Triturus newts.

Ben Wielstra introduces Eurasian newts of the genus Triturus.

The ontogenetic origins of skull shape disparity in the Triturus cristatus group.

Comparative studies of ontogenies of closely related species provide insights into the mechanisms responsible for morphological diversification. Using geometric morphometrics, we investigated the ontogenetic dynamics of postlarval skull shape and disparity in three closely related crested newt species. The skull shapes of juveniles just after metamorphosis (hereafter metamorphs) and adult individuals were sampled by landmark configurations that describe the shape of the dorsal and ventral side of the newt skull, and analyzed separately. The three species differ in skull size and shape in metamorphs and adults. The ontogenies of dorsal and ventral skull differ in the orientation but not lengths of the ontogenetic trajectories. The disparity of dorsal skull shape increases over ontogeny, but that of ventral skull shape does not. Thus, modifications of ontogenetic trajectories can, but need not, increase the disparity of shape. In species with biphasic life-cycles, when ontogenetic trajectories for one stage can be decoupled from those of another, increases and decreases in disparity are feasible, but our results show that they need not occur.

Proliferative events experimentally induced by transient cold shock in the brain of adult terrestrial heterothermic vertebrates: preliminary analysis of PCNA expression in Triturus carnifex.

Experimental procedures used to investigate the persistence, location and abundance of scattered ("matrix cells") and/or clustered ("matrix areas") stem cells in the brain, responsible for proliferation in adult terrestrial heterothermic vertebrates have included an induced transient drop in body temperature in specimens subsequently deprived of encephalic areas. In a set of coordinated investigations focused on the influence of an exposure to a drastic thermally environment on these activities, we gave priority to Triturus carnifex, since there is a much larger amount of detailed, unequivocal experimental evidence available for this species than for other vertebrates of the same evolutionary level. In the present study, cold-shocked newts were examined after a stay at external temperature (the most suitable one based on previous experience) to allow the maximal expression of cerebral proliferation. In a qualitative evaluation, the brain of experimental specimens compared with that of normal individuals seemed not to show, contrary to expectations, more pronounced cell proliferation as assessed by Proliferating Cell Nuclear Antigen immunolabelling of neural-like cells in the S phase of cell cycle. This discrepancy with previous reports from other authors may depend on having used cold stress alone, while other traumatic stimuli (operatory shock, encephalic injury) administered by the previous authors might have induced a greater number of cells to move from a stand-by condition to proliferation, allowing for reparative and/or regenerative phenomena.

Is the prefrontal bone in Alpine newt (Triturus alpestris Laurenti, 1768) of dual origin?

According to current knowledge, only the prefrontal bone (os prefrontale) of the circumorbital series is preserved in the family Salamandridae. However, the exact origin and number of ossification centres creating this bone is unknown. Detailed examination of the prefrontal bone during ontogeny of juvenile and adult specimens of the Alpine Newt (Triturus alpestris) indicates its dual origin (prefrontal and lacrimal). We found that the prefrontal bone originates from four ossification centres, i.e. three prefrontal centres and one posterior lacrimal centre. The anterior lacrimal centre participates in the maxillar ossification. The development of these ossification centres occurs very late in ontogeny (at stage 54), and starts after differentiation of the nasal capsules. The total fusion of the lacrimal ossification centre with the prefrontal bone of T. alpestris is distinct from the fully differentiated lacrimal bone attached to the prefrontal bone of the fossil family Branchiosauridae (Temnospondyly). We propose that heterochrony, observed in the recent species, is a delayed development followed by accelerated ossification that resulted in the fusion of the anterior lacrimal centre with the maxilla and the posterior lacrimal centre with the prefrontal bone.

Structural diversification of the gustatory organs during metamorphosis in the alpine newt Triturus alpestris.

Gustatory organs of the taste bud type occur in the epithelial lining of the oropharyngeal cavity of alpine newt larvae. They resemble the taste buds of bony fish, both in appearance (as revealed by scanning electron microscopy) and in detailed internal structure (seen on transmission electron micropscopy). During metamorphosis, at stage 55 of development, the secondary tongue (i.e. the soft tongue) is well formed and the anlages of taste discs are clearly apparent. Somewhat later, taste discs also appear in the epithelial lining outside the tongue, paralleling the disappearance of the taste buds. Well-developed taste discs of the newt differ from taste buds mainly by their structurally diversified set of 'associate cells' (mucous, wing and glial cells), which have no synaptic contact with nerve fibres. These cells accompany the neurosensory cellular components of the taste disc, i.e. the taste receptor cells and basal cells. This indicates that gustatory organs in metamorphosed newts, regardless of their small dimensions, fulfil the criteria established for taste discs previously defined in other Caudata and Anura species. Therefore, in the development of the newt there are two subsequent types of gustatory organs and two generations of the tongue: primary, in the larvae, and secondary, in metamorphosed animals.

Localization of two nitric oxide synthase isoforms, eNOS and iNOS, in the skin of Triturus italicus (Amphibia, Urodela) during development.

We analyzed the skin of Triturus italicus (Amphibia, Urodela) histologically during the larval, pre-metamorphic and adult phases in parallel with the immunohistochemical evaluation of the expression of two nitric oxide synthase (NOS) isoforms, i.e. the inducible NOS (iNOS) and the endothelial NOS (eNOS). Our results indicate that, during the larval and adult phases, substantial changes in the intensity and localization of both iNOS and eNOS are present. In contrast, the pre-metamorphic newts show a labelling pattern similar to that found in the skin of adult individuals. These data suggest an involvement of the NOS system in the larval epidermis during functional maturation phase starting at the climax and preceding structural rearrangements during metamorphosis, emphasizing the putative functional importance of differential isoform expression related to a distinct phase of the biological cycle.

Sex reversal of the newt Triturus cristatus reared at extreme temperatures.

Crested newt larvae were reared at defined temperatures, either from uncleaved eggs or from early feeding larvae, until metamorphosis when sexual differentiation had occurred. Trials at 18-24 degrees C showed a 1:1 sex ratio. A higher temperature trial produced more males than females, including some XX neomales. Lower temperatures resulted in a significant excess of females, including XY neofemales. Sex reversal only occurred in about half the possible cases on average. Extreme temperatures must perturb the normal XX/XY system of sex determination, to reveal either an ancestral ZZ/ZW system or a still more primitive environmental control. It is suggested that neofemales (but not neomales) could occur in nature.

Myotomal myogenesis in Triturus vulgaris L. (Urodela) with special reference to the role of mesenchymal cells.

Two stages can be distinguished in the differentiation of myotomal muscle fibres in Triturus vulgaris. In the first stage only synchronously differentiating myotomal cells are engaged; in the second stage mesenchymal cells also take part in the process. Myotomal cells (primary myoblasts) fuse to form 2-3 nucleate myotubes. Only in the caudal part of the embryo mononucleate myotubes persist. The mononucleate myotubes, like polynucleate ones, occupy the whole length of the myotome. The differentiation of myotubes is accompanied by vitellolysis. At further development stages mesenchymal cells enter the intermyotomal fissure, after which they migrate to the myotomes, between the myotubes. The cells that remain in the intermyotomal fissures retain their fibroblastic potential (they synthesise collagen). Their daughter cells adjoining the myotubes acquire myogenic abilities. Their myoblastic potential is evidenced by their ability to fuse with the myotube. Fusion of secondary myoblasts (of mesenchymal origin) with the myotube results in further growth of the myotubes. In T. vulgaris myotomal myotubes and muscle fibres developing from them are of myotomal-mesenchymal origin.

Planar signalling is not sufficient to generate a specific anterior/posterior neural pattern in pseudoexogastrula explants from Xenopus and Triturus.

Early observations on the morphology of total exogastrulae from urodeles (Axolotl) had provided evidence for essential vertical signalling mechanisms in the process of neural induction. Conversely, more recent studies with anurans (Xenopus laevis) making use of molecular markers for neural-specific gene expression appear to support the idea of planar signalling as providing sufficient information for neural differentiation along the anterior-posterior axis. In an attempt to resolve this apparent contradiction, we report on the comparative analysis of morphology and gene expression characteristics with explants prepared from both urodeles (Triturus alpestris) and anurans (Xenopus laevis). For this purpose, we have made use of a refined experimental protocol for the preparation of exogastrulae that is intended to combine the advantages of the Holtfreter type exogastrula and the Keller sandwich techniques, and which we refer to as pseudoexogastrula explants. Analysis of histology and expression of several neural and ectodermal marker genes in such explants suggests that neural differentiation is induced in both species, but only within the intermediate zone between ectoderm and endomesoderm. Therefore, experiments with Xenopus and Triturus explants described in this communication argue against planar signalling events as being sufficient to generate a specific anterior/posterior neural pattern.

Immunohistochemical localisation of amelogenin-like proteins and type I collagen and histochemical demonstration of sulphated glycoconjugates in developing enameloid and enamel matrices of the larval urodele (Triturus pyrrhogaster) teeth.

The presence of collagen in enameloid distinguishes it clearly from true enamel, but little is known about the phylogenetic relationship between these 2 tissues. It has previously been reported that amelogenins are the principal proteins of the enamel matrix, that type I collagen and chondroitin sulphates are the predominant matrices in dentine, and that amphibian and reptilian aprismatic enamels, contain no sulphated glycoconjugates, although certain sulphated substances are secreted into mammalian prismatic enamel during matrix formation. The larval urodele (Triturus pyrrhogaster) teeth are known to be composed of enameloid, dentine, and enamel-like tissue. To characterise the tooth matrices, the localisation of amelogenin-like proteins, type I collagen, and sulphated glycoconjugates was investigated. Chondroitin sulphates and fine fibrils immunoreactive for type I collagen were elaborated as the enameloid matrix inside the dental basement membrane. After the matrix had been deposited in full thickness, coarse collagen fibrils also immunoreactive for type I collagen and chondroitin sulphates were deposited below as the first dentine matrix. Further, enamel-like matrix with no collagen fibrils or sulphated glycoconjugates but strongly immunoreactive for amelogenins was deposited on the dentine. Although no immunolabelling for amelogenins was found over the enameloid matrix, at least at the formation stage, the zone of coarse collagen fibrils of dentine was partially immunoreactive as observed in mammalian mantle dentine. From the ontogeny and matrix constituents of larval urodele teeth, it is suggested that enameloid is originally a dentine-like tissue.

B24 protein stored in lampbrush spheres is involved in early cleavage in urodele amphibians.

A cDNA encoding a protein (B24) belonging to the Mcm/P1 family was isolated from the newt Triturus carnifex. In eukaryotes, the members of the Mcm/P1 family are essential factors in the DNA replication process. B24 protein (TcMcm3) is present in salamandrid ovarian oocytes and early embryos; its role was tested by injecting specific anti-B24 monoclonal antibodies into the cytoplasm of one blastomere of two-cell stage embryos. The injected blastomere encountered cleavage arrest either soon after the injection or following one or two divisions; later, it degenerated. Instead, the uninjected blastomere went on developing and organizing a hemi-embryo, which does not grow beyond the tailbud stage. These results are consistent with the hypothesis that the B24 protein is involved in DNA replication at cleavage. The B24 protein studied here appears to play a specific role in early development; other variants of the Mcm3 group seem to be employed by different adult tissues.

Factors responsible for the establishment of the body plan in the amphibian embryo.

A central topic of embryology is the establishment of the body plan during embryogenesis. Starting with maternal factors distributed in the early cleavage stages in distinct patterns and gradients cell-to-cell interactions including early embryonic induction result in the formation of mesoderm and the organizer area. While many facts are known about the role of growth factors like activin (closely related to the vegetalizing factor), processed Vg1, BMPs and FGF for mesoderm formation, the establishment of the central nervous system is not yet well understood. However, there is growing evidence that neural induction is a multistep process at the level of the dorsal mesoderm (organizer) and the reacting neuroectoderm. Therefore the existence of only one neuralizing factor is unlikely. We report about data that follistatin protein is not a direct neural inducer. Furthermore our comparative studies of Xenopus and Triturus exogastrulae indicate that planar signals are unlikely in the Triturus embryo (urodeles) during the early steps of neural induction. Vertical signals emanating from the chordamesoderm are essential for the terminal neuralization and regionalization of the central nervous system during gastrulation for both Xenopus and Triturus. The putative role of neuralizing factors and BMP/activin-like molecules for the stabilization or shift of neuroectoderm into different pathways of differentiation (epidermis or neural default state) is discussed.

[Mirror symmetrical and "left dominant" organization in the gastrula stage molted parabiotic larva of mountain salamander]. / Spiegelsymmetrische und "linksdominante" Organogenese bei im Gastrulastadium verschmolzenen Parabioselarven des Bergmolches (Triturus alpestris).

Gastrulae of Triturus alpestris were laterally fused (body axes in equal directions). The parabiotic larvae mostly showed an undivided body with tail, but exhibited two heads. Defects of organogenesis were frequently observed in the gut, and, to some fewer extent, in the heart. The gut mostly develops undivided, the heart in singular or (with transitions) as two organs. The number of organ-inversions and partial inversions was high (table 1). In addition a transindividual organ-symmetry (mirror-image symmetry of the "pair situs") was abundant (table 3, fig. 6) and the gut and the habenular nuclei showed a strong dominance for that phenotype. The organ inversions were more abundant in the right organ or part of the organ. These right-left-differences were statistically significant referring to the inversion tendency of the heart and the habenular nuclei (table 2). This "left-dominance" observed in the organ Anlagen investigated is discussed in regard to results published previously.

The role of vertical and planar signals during the early steps of neural induction.

The classical Einsteck-test (Spemann and Mangold, Roux Arch. Dev. Biol. 100: 599-638, 1924) and data from total exogastrulae (Holtfreter, 1933) suggest that vertical signals are transmitted between the chordamesoderm (organizer) and reacting ectoderm in the early phase of neural induction. In contrast to these results with Axoloti (urodeles), several authors observed the expression of neural specific genes in Xenopus exogastrulae, isolated dorsal blastopore lip with adjacent ectoderm (open-face explants) and Keller-sandwiches. Our data with Xenopus (anurans) also show that the expression of neural specific genes takes place in exogastrulae. However, when we prepared open face explants and exogastrula-like structures by microdissection at very early gastrula stage, the signal of a class II beta-tubulin, characteristic of terminal neural differentiation, is not found in the ectoderm. These results suggest that planar signals transmitted from the chordamesoderm into the ectodermal part can fairly be excluded under these experimental conditions. In similar experiments with Triturus alpestris we could not observe either the differentiation of neural structures in the ectodermal part of exogastrulae. These results confirm earlier experiments of Holtfreter performed with Ambystoma mexicanum (Axoltl) embryos. On the basis of the published data of different authors and our results, we cannot exclude the existence of planar signals for early and/or transient expressed genes before the onset of gastrulation in Xenopus, which make the neuroectoderm susceptible for the response to vertical signals during gastrulation. On the other hand our experiments with Triturus alpestris suggest that planar neural signals are unlikely in this species. These differences between Triturus and Xenopus embryos are discussed in the context of the peculiarities in morphological structure, competence and speed of development of the two species.

Partially purified factor from embryonic chick brain can provoke neuralization of Rana temporaria and Triturus alpestris but not Xenopus laevis early gastrula ectoderm.

A high neuralizing activity has been determined in forebrain of 7.5-day old chick embryos using Rana temporaria early gastrula ectoderm as reacting tissue (Mikhailov and Gorgolyuk, Soviet Scientific Reviews, Section of Physiology and General Biology, Vol. 1: 267-306, 1987). The corresponding protease-sensitive agent was extracted, partially purified by chromatography on DEAE-Toyopearl and Heparin-Ultragel columns, and its neuralizing activity was tested in vitro on ectoderm isolated from early gastrulae of R. temporaria, Triturus alpestris, and Xenopus laevis at different concentrations and for different periods of time (animal cap assay). Induction of neural structures was found in R. temporaria and T. alpestris explants (up to 100 and 60%, respectively), but not in cultures of X. laevis ectoderm. Under our experimental conditions, so-called "autoneuralization" of the ectoderm explants can safely be excluded. The results are discussed in relation to the neural competence of amphibian ectoderm and the mechanisms of neuralizing actions of different factors which might be involved in neural induction and patterning.

[The symmetrical relation of intestines, the habenular nuclei and the intestinal venous system in dorsal-doral and ventral-ventral grown parabiontic larva of mountain salamanders (Triturus alpestris)]. / Die Symmetrieverhältnisse der Eingeweide, der Habenulakerne und des Darmvenensystems bei dorsal-dorsal und ventral-ventral verwachsenen Parabiose-larven des Bergmolches (Triturus alpestris).

Embryos of the newt Triturus alpestris were fused in their dorsal regions (DD-parabionts) of their ventral regions (VV-parabionts), while the longitudinal axes were in equal direction. In these parabionts situs and symmetry of the gut, the heart, the habenular nuclei and the vitelline vein were studied. The fusion of the embryos of the DD-parabionts was performed in three developmental stages (after Harrison): phase of neurulation (14-18, N-parabionts), late phase of neurulation (19-22, E-parabionts) and tail bud stage (23-27, S-parabionts). VV-parabionts were only fused in phase of neurulation (14-18). The external development of the parabionts in most cases was normally. DD-parabionts showed a more or less pronounced deformation of their longitudinal axes. In DD-parabionts all organs developed separately in nearly all cases. In VV-parabionts a partial fusion of the heart was sometimes observed, while fusion of large part of gut occurred regularly. In DD-parabionts complete and incomplete inversions of all organs and the vitelline vein are extremely abundant in the N-parabionts, much lower in E-parabionts and nearly absent in S-parabionts. The differences are statistically of high significance. The "pair situs" symmetries of N-parabionts (DD-parabionts) show always a strong dominance of a transindividual organ- and vitellin vein-symmetry. In VV-parabionts (N-parabionts) the tendency for inversion of the asymmetric organs is statistically significant lower as in DD-parabionts. In regard to this observation there is a preference for an asymmetric "pair situs" in the heart, habenular nuclei and vitelline vein system. In "polarisied" DD-parabionts ("left" as well as "right" parabionts) the asymmetric organs as well as the vitelline vein system inversions were more often observed in the "left" parabiont compared with the "right" parabiont in N- and E-parabionts. These right-left differences partial are statistically significant (P < 0.05). The organ situs-correlation as well as the situs-correlation between vitelline vein and gut or heart, respectively, are mostly positive (concordant). The frequency of dominance of "left" organ inversions argues against a morphogenetic "left dominance" of the amphibian embryo. The determination of organ-asymmetries is irreversible only after the end of neurulation in tail-bud-stage. The results suggest the efficiency of a "symmetry-factor", which determines the organ symmetry during neurulaphase. The differences between DD- and VV-parabionts are statistically significant and point to a dorso-ventral polarity of the embryo, with a maximal morphogenetic potency in the dorsal region.

Spinal cord neuron classes in embryos of the smooth newt Triturus vulgaris: a horseradish peroxidase and immunocytochemical study.

Spinal cord neurons were investigated in embryos of Triturus vulgaris, the smooth newt, just prior to hatching. These embryos can swim if freed from their egg membranes. Horseradish peroxidase (HRP) labelling, together with GABA and glycine immunocytochemistry (ICC), revealed nine distinct anatomical classes of neuron. 1. Ventrolateral motoneurons with mainly dorsal dendrites, sometimes a descending central axon and peripheral axon innervating the trunk muscles. 2. Dorsal primary sensory Rohon-Beard neurons innervating skin and with dorsal ascending and descending axons in spinal cord. 3. Commissural interneurons with mid-cord unipolar soma, glycine-like immunoreactivity, dendrites on initial segment of ventral axon which crosses cord to ascend or branch. 4. Dorsolateral commissural interneurons with multipolar soma in dorsolateral position with dorsal dendrites and ventral axon which crosses and ascends or branches. 5. Giant dorsolateral commissural interneurons with large dorsolateral somata widely spaced (130-250 microns spacing) with process projecting dorsally to other side, dorsolateral dendrites and ventral axon which crosses to ascend and branch. 6. Dorsolateral ascending interneurons in dorsolateral position with multipolar soma and ascending axon on same side. 7. Ascending interneurons with unipolar soma, GABA-like immunoreactivity and ascending axon on same side. 8. Descending interneurons with bi- or multi-polar soma, extensive dorsal and ventral dendrites, and descending axon on same side. They may also have ascending axons. 9. Kolmer-Agduhr cerebrospinal fluid contacting neurons with cilia and microvilli in lateral corners of neural canal. GABA-like immunoreactivity, no dendrites and ascending axon. Eight of the nine cells classes were found to bear a marked resemblance to neurons previously described in zebrafish and Xenopus embryos in terms of their anatomy, distribution and immunoreactivity to GABA and glycine. Homologies and possible functions are discussed. Giant dorsolateral commissural neurons, were not found in Xenopus or teleosts but were present in Ambystoma mexicanum and Neoceratodus. The regular, possibly segmental longitudinal distribution pattern of these cells within the cord is unusual among amphibian spinal neurons.