Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the “agarose-embedded heat shock (AeHS) method” involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.     

Morphogenetic changes during newt tail regeneration under changed gravity conditions

Gravity-dependent shape alterations in newt tail regenerates are described, which were previously noticed in experiments onboard satellites Foton M2, M3 and in corresponding laboratory controls. Laboratory conditions were developed that allow reproducing this phenomenon persistently in the adult newts Pleurodeles waltl (Michahelles, 1830). The newts kept in an aquarium (in partial weightlessness) after 1/3 tail amputation developed normal lanceolate regenerates, while those that stayed on a moist mat (exposed to greater gravity than in aquarium) developed curved tail regenerates. Dynamics of the shape alterations were described using computer morphometric analysis. The curve was shown to develop at stage III of regeneration and to be caused by bending of the developing axial structures: the ependymal tube and the cartilage rode. Cellular processes were described that accompany the tail shape changes, such as cell migration and formation of dense aggregates. Unequal proliferation throughout the wound epidermis and blastema was revealed using BrdU assay. Proliferation increased within dorsal and apical regions of the regenerates in the newts kept on the mat cell compared with the aquarian animals.     

Hormone action in newt limb regeneration: insulin and endorphins

Although several hormones have been linked to newt limb regeneration, a cohesive hypothesis as to how these hormones control the process is yet to emerge. A critical review of the traditional approaches and a reevaluation of currently operative assumptions and interpretations of results precede the data on insulin and beta-endorphin. Results from in vivo and in vitro experiments on insulin are summarized, showing that insulin not only promotes various cellular events but also is essential for the expression of the mitogenic effect of nerves on cultured newt limb blastemata. Furthermore, the strong likelihood that insulin may be the common link in promoting limb regeneration in hypophysectomized newts that received pituitary hormone replacement therapy or a nutritional supplement is discussed. The status of beta-endorphin in regeneration is also explored. Data are presented to show that vertebrates with regenerating capacity (newts, tadpoles) have higher levels of plasma beta-endorphin than that found in species where the capacity to regenerate is either restricted (frogs) or totally lost (mammals). beta-Endorphin-like immunoreactivity has been localized in the epidermis of a regenerating newt blastema, as well as in the intermediate lobe of the pituitary gland of axolotl, newt, and Xenopus. A possible opiate connection in vertebrate limb regeneration, in particular, wound healing, is discussed.     

Cytogenetic Maps of Lampbrush Chromosomes of Newts of the Genus Pleurodeles: An Algorithm of Lampbrush Chromosome Identification inPleurodeles waltl by Immunocytochemical Staining of Landmark Loops with Polyclonal Anti-Ro52 Antisera

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

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory. In these animals, survival drops drastically after metamorphosis as their foraging behaviour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined metamorphosis inhibition (Ml) protocols to bypass the period (four months to two years after hatching) in which the animal feeds exclusively on moving foods. We found that approximately 30% of animals survived after 2-year Ml, and that the survivors continuously grew, only with static food while maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamorphosis with TU allows newts to be reared with the same static food under aqueous conditions, providing an alternative rearing protocol that offers the advantage of bypassing the critical period and obtaining animals that have grown sufficiently for use in regeneration studies.     

Changes in the Relative Number of Bipolar-Like Cells in the Retina of Pleurodeles waltl as a Function of Age and as a Result of Light Exposure

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

Development of Notophthalmus viridescens embryos

Although the North American, adult, red-spotted newt has been an excellent model in the study of appendage regeneration at the tissue and cell levels, experiments involving embryonic forms have been essentially impossible to perform at the molecular level due to the great difficulty in breeding newts in the laboratory. Recently, we reported our methods to successfully spawn many thousands of embryos of Notophthalmus viridescens for developmental studies on a year round basis. As no detailed examination of embryogenesis of this amphibian exists, we provide a scanning electron microscopic overview of N. viridescens development. Furthermore, we chronicle the life cycle of the newt, when produced under laboratory conditions, which also undergoes two metamorphoses as newts in the wild.     

Tolerance, limb regeneration and collagen fibrogenesis under high oxygen concentrations by the adult newt, Diemictylus viridescens

Adult newts placed in an atmospheric environment of 85% oxygen, saturated humidity, and at a temperature of 20 ± 1°C survived particularly well a 44-day test period. They did not succumb to “oxygen toxicity” as has been frequently reported for other vertebrate species. Having established the newt's tolerance of high oxygen atmosphere, the effect of oxygen on growth and development in the regenerating newt limb was investigated. Under the atmospheric conditions described above, and under 92% oxygen, the regeneration of adult newt limbs appeared to be retarded during the first 25 days after amputation when compared with regenerating limbs of control animals kept under a normal atmosphere of 21% oxygen (air). Thereafter, little or no difference could be discerned between the regeneration of experimental and control limbs. It is known that molecular oxygen participates directly in the hydroxylation of proline to hydroxyproline in the synthesis of collagen. Sectioned regenerates stained specifically for collagen were examined to determine if collagen synthesis was induced in experimental animals. Two regeneration-inhibited limbs of oxygenated newts showed cicatrical repair of the apical limb stump 25 days after amputation. However, the majority of the experimental animals revealed no obvious increase in collagen fibers. These results contraindicate any marked “oxygen toxicity” affecting the life of the newts, or regeneration of their limbs. It is suggested that a change in collagen fiber type might have been induced by the high-oxygen atmosphere. Investigations to test this hypothesis are currently underway.     

Lifelong testicular differentiation in Pleurodeles waltl (Amphibia, Caudata)

Background  

In numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented.     

Application of local gene induction by infrared laser‐mediated microscope and temperature stimulator to amphibian regeneration study

Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three‐dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single‐cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR‐LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature‐controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR‐LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre‐LoxP system as well as to analyze gene function in a target region or cells with less off‐target effects in the study of amphibian regeneration.     

Lens regeneration in axolotl: new evidence of developmental plasticity

Background

Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens.

Results

Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible.

Conclusions

Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.

     

From lens regeneration in the newt toin-vitrotransdifferentiation of vertebrate pigmented epithelial cells

Through studies to clarify the cellular origin of lens regeneration in the newt, the pigmented epithelial cells of the iris and the retina of many vertebrate species have been shown to possess a dormant potency to transdifferentiate into the lens. The method ofin-vitroculture of pigmented epithelial cells has been optimized to enable detailed studies of the transdifferentiation process by molecular techniques. Growth factors and extracellular matrix components are found to be important in the control of the transdifferentiation process. New systems forin-vitroculture are introduced, while prospects for renewedin-vivostudies using newts are given.     

Increased protein kinase C activity in the central nervous system of the newt during limb regeneration.

Protein kinase C (PKC) activity was examined in the CNS of the newt Pleurodeles waltlii undergoing regeneration after limb amputation. In the spinal cord and brain of control newts, the level of PKC activity was virtually the same for the cytosolic and the particulate fractions. At days 7 and 14 after amputation of two limbs, a twofold increase in overall PKC activity occurred in the spinal cord and accounted for increased membrane-bound activity, while cytosolic activity was not significantly impaired. In contrast, overall PKC activity was not affected in brain. However, a twofold increase in the brain particulate fraction occurred at day 14 while cytosolic activity decreased proportionately. Similar alterations were observed in newts undergoing one or multiple limb amputations. Such changes in PKC activity neither occurred in the CNS of newt after limb denervation nor in the CNS of limb amputated frog Rana temporaria, an Amphibian which is unable to regenerate. Taken together, these results provide evidence that PKC of the CNS is involved in the regeneration process of newts. Changes in activation-associated PKC distribution proceeded through different mechanisms: long-lasting increase in membrane bound activity with a net increase of overall activity in the spinal cord, and long-term redistribution of enzyme activity to the particulate fraction in brain.     

Study of a new group of bioregulators isolated from the greater plantain (<Emphasis Type="Italic">Plantago major</Emphasis> L.)

Proteins with physicochemical properties and biological activity similar to those of membranotropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.     

Influence of indomethacin on lens regeneration in the newt notophthalmus viridescens

Summary Following lentectomy newts were injected with indomethacin in a variety of carrier solutions at doses ranging from 1.2–120 mg/kg body weight every other day for 15–17 days. The results show that injection of this drug according to the regimen used has no significant effect on regeneration of the lens. The data suggest, but do not prove, that prostaglandins may not play a major role in the early phases of lens regeneration in the newt.     

Temporal analysis of the role of growth hormone in the initiation and maintenance of limb regeneration in the hypophysectomized newt Notophthalmus viridescens

This study was designed to investigate and determine for how long, after either hypophysectomy or the third (last) growth hormone injection (to previously hypophysectomized newts), the circulating and now declining titers of endogenous or exogenous hormone remained at a sufficient concentration to permit a morphologically normal forelimb regeneration response in the adult newt Notophthalmus viridescens. To examine the declining levels of endogenous hormone (hormone withdrawal series [HW]), left forelimbs were amputated at specific times following hypophysectomy. Right forelimbs were amputated 5 days prior to hypophysectomy. The declining levels of exogenous hormone (hormone replacement series [HR] were examined in newts whose left forelimbs were amputated at specific times following the last of three consecutive alternate-day growth hormone injections that were initiated 5 days post hypophysectomy. Right forelimbs were amputated immediately following the first hormone injection. All experimental animals were sacrificed when their right forelimbs regenerated to an advanced digitiform regenerate. In both series right forelimbs regenerated normally. In the HW series normal regeneration resulted only when forelimbs were amputated within 48 hours post hypophysectomy, whereas in the HR series normal regeneration occurred in only those newts whose forelimbs were amputated within 12 hours of the last hormone injection. The regeneration response of left forelimbs in both series gradually declined with the time interval between either hypophysectomy or hormone injection and forelimb amputation. As the hormone titer declined, fewer limbs initiated a normal response; they became progressively more hypomorphic and eventually failed to undergo typical regeneration.     

Limb regeneration and survival of prolactin treated hypophysectomized adult newts

Hypophysectomized adult newts exhibited 98% survival and limb regeneration at 23 days post-hypophysectomy when injected intraperitoneally every other day with prolactin (0.015 U/newt) and kept continuously in aquaria with 1 × 10^?7 concentration of thyroxine. Thyroxine alone was no more effective than saline injections. Prolactin (1.2 U/newt every other day) alone increased survival and limb regeneration, but less effectively than did the prolactin-thyroxine combination.     

Subtractive screen of potential limb regeneration related genes from Pachytriton brevipes

Regeneration capacity varies greatly among different animal species. In vertebrate, amphibian especially the Urodela, has been used as a powerful model system to study the mechanism of tissue regeneration because of the strong ability to regenerate their damaged or lost appendages. Pachytriton brevipes, a species of newt, which is widely distributed in south of China, can completely restore their damaged limbs within several months. In this study, we use modified suppression subtractive hybridization assay and dot-blot screening to identify candidate genes involved in tissue regeneration in P. brevipes. We successfully isolated 81 ESTs from a forward regeneration subtraction library. And we further verified the differential expression of four candidate genes, Rpl11, Cirbp, Ag2 and Trimx, between regenerating blastema and non-regeneration tissues by in situ hybridization. These genes were also be further characterized by phylogenetic and bioinformatic analysis. In general, we provided a comparative experimental approach to study the mechanisms of vertebrate regeneration.