Adult Newt Research Articles

A newt's eye view of lens regeneration

In this paper we describe the basic process of lens regeneration in adult newt and we pinpoint several issues in order to obtain a comprehensive understanding of this ability, which is restricted to only a few salamanders. The process is characterized by dynamic changes in the organization of the extracellular matrix in the eye, re-entering of the cell cycle and dedifferentiation of the dorsal iris pigment epithelial cells. The ability of the dorsal iris to contribute to lens regeneration is discussed in light of iris-specific gene expression as well as in relation to factors present in the eye.

Effects of a CDK inhibitor on lens regeneration.

Lens regeneration in adult newts is always initiated from the dorsal iris by transdifferentiation of the pigment epithelial cells. One of the most important early events should be the ability of pigment epithelial cells to dedifferentiate and re-enter the cell cycle. As a first step in an attempt to study this event, we have decided to examine the effects of a cyclin-dependent kinase-2 inhibitor on lens regeneration. At the appropriate concentration, this inhibitor completely abolished the ability of pigment epithelial cells to form a new lens, but it did not stop them from dedifferentiating and forming a small lens vesicle. The effects of this inhibitor seem to be mediated by its opposite effects on cell proliferation and apoptosis. The inhibitor significantly reduced cell proliferation and enhanced apoptosis of pigment epithelial cells both in vitro and in vivo and of the regenerating lens in vivo.

Use of chemical cues in detection of conspecific predators and prey by newts, Notophthalmus viridescens

Detection of heterospecific predators and prey via chemical cues is well known, but only a few studies have examined the potential for such discrimination in cannibalistic systems. In newts, Notophthalmus viridescens, adults are opportunistic predators of conspecific larvae. I used a laboratory bioassay to determine whether larvae and adults distinguish between chemical stimuli from members of the different age classes. Larvae distinguished between chemical stimuli from larvae and adults by decreasing their activity only following exposure to stimuli from adults. Decreased activity is consistent with an antipredator response in many prey species, including larval newts. In contrast, adults increased their activity and increased time spent in open areas in response to stimuli from larvae, but not to stimuli from adults. Increased activity is consistent with a feeding response; adults also showed increased activity and increased time in open areas in response to chemical stimuli from familiar heterospecific prey (brine shrimp). The proximate cue that allowed the newts to distinguish between the different age classes is not associated with short-term dietary differences because all stimulus animals were fed the same prey. Stimulus “strength” was controlled by diluting the stimulus solutions according to the volume of the stimulus animal. Therefore, there appear to be intrinsic differences in the chemical signatures of larval and adult newts.

The spatial restrictions of 5' HoxC genes expression are maintained in adult newt spinal cord

Urodele amphibians are the only adult vertebrates possessing the capacity to regenerate their limbs and tail after amputation. Epimorphic regeneration is characterized by the accumulation of undifferentiated and dividing mesenchymal cells originating from the tissues of the stump, which form a blastema. It has been proposed that the ability to regenerate precisely the amputated structures depends on a ‘positional memory’ of the cells at the level of amputation plane and that a continuum of positional value would be present in adult urodeles along the appendages able to regenerate. Hox genes are good candidates for playing a role in providing the capacity for regeneration and for carrying positional information. Here, we report the cloning of four AbdB-like genes ( Hoxa9, Hoxc10, Hoxc12 and Hoxc13) in the newt Pleurodeles waltl (Pw). To analyse their expression pattern along the antero-posterior (AP) axis of adult urodele central nervous system (CNS), we used the reverse transcription-polymerase chain reaction (RT-PCR) and showed that the 5’ HoxC genes expression pattern conforms to the usual spatial colinearity rule. In addition, the expression level in tail regenerates of PwHoxc13, PwHoxc12, and PwHoxc10 was respectively 20, 7 and 2 fold higher than in adult tail. These last results suggest that 5' HoxC genes could specify positional memory in adult spinal cord (SC) and could be involved in axial patterning of the tail during regeneration.

A decay of gap junctions associated with ganglion cell differentiation during retinal regeneration of the adult newt

Changes in the gap junctional coupling and maturation of voltage-activated Na + currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na + currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na + currents were detected in premature ganglion cells with round somata in the ‘intermediate-II’ regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na + current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na + current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na + currents in ganglion cells.

Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes.

Adult newt cardiomyocytes, in contrast to their mammalian counterparts, can proliferate after injury and contribute to the functional regeneration of the heart. In order to understand the mechanisms underlying this plasticity we performed longitudinal studies on single cardiomyocytes in culture. We find that the majority of cardiomyocytes can enter S phase, a process that occurs in response to serum-activated pathways and is dependent on the phosphorylation of the retinoblastoma protein. However, more than half of these cells stably arrest at either entry to mitosis or during cytokinesis, thus resembling the behaviour observed in mammalian cardiomyocytes. Approximately a third of the cells progress through mitosis and may enter successive cell divisions. When cardiomyocytes divided more than once, the proliferative behaviour of sister cells was significantly correlated, in terms of whether they underwent a subsequent cell cycle, and if so, the duration of that cycle. These observations suggest a mechanism whereby newt heart regeneration depends on the retention of proliferative potential in a subset of cardiomyocytes. The regulation of the remaining newt cardiomyocytes is similar to that described for their mammalian counterparts, as they arrest during mitosis or cytokinesis. Understanding the nature of this block and why it arises in some but not other newt cardiomyocytes may lead to an augmentation of the regenerative potential in the mammalian heart.

The appearance and maturation of excitatory and inhibitory neurotransmitter sensitivity during retinal regeneration of the adult newt

Using living slice preparations from newt retinas at different stages of regeneration, we examined the time course of appearance and maturation of neurotransmitter-induced currents with whole-cell patch-clamp methods. Neurons from which currents were recorded were identified by Lucifer Yellow fills. All progenitor cells examined at the regenerating retinas did not express any voltage-gated Na + currents and responsiveness to excitatory amino acid analogues (AMPA and NMDA) and inhibitory amino acids (GABA and glycine). Voltage-gated Na + currents were first detected in premature ganglion cells with round cell body located at the most proximal level of the ‘intermediate-II’ regenerating retina. AMPA- GABA- and glycine-induced currents were simultaneously observed in many premature ganglion cells expressing Na + channels, but not all, suggesting that the onset of the Na + channels is slightly earlier than that of excitatory and inhibitory amino acid receptors in regeneration. NMDA-evoked currents were first observed in the ‘intermediate-III’ regenerating retina just before the synaptogenesis. Pharmacological properties and reversal potential values of the excitatory and inhibitory amino acid responses did not change substantially between regenerating ganglion cells and mature ganglion cells, while rectification properties of current–voltage relations for AMPA and NMDA responses were somewhat different between them.

Occurrence of 11-oxotetrodotoxin in the red-spotted newt, Notophthalmus viridescens, and further studies on the levels of tetrodotoxin and its analogues in the newt's efts

An analogue of tetrodotoxin (TTX), 11-oxoTTX, was semi-purified from the red-spotted newt, Notophthalmus viridescens, and identified by ESI-MS and 1H NMR spectra. The levels of TTX, 11-oxoTTX and 6- epiTTX in early stage of development (efts) of this newt were investigated by a post-column fluorescent-HPLC system, and compared with those of adult newts. The level of 11-oxoTTX in both of adults and efts were remarkably high, almost close to those of TTX, while 6- epiTTX was a minor component in both stages. The level of 6- epiTTX in efts (1.8±1.3 μg/g, SD, n=10) was significantly larger than that in adults (0.51±0.26 μg/g, SD, n=12) ( p<0.05), while no significant difference was observed in the levels of TTX and 11-oxoTTX between efts (13±7.4 and 9.1±5.6 μg/g) and adult newts (16±6.3 and 13±6.2 μg/g, respectively) ( p>0.05).

Morphological and biochemical changes in carotenoid granules in the ventral skin during growth of the Japanese newt Cynops pyrrhogaster.

The color of the ventral skin of the Japanese adult newt Cynops pyrrhogaster is red, whereas that of the small juvenile newts at metamorphosis is creamy. Xanthophores in the red skin have many ring carotenoid vesicles (rcv) and a few homogenous carotenoid granules (hcg), as reported earlier. To understand the reason for this change in coloration of the ventral skin of the newt, we carried out histological and biochemical studies to see whether the size and the number of carotenoid granules (hcg and rcv) in the xanthophores and also carotenoid content in the ventral skin change during the growth of this animal. By electron microscopic observation, only hcg were observed in the creamy skin of larvae at stage 59. The diameter of the hcg in the skin of the larvae was approximately 0.85 microm, but significantly decreased to 0.35 microm in the skin of the small juvenile newt. However, the number of the hcg/100 microm (2) of a xanthophore in the ventral skin was very low in the larva at stage 59, but increased in the small juvenile. The carotenoid content was very low in the creamy skin of small juveniles, but dramatically high in the red skin of the adult newts. In the red skin of the adult newt, many rcv (85%) and a few hcg (15%) were observed. However, the number of carotenoid granules (rcv and hcg)/100 microm(2) of a xanthophore in the red skin of adult newts was not different from that of hcg/100 microm (2) of a xanthophore in the creamy skin of small juveniles. The results, taken together, suggest that the increase in the size and the number of carotenoid granules and also carotenoid content in the ventral skin is very important for red body coloration during the growth of the Japanese newt Cynops pyrrhogaster.

Dryness increases predation risk in efts: support for an amphibian decline hypothesis

One hypothesis for amphibian declines is that increased dryness attributed to global climate change exposes amphibians to greater biotic threat and, consequently, greater mortality. But, little is known about behavioral responses of terrestrial amphibians to dry conditions alone or in combination with biotic threats. We used field observations and laboratory experiments to test the response of efts (terrestrial juveniles) of the eastern red-spotted newt, Notophthalmus viridescens, to separate and combined desiccation and predation risks. When only at risk of desiccation, efts moved into shade, traveled down slope, decreased activity, and adopted water-conserving postures. Efts also significantly reduced the rate of water loss by huddling and were attracted to chemical cues from conspecific efts but not from conspecific adults. Thus, efts have a variety of behaviors that reduce the risk of dehydration associated with climate change. When faced only with a predation risk, represented by adult and eft newt tissue extracts (alarm chemicals), efts reduced their activity and avoided alarm cues from both sources. When exposed to combined desiccation and predation risks, efts were less active than when exposed to either risk separately and avoided adult tissue extracts, but not eft extracts. These results suggest that under dry conditions, conspecific tissue extracts contain both attractive (huddling) and repulsive (predator-related) chemical components that induce offsetting behavioral responses. This is the first study to demonstrate moisture-dependent responses to conspecific rinses and alarm substances, underscoring the importance of considering environmental moisture and animal hydration in studies examining responses to conspecific odors and/or alarm chemicals. These results support the hypothesis that elevated dehydration risk may compromise anti-predator behavior and exacerbate amphibian population declines.

Morphological and functional organization of ON and OFF pathways in the adult newt retina.

Morphological and functional organization of ON and OFF pathways in the adult newt retina were examined by intracellular recording and staining techniques and immunohistochemistry. Synaptotagmin immunoreactivity discriminated three broad bands within the IPL: the distal band (sublamina I), the middle band (sublamina II) consisting of two dense punctate bands (sublaminae II(a) and II(b)), and proximal band (sublamina III). The Lucifer-yellow labeled OFF amacrine and ganglion cells send their processes mainly in sublamina I and/or II(a) where OFF bipolar cells extend their axon terminals, while ON amacrine and ganglion cells send their processes in sublamina III and/or II(b) where ON bipolar cells extend their axon terminals. Processes of ON-OFF amacrine and ganglion cells ramify broadly in the whole thickness of the IPL. Many bipolar cells responded to light spot with a transient hyperpolarization at both light onset and offset. They are probably subtypes of ON bipolar cells, because their axon terminals branch mainly in sublaminae III and/or II(b), although a few cells ramified the axon at both sublaminae II(a) and III. Two immunohistochemical markers for bipolar cells, PKC and RB-1, identified axon terminals in sublaminae III and/or II(b). From the ramification pattern of axon terminal, they are probably subtypes of ON bipolar cells. ChAT-ir amacrine cells ramified their dendrites in either sublamina I or II(b). Altogether, present studies support the general idea of segregation of ON and OFF pathways in sublaminae a and b of the IPL.

Somatostatin receptor immunoreactivity in the eye of the adult newt (Pleurodeles waltlii Michan)

The neuropeptide somatostatin is found in the retina of many species, yet its role in the visual process remains to be elucidated. The aim of the present study was to examine the expression and cellular localization of somatostatin receptor subtypes (sst; sst 2A, sst 2B and sst 3) in the eye of the adult newt Pleurodeles waltlii using immunohistochemistry. sst 2A immunoreactivity was observed in bipolar cells, in the inner segments of cone photoreceptors, as well as in the region corresponding to connecting cilia of rods. sst 2B immunoreactivity was not detected. sst 3 immunostaining was localized most intensely in the inner segments of cones, and in cilia of rods. These results suggest that somatostatin acting via sst 2A and sst 3 receptors may play an important role in retinal physiology of the lower vertebrates.

An organotypic culture of the newt retina together with other tissues of the posterior eye segment as a model for studying the effects of the cell adhesion glycoproteins

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins and the localization of acetylcholinesterase activity were found to appear during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.

A Role for Endogenous Electric Fields in Wound Healing

This review focuses on the experimental evidence supporting a role for endogenous electric fields in wound healing in vertebrates. Most wounds involve the disruption of epithelial layers composing the epidermis or surrounding organs in the body. These epithelia generate a steady voltage across themselves that will drive an injury current out of the wounded region, generating a lateral electric field that has been measured in four different cases to be 40-200 mV/mm. Many epithelial cells, including human keratinocytes, have the ability to detect electric fields of this magnitude and respond with directed migration. Their response typically requires Ca2+ influx, the presence of specific growth factors and intracellular kinase activity. Protein kinase C is required by neural crest cells and cAMP-dependent protein kinase is used in keratinocytes while mitogen-activated protein kinase is required by corneal epithelial cells. Several recent experiments support a role for electric fields in the stimulation of wound healing in the developing frog neurula, adult newt skin and adult mammalian cornea. Some experiments indicate that when the electric field is removed the wound healing rate is 25% slower. In addition, nearly every clinical trial using electric fields to stimulate healing in mammalian wounds reports a significant increase in the rate of healing from 13 to 50%. However, these trials have utilized many different field strengths and polarities, so much work is needed to optimize this approach for the treatment of mammalian wounds.

Spatial and temporal patterns of distribution of the gap junctional protein connexin43 during retinal regeneration of adult newt

Newts possess the ability to regenerate a functional retina after complete removal of the original retina. We performed immunoblot and immunohistochemical analyses of newt retinas at different stages of regeneration by using an antibody against a gap junction channel protein, connexin43 (Cx43). The specificity of the antibody was shown on immunoblots as well as immunohistochemical staining pattern in the normal retina. Punctate Cx43 immunolabeling was detected intensely between proliferating cell nuclear antigen-immunoreactive progenitor cells in the regenerating retinas, and the amount of this labeling tended to be prominent along both scleral and vitreal sides. The amount of Cx43 became less abundant as regeneration proceeded. This temporal loss of Cx43 during regeneration was also shown on the immunoblot analysis. Furthermore, the loss of Cx43 was observed in a spatial manner in the peripheral retina, where progenitor cells clustered at the ciliary marginal zone (CMZ) are adding new cells of all types in order toward the central retina. Immunolabeling often extended longitudinally throughout the retina when regenerating retinas became thick. Double immunolabeling with Cx43 and glial fibrillary acidic protein indicated the overlapping between the Cx43 and Müller cell processes. At the beginning of the synaptic formation, immunolabeling almost disappeared in the entire retina. However, in the completely regenerated retina, Cx43 reappeared in the distal end of Müller cells and pigment epithelial cells in the same pattern as in the normal retina. The above observations lead us to speculate that Cx43-mediated gap junctions may play an important role in regenerating events. Possible roles of Cx43 during regeneration are discussed.

Thrombin Activation of S-Phase Reentry by Cultured Pigmented Epithelial Cells of Adult Newt Iris

Following local injury or tissue removal, regeneration in urodele amphibians appears to be dependent on cell cycle reentry and dedifferentiation of postmitotic, terminally differentiated cells in the remaining tissues. Regeneration of the lens of the eye occurs by the dedifferentiation of pigmented epithelial cells (PEC) of the iris and their subsequent transdifferentiation into lens cells. A key question is how cell cycle reentry is regulated. Here we demonstrate that thrombin activates S-phase reentry of newt PEC in vitro. Based on these findings, and on previous experiments showing that newt skeletal myotubes reenter the cell cycle following thrombin stimulation, we suggest that thrombin is a critical signal for initiation of vertebrate regeneration.

Thrombin Activation of S-Phase Reentry by Cultured Pigmented Epithelial Cells of Adult Newt Iris

Following local injury or tissue removal, regeneration in urodele amphibians appears to be dependent on cell cycle reentry and dedifferentiation of postmitotic, terminally differentiated cells in the remaining tissues. Regeneration of the lens of the eye occurs by the dedifferentiation of pigmented epithelial cells (PEC) of the iris and their subsequent transdifferentiation into lens cells. A key question is how cell cycle reentry is regulated. Here we demonstrate that thrombin activates S-phase reentry of newt PEC in vitro . Based on these findings, and on previous experiments showing that newt skeletal myotubes reenter the cell cycle following thrombin stimulation, we suggest that thrombin is a critical signal for initiation of vertebrate regeneration.

Celestial orientation in the marbled newt ( Triturus marmoratus )

Orientation toward breeding ponds plays an important role in the seasonal movements of amphibians. In this study, adult marbled newts were tested in a circular arena to determine sensory cues used to locate breeding ponds. Animals were collected from a temporary pond situated in northern Spain, taken to the experimental site 340 m distant, and tested for orientation under a variety of conditions (i.e., orientation under a clear night sky, orientation under an overcast night sky, and orientation under a clear night sky in the presence of an altered geomagnetic field). These investigations have demonstrated that the marbled newt is able to orient using celestial cues. Animals chose a compass course in the direction of their breeding pond only when celestial cues were available. Conversely, the ambient geomagnetic field does not seem to be relevant to orientation of marbled newts since they were unable to orient themselves using the ambient geomagnetic field in the absence of celestial cues.

Egg transport in the coelom of the newt cynops pyrrhogaster. I. The ovulated egg is transported to the ostium by the ciliary movement of coelomic epithelial cells.

We investigated the mechanism of egg transport in the newt not only by inserting various conditioned eggs into the recipient's body but also by placing them on the coelomic epithelia of the opened body cavity in the adult female newt. Most of the inserted coelomic eggs were oviposited, while 4 of 14 inserted de-jellied uterine eggs and 3 of 10 inserted de-jellied fertilized eggs were oviposited. The coelomic eggs placed on the coelomic epithelia were transported toward the ostium and entered the ostium. The de-jellied uterine eggs and the de-jellied fertilized eggs were transported to the ostium as well. Of all the eggs examined, the coelomic egg was transported the fastest. The transport speeds of coelomic eggs treated with periodic acid and the speed of boiled coelomic eggs were less than those of untreated coelomic eggs. In contrast, the transport speeds of coelomic eggs treated with trypsin and the speed of coelomic eggs removed from their vitelline envelopes (naked eggs) were faster than those of untreated coelomic eggs. Other experiments were carried out in order to ascertain the dependence of sexual activity on egg transport. The speed of coelomic egg transport in artificially sexually activated females was faster than in sexually inactive females, although the ciliary movement could always be observed in both sexually active females and sexually inactive females. This suggests that the speed of egg transport on the coelomic epithelia is controlled by the sexual activity of the female.

An ultrastructural and carotenoid analysis of the red ventrum of the Japanese newt, Cynops pyrrhogaster.

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)-reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab-reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, beta-carotene and five other kinds of carotenoids were detected by thin-layer chromatography (TLC). In the ventral yellow skin of lab-reared newts, however, only beta-carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab-reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.