Chemical Ecology of the North American Newt Genera Taricha and Notophthalmus.

The North American newt genera Taricha and Notophthalmus (order Caudata) are well known for the combination of potent toxicity, aposematic coloration, and striking defense postures that protects these animals from predation. This suite of traits is centered around the neurotoxin tetrodotoxin, which causes paralysis and death in metazoans by disrupting the initiation and propagation of electrical signals in the nerves and muscles. Tetrodotoxin defends newts from predation across multiple life history stages and its role in generating arms-race coevolution between Taricha newts and garter snake (genus Thamnophis) predators is well studied. However, understanding the broader picture of chemical defenses in Taricha and Notophthalmus requires an expanded comprehension of the defensive chemical ecology of tetrodotoxin that includes possible coevolutionary interactions with insect egg predators, protection against parasites, as well as mimicry complexes associated with tetrodotoxin and aposematic coloration in both genera. Herein the authors review what is known about the structure, function, and pharmacology of tetrodotoxin to explore its evolution and chemical ecology in the North American newt. Focus is made specifically on the origin and possible biosynthesis of tetrodotoxin in these taxa as well as providing an expanded picture of the web of interactions that contribute to landscape level patterns of toxicity and defense in Taricha and Notophthalmus.

Cellular basis of brain maturation and acquisition of complex behaviors in salamanders.

The overall bauplan of the tetrapod brain is highly conserved, yet significant variations exist among species in terms of brain size, structural composition and cellular diversity. Understanding processes underlying neural and behavioral development in a wide range of species is important both from an evolutionary developmental perspective as well as for the identification of cell sources with post-developmental neurogenic potential. Here, we characterize germinal processes in the brain of Notophthalmus viridescens and Pleurodeles waltl during both development and adulthood. Using a combination of cell tracking tools, including clonal analyses in new transgenic salamander lines, we examine the origin of neural stem and progenitor cells found in the adult brain, determine regional variability in cell cycle length of progenitor cells, and show spatiotemporally orchestrated neurogenesis. We analyze how maturation of different brain regions and neuronal subpopulations are linked to the acquisition of complex behaviors, and how these behaviors are altered upon chemical ablation of dopamine neurons. Our data analyzed from an evolutionary perspective reveal both common and species-specific processes in tetrapod brain formation and function.

Disease dynamics of red-spotted newts and their anuran prey in a montane pond community.

Long-term monitoring of amphibians is needed to clarify population-level effects of ranaviruses (Rv) and the fungal pathogen Batrachochytrium dendrobatidis (Bd). We investigated disease dynamics of co-occurring amphibian species and potential demographic consequences of Rv and Bd infections at a montane site in the Southern Appalachians, Georgia, USA. Our 3-yr study was unique in combining disease surveillance with intensive population monitoring at a site where both pathogens are present. We detected sub-clinical Bd infections in larval and adult red-spotted newts Notophthalmus viridescens viridescens, but found no effect of Bd on body condition of adult newts. Bd infections also occurred in larvae of 5 anuran species that bred in our fishless study pond, and we detected co-infections with Bd and Rv in adult newts and larval green frogs Lithobates clamitans. However, all mortality and clinical signs in adult newts and larval anurans were most consistent with ranaviral disease, including a die-off of larval wood frogs Lithobates sylvaticus in small fish ponds located near our main study pond. During 2 yr of drift fence monitoring, we documented high juvenile production in newts, green frogs and American bullfrogs L. catesbeianus, but saw no evidence of juvenile recruitment in wood frogs. Larvae of this susceptible species may have suffered high mortality in the presence of both Rv and predators. Our findings were generally consistent with results of Rv-exposure experiments and support the purported role of red-spotted newts, green frogs, and American bullfrogs as common reservoirs for Bd and/or Rv in permanent and semi-permanent wetlands.

Superresolution imaging of transcription units on newt lampbrush chromosomes.

We have examined transcription loops on lampbrush chromosomes of the newt Notophthalmus by superresolution microscopy. Because of the favorable, essentially two-dimensional morphology of these loops, an average optical resolution in the x-y plane of about 50 nm was achieved. We analyzed the distribution of the multifunctional RNA-binding protein CELF1 on specific loops. CELF1 distribution is consistent with a model in which individual transcripts are tightly folded and hence closely packed against the loop axis.

Comparison of buprenorphine and butorphanol analgesia in the eastern red-spotted newt (Notophthalmus viridescens).

The experimental use of amphibian models in biomedical research increases yearly, but there is a paucity of reports concerning analgesic use in many of these species. In this study, buprenorphine given by intracoelomic injection and butorphanol added to the tank water were compared for analgesic effect in the eastern red-spotted newt after bilateral forelimb amputations. Newts undergoing anesthesia but not surgery and newts having surgery but not given analgesia postoperatively were used as control groups. Animals were tested for food consumption, spontaneous movement, response to tapping on the tank, response to being touched, and body posture. Both buprenorphine by intracoelomic injection and butorphanol in tank water significantly promoted resumption of normal behavior after bilateral surgical amputation of the forelimbs. The difference between analgesic treatment and no analgesic treatment was maintained until 72 h after surgery.

Correlation of seasonal acclimatization in metabolic enzyme activity with preferred body temperature in the Eastern red spotted newt (Notophthalmus viridescens viridescens).

Eastern red spotted newts, as aquatic adults, are active year round. They are small and easy to handle, and thus lent themselves to a laboratory study of seasonal changes in preferred body temperature and biochemical acclimatization. We collected newts in summer (n=20), late fall (n=10) and winter (n=5). Ten each of the summer and late fall newts were subjected to an aquatic thermal gradient. Summer newts maintained higher cloacal temperatures than late fall newts (26.8+/-0.5 degrees C and 17.2+/-0.4 degrees C, respectively). In addition, the activity of three muscle metabolic enzymes (cytochrome c oxidase (CCO), citrate synthase (CS) and lactate dehydrogenase (LDH)) was studied in all newts collected. Newts compensated for lower late fall and winter temperatures by increasing the activity of CCO during those seasons over that in summer newts at all assay temperatures (8, 16 and 26 degrees C). The activity of CS was greater in winter over summer newts at 8 and 16 degrees C. No seasonal differences in LDH activity were demonstrated. These data in newts indicate that this amphibian modifies some muscle metabolic enzymes in relation to seasonal changes and can modify its behavioral in a way that correlates with those biochemical changes.

Effects of exogenous FGF-1 treatment on regeneration of the lens and the neural retina in the newt, Notophthalmus viridescens.

Experiments were designed to compare the effects of recombinant newt fibroblast growth factor-1 (rnFGF-1) and recombinant human glial growth factor (rhGGF) on lens and retina regeneration in the eyes of adult newts. Both eyes were retinectomized and lentectomized. Beginning 3 days after the operation, one eye was given either 0.1 microg of rnFGF-1 or 0.1 microg of rhGGF in 1 microl of phosphate-buffered saline (PBS) per injection, three per week. Contralateral operated eyes served as controls and were treated with PBS alone or were not injected. In eyes that were not injected, injected with PBS alone, or with PBS containing rhGGF, regeneration of both the retina and the lens proceeded normally as described in the literature. In these control eyes, the entire retinal pigmented epithelium (RPE) depigmented/dedifferentiated and a retina rudiment formed from which a new retina regenerated by the end of the experiment at day 41 post-operation. Likewise, only a small area of dorsal iris depigmented/dedifferentiated and formed a lens vesicle from which a lens subsequently regenerated. The vitreous remained relatively free of loose cells. In eyes given rnFGF-1, the RPE depigmented/dedifferentiated and formed what appeared to be a retina rudiment but a new retina did not regenerate. Instead, vesicles were seen associated with the retina rudiment. In eyes given rnFGF-1, both the dorsal iris and ventral iris depigmented/dedifferentiated and lens regeneration occurred but the new lenses had abnormal fiber cells and the lens epithelium was very thin or absent. In addition, ectopic lenses usually regenerated in rnFGF-1-treated eyes. An abundance of loose cells were present in the vitreous of rnFGF-1-treated eyes associated largely with the RPE and the dorsal and ventral irises. The results are consistent with the view that the timely expression of FGFs is involved in the depigmentation/dedifferentiation of the RPE and dorsal iris and is necessary for proper regeneration of the lens and neural retina. Continued presence of FGF results in continued and excessive dedifferentiation, resulting in the lack of retina regeneration and abnormal lens regeneration.

Behavioral titration of a magnetic map coordinate.

Spatial variation in the inclination of the geomagnetic field has been implicated in the map component of homing by eastern red-spotted newts Notophthalmus viridescens. Here we show that when newts are exposed to small changes in magnetic inclination, the most dramatic effects on homing orientation occur at values close to the 'home value', as predicted by the magnetic map hypothesis (Phillips 1996). Newts reverse the direction of homing orientation over a range of inclination of 0.5 degrees spanning the home value, providing further evidence that magnetic inclination or one of its components (i.e., vertical or horizontal intensity) is used to derive map information.

Repellent function of male pheromones in the red-spotted newt.

Pheromones act as attractants and sexual stimulants in most vertebrates. For example, in red-spotted newts, Notophthalmus viridescens, female pheromones attract males, and male pheromones increase female receptivity. However, no studies have determined whether male vertebrates produce a pheromone that repels competing males. Through a series of olfactory mate selection tests, we found that sexually motivated male red-spotted newts produce a pheromone that functions to repel other approaching males. Our finding is the first report of a repelling function for pheromones in male vertebrates. The pheromones may act to increase both the sender's and receiver's mating success when the operational sex ratio (OSR) is male biased.

Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles.

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.

Assembly of lampbrush chromosomes from sperm chromatin.

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24-48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific "landmarks. " Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.

Chromosome dynamics: yeast pulls it apart.

Sister chromatid cohesion and chromosome condensation are both essential to the successful completion of mitosis. The recent identification and characterization of the yeast Mcd1p/Scc1p protein reveals a previously unsuspected mechanistic link between these processes.

The newt ribozyme is part of a riboprotein complex.

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a "hammerhead" domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Use of enzyme overlay membranes to survey proteinase activity in frozen sections: cathepsin-like and plasmin-like activity in regenerating newt limbs.

We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems.

Keratin 8 and 18 expression in mesenchymal progenitor cells of regenerating limbs is associated with cell proliferation and differentiation.

Keratins are considered markers of epithelial differentiation. In lower vertebrates, however, immunoreactivity for keratin 8 and 18 has been reported in nonepithelial cells, particularly in mesenchymal progenitor cells of regenerating complex body structures. To confirm that such reactivity does indeed reflect keratin expression and to investigate their possible role in regeneration, we have isolated clones coding for the newt homologues of keratin 8 and 18 (NvK8 and NvK18, respectively) and studied their distribution and changes in their expression following experimental manipulations. Analysis of NvK8 and NvK18 transcripts confirms that K8 and K18 are expressed in the blastemal cells of regenerating newt limbs and that their expression is first observed 3-5 days after amputation, when the blastemal cells start to proliferate under the influence of the nerve, whose presence is essential for regeneration to proceed. In contrast, no induction of these keratins is observed following amputation of a larval limb at a stage when organogenesis is proceeding in a nerve-independent manner. To establish whether there is a causal relationship between keratin expression and cell proliferation in the adult limb blastema, we have investigated whether their expression is nerve-dependent and whether suppression of their expression in cultured blastemal cells affects cell division and differentiation. Analysis of keratins in denervated limbs demonstrates that the nerve is not necessary to induce their expression. However, treatment of cultured blastemal cells with K8 and K18 anti-sense oligonucleotides significantly decreases DNA synthesis and induces changes in cell morphology, suggesting that expression of these keratins during regeneration may be necessary for the maintenance of the undifferentiated and proliferative state of blastemal cells.

Regeneration of lower and upper jaws in urodeles is differentially affected by retinoic acid.

The vitamin A derivative retinoic acid (RA) is a powerful teratogen which can induce severe craniofacial and limb malformations if administered at certain stages of gestation. In addition this compound has been shown to affect patterning in regenerating systems. A classical example is the induction of supernumerary structures along the proximodistal axis of the regenerating amphibian limb. We have investigated the effect of RA on other regenerating systems, the amphibian lower and upper jaws, both in developing and adult animals. We report here that RA does not induce formation of extra structures either in the lower or in the upper jaw of adult newts under experimental conditions where duplications of the regenerating limb occur. However, RA selectively induces severe malformations in the upper jaw regenerate that resemble those induced in avian and mammalian embryos. Analysis of the expression of the newt retinoic acid receptors RAR alpha and delta in upper and lower jaws showed that RAR alpha was expressed at a significant level in the wound epidermis, but not in blastemal cells, whereas no RAR delta could be detected in the regenerate either by in situ hybridization or by using an anti-RAR delta antibody. Therefore, unlike in the limb, in jaws RAR delta is not up-regulated following amputation, and this difference in expression may be causally related to the different effects induced by RA on jaws and limbs. In order to establish whether retinoids affected regeneration of developing jaws in a similar fashion, their effects were studied in animals whose jaws had been amputated at different developmental stages. Under the experimental conditions used overall growth retardation and head defects were observed in the majority of embryos which had been amputated and treated with retinol palmitate (RP) between stages 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 45 was not significantly affected by the treatment, although the early phase of regeneration was slower than in controls. The different responses to retinoids of regenerating facial structures in embryos, larvae and adults will be discussed.