Search

Urodelan amphibian larvae develop three pairs of branching external gills on both sides of the pharyngeal region, and this study focuses on motile cilia on the gill surface. High-speed camera was used to observe ciliary strokes on the surface of the external gills of Pleurodeles waltl larvae. We found that the directionality of ciliary beating is position-dependent on the gill surface, and this rule is applicable to all the samples examined. For visualizing water flow around the external gills, we used bead suspensions. We revealed continuous anterior-to-posterior water flow generated by coordinated ciliary beating. Around the frontal surface of the gill stem (gill rachis), water flows countercurrent to the bloodstream beneath the gill epidermis. These results suggest that ciliary beating in each ciliated cell is coordinated, which cooperatively generates continuous and directional ciliary flow. We next visualized the overall distribution of ciliated cells on the gill surface by immunostaining of acetylated alpha-tubulin. Our results showed that the fine branches of external gills (fimbriae) have a circumferential distribution of cilia aligned orthogonal to the longitudinal axes of fimbriae, which facilitates water flow from proximal to the distal part of the fimbriae through the gills. This ciliary distribution pattern and directionality of cilia-driven flow are shared among five urodelan and two anuran species. Taken together, our findings suggest that the distribution and motility of ciliated cells on the surface of external gills is finely controlled, and this might support efficient respiration by the gills in urodeles.

One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration. Scaling and shaping of vertebrate muscles and skeletal elements has always been enigmatic and required an advanced technical level in order to analyse the cell distribution in 3D. In this work, synchrotron X-ray computed microtomography (µCT) and chemical contrasting has been exploited for a quantitative analysis of the 3D-cell distribution in tissues of a developing salamander (Pleurodeles waltl) limb – a key model organism for vertebrate regeneration studies. We mapped the limb muscles, their size and shape as well as the number and density of cells within the extracellular matrix of the developing cartilage. By using tomographic approach, we explored the polarity of the cells in 3D, in relation to the structure of developing joints. We found that the polarity of chondrocytes correlates with the planes in joint surfaces and also changes along the length of the cartilaginous elements. Our approach generates data for the precise computer simulations of muscle-skeletal regeneration using cell dynamics models, which is necessary for the understanding how anisotropic growth results in the precise shapes of skeletal structures.

Immunohistochemical study of the expression of recoverin (photoreceptor protein) in the retina of Pleurodeles waltl adult triton was carried out in health, during regeneration after removal, and under conditions of long-lasting detachment. Studies with polyclonal (monospecific) antibodies to recoverin showed that normally it is present in the internal segment, connective cilium, in distal portions of the external segments of cones and rods, and in Landolt clubs of displaced bipolar cells. Detachment of the retina is associated with translocation of recoverin from the photoreceptor processes to perikaryons, and the content of recoverin-positive displaced bipolar cells increases. During regeneration of the retina after its excision via conversion of the pigmented epithelial cells, recoverin is synthesized in the prospective photoreceptor perikaryons and then accumulates in the forming inner segments. Hence, recoverin can serve as a reliable marker in studies of photoreceptor differentiation and functioning during regeneration or survival of the retina.

The effect of the adhesion protein isolated from the bovine cornea was studied on the model of mechanical injury (cross cutting of the cornea). In the concentration of 10−12 mg/ml, the protein influenced the proliferation of corneal epithelial cells in newt Pleurodeles waltl in vivo. Experiments were conducted using autoradiography, and the nuclear labeling index (NLI) was determined at different times after surgery and in different corneal regions. This adhesion protein significantly induced proliferation of corneal epithelial cells relative to control groups with the injured eyes treated with the serum adhesion protein at the same concentration or water. The differences between the experimental and control animals were most pronounced 7 days after surgery. By day 14, they were less pronounced but still significant. On day 28, no significant differences in NLI were observed between the three groups, although these values remained higher than in intact animals. An increased pool of proliferating cells in the corneal epithelium was observed both in the affected and intact areas. The data obtained indicate that the biological activity of this protein is not species specific and that it can be a proliferation factor for corneal epithelial cells.

Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodelesnewts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoproteins of landmark lateral loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain lampbrush chromosome preparations in newts of the genes Pleurodeles. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker landmark loops along the chromosome length were constructed.

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.

The presence and localization of the calcium-binding protein recoverin, initially found in photoreceptors of the bovine eye, were immunochemically studied in retina of the new Pleurodeles waltl. Polyclonal monospecific antibodies against recoverin were raised and the methods of immunoblotting and indirect immunofluorescence were used. A protein with an apparent molecular mass of 26 kDa was found in the retina extract, which was specifically stained by the antibodies against recoverin. Localization of recoverin was studied on the retina sections: an intense reaction was found in the inner segments and a weak reaction was found in the basal part of the outer segments of photoreceptors and in Landolt's clubs of displaced bipolars. The results we obtained suggest for the first time the presence of recoverin in the retina of a representative of the Urodeles and indicate to interspecific conservativeness of this protein and differences of its localization in the retina photoreceptors in different species. The data obtained open a possibility of using recoverin as a marker protein of photoreceptors and displaced bipolars in studies of retina regeneration in newts.

The study of the population of bipolar-like cells (displaced bipolars) was continued in order to establish their role in development and regeneration of the retina in lower vertebrates. The size of the population of these cells was estimated on serial semithin sections in the retina of normal eyes in adult and young newt Pleurodeles waltl, as well as in adult newts subjected to long-term bright illumination. The population of displaced bipolars was significantly increased with reference to all cells of the outer nuclear layer. In young and illuminated animals, their numbers were approximately 1.3 and 1.4 times that in the adult animals not exposed to constant light. The results obtained favor the earlier suggestion of the involvement of the displaced bipolars in growth and restoration of the outer nuclear layer in the retina of newts during development and after trauma.

The recombination-activating gene 1 (RAG1) product is required for the somatic rearrangement of immunoglobulin and T-cell receptor genes. We cloned and sequenced the large continuous open reading frame coding for the salamander Pleurodeles waltl RAG1 protein. Semi-quantitative RT-PCR experiments were performed to quantify the expression of RAG1 in different tissues. The strongest signal was observed in the thymus of juvenile animals, confirming the primary lymphoid nature of that organ. Weaker expression was observed in the spleen, brain, and eyes of adults. Signals in these tissues represented 5.5%, 4.6%, and 2.0%, respectively, of the signal detected in the thymus. Expression in brain was confirmed by in situ hybridization. Similarly, low amounts of RAG1 transcripts were previously detected in the mouse brain. Moreover, the transcription of RAG1 begins as early as the neurula stages of development. These data suggest that the RAG1 protein could play a role in the central nervous system of vertebrates.

Experiments on stationary culture of posterior eye and roll-bottle culture of the whole eye from adult water lizards Pleurodeles waltl showed that sclera bioregulator produces a stabilizing effects on adhesion interactions between the sclera, choroid, and pigment epithelium and on the maintenance of viability of sclera fi broblasts and pigment epithelium cells.

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epitheliumin vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10−12 M solutions.

The aim of the present work was to study the carbohydrate moieties present on N- and O-linked oligosaccharides of duct cells of a urodele amphibian testis, by means of lectin histochemistry. It was found that duct cells have a carbohydrate composition that includes ensuremath α(1,3)-, α(1,4)- or α(1,6)-linked Fuc and Man on N-linked oligosaccharides, Gal and GlcNAc on O-linked oligosaccharides, and DBA-positive GalNAc, α(1,2)-linked Fuc and Neu5Acα(2,3)Gal β(1,4)GlcNAc on both N- and O-linked oligosaccharides. All the duct cells showed the same lectin labelling pattern, the only exception being some sparse duct cells that showed the sequence Neu5Acα(2,6)Gal/GalNAc. The possible roles of duct cells in sperm maturation and the hypothesis for a common origin of duct and follicle (Sertoli) cells in the urodele testis are discussed.

After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube.

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Galβ1,3GalNAc. Sialic acid was scarcely observed, the Neu5Acα2,-3Galβ1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.

The changes of myosin isoform pattern and of its associated light chains in relation to the myosin ATPase profile were analysed in different muscles of the hypothyroidian amphibian Pleurodeles waltlii submitted to terrestrial stepping, using electrophoretic and histochemical techniques. These changes were specific to the muscle type but appeared globally characterized by a type-IIB to type-IIA/I fibre transition associated with a transition from fast to intermediate and/or slow myosin isoforms. These results are similar to the effects of endurance training on locomotor muscles of mammals. The diaphragm of experimental animals was also characterized by a complete disappearance of the larval myosin isoforms which were detected in the diaphragm of control animals. The myosin pattern of ventricular muscle did not change following terrestrial stepping. This work indicates that thyroid hormone does not regulate the muscle adaptations that occur following terrestrial stepping and suggests a more complex mechanism of regulation in which innervation could be implicated.

The distribution of 5-HT-like-immunoreactive cell bodies and fibres was studied in the brains of the salamanders Hydromantes italicus, H. genei, Plethodon jordani, Desmognathus ochrophaeus (family Plethodontidae), and Pleurodeles waltl (family Salamandridae). In addition, double-labelling experiments with biocytin were carried out to identify the relationship between serotonergic fibres and neurons involved in the processing of sensory and sensorimotor information. In all species, 5-HT-immunopositive somata are found in the ventral thalamus close to the ventricle forming the paraventricular organ. In the hypothalamus, cells are labelled in the ependymal layer around the infundibular recess and at the lateral edge of the periventricular grey. In the pretectum, a few immunoreactive cells are situated dorsolaterally in the grey matter. In the tegmentum and medulla oblongata, cells of the raphe nuclei are regularly distributed along the midline; labelled perikarya are occasionally found in the cervical spinal cord. 5-HT-like-immunoreactive fibres are widely distributed throughout the nervous system. Densely arborizing fibres are found in the olfactory bulb, striatum and amygdala. Distinct fibre projections extend in the ventral thalamus and tectum. Biocytin tracing of striatal and tectal projection neurons and ascending reticular neurons combined with the demonstration of 5-HT suggest that the striatum, the tectum and the ascending activating system are strongly influenced by 5-HT.

The effects of cyclic AMP (cAMP) and cyclic GMP (cGMP) on dihydropyridine sensitive Ca2+ channels were investigated under voltage-clamp in defolliculated Pleurodeles oocytes. Intracellular injection of cAMP or extracellular application of the permeable cAMP analogue (8-Bromo cAMP, 8Br-cAMP) decreased the Ba current (IBa). This effect on IBa was blocked by the injection of protein kinase A inhibitor. Similar results were found upon internal application of the catalytic subunit of protein kinase A. In contrast, the injection of cGMP or perfusion of 8Br-cGMP increased IBa amplitude. The increase of IBa by 8Br-cGMP was blocked by the injection of the selective inhibitor of protein kinase G (KT5823). These results support the hypothesis that the basal Ba current amplitude of Pleurodeles oocytes is under the control of Protein Kinases A (PKA) and G (PKG) activity. This regulation of Ca2+ channels by the second messengers, and particularly by cAMP may reflect an important step in the maturation processus of Pleurodeles oocytes.

By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodeles. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of approximately Mr 180000 and a structurally nonrelated cytoplasmic protein of Mr 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.

The distribution of GABAergic neurons in brains of the family Salamandridae (Pleurodeles waltli, Triturus alpestris) has been investigated immunohistochemically with an antibody against gamma-aminobutyric acid (GABA). In adult animals, immunoreactive neurons, fibers, and terminals are abundantly labeled. In the telencephalon, pallial areas contain fewer GABAergic neurons and fibers than basal forebrain areas. The amygdalar complex and the habenulae have a complex pattern of GABA-immunoreactivity that is especially pronounced within the neuropil. The pretectal and basal optic systems are provided with GABAergic neurons, corroborating electrophysiological results. The dorsal thalamus and parts of the torus semicircularis are almost completely devoid of GABA-immunoreactive neurons. In the torus, magnocellular neurons known to project to the contralateral counterpart are distinctly GABA-immunoreactive. During ontogeny, GABAergic neurons arise early when the first reflexive movements occur after mechanical stimulation. At stage 28, cells are labeled initially near the nucleus of the medial longitudinal fasciculus, which is the first supraspinal tract to appear in ontogeny. At stage 30 (still before hatching), GABAergic neurons are found in the pretectum, immunoreactive neurons arising in the dorsal tegmentum slightly later. Both systems are known to mediate basic reflexes in gaze stabilization. The commissura posterior is GABAergic at early stages suggesting an important functional role in homonymous inhibition between both sides. Thus in salamanders, the neurotransmitter GABA displays a complex distribution, similar to that in other vertrebrates. This pattern emerges early in ontogeny.

The mitotic Z and W sex chromosomes in Pleurodeles seem to be identical. Earlier morphological and molecular analyses of lampbrush paired chromosomes in the female meiosis showed clearly that 20% of the chromosomal length located in the middle part of the sex bivalent (bivalent IV) is heteromorphic. We investigated here the base content and composition of the DNA axes in the heteromorphic region by quantitative fluorescence imaging using various base-specific (DAPI, Hoechst 33342 and chromo-mycin A3) or base-nonspecific (ethidium bromide) fluorescent DNA probes. Our results show a significantly higher percentage of AT bases in Z than in W differential sectors. In addition the entire base content of Z appears slightly higher than that of W.

The entire 1731 retrotransposon of Drosophila melanogaster, tagged with the E. coli lac Z gene inserted in its gag sequence, was injected into oocytes and fertilized eggs of the urodele amphibian Pleurodeles waltl. Expression of the reporter gene indicated that the 1731 promoter (its 5'LTR) is active in the embryos and not in the oocytes. It appeared that this element is regulated as amphibian genes are at the beginning of the development, i.e. that expression was detected after the mid blastula stage and maintained up to four or five days after injection. Another construction associating the modified 1731 promoter with the CAT gene is also expressed in Pleurodeles embryos during the same period of development. This indicated that the 1731 promoter issued from a Drosophila species is activated as promoting sequences of amphibian zygotic genes are, suggesting that in the case of horizontal transfer, 1731 can be expressed into vertebrate organisms.

An antiserum was prepared against the recently purified bullfrog (bf) growth hormone (GH); it was applied to sections of brain and pituitary of three urodele (Ambystoma, Pleurodeles and Cynops) and three anuran (Xenopus, Bufo vulgaris and B. japonicus) species. No immunostaining was obtained in the urodele pituitary, being consistent with the results of immunoblot analysis of the pituitary homogenate. In the three anuran species, strong immunoreactivity was observed in GH cells that were concentrated in the posterodorsal region of the pars distalis. No GH-like immunoreactivity was detectable in the brain of any of the species. A comparison using adjacent sections stained with anti-bf prolactin (PRL) confirmed the anteroventral localization of PRL cells. Colocalization of GH and PRL was not apparent. These data suggest that the molecular structure of amphibian GHs is considerably different between anurans and urodeles. The antiserum used in the present work shows a high species specificity, recognizing only anuran GHs. In contrast anti-bfPRL labeled PRL cells in all the amphibian species studied in the present work, suggesting that PRLs possess common amino acid sequences recognized by the anti-bfPRL.

The 180 kDa component of the neural cell adhesion molecule (NCAM-180), total NCAM (NCAM-total) and the polysialic acid modification of NCAM (PSA) show similar temporal and spatial regulation in the developing tecta of Pleurodeles waltl (salamander) and Discoglossus pictus (frog). Whereas NCAM-total is found throughout the tectal tissue on neurons and glia, NCAM-180 is only found on non-proliferating neurons and in fiber layers. PSA is expressed by a subset of NCAM-180-positive cells. Western blots show that there is little polysialylated NCAM-140 in the developing amphibian tectum. Regions unstained for PSA and NCAM-180 correspond precisely to the growth zones of the tectum. NCAM-180 and PSA are not present in tecta of early larvae. Staining intensity is strongest at midlarval stages for both antigens. At metamorphosis, PSA is strongly downregulated, whereas NCAM-180 is downregulated in juvenile animals. Both antigens are still present in fiber layers of adult animals. In dissociated tissue culture of the frog tectum, NCAM-180 is not present on astrocytes, but on neuronal cells. Expression is enhanced at cell contact sites, suggesting that NCAM-180 is involved in cell contact stabilization. This study shows that general features of temporal and spatial regulation of NCAM isoforms and PSA are highly conserved in frog and salamander tecta, despite large differences in the rate of cell migration and the degree of lamination in these homologous brain regions.

The development of the tectum mesencephali was studied in the frog Rana temporaria and the salamander Pleurodeles waltl by means of nuclear staining and by labeling of cells with bromodeoxyuridine (BrdU). The general spatial and temporal pattern of cell proliferation and cell migration is the same in both species, despite drastic differences in overall tectal morphology. However, the salamander species differs from the frog species by (1) a generally lower cell proliferation rate, (2) a reduction in the activity of the lateral proliferation zone, and (3) a reduction in the formation of superficial cellular layers. Because point (3) affects processes that occur late in ontogeny, our experiments provide evidence that the simple morphology of the tectum of Pleurodeles waltl, compared with the multilayered tectum of Rana, is a consequence of a paedomorphic alteration of the ancestral developmental pattern of the amphibian tectum.

in amphibian development are nowbeyond doubt; the ontogeny of almost any system ismore or less thyroid-hormone-dependent. Thyroid hor-mones are generally believed to control skeletal devel-opment in amphibians. Development of all systems andorgans depends on the interplay of two factors: the sen-sitivity of a developing tissue (or structure) to thyroidhormones and their concentration in plasma [1]. Thesame is true for skeletal development in general and thesequence of skull ossification in particular. However,the regulatory role of thyroid hormones in the skullossification has been experimentally demonstrated onlyin some anuran [2] and caudate (plethdontids) amphib-ians. Moreover, in the latter, only a few bones withmetamorphic development have been studied [3–5].However, the revealed effects of thyroid hormones areusually extrapolated to all cranial ossifications in allcaudate amphibians [6, 7], although it is unclearwhether such a broad extrapolation is justified, at leastas far as caudate amphibians are concerned. In them,extirpation of the thyroid gland, a procedure widelyused in experimental embryology, does not eliminatecranial ossification [8]. Hypophysectomy, whichblocks thyroid hormone synthesis despite the presenceof the intact thyroid gland, also fails to inhibit cranialossification. In addition, although some ontogeneticprocesses in caudate amphibians (e.g., hemoglobintransformation from the larval to the adult form) arebelieved to be induced by thyroid hormones, there isexperimental evidence suggesting that they are in effectindependent of these hormones [9].These findings facts described determined the goalsand the methods of our study. We examined the role ofthyroid hormones in cranial ossification in the ribbednewt by comparing the cranial ontogeny in normal ani-mals and animals with thyroid hypofunction. To inducethyroid hypofunction, ribbed newt larvae were rearedin thiourea beginning from the stage when they beganactive feeding. Thiourea is the so-called goitrogen, achemical inhibitor of thyroid hormone synthesis. Atspecified intervals, the larvae were fixed in formalinand stained in toto with an alcoholic solution of alizarinred “for bone” (bone-specific staining). The data onnormal cranial development were obtained by examin-ing the material kindly provided by Lebedkina and bysearching the literature for relevant information [10, 11].Until the premetamorphic stage at which the larvaldevelopment is completed, no difference between con-trol and thiourea-exposed animals could be detected inthe growth characteristics and the external morphology.After this stage, “normal” larvae underwent metamor-phosis, during which they lost external gills and the finfold, whereas thiourea-treated counterparts continuedto grow, retaining both structures and the overall larvalmorphology. As a result, giant larvae developed, mea-suring 120 mm in length on the seventh month of life.Normal P. waltl larvae at the premetamorphic stage,when their size reaches its maximum, are only 40–50 mm long [10–12].In the ribbed newt, all but three (maxillary, prefron-tal, and nasal) dermal bones of the skull develop duringthe larval period. The three elements indicated developlater in metamorphosis [10, 11]. Endochondral replace-ments take place at the end of the larval period and dur-ing metamorphosis, with the articular and mental bonesarising after completion of metamorphosis. During thelarval period (from the beginning of active feeding tothe end of the premetamorphic stage), the thiourea-exposed animals exhibited a normal pattern of dermalskull development: the sequence of ossifications, theonset time of their calcification, and the growth rates ofindividual bones were the same as in control larvae. Bythe end of the premetamorphic stage, the skull was cov-ered by all the dermal bones that it had to have at thisstage. The maxillary, prefrontal, and nasal dermalbones develop later in normal larvae, concurrently with

Galanin-like immunoreactivity was localized in the brain of Urodela (Ambystoma, Pleurodeles) and Anura (Bufo, Xenopus) by immunocytochemistry with anti-porcine galanin antiserum. In the four species, immunoreactive perikarya were observed in the telencephalon (striatum, amygdala), diencephalon preoptic area mainly along the anterodorsal wall of the preoptic recessus, suprachiasmatic nucleus, lateral hypothalamus, ventral and dorsal infundibular nuclei, paraventricular organ, and rhombencephalon (nucleus of the solitary tract). Galaninergic fibres extended in similar regions and in the medial septum, ventral telencephalon, ventral hypothalamus, median eminence, and various mesencephalic and rhombencephalic regions. Contacts with the cerebrospinal fluid cavity occurred along the preoptic recessus (Ambystoma) and the ventral infundibular wall (all species). Fibres were scarce in the neurohypophysis. The distal and intermediate lobes of the pituitary were virtually devoid of immunoreactivity. The galaninergic system appeared more developed in adult amphibia than in young animals, suggesting the stimulating influence of sex steroids on the expression of galanin as previously described in Anguilla. The extensive distribution of the galanin-like immunoreactive neurons in amphibian brains suggests that this peptide may act as a neuromodulatur and/or neurotransmitter.

Although the photomechanical response of the iris in amphibians has been characterized, the central pathway of reflex control is unknown. We investigated this pathway by electrophysiological and anatomical techniques in urodeles. Sustained responding neurons in the pretectum were localized and characterized. Three neuron types were found and compared with tonic neurons in mammals. They were driven by the contralateral retina and their dendrites arborized in the pretectal neuropil. The localization of cell bodies resulted in the identification of a physiological nucleus comparable with the nucleus praetectalis olivaris. It was shown that the tonic neurons project ipsi- and contralaterally to the oculomotor region, where axons terminate in deep layers of the stratum album. Using horseradish peroxidase transport the ciliary ganglion in urodeles was identified. It occurs in ontogenesis parallel to the first detectable pupillary light reflex corresponding to the onset of metamorphosis.

We analyzed by scanning electron microscopy (SEM) the intact lampbrush chromosomes of the Pleurodeles oocyte karyotype. Each of the 12 bivalents was identified and photomicrographed in phase contrast and at low SEM magnification before the structure of chromomeres and lateral loops was analyzed at higher magnifications. Stereographic examination showed that the chromomere structure is not as well defined as it appears in the light microscope. The characteristic morphology of typical landmarks such as granular or globular loops seems to be due to a more or less tight packing and coiling of matrix components. This packing probably corresponds to the storage of transcription products and therefore of genic information.

The effects of the divalent cation ionophore A 23187 and papaverine on the in vitro differentiation of isolated embryonic cells from young neurulae (Pleurodeles waltlii) were observed. These experiments suggest that an intracellular increase or decrease of the divalent cation concentration (Ca++) stimulates or disturbs the morphological differentiation of already determined embryonic cells from neurulae but does not change their developmental pathway.

The organization of the lampbrush chromosomes of Pleurodeles waltl was studied by fixation and embedding of oocytes in toto and correlated with that observed in end-embedded preparations of isolated chromosomes. Particular attention was focused on marker loops, like the granular and globular loops, and atypical structures known as spheres (S) and M. In both types of preparations, the majority of the loops, the so-called normal loops, and the granular loops appeared to share a common basic organization, with ribonucleoprotein (RNP) fibrils appearing as strings of 30 nm particles, as described by earlier authors. Some new types of loop organization were observed: (i) P loops with 45 nm RNP particles; (ii) dense granular loops; (iii) loops with a cylindrical organization. RNP fibrils formed by 60 nm particles were found to occur in association with the globular loops. EDTA staining suggested the presence of large amounts of RNP in the sphere but very little in M. Three morphologically different types of RNP granules could be observed free in the nucleoplasm.

In vivo irradiation of ovaries of Pleurodeles poireti by gamma-rays leads to structural rearrangement of lampbrush chromosomes in late vitellogenic oocytes (stages V and VI). The loops collapse into the chromomeres and the axes condense. Doses between 200 and 2,000 rads have been tested. We observed that such changes were dependent on the irradiation dose though the chronological order of the events was irrespective of the dose. The maximum effect was attained about 10 h after irradiation. The alterations are totally reversible. Over a period of 3 days chromosomes gradually relax regenerate loops and recover their normal appearance. In mid vitellogenic oocytes (stages III and IV) lampbrush chromosomes do not undergo radiation induced alterations. It seems that only full-grown oocytes are competent to respond to the ionizing-flow.

Monoclonal antibodies against germinal vesicle antigens from Pleurodeles oocytes crossreact with lampbrush chromosomes of various Triturus species: C36/6, A33/22 and B71/22 bind to most lateral loops, B24/3 labels the spheres, while A1/5 and B81 give a distribution of fluorescent loops which is highly reproducible and species specific. — The antigens involved were characterized by immunoblotting of electrophoretic gels of germinal vesicle proteins and the molecular weights of those that bound to monoclonal antibodies C36/6, A33/22, B24/3 and C3/1 were determined. — The possible relationship between sites immunostained by some monoclonal antibodies and given DNA sequences distributed along the chromosomes is discussed. A new approach to cytotaxonomic and cytogenetic studies through the use of monoclonal antibodies on lampbrush chromosomes is offered, which can give new insight into the molecular mechanisms of speciation and karyological evolution in European newt species.

The method of spreading transcription complexes has been applied to amphibian oocytes of Pleurodeles genus. Complexes of nucleolar origin show a regular and homogeneous organization similar to that described in other materials. The observations add to the interpretation as an amplification of nucleolar DNA and a redundancy of ribosomal cistrons in the two species studied. -- On the other hand, complexes of chromosomal origin display a great diversity. Two main characteristics can be drawn: the existence of several transcription units in a chromosomal organization unit and the possibility to point out a special architecture at the RNP fibril level. Applying a shadowing technique used for isolated molecules is an improvement compared with earlier methods based on PTA coloration.

A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.

In eggs of Pleurodeles treated with chloralhydrate (0.1 M) spindle and astral fibers are progressively destroyed after 4 hours, leading to apolar nuclei, apolar mitoses and “monopolar” mitoses, the so-called star metaphases. After 1 hour the spindle is shortened, but not narrowed and separated from the poles and asters. Its microtubules, grown before metaphase, are first inhibited at their ends near the centrospheres. After 4 hours, defibrillated achromatic material, stained by methyl blue, surrounds a clearer zone originating from the nucleoplasm, in which chromosomes are embedded. At the EM level the treatment induces the formation of unusual tubular bodies connected with the centrospheres and of similar bodies related to kinetochores and chromosomes. These bodies are formed of tubular residues, parallel or in concentric systems, the latter embedded in a matrix containing tightly packed filaments of 170 A diameter. The star metaphase is characterized by homogeneous centrospheres formed only of filaments and completely independent from kinetochores and chromosomes. Chromosomes are radially distributed around a central common mass, which keeps the chromosomes together; it is formed of a finely fibrous matrix containing disordered microtubular residues; kinetochores are embedded in the common mass. Fuzziness and alteration of chromosomes proceed as a direct action of the chloralhydrate. The star metaphase is not a real “monopolar” mitosis.

L'evolution des differents elements de la region cervicale du spermatozoide dePleurodeles waltlii a ete suivie et decrite depuis le tout debut de la spermiogenese. Leur origine est parfois delicate a definir. Certains dictyosomes restent en relation constante avec les granules pericentriolaires et les deux anneaux differents, meme lorsque ces derniers quittent la region acrosomienne.

We observed in Pleurodeles embryos, stage 34, that the duration of the cell cycle and its phases was approximately the same for every tissue but was easily modified by varying the temperature. The generation time and the duration of S phase in embryos submitted to a 12° C temperature instead of 26° C are tripled or quadrupled. A temperature rise produced a proportionale shortening inG 2 andM phases and a lengthening inG 1 phase. ThisG 1 phase is not detectable at 12° C but represent a 1/4 of the total generation time at 26° C. The more differentiated the cells are, the longer is theG 1 time. The cell population studied during these experiments are growing exponentially. Growth fraction, which represents the exponential growth basis, is temperature independent but has a tissue specificity. This growth fraction is smaller the more the tissue is differentiated. However, the relative rate of cell division, inversely proportional to the generation time, is temperature dependent and appears to control the embryo's relative rate of growth under different temperatures.

The fine structure of the ultimobranchial (UB) glands of two common laboratory urodeles, viz., larval axolotls, Ambystoma mexicanum Shaw and adult Pleurodeles waltlii Micahelles, is described and compared in what is believed to be the first ultrastructural report on urodele UB glands. The axolotl UB gland shows a wide variety of form, being represented by an elongated diffuse series of follicles and sometimes by one or two large discrete terminal follicular bodies. In these axolotl UB glands up to four cell categories are distinguishable including a tonofilamentous cell and a secretory cell that is possibly homologous with calcitonin-producing C cells of anurans or other vertebrates. These two cell categories are also found in the Pleurodeles gland. The possible significance of the various cells is considered.

The immunocytochemical localization of several substances with putative neurotransmitter or modulator properties was investigated in the retinae of three urodele species. Gamma-aminobutyric acid-like immunoreactive labelling appeared in different types of amacrine and horizontal cells. In addition, labelled fibres in the optic nerve were detected. It was not possible to determine whether these fibres were ganglion-cell axons or part of an efferent projection. Endogenous serotonin was found in several populations of amacrine cells including stratified and diffuse types. Glucagon-like immunoreactivity appeared in one bistratified amacrine cell type, and neurotensin-like immunoreactivity was detected in a single monostratified amacrine cell type. Metenkephalin-like-immunoreactive labelling was type. Metenkephalin-like-immunoreactive labelling was rare but found in several sublaminae of the inner plexiform layer. Thus each peptide-like-immunoreactive cell type makes up a distinct and unique population of cells and probably has a special functional role in retinal processing. There are striking similarities in the peptide-like immunoreactive patterns of Triturus alpestris and Necturus maculosus whereas in Ambystomatidae the peptide-like-immunoreactive systems appear to be differently organized. This supports the hypothesis that Salamandridae and Proteidae are more closely related to each other than to the Ambystomatidae.

The three-dimensional organisation of fibronectin (FN) in early amphibian embryos (Ambystoma mexicanum, Pleurodeles waltlii) was studied with the use of antibodies against amphibian-FN. Immunofluorescence labelling was performed on whole-mount specimens. It was shown that before gastrulation an extensive extracellular network consisting of anastomosed FN-fibrils underlies the roof of the blastocoel prior to the migration of mesodermal cells. Initially, FN-fibrils develop radially on the inner surface of ectodermal cells (early blastula stage) and become confluent there to elaborate a fibrillar network. During the late blastula stage the entire surface of the blastocoel roof (presumptive ectoderm and mesoderm) is covered by the FN-rich extracellular matrix. As gastrulation proceeds, the migrating mesodermal cells interact with the FN-fibrillar network. The results suggest that the FN-containing extracellular matrix plays an important role in mediating mesodermal cell-substratum contacts in gastrulating embryos. No FN-fibrils were found in other parts of the embryo.

The distribution of somatostatin (SRIF) - and corticotropin-releasing factor (CRF)-like - immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.

Injection of 5-hydroxytryptophan (5-HTP), a precursor of serotonin, induces dispersion of melanin in the amphibians, Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura), in the goldfish, Carassius auratus, and in the carp, Cyprinus carpio. It is accompanied by a dispersion of erythrophore pigments. In the pituitaries of Pleurodeles and goldfish, a stimulation of MSH cells, characterized by a significant nuclear hypertrophy, is also observed; in Carassius, MSH cells may become degranulated. Serotonin appears to exert a stimulating effect on MSH release in lower vertebrates. Swimming behavior is disturbed in the goldfish and the carp; gaseous metabolism in the swim-bladder may be affected by injection of 5-HTP, as previously reported in the eel. Prolactin (PRL) cells appear activated, but remain granulated in the treated goldfish. No clear response of PRL cells to injection of 5-HTP can be observed in Pleurodeles. A possible role of serotonin in Pleurodeles submitted to an experimental aeroionization is briefly discussed.

THE experiments referred to below are concerned with the problem of the regulative power of embryonic anlage, the study of which has been taken to prove the equipotentiality of the eye primordia in amphibian embryos in the stages following the closure of the medullary folds. In Pleurodeles waltli it has been shown1 that two optic vesicles may fuse together when that of the donor is transplanted in the close proximity of the optic vesicle of the host, keeping the same orientation of the normal position in the implanted eye. The degree of fusion depends upon the size of the removed portion of the vesicle in the host, and varies from that of double eyes partially fused to single eyes of larger size than the normal, which have developed by complete fusion of the two optic vesicles, which regulate its size afterwards, provided that the lens is not double.

Specimens of Pleurodeles waltl which were kept in deep water and shocked whenever they ascended to the surface for air soon ascended less frequently than yoked controls which were shocked independently of their own behavior whenever the experimental animals were shocked. The results suggest that, under suitable conditions, amphibians are capable of rapid learning.

The complexes of organs induced by centrifugation of Amphibia blastulas or gastrulas interfere with the primary axis of the embryo: (a) explantates of centrifugated ectoblast are always poorer in differentiation than the same ectoblast leftin situ; (b) the complexes arising in the ventral part of the embryo have the tendency to elongate parallel to the main axis of the embryo and become connected with the head of the primary embryo; (c) grafts made on Axolotl eggs show that, when the reaction is poor, it cannot express itself except in cases of close contact with the normal organizer; (d) reciprocally, implants of strongly reacting centrifugated ectoblast placed on the ventral part of a normal gastrula of Pleurodeles induce modifications of the primary organs, viz. the transformation of the first somites into chorda.