cooler embryonic temperatures did not achieve the larval sizes of individuals from the warmer embryonic treatments, regardless of larval temperature. Our investigation of growth and development at different temperatures demonstrates carry-over effects and provides a more comprehensive understanding of how organisms respond to temperature changes during early development.

Tetrodotoxin concentrations within a clutch and across embryonic development in eggs of the rough-skinned newts (Taricha granulosa).

Tetrodotoxin is an enigmatic neurotoxin that is found in a wide-variety of organisms. Unfortunately, tetrodotoxin (TTX) toxicity across life-history stages is poorly understood in most organisms. Rough-skinned newts (Taricha granulosa) possess the greatest known quantities of TTX of any organism and numerous studies have begun to elucidate these patterns in this species. We conducted a series of studies to answer the following questions: (1) do eggs from a single female's clutch vary in toxicity? (2) does TTX concentration change during embryonic development? and (3) does the jelly coat from newt eggs possess TTX? We found that the amount of TTX in newt eggs depended on the relative "position" of the egg within a clutch; eggs deposited at the beginning of the clutch had substantially more TTX than those at the end. During development egg toxicity remained consistent until hatching. The jelly coat contained small quantities of TTX, but these were not correlated with the toxicity of the embryo. These results clarify several long-held interpretations about embryo toxicity and continue to elucidate the life-history patterns of tetrodotoxin toxicity in this amphibian.

Increased frequency and severity of developmental deformities in rough-skinned newt (Taricha granulosa) embryos exposed to road deicing salts (NaCl & MgCl2).

Road-side aquatic ecosystems in North America are annually polluted with millions of tons of road deicing salts, which threaten the survival of amphibians which live and breed in these habitats. While much is known of the effects of NaCl, little is known of the second most-commonly used deicer, MgCl(2), which is now used exclusively in parts of the continent. Here we report that environmentally relevant concentrations of both NaCl and MgCl(2) cause increased incidence of developmental deformities in rough-skinned newt hatchlings that developed embryonically in these salts. In addition, we provide some of the first quantification of severity of different deformities, and reveal that increased salt concentrations increase both deformity frequency and severity. Our work contributes to the growing body of literature that suggests salamanders and newts are particularly vulnerable to salt, and that the emerging pollutant, MgCl(2) is comparable in its effects to the more traditionally-used NaCl.

High resolution three-dimensional imaging: Evidence for cell cycle reentry in regenerating skeletal muscle.

Newts and other urodele amphibians can replace lost structures including limbs, providing a vertebrate model for the study of regeneration of complex tissues. The composite of different cell and tissue types in the limb, however, presents a challenge for their imaging in three-dimensions (3D) at cellular level resolution. To observe myofibers in vivo without distortion, we developed a streamlined protocol whereby 80 µm thick cryosections are mounted on slides, processed for immunohistochemistry, imaged using confocal microscopy and z-stacks rendered in 3D. This methodology enabled precise in situ rendering of regenerating muscle, demonstrating cell cycle reentry of nuclei within the myofiber syncytium. The high resolution imaging of muscle or comparable tissue types as intact 3D entities in the context of extracellular and intracellular molecules allows for the determination of signaling and cell response pathways, making this method useful for studies that attempt to characterize rare physiological events in vivo.

Development of Ca²(+) signaling mechanisms and cell motility in presumptive ectodermal cells during amphibian gastrulation.

This study investigated the development of Ca²(+) signaling mechanisms and their role in initiating morphogenetic cell movement in the presumptive ectoderm of Japanese newt (Cynops pyrrhogaster) during gastrulation. Histochemical staining using fluorescently labeled ryanodine and dihydropyridine probes revealed that dihydropyridine receptor (L-type Ca²(+) channels) appeared in stage 12b embryos, while ryanodine receptors were expressed in both stage 11 and 12b embryos. Transmission electron microscopy of stage 12b embryos showed abundant peripheral couplings, which are couplings of the endoplasmic reticulum and cell membrane with an approximate 12 nm gap. Caffeine increased the intracellular free Ca²(+) concentration ([Ca²(+)](i)) in presumptive ectodermal cells isolated from both stage 11 and 12b embryos, while (±)-Bay K 8644 ((±)-BayK) increased [Ca²(+)](i) in cells isolated from stage 12b embryos, but not in cells isolated from stage 11 embryos. Dantrolene and nifedipine completely inhibited increases in [Ca²(+)](i) after treatment with caffeine and (±)-BayK, respectively. Caffeine activated the motility of cells isolated from both stage 11 and 12b embryos, but (±)-BayK only activated the motility of cells isolated from stage 12b embryos. These findings suggested that formation of the Ca²(+) -induced Ca²(+) release system in presumptive ectodermal cells during gastrulation plays an important role in the initiation and execution of epibolic extension.

Sexual size dimorphism in the evolutionary context of facultative paedomorphosis: insights from European newts.

BACKGROUND: Sexual size dimorphism (SSD) is a key evolutionary feature that has been studied in many organisms. In a wide range of species, this pattern is more complex because of polymorphism within each sex. However, it is not known whether the magnitude and direction of SSD could be affected by alternative developmental trajectories within sexes. Our aim was to test whether an intrasexual polymorphism, facultative paedomorphosis (a process in which the development of somatic and gonadal tissues differs in alternative morphs), could affect SSD variation patterns in European newts. RESULTS: We report here the first evidence that SSD varies depending on the paedomorphic or metamorphic ontogenetic pathway. In species with a consistent female-biased SSD, paedomorphosis decreased the SSD level, but did not affect its direction. In species with moderate female-biased SSD or variable SSD patterns, paedomorphosis changed the magnitude, or both the magnitude and the direction, of SSD. CONCLUSION: Our study highlights the importance of developmental processes for shaping SSD patterns in populations in which contrasting life-history pathways evolved. European newts express different SSD patterns depending on their developmental pathway (i.e., metamorphosis versus paedomorphosis), as well as their species and population. These findings emphasize the importance of studying alternative morphotypes, which are found in a wide range of animal groups, to understand the evolution of SSD.

Origin of the prechordal plate and patterning of the anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster.

We analyzed the notochord formation, formation of the prechordal plate, and patterning of anteroposterior regional specificity of the involuting and extending archenteron roof of a urodele, Cynops pyrrhogaster. The lower (LDMZ) and upper (UDMZ) domains of the dorsal marginal zone (DMZ) of the early gastrula involuted and formed two distinct domains: the anterior fore-notochordal endodermal roof and the posterior domain containing the prospective notochord. Cygsc is expressed in the LDMZ from the onset of gastrulation, and the Cygsc-expressing LDMZ planarly induces the notochord in the UDMZ at the early to mid gastrula stages. At the mid to late gastrula stages, part of the Cygsc-expressing LDMZ is confined to the prechordal plate. On the other hand, Cybra expression only begins at mid gastrula stage, coincident with notochord induction at this stage. Anteroposterior regional specificity of the neural plate was patterned by the posterior domain of the involuting archenteron roof containing the prospective notochord at the mid to late gastrula stages. Cynops gastrulation thus differs significantly from Xenopus gastrulation in that the regions of the DMZ are specified from the onset of gastrulation, while the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Thus we propose that Cynops gastrulation is divided into two phases: a notochord induction phase in the early to mid gastrula, and a neural induction phase in the mid to late gastrula.

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis.

We have previously shown that mammalian follicle-stimulating hormone (FSH) promotes the proliferation of spermatogonia and their differentiation into primary spermatocytes in organ culture of newt testis. In the current study, we performed microarray analysis to isolate local factors secreted from somatic cells upon FSH treatment and acting on the germ cells. We identified neuregulin 1 (NRG1) as a novel FSH-upregulated clone homologous to mouse NRG1 known to control cell proliferation, differentiation and survival in various tissues. We further isolated cDNAs encoding two different clones. Amino acid sequences of the two clones were 75% and 94% identical to Xenopus leavis immunoglobulin (Ig)-type and cysteine-rich domain (CRD)-type NRG1, respectively, which had distinct sequences in their N-terminal region but identical in their epidermal growth factor (EGF)-like domain. Semi-quantitative and quantitative PCR analyses indicated that both clones were highly expressed at spermatogonial stage than at spermatocyte stage. In vitro FSH treatment increased newt Ig-NRG1 (nIg-NRG1) mRNA expression markedly in somatic cells, whereas newt CRD-NRG1 (nCRD-NRG1) mRNA was only slightly increased by FSH. To elucidate the function of newt NRG1 (nNRG1) in spermatogenesis, recombinant EGF domain of nNRG1 (nNRG1-EGF) was added to organ and reaggregated cultures with or without somatic cells: it promoted spermatogonial proliferation in all cases. Treatment of the cultures with the antibody against nNRG1-EGF caused remarkable suppression of spermatogonial proliferation activated by FSH. These results indicated that nNRG1 plays a pivotal role in promoting spermatogonial proliferation by both direct effect on spermatogonia and indirect effect via somatic cells in newt testes.

Cellular distribution of Mr 25,000 protein, a protein partially overlapping phosvitin and lipovitellin 2 in vitellogenin B1, and yolk proteins in Xenopus laevis oocytes and embryos.

A phosphorylated protein with molecular mass of 25,000 (pp25) can be derived from Xenopus laevis vitellogenin B1. In order to clarify the distribution of pp25, the changes in the concentration and localization of this protein in oocytes and embryos were examined by immunoblotting and immunohistochemistry using anti-pp25 antibodies, and compared with those of yolk proteins. In oocytes, pp25 was shown to localize characteristically at the surface just below the plasma membrane by immunohistochemical analysis. Interestingly, during embryogenesis, immunocytochemical staining revealed a transition of the pp25 distribution from beneath the outer surface of each germ layers to endoderm during tailbudding. In contrast, yolk proteins were localized in endoderm constantly throughout the developmental stages. However, the level of pp25 in the cytoplasm gradually decreased following the growth of embryos at the tailbud stage and disappeared at the tadpole stage, as shown by immunoblot analysis. These results suggest that pp25 could play different roles from those of yolk proteins such as lipovitellin and phosvitin in X. laevis oocytes and developing embryos.

Heart formation and left-right asymmetry in separated right and left embryos of a newt.

During vertebrate cardiac development, the heart tube formed by fusion of right and left presumptive cardiac mesoderms (PCMs) undergoes looping toward the right, resulting in an asymmetrical heart. Here, we examined the right and left PCMs with regard to heart-tube looping using right- and left-half newt embryos (Cynops pyrrhogaster ). In the half embryos, the rightward (normal) loop of the heart tube was formed from the left PCM, irrespective of the timing of its separation, while the leftward (reversed) loop of the heart tube was formed from the right PCM, separated by stage 18. In addition, the direction of the leftward loop was inverted to the rightward direction in right-half embryos bisected after stage 18. Incision or resection of the embryonic caudal region implicated interactions between the right and left sides of this region as crucial for inverting the direction of the heart-tube loop from leftward to rightward in the right-half embryos. In situ hybridization of CyNodal (Cynops nodal-related gene) suggested that the inversion of heart looping in the right-half embryos has no association with the CyNodal expression pattern. Based on these findings, we propose a mechanism for the rightward looping underlying normal amphibian cardiac development.

CyNodal, the Japanese newt nodal-related gene, is expressed in the left side of the lateral plate mesoderm and diencephalon.

The nodal and nodal-related genes play fundamental roles during deuterostome left-right axis formation. Several of these genes show left-sided expression in the lateral plate mesoderm and brain region. We have isolated the nodal-related gene, CyNodal, from Cynops pyrrhogaster. CyNodal mRNA is detected at the marginal zone and left side of several tissues. The left-sideness of CyNodal mRNA expression is highly conserved throughout vertebrate evolution. However, CyNodal mRNA expression shows little variation from the Xenopus nodal-related gene 1, in that CyNodal gene expression in the left lateral plate mesoderm shifts from posterior to anterior at least twice.

Downregulation of Otx2 in the dedifferentiated RPE cells of regenerating newt retina.

Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Muller glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented monolayer (the outer layer) attached to Bruch's membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers, expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented and the de-pigmented layers. This observation implicates the appearance of Muller glial cells in an early phase of regenerating retina.