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At least 19 recordsLinked to original sources

PubMed1986

Stress and changes in the blood of newts, Notophthalmus viridescens, during early regeneration.

During the summers of 1984 and 1985, adult red-spotted newts, Notophthalmus viridescens, were maintained in the laboratory at 23 degrees +/- 0.5 degrees C under natural photoperiods. From each of the experimental animals, the right forelimb was amputated just proximal to the elbow. Control newts were not manipulated surgically. Eight, 15, and 22 days after the time of amputation, equal numbers of regenerating and control animals were sacrificed, and blood smears of each individual were prepared with Wright's stain. Mean differential counts of leukocytes of the two groups of newts indicated that the relative number of neutrophils increased and the relative number of lymphocytes decreased in the regenerating animals as compared to their controls (Fig. 1 and Fig. 2). Earlier studies had shown that lymphopenia and neutrophilia occur in red-spotted newts treated with hydrocortisone or with ACTH or subjected to environmental stress (Bennett and Daigle 1983). Consequently, it is suggested that amputation and/or early regeneration may stimulate the increased production of hormones associated with stress in vertebrates, which may, in turn, influence regeneration, itself, and that the detailed study of the distribution of leukocytes in Notophthalmus viridescens may provide an assay with which to study the regulation of regeneration in this species.

Animals↗

PubMed1976

On the diversity of sperm histones in the vertebrates. I. Changes in basic proteins during spermiogenesis 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.

Animals↗

PubMed1990

Renal responses to mesotocin in adult Ambystoma tigrinum and Notophthalmus viridescens.

Renal clearance methods were used to observe responses to the posterior pituitary peptide, mesotocin, by the kidneys of adult Ambystoma tigrinum and Notophthalmus viridescens. Ambystoma tigrinum responded to hypophysectomy with a decrease in urine flow and glomerular filtration rate (GFR); however tubular water reabsorption was not affected. Treatment with 5 ng/g mesotocin returned both urine flow and GFR to control levels. Notophthalmus viridescens responded to mesotocin treatment with an increased GFR. Ambystoma tigrinum decreased both plasma [Na+] and [K+] following hypophysectomy, however mesotocin did not reverse these decreases. Urine [Na+] did not change as a result of hypophysectomy alone. This ion increased in urinary concentration following mesotocin treatment of hypophysectomized salamanders. Renal tubular reabsorption of K+ decreased following hypophysectomy; however mesotocin replacement did not reverse this. We consider it unlikely that mesotocin is involved in these tubular responses because of the failure of hypophysectomy and mesotocin replacement to consistently mirror each other.

Adaptation, Physiological↗

PubMed1996

Electric field strength and epithelization in the newt (Notophthalmus viridescens).

There is convincing evidence that endogenous electric fields are necessary for normal wound epithelization, but it is unclear whether normal epithelization rates can be accelerated increasing normal field strengths. Although we confirmed that normal lateral fields are required for normal Notophthalmus viridescens epithelization rates, significant increases in epithelization were not achieved by increasing normal field strengths with direct current. With increases of less than 50%, epithelization rates were slightly increased. When the field strengths were augmented by 50%, epithelization was significantly retarded. This pattern was also observed when we applied direct current to wounds whose fields were nullified with benzamil. Epithelization was more rapid in wounds with field strengths raised to 20, 40, and 60 mV/mm than in benzamil-treated wounds without field augmentation. Epithelization was most rapid at 40 mV/mm, the normal value. When fields were augmented to 80 and 100 mV/mm, the epithelization rate diminished significantly. We also augmented field strengths by increasing the Na+ concentration in the medium surrounding the digits. Wounds healed more slowly in 10 mM Na+ than in 1.5 mM Na+, the normal pond water concentration. When field strengths in 10 mM Na+ were diminished to levels found in contralateral digits at 1.5 mM Na+ by applying direct current, the epithelization rate was restored to normal. We conclude that newt wound epithelization rates are nearly maximal at normal wound field strengths. Field strengths significantly higher than normal (50% more) result in significantly diminished rates of epithelization.

Amiloride↗

PubMed1999

Molecular cloning of the Notophthalmus viridescens radical fringe cDNA and characterization of its expression during forelimb development and adult forelimb regeneration.

Larval and adult newts provide important experimental models to study limb development and regeneration. These animals have exceptional ability to regenerate their appendages, as well as other vital structures. Our research examines the role of the fringe gene (fng) in the developing and regenerating adult newt forelimb. Fringe codes for a secretory protein. It was first discovered in Drosophila, and later homologues were isolated in Xenopus laevis, chick and mouse. This gene has been highly conserved throughout evolution, indicating its crucial role in vertebrate and invertebrate development. We have isolated, cloned, and sequenced the full length of the Notophthalmus viridescens radical fringe cDNA (nrFng) by screening a newt forelimb blastema cDNA library with a 500-bp fragment of the Xenopus lunatic fringe cDNA. The newt fringe cDNA codes for a 396 amino acid protein with a predicted N-terminal signal sequence. Newt fringe shows high homology with radical fringe homologues of many species. Whole mount mRNA in situ hybridization on several stages of newt limb development reveals that nrFng is first expressed in the limb field, with intense expression as the limb bud develops. However, gene expression diminishes with more advanced digit development. A significant role in adult forelimb regeneration is also evident, as we isolated the cDNA from a regeneration-specific library and found it highly expressed during the regenerative phases of active cell division and then down regulated at sites undergoing differentiation and morphogenesis.

Amino Acid Sequence↗

PubMed1980

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens.

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.

Animals↗

PubMed1995

Re-programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens).

We have previously shown, by in situ hybridization, that fibroblast growth factor receptor 2 (FGFR2) is present in the basal layer of wound epithelium during limb regeneration in newts (Notophthalmus viridescens). In contrast, FGFR1 expression is observed throughout the blastema mesenchyme but is distinctly absent from the wound epithelium (Poulin et al. [1993] Development 119:353-361). Sequence analysis revealed that we have isolated both the KGFR and bek variants of FGFR2. These two variants differ only in the second half of the last of their three (or two) Ig-like domains. In this report, we show the expression patterns of FGFR2 variants during limb regeneration by in situ hybridization. During the pre-blastema stages of regeneration, FGFR2 expression was observed in the basal layer of the wound epithelium and in the cells of the periosteum. The wound epithelial hybridization was observed when the KGFR-specific probe was used while the bek-specific probe hybridized to mRNA in the cells of the periosteum. As regeneration progresses to the blastema stages, KGFR expression continued to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. The bek-specific hybridization pattern observed at this stage corresponds specifically to the mesenchymal hybridization. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. Interestingly, there appears to be a dorsoventral gradient of the expression of both KGFR and bek variants of FGFR2, which are opposite each other at the later stages of regeneration. Thus, re-programming of expression of the two FGFR2 variants is required during the initial wound closure of limb regeneration. Remarkably, the expression patterns of KGFR and bek mimic those observed in the mouse limb bud during early embryonic development (Orr-Urtreger et al. [1993] Dev. Biol. 18:475-486). Moreover, our results suggest that the two FGFR2 variants have distinct roles in limb regeneration. Further investigation regarding the potential sources of the FGF ligands will help establish the roles that FGFs and FGFRs play in limb regeneration.

Amino Acid Sequence↗

PubMed1991

Tenascin localization in skin wounds of the adult newt Notophthalmus viridescens.

Earlier studies have shown that the extracellular matrix (ECM) protein tenascin (TN) is present between uninjured epidermal cells of urodele appendages, but is absent from most of the mesenchymally derived ECM. Following appendage amputation, this distribution is reversed. TN is lost from the epidermis and appears in the ECM of the stump and the regeneration blastema. In the present study, monoclonal and polyclonal antibodies to TN were used to localize this protein immunohistochemically in limbs of the adult urodele Notophthalmus viridescens at various stages following skin removal with or without damage to underlying muscle to determine 1) if the loss of TN by the epidermis and its gain by mesenchymal tissues occurs in wounds that do not require regulation by epigenetic mechanisms, and 2) if TN is present in the provisional wound matrix beneath migrating epidermal cells. In addition, skin explants were cultured on TN-coated dishes to learn if TN possesses active sites that can support epidermal cell migration. The results indicate that simple wounding leads to the same TN patterns as occurs following limb amputation. Tenascin loss from the epidermis could be seen as early as 6 hr after wounding, a time during which migrating epidermal cells are moving over the wound bed. During this period, there was no evidence of TN in the provisional wound matrix. In contrast to collagen, which supports considerable epidermal cell migration from skin explants, TN allowed no more migration than did the inactive protein, myoglobin.

Animals↗

PubMed1987

Influence of chromatographic fractions of extracts derived from bovine neural retina on newt (Notophthalmus viridescens) lens regeneration in vitro.

Removal of the ocular lens in adult newts (Notophthalmus viridescens) is followed by a series of cellular events leading to regeneration of a new lens by cell type conversion of pigmented iris epithelial cells at the dorsal pupillary margin (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). Following depigmentation and five to seven cell divisions, iris epithelial cells redifferentiate into lens fiber cells and synthesize crystallin proteins (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). This process is dependent upon neural retina in vivo (Stone, Anat. Rec. 131:151-172, 1958; Reyer, Dev. Biol. 14:214-225, 1966) and in vitro (Yamada et al., Differentiation 1:65-82, 1973). Acting on the hypothesis that the role of the neural retina is to promote passage of iris epithelial cells through the requisite number of cell cycles which will then allow them to redifferentiate as lens fiber cells (Yamada, in: Cell Biology of the Eye. Academic Press, New York, 1982), we undertook testing of the effects of eye-derived mitogenic substances, as well as other mitogens, on regeneration of lens from iris in organ culture. We have previously defined a critical period for the retinal influence in vivo and in vitro, and have shown that crude extracts of retina can enhance regeneration of lenses in culture (Connelly et al., J. Exp. Zool., 240:343-351, 1986). In this paper, we report on the lens regeneration enhancing activity (LRA) of more highly purified fractions of the retinal extracts. Heparin-sepharose chromatography of the crude retinal extract yields three fractions (Courty et al., Biochemie 67:265-269, 1985) called EDGF I, II, and III. EDGF I and II have affinity for heparin, while EDGF III does not. In our bioassay, LRA appears only in the EDGF III fraction. Dialysis of EDGF III against 0.1 N acetic acid yields a fraction which has affinity for cibacron blue sepharose (eluting at 2.15 M salt) and also has significant LRA. Because insulin at high doses has a marginal effect on lens regeneration in culture (Williams and McGlinn, Am. Zool. 19:923, 1979; Connelly, Differentiation 16:85-91, 1980), we tested IGF-I. Because of the putative neurotrophic effects of transferrin (Tf) (Mescher and Munaim, J. Exp. Zool., 230:485-490, 1986), we tested Tf for its ability to enhance regeneration of the lens in culture. IGF-I seems to have an enhancing effect on lens regeneration; Tf does not.

Animals↗

PubMed1988

Treatment of brachial nerves with colchicine inhibits limb regeneration in the newt Notophthalmus viridescens.

In urodele amphibians, limb regeneration is dependent on innervation and is blocked by the administration of colchicine. The objective of this experiment was to determine if colchicine blocks limb regeneration by a direct action on the blastema cells or by an indirect action on the nerves, specifically, if colchicine treatment of the brachial nerves would inhibit limb regeneration in the newt Notophthalmus viridescens. Colchicine was applied to the nerves by implanting a colchicine-loaded silastin block adjacent to the brachial nerves of an amputated newt limb. With appropriate dose levels of colchicine, limb regeneration was completely inhibited. Contralateral control limbs, carrying unloaded silastin blocks, and control limbs with colchicine-loaded blocks implanted equidistant from the blastema, but not adjacent to the brachial nerves, regenerated normally. Thus, the results indicate that the colchicine inhibition of limb regeneration is mediated by colchicine effects on the nerves. The possible mechanism of colchicine action on nerves may involve either wallerian degeneration, or inhibition of axoplasmic transport, or both.

Animals↗

PubMed1988

Appearance and regulation of an antigen associated with limb regeneration in Notophthalmus viridescens.

Previous reports from this laboratory have documented a cellular antigen, 22/18, associated with regeneration of forelimbs in the newt Notophthalmus viridescens (Kintner and Brockes: Nature 308:67-69, 1984; Journal of Embryology and Experimental Morphology 89:37-55, 1985; Brockes: Science 225:1280-1287, 1984; Kintner et al.: Molecular Basis of Neural Development, 1985). The antigen is expressed at various stages of regeneration in several types of cells including Schwann cells and muscle cells. We report here that, in addition to the previous results, the expression of 22/18 occurs very early in the regeneration process and is initially confined to an apparently nonmuscle, non-neural population of cells. The well-defined context of the antigen's first appearance has allowed us to investigate the regulation of its expression. In particular, injury is sufficient to elicit the appearance of 22/18. It can appear prior to mitosis and despite denervation. In circumstances where regeneration is inhibited, expression of 22/18 can persist.

Animals↗

PubMed1989

Delayed carpal ossification in Notophthalmus viridescens Efts: Relation to the progress of mesopodial completion in newt forelimb regenerates.

The persistence of cartilage in the adult newt (Notophthalmus viridescens) forelimb skeletal regenerate has recently been reported by Libbon et al. It is particularly evident in the carpal group, which remains cartilaginous for at least 9 months while all other regrown skeletal parts are either ossified or ossifying. The present investigation was undertaken to determine whether delayed ossification in the adult regenerate recapitulates a pattern evident during the development of the forelimb of the red eft (N. viridenscens' immature terrestrial phase). Right upper limbs of 20 efts were examined at low magnification to establish carpal composition and organization. Among five efts of the smallest size (26.54 plus or minus 2.20 mm snout-to-vent length), and displaying bright orange dorsal skin coloration, all carpal rudiments were cartilaginous. Eleven efts, intermediate in length (33.88 plus or minus 0.81 mm), displaying orange-green coloration, all carpals had ossified. These observations demonstrate that during adult newt forelimb regeneration, the failure of the carpal elements to begin ossification in synchrony with other regrown skeletal parts duplicates a similar schedule of delayed mesopodial ossification which is evident during the development of the red eft's forelimb.

Animals↗

PubMed1992

The effect of environmental pH on sodium balance in the red-spotted newt, Notophthalmus viridescens.

Sodium influx and efflux of the red-spotted newt, Notophthalmus viridescens, at two pHs were investigated in efts and adults in water and on land. The sodium efflux rate constant was not affected by pH. Efflux was generally greater under aquatic than terrestrial conditions, and for efts than for adults. In an aquatic setting, both adults and efts were able to maintain sodium balance at pH 5, but exhibited net sodium loss at pH 3. Sodium influx was greater in efts at pH 5 than in any other group. Adults in all treatments lost mass during the experiment, while efts in all treatments were able to maintain body mass. In their normal terrestrial environment, red efts probably avoid substantial sodium fluxes by maintaining a hydrophobic skin, which decreases contact with soil solutions.

Animals↗

PubMed1994

Factors altering DNA synthesis in the cardiac myocyte of the adult newt, Notophthalmus viridescens.

To better understand the mechanisms governing the proliferation of cardiac myocytes it is important to identify the factors controlling this phenomenon, and to characterize their actions. DNA synthesis was quantified in vitro in ventricular myocytes from the adult red-spotted newt, Notophthalmus viridescens. Ventricles were enzymatically separated and plated onto laminin. Myocytes were fed modified L-15 medium with 10% fetal bovine serum, and were variously treated with transforming growth factor-beta, transforming growth factor-beta combined with platelet-derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, 12-0-tetradecanoylphorbol-13-acetate, heparin, or conditioned medium from ventricular myocytes or non-myocytes (primarily endothelial cells). With their final feeding the cells were given 1 mu Ci/ml of tritiated thymidine, and 24 hours later were fixed and stained. Dishes were coated with photographic emulsion, exposed, and developed. The percent of cells with labeled nuclei was determined. Experimental media that significantly increased DNA synthesis included those containing acidic fibroblast growth factor (121% of control), basic fibroblast growth factor (119% of control), 12-0-tetradecanoylphorbol-13-acetate (233% of control) and conditioned medium from ventricular myocytes (230% of control) or non-myocytes (128% of control). Media significantly inhibiting DNA synthesis were those containing heparin (31% of control), transforming growth factor-beta (38% of control), non-myocyte conditioned medium and heparin (75% of control), or transforming growth factor-beta and platelet-derived growth factor (63% of control).

Animals↗

PubMed1987

Transcription of the histone loci on lampbrush chromosomes of the newt Notophthalmus viridescens.

We have investigated transcription of the histone gene cluster on lampbrush chromosomes of the newt Notophthalmus viridescens. Clusters of the five histone genes are separated by long tracts of a DNA repeat called satellite 1. Previous in situ hybridization results demonstrated the presence of histone gene coding regions, intergene spacers, and flanking satellite repeats in giant transcription units on lampbrush chromosomes. These results suggested a model in which transcription initiates at histone gene promoters and continues through the rest of the gene cluster into satellite repeats. The readthrough transcription model predicts that spacer regions upstream of the first promoter in the gene cluster should be absent from nascent transcripts on chromosome loops. We have used such upstream spacer probes for in situ hybridization to lampbrush chromosomes. Contrary to our expectation, the results show that upstream spacer regions are transcribed, and indicate that transcription often initiates upstream of the first histone gene promoter. The relationship of giant transcription units in oocyte nuclei to cytoplasmic histone mRNA is discussed.

Animals↗

PubMed1986

Magnetic compass orientation in the Eastern red-spotted newt (Notophthalmus viridescens).

Laboratory tests were carried out to examine the orientation behavior of adult Eastern red-spotted newts (Notophthalmus viridescens) to earth-strength magnetic fields. Groups of 30 to 40 newts were housed in water-filled, all-glass aquaria with an artificial shoreline at one end. The aquaria were located in a greenhouse or outdoors adjacent to the laboratory building, and aligned on either the magnetic north-south or east-west axis. Tests were carried out in an enclosed indoor arena. Newts were tested in four horizontal alignments of the magnetic field: the ambient magnetic field (magnetic north at North) and three altered fields (magnetic north rotated to East, South or West). Data were analyzed after pooling the magnetic bearings from all four conditions in such a way as to retain the component of the newts' orientation that was a consistent response to the magnetic field. Elevation of training tank water temperature was used to increase the newts' motivation to orient in the direction of shore. Newts exposed to a training tank water temperature of 33-34 degrees C just prior to testing exhibited consistent unimodal magnetic compass orientation. The direction of orientation was altered predictably by changing training tank alignment and location relative to the laboratory building. The results provide the first evidence of a strong, replicable magnetic compass response in a terrestrial vertebrate under controlled laboratory conditions. Further, the present study demonstrates that the Eastern newt is able to learn a directional response relative to the earth's magnetic field.

Animals↗

PubMed1990

The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt, Notophthalmus viridescens, during limb regeneration.

Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt, Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.

Animals↗

PubMed2004

Growth and apoptosis during larval forelimb development and adult forelimb regeneration in the newt ( Notophthalmus viridescens).

Many of the genes involved in the initial development of the limb in higher vertebrates are also expressed during regeneration of the limb in urodeles such as Notophthalmus viridescens. These similarities have led researchers to conclude that the regeneration process is a recapitulation of development, and that patterning of the regenerate mimics pattern formation in development. However, the developing limb and the regenerating limb do not look similar. In developing urodele forelimbs, digits appear sequentially as outgrowths from the limb palette. In regeneration, all the digits appear at once. In this work, we address the issue of whether regeneration and development are similar by examining growth and apoptosis patterns. In contrast to higher vertebrates, forelimb development in the newt, N. viridescens, does not use interdigital apoptosis as the method of digit separation. During adult forelimb regeneration, apoptosis seems to play an important role in wound healing and again during cartilage to bone turnover in the advanced digits and radius/ulna. However, similar to forelimb development, demarcation of the digits in adult forelimb regeneration does not involve interdigital apoptosis. Outgrowth, rather than regression of the interdigital mesenchyme, leads to the individualization of forelimb digits in both newt development and regeneration.

Animals↗