Newt Notophthalmus Viridescens Research Articles

In vitro studies of the influence of prolactin on tail regeneration in the adult newtNotophthalmus viridescens.

In vitro experiments were carried out to determine the effects of prolactin, and prolactin in combination with other hormones on the regeneration of adult newt tail blastemata. A total of 271 blastemata were explanted 13 days postamputation and were organ cultured for 96 h at 20 (±1)°C. Treatment with prolactin alone resulted in an increase in the blastema cell density of the tail regenerates. Cell accumulation and cell alignment were observed ventral to the reconstituted spinal cord. Prolactin and thyroxine, in combination, improved development of tail regenerates as compared with treatment with prolactin or thyroxine singly, supporting the results of earlier in vivo studies. Optimal development was obtained only when prolactin, insulin, thyroxine and hydrocortisone were added to the culture medium. Regeneration of tail explants maintained in medium augmented with the four hormones closely resembles that of in vivo tail blastemata 17 days post-amputation.

The effect of pH on tricaine methanesulfonate induced anaesthesia of the newt Notophthalmus viridescens

The anaesthetic tricaine methanesulfonate (TMS), when dissolved in distilled water, yields an acidic solution. To determine if pH affected the anaesthetic efficacy of TMS in the newt Notophthalmus viridescens, newts were anaesthetized in 0.1% TMS solutions adjusted to pH's of 3, 4, 5, 6, 7, or 8 with NaOH. As the pH increased, there was a significant decrease in the time required to induce anaesthesia and an increase in the time required to recover from the anaesthetic. We conclude that adjusting the pH of TMS solutions towards neutrality increases their anaesthetic efficacy.

Levels of cyclic GMP and cyclic AMP in regenerating forelimbs of adult newts following denervation.

The effect of short-term denervation (0, 12, 24, and 72 hours) on the levels of cyclic 3'5'-guanosine monophosphate (cGMP) and cyclic 3'5'-adenosine monophosphate (cAMP) in adult newt (Notophthalmus viridescens) forelimbs at 15, 22, and 35 days of regeneration was investigated. Regenerate blastema and stump cyclic nucleotide levels were compared with those of the contralateral intact forelimb and hindlimb, with levels in the normally regenerating blastema, and with levels measured in the forelimbs of intact, nonoperated animals. Variations in cyclic nucleotide levels occurred according to regeneration stage and tissue type. Changes in level were noted immediately upon denervation and subsequently at other sample times in all regenerate and control series. Parallel fluctuations occurred in regenerate stump and contralateral intact forelimbs. Our results from nonamputated denervated and sham-denervated animals indicate that short-term, denervation-associated cyclic nucleotide fluctuations cannot be attributed solely to the loss of innervation.

Activation and partial characterization of prolyl hydroxylase from regenerating limbs of the adult newt Notophthalmus viridescens

1. 1. Prolyl hydroxylase activity extracted from regenerating newt ( Notophthalmus viridescens) limb tissues can be increased by brief preincubation with cofactors (ascorbate, α-ketoglutarate, Fe 2+ and O 2) prior to assay with [ 3H]proline-labeled collagen substrate. 2. 2. Newt prolyl hydroxylase is optimally active at 30°C, but loses activity rapidly at 37°C. The presence of cofactors or substrate decreases enzyme heat lability. 3. 3. Detergents (Triton X-100 and octyl glucoside) do not aid enzyme extraction and inhibit enzyme activity. Activity solubilized during homogenization without detergent remains soluble following high speed centrifugations. 4. 4. Comparative studies are reported for enzyme extracted from chick embryo tissues.

Preparation of cell cultures attached to ultrathin plastic films for EM analysis

This chapter discusses the stereoscopic electron microscopy of chromosomes. The studies on the higher order structure of chromosomes suggest that stereomicroscopy at 1MeV is at present the most useful method of obtaining information on the complex three-dimensional structure of chromosomes. The chromatin fibers are suspended in the hypophase from a protein surface film and can be picked up by touching a grid covered with a carbon-coated Formvar film to the surface. They are fixed in 4% aqueous paraformaldehyde. The native 20-nm fiber is a supercoil of the closely packed string of nucleosomes stabilized by Ca2+. Polytene chromosomes can be isolated from dipteran larval tissue by squashing in 50% acetic acid between a slide and a coverslip. The supercoil in the 20-nm fibers released from erythrocyte nuclei of the newt Notophthalmus viridescens by spreading on a water surface is shown. It is found that in this nucleus, all chromatin is transcriptionally inactive and is in the form of 20-nm fibers.

Cyclic AMP and cyclic GMP levels in regenerating adult newt forelimbs following anaesthetization

Levels of cyclic AMP and cyclic GMP in adult newt (Notophthalmus viridescens) intact and 22- and 35-day regenerate forelimbs were studied, as were the levels in the contralateral intact forelimbs and hind limbs. MS 222 anaesthetization results were compared with those of liquid nitrogen fixation. We observed an elevation in the levels of cAMP in animals having undergone MS 222 anaesthetization as compared with those immersed in liquid nitrogen. Cyclic GMP levels appear to be comparatively unaffected. Both the anaesthetic and liquid nitrogen animal series displayed differences in cAMP and cGMP levels according to the particular tissue sample taken and the stage of regeneration.

Histone gene clusters of the newt notophthalmus are separated by long tracts of satellite DNA

The genomic organization of the histone genes of the newt Notophthalmus viridescens is described. Genes for the five proteins are clustered on a 9.0 kb segment of cloned DNA which is part of a homogeneous family of sequences containing 600–800 members per haploid genome. The 9.0 kb histone gene clusters are not adjacent in the genome, but are separated from neighboring clusters by up to 50 kb or more of cluster spacer sequences; some or all of these spacer sequences are members of a predominantly centromeric satellite DNA with a 225 bp repeating unit.

Effect of bovine pituitary hormone preparations on newt lens regeneration in vitro: Stimulation by thyrotropin

The anterior lobe of the pituitary gland can stimulate lens regeneration from the dorsal iris in the newt Notophthalmus viridescens. We have studied the effect of pituitary hormone preparations on this process. Dorsal irises were cultured for 20 days in diluted Medium 199 supplemented with 10% fetal calf serum. Bovine thyrotropin TSH-B8 at concentrations of 30 to 3000 μg/ml significantly stimulated lens regeneration in these dorsal irises. Well-developed lenses, up to stage 9, were formed, in which γ-crystallin, a protein specific for lens fibers of young lenses, was detected by immunofluorescence. Additionally, the mitotic index was 5.5 times elevated in these explants when compared to their controls. Lutropin LH-B10 at concentrations of 30 to 3000 μg/ml, prolactin PRL-B4 at concentrations of 23 to 1600 μg/ml, and porcine adrenocorticotropin ACTH-6002 at concentrations of 3 to 300 μg/ml did not stimulate lens regeneration. A weak stimulation of lens formation was observed in iris cultures with 2700 μg/ml of follitropin FSH-B1 or 3000 μg/ml somatotropin GH-B18, but not at concentrations of 30 μg/ml. Our results suggest that the inherent ability of the dorsal iris to form lens can be activated by the bovine thyrotropin preparation TSH-B8.

Role of prostaglandins in amphibian limb regeneration

Following limb amputation in the newt (Notophthalmus viridescens), the animals were given daily intraperitoneal injections of 0.1 or 1.0 mg of either indomethacin or acetylsalicylic acid. This had no observable influence on the rate or extent of limb regeneration or the histological appearance of the regenerating limb when compared with controls. Following limb amputation in toads (Bufo americanus), the limb stumps were injected daily for the first 15 days after amputation with 5 μg of either prostaglandin E1 or prostaglandin E2. Neither of these treatments had any observed effect on subsequent wound healing of the limbs when compared with controls. These observations suggest that prostaglandins do not play a major role in control of amphibian limb regeneration.

Characterization of a cloned histone gene cluster of the newt Notophthalamus viridescens.

We report the cloning and characterization of a histone gene cluster of the newt Notophthalamus viridescens. Fragments containing newt histone genes were identified in whole genome Southern blots; these fragments were cloned into a bacteriophage lambda cloning vector constructed for this purpose. The positions of most of the histone genes were determined by hybridizing subcloned sea urchin histone genes to digests of the cloned newt gene cluster. The position of each gene was verified, and its polarity determined by sequencing a portion of each. The order of the genes in the cloned segment is H1-H3-H2B-H2A-H4, with each of the genes but H2B being transcribed in the same direction. Subcloned segments of the histone gene repeat were used to determine the size of each newt oocyte histone mRNA.

Hyperpigmented patches in the skin of the newt Notophthalmus viridescens

In the integument of the red-spotted newt there occasionally appear patches of skin which are at the same time melanistic and iridescent. Such hyperpigmented patches have been found on the back, on the tail and on the dorsal surface of both fore and hind limbs. Cytological examination of several such areas revealed the presence of large numbers of chromatophores distributed throughout the dermis. The majority of the chromatophores consisted of atypically large and dendritic melanophores, which contained typical pigment granules. The iridescence resulted from a high incidence of iridophores. Xanthophores also were found in considerable abundance. This extensive and apparently random intermingling of melanophores, iridophores and xanthophores in limited areas constitutes a striking exception to the usual distributional patterns of pigment cells in this animal.

Chapter 5 Stereoscopic Electron Microscopy of Chromosomes

Publisher Summary This chapter discusses the stereoscopic electron microscopy of chromosomes. The studies on the higher order structure of chromosomes suggest that stereomicroscopy at 1MeV is at present the most useful method of obtaining information on the complex three-dimensional structure of chromosomes. The chromatin fibers are suspended in the hypophase from a protein surface film and can be picked up by touching a grid covered with a carbon-coated Formvar film to the surface. They are fixed in 4% aqueous paraformaldehyde. The native 20-nm fiber is a supercoil of the closely packed string of nucleosomes stabilized by Ca 2+ . Polytene chromosomes can be isolated from dipteran larval tissue by squashing in 50% acetic acid between a slide and a coverslip. The supercoil in the 20-nm fibers released from erythrocyte nuclei of the newt Notophthalmus viridescens by spreading on a water surface is shown. It is found that in this nucleus, all chromatin is transcriptionally inactive and is in the form of 20-nm fibers.

Fine structure of the surfaces of the epithelium and mesenchyme of the newt limb regenerate, separated in salt solution: 14 to 25 days.

Forelimbs of the newt Notophthalmus (Triturus) viridescens were amputated at the wrist, and at 14 to 25 days, the new limb regenerates were removed by a razor-blade cut. After incubation of the isolated regenerates in a medium containing only inorganic salts, the epithelium was removed easily from the underlying mesenchyme. The surfaces separated in the salt solution were examined using scanning and transmission electron microscopy to evaluate the completeness of the separation and the extent of tissue damage near the plane of separation. Rarely was an epithelial cell found clinging to the mesenchymal surface, supporting previous light microscopic observations. The most apparent morphological damage was sustained by the basal epithelial cell surfaces, which exhibited blebbing; the formation of vacant vesicles within the cytoplasm near the newly exposed plasma membranes; the structural disruption of some organelles; and the tearing away of fragments of the basal epithelial cell plasma membranes, which remained adherent to the mesenchymal surface during separation procedure. Examination of the structure of the epithelial-mesenchymal interface of intact regenerates showed that the plane of epithelial-mesenchymal separation was, for the most part, between the basal epithelial cell membrane and its substrate, an extracellular material which remained at the mesenchymal surface after separation. This substrate was in the form of an extracellular material closely adherent to the basal epithelial cell membrane at the distal tips of the regenerates; a developing basal lamina at the periphery of regenerates undergoing blastema accumulation; or a mature basal lamina at the base of digit stage regenerates. The usefulness of these separated tissues for future experimentation and the evaluation of the nature of the epithelial substratum throughout the course of limb regeneration are discussed.

Neural and hormonal stimulation of DNA and protein synthesis in cultured regeneration blastemata in the newt Notophthalmus viridescens

Distal portions of cone-stage newt forelimb blastemata were cultured transfilter to spinal ganglia for 36 or 72 hr. Addition of insulin to the medium consistently resulted in a significant increase (250% in ganglionated and 238% in nonganglionated blastemata) in the incorporation of [ 3H]thymidine into DNA, as compared to nontreated controls. When blastemata were cultured without ganglia for 36 or 72 hr, DNA synthesis decreased to 73 and 71%, respectively, of that achieved by ganglionated explants. When insulin was excluded from the medium, DNA synthesis decreased to 40% of insulin-treated explants, and, in the absence of both nerves and insulin, it declined to 31% of insulin-treated, innervated explants. The presence of insulin in the medium also resulted in an augmentation of ( 14C)-labeled amino acid incorporation into proteins; the average increase was 168%, as compared to untreated controls. l-thyroxine, growth hormone and hydrocortisone in combination with insulin, did not enhance the effects on DNA or protein synthesis of insulin alone. Also, exogenous cyclic nucleotides (cAMP, cGMP) and alterations of their endogenous levels with acetylcholine, sodium azide, theophylline or prostaglandins failed to elicit significant changes in DNA or protein synthesis. The existence of a synergistic action on DNA synthesis between nerves and insulin is suggested.

The Effect of Colchicine on the Induction of the Stellate Configuration in Dorsal Iris Epithelial Cells of the Newt Notophthalmus viridescens, in vitro

Summary Treatment of cultured cells derived from the dorsal iris of the newt, Notophthalmus viridescens, with cAMP or substances which increase intracellular levels of cAMP induces a transient but striking change in the cells’ morphology [1, 2]. The resultant formation of fine cytoplasmic projections radiating from the cell centre has been called the stellate configuration [1, 2], and it has been suggested that similar processes occur during depigmentation and cellular dedifferentiation during lens regeneration in vivo [1, 2]. The present study was undertaken to determine if microtubules play a role in the formation of the stellate configuration in iris epithelial cells in vitro. Thus, cells derived by outgrowth from iris tissue explants were treated with a variety of agents to induce the stellate configuration. The most effective agents tested were papaverine and adenosine. Subsequently cells were treated with the inducing agents adenosine and papaverine, and the drug colchicine. Colchicine caused a disruption or modification of the stellate configuration, except in cases where very high doses of the inducing agents were used. When cells were first treated with the inducing agents, followed 80 min later by colchicine treatment, the stellate configuration was rapidly lost. These results strongly suggest that microtubules are involved in the production of the stellate configuration in iris epithelial cells in vitro and are thus implicated in the depigmentation and dedifferentiation responses in lens regeneration in vivo.

Redifferentiation, cellular elongation and the cell surface during lens regeneration

ABSTRACT Dorsal irises from normal and lentectomized eyes of the newt Notophthalmus viridescens were cultured in vitro in the presence of colchicine (1·25 × 10−3 to 2·5 × 10−6 M), vinblastine sulfate (4·4 × 10−8) and cytochalasin B (20 μg/ml). Explants were treated for 24 h and implanted into host eyes of previously lentectomized newts. In regenerates treated with colchicine at stages prior to the onset of lens fiber formation, cellular elongation was inhibited or considerably delayed. Cytochalasin B has no effect on lens fiber differentiation when compared with regenerates treated with its solvent, dimethyl sulfoxide. Colchicine-treated regenerates showed no detectable fluorescence when stained with anti-newt lens fluorescent antibodies, suggesting that cells in which cellular elongation was inhibited were unable to synthesize lens specific proteins. Ultrastructural observations of elongating lens regenerate cells showed microtubules present in the cytoplasm parallel to the axis of elongation. Microtubules were not observed in depigmented cells from colchicine-treated regenerates. Characteristic vinblastine-induced microtubule crystals were present in depigmented cells from vinblastine-treated regenerates. Experiments using cell electrophoresis showed that neuraminidase-sensitive groups were detectable at the ionic double layer of cells from implanted regenerates at stages where lens fiber differentiation occurs. Cells from colchicine-treated regenerates showed no significant reduction of their electrophoretic mobility after neuraminidase treatment. This suggests that the appearance of some cell surface components in the redifferentiated cell may also be colchicine sensitive and probably mediated by microtubules.

On the Diversity of Sperm Histones in the Vertebrates: I. Changes in Basic Proteins during Spermlogenesis in the Newt Notophthalmus viridescens

Sperm histones display great variability in contrast to the conservation of most classes of somatic histones. To study this paradox, this series of papers examines the variation of histone patterns in the testis and sperm of vertebrates, particularly amphibians and reptiles, and attempts to relate such variation to genetically based sex determination as hypothesized by Bloch [Genetics Supplement 61, 93 (1969)]. In the present study we have investigated spermiogenesis in the newt Notophthalmus viridescens viridescens. Cytochemical experiments indicate that the basic nuclear proteins undergo progressive shifts from somatic type histone leads to very arginine-rich "stable protamine" in the later spermatids leads to protamine in the mature sperm. Electrophoresis of Notophthalmus histones extracted from chromatin reveals that the pattern of testes specific basic proteins in the urodele is distinct from the pattern of testicular proteins in the anurans Bufo americanus and Xenopus laevis. Species within the class Amphibia therefore exhibit considerable diversification in their type of basic sperm proteins.

Inversion heterozygosity in females of the newt Notophthalmus viridescens and its influence on chiasma distribution.

In a wild population of the American newt Notophthalmus viridescens 15 females out of a total of 94 were found to be heterozygous for a paracentric inversion which includes almost the whole of the longer arm of the smallest chromosome (XI). The inversion was recognized in preparations of lampbrush chromosomes because it transfers the sequential loops, which normally lie close to the telomere, to a position neighbouring the centromere. Because of inversion the transcriptional polarity of the sequential loops is reversed vis-à--vis the chromosome as a whole. In normal bivalents XI (both in male and female meiosis) each arm pair generally forms a single chiasma close to the telomeres (proterminal localization). In bivalents XI heterozygous for the inversion no chiasmata are formed between the mutually inverted longer arm pairs, presumably because they fail to synapse, but chiasma frequency in the non-inverted shorter arm pairs is increased, and the normal restraint on chiasma distribution in this arm pair is lifted. An explanation is offered in terms of the availability of recombination nodules, and the time of their association with the synaptonemal complex.

Ribosomal RNA Synthesis in the Eastern North-American Newt, Notophthalmus viridescens

Ribosomal RNA (rRNA) synthesis, the initiation of which is an early major event during the transformation of iris into lens in the newt, was characterized in the TVI cell-line derived from the eastern North-American newt Notophthalmus viridescens. Employing the technique of polyacrylamide gel electrophoresis, molecular-weight measurements were made on newt rRNAs using Xenopus laevis and E. coli rRNAs as standards. The molecular weights of N. viridescens 28S and 18S rRNA were found to be 1.4 X 10(6) and 0.7 X 10(6) respectively. The precursor to these RNAs had a molecular weight of 3.1 X 10(6). Three probable intermediates in the processing of precursor to mature rRNA were also identified. On the basis of the molecular weights of all species of RNA identified, a processing pathway, similar to that of Xenopus, has been suggested. Some unusual features in the kinetics of precursor rRNA labelling and processing suggest the possibility that newt-cell rRNA synthesis may be controlled by the availability of essential amino acids in a manner similar to that observed in mammalian cells. A possible relationship between the availability of essential amino acids, the initiation of rRNA synthesis in the newt iris, and the control of lens regeneration is discussed.

An Established Cell Line from the Newt Notophthalmus viridescens

An established cell line, derived from the dorsal iris of the eastern North American newt, Notophthalmus viridescens, is described. The cells display an epithelial-like behaviour in culture, grow relatively slowly, possess considerably larger chromosomes than mammals and are heteroploid, although some near-diploid cells are present in the culture. The line is characterized by a strong tendency to overlapping and aggregation in spite of its origin from adult tissue.