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.     

Bioengineered human growth hormone supports limb regeneration in the hypophysectomized newt Notophthalmus viridescens

It is well documented that growth hormone (GH) replacement therapy will restore normal limb regeneration to hypophysectomized adult newts. However, it is also known that the GH preparations used in previous reports were contaminated by other pituitary hormones shown to support regeneration when administered free of GH. The recent availability of bioengineered human GH was studied for its ability to restore the regenerative capacity to hypophysectomized newts. Five days posthypophysectomy adult newts were subject to forelimb amputation distal to the elbow. Animals were divided into three groups (n greater than 20). Each received one of three GH preparations: pituitary-derived bovine GH, pituitary-derived human GH, or bioengineered human GH. GH was administered via intraperitoneal injection (0.029 IU/50 microliters) on alternate days for either the first 5 days (total of 3 injections) or for 35 days (total of 18 injections). Pituitary-intact and hypophysectomized control newts were subjected to forelimb amputation and injected with hormone diluent. All newts that received GH demonstrated normal limb regeneration to the early digitiform stage by 35 days postamputation. None of the hypophysectomized control newts showed any evidence of regeneration. We conclude that GH alone can restore the ability to undergo normal limb regeneration to hypophysectomized newts.     

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.     

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.     

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

Summary In vitro experiments were carried out to determine the effects of prolactin, and prolactin in combination with other hormones on the regeneration of adult newt tail blastemata. A total of 271 blastemata were explanted 13 days postamputation and were organ cultured for 96 h at 20 (±1)°C. Treatment with prolactin alone resulted in an increase in the blastema cell density of the tail regenerates. Cell accumulation and cell alignment were observed ventral to the reconstituted spinal cord. Prolactin and thyroxine, in combination, improved development of tail regenerates as compared with treatment with prolactin or thyroxine singly, supporting the results of earlier in vivo studies. Optimal development was obtained only when prolactin, insulin, thyroxine and hydrocortisone were added to the culture medium. Regeneration of tail explants maintained in medium augmented with the four hormones closely resembles that of in vivo tail blastemata 17 days post-amputation.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

A dynamic spatiotemporal extracellular matrix facilitates epicardial-mediated vertebrate heart regeneration

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.     

β- and γ-Crystallin Synthesis in Rat Lens Epithelium Explanted with Neural Retina

Culturing the dorsal iris epithelium of a newt with a pituitary gland in organ culture greatly enhances the ability of the iris epithelium to produce advanced lens regenerates in vitro. In an attempt to elucidate the mechanism by which the pituitary enhances lens regeneration irido-corneal complexes from adult newts were cultured in medium to which various substances had been added either singly or in numerous combinations. Prolactin, insulin, hydrocortisone, and thyroxine failed to enhance the production of advanced lens regenerates in any of the doses or combinations tested. Similarly, addition of 50 μg/ml of sodium or calcium ascorbate had no effect on the progress of lens regeneration in vitro. Addition of dibutyryl cyclic-AMP caused an inhibition of depigmentation and regeneration at high doses. The results of these experiments show that the effects of the pituitary cannot be duplicated by hormones which other authors have asserted to be beneficial to limb or tail regenerates in vitro. The results with cyclic AMP suggest that prolonged exposure to high doses of cyclic AMP inhibit regeneration and indicate that further studies on the fluctations in cyclic AMP levels throughout the process of lens regeneration must be done.     

The influence of hormones and other substances on lens regeneration in vitro

Culturing the dorsal iris epithelium of a newt with a pituitary gland in organ culture greatly enhances the ability of the iris epithelium to produce advanced lens regenerates in vitro. In an attempt to elucidate the mechanism by which the pituitary enhances lens regeneration irido-corneal complexes from adult newts were cultured in medium to which various substances had been added either singly or in numerous combinations. Prolactin, insulin, hydrocortisone, and thyroxine failed to enhance the production of advanced lens regenerates in any of the doses or combinations tested. Similarly, addition of 50 microgram/ml of sodium or calcium ascorbate had no effect on the progress of lens regeneration in vitro. Addition of dibutyryl cyclic-AMP caused an inhibition of depigmentation and regeneration at high doses. The results of these experiments show that the effects of the pituitary cannot be duplicated by hormones which other authors have asserted to be beneficial to limb or tail regenerates in vitro. The results with cyclic AMP suggest that prolonged exposure to high doses of cyclic AMP inhibit regeneration and indicate that further studies on the fluctations in cyclic AMP levels throughout the process of lens regeneration must be done.     

Molecular genetic system for regenerative studies using newts

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.     

Abnormal Limb Regeneration without Tumor Production in Adult Newts Directed by Carcinogens, 20-Methylcholanthrene and Benzo (α) pyrene

The effects of potent carcinogens, 20-methylcholanthrene (MC) and benzo(α)pyrene (BP), on limb regeneration were studied in adult newts. A microcrystal of these carcinogens was administered directly to the blastema of forelmibs on day 7 after amputation. The formation of the regeneration cone was delayed and the cone was shifted in abnormal polarity depending upon the site of micro-crystal administration. These carcinogens affected morphogenesis of skeletal structures of regenerating limbs. Subregeneration and superregeneration of either or both carpals and digits, absence of either or both ulna and radius, and accessory limb formation have been recorded as abnormalities caused by these carcinogens. Non-carcinogenic benzocompounds did not show such effects as those of MC and BP. The regeneration blastema of the limb appears to be resistant to carcinogenic effects of the carcinogens used since tumor formation has never been observed in our study so far.     

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.     

Influence of the pituitary on Wolffian lens regeneration

Removal of the pituitary 3 days before lentectomy retards Wolffian lens regeneration in the adult newt, Notophthalmus viridescens, by two stages over a 21-day period. Hypophysectomy 5 or 10 days after lentectomy does not alter the progress of regeneration during the subsequent 10-day period. Hypophysectomy 3 days before lentectomy also significantly decreases the incorporation of [³H]thymidine by iris epithelial nuclei 5 days after lentectomy but has no statistically significant effect on the incorporation 7 days after lentectomy.Pituitary tissue from newts or frogs enhances the regenerative activity of newt iris epithelial cells in vitro and in many cases promotes lens fiber formation. To a lesser extent, other tissues, such as nerve ganglion, also enhance the production of lens fiber cells from iris epithelium in vitro, whereas muscle tissue does not; and under certain conditions iris epithelial cells were found to depigment and redifferentiate into lens cells in the absence of other tissues in vitro.     

Cell proliferation in the amputated limb of lizard leading to scarring is reduced compared to the regenerating tail

After amputation, the tail of lizards regenerates while the limb forms a short scarring outgrowth. Using phospho‐histone‐H3 immunohistochemistry the mitotic activity of limb tissues at 12–25 days after amputation has been studied, when a limb outgrowth of 0.5–2 mm in length is covered by wound epidermis and the underlying connective is turning into a dense scar. In comparison with a regenerating tail of 3–5 mm in length, the number of dividing cells is reduced of 40–70% in different tissues of the scarring limb 1–2 mm in length at 18 days postamputation. Dividing cells are still present at 12–25 days postamputation in the cartilaginous epiphyses of the transected tibia and fibula and of the untransected femur. Also, the injured muscles present at the base of the scarring outgrowth still contain sparse dividing cells after 25 days postamputation of the limb. Together previous studies, the present observations suggest that after the initial proliferation of fibroblasts deriving from the injured tissues, especially from the dermis and intermuscle connectives during the initial 7–15 days postinjury, these cells cover the injured tissues underneath the wound epidermis, but rapidly produce high levels of collagen turning the initial blastema into a scar.     

Limb regeneration following the discontinuation of growth hormone therapy in the hypophysectomized newt,Notophthalmus viridescens

Untreated adult newts do not undergo normal limb regeneration following hypohysectomy. A fibrocellular dermal barrier (cicatrix) atypically forms between the apical epithelium and the underlying mesenchymal tissues. Historically, continuous administration of growth hormone or of prolactin in combination with thyroxine restored regenerative capacity to these newts. In a previous investigation, we demonstrated that the initial effect of these two hormone treatments, when administered on alternate days to hypophysectomized newts beginning eight days post-amputation, was to facilitate the erosion of the fibrocellular barrier and establish the epithelial mesenchymal interface that is observed in a regenerating limb. The present investigation was designed to evaluate the necessity of continuous hormone therapy to maintain limb regeneration in hypophysectomized newts. One, two, or three injections of growth hormone or of prolactin in combination with thyroxine was administered on successive alternate days to hypophysectomized newts either immediately following limb amputation (ID) or beginning eight days post-amputation (DD). The ID and DD newts receiving one, two, or three injections of growth hormone showed evidence of regeneration to the digitiform stage by day 30 post-amputation, while those receiving prolactin and thyroxine underwent wound healing. While both hormone treatments initially promoted a dermis-free apical epithelium, only hypophysectomized newts that had received growth hormone were able to continue regenerating. We have, therefore, concluded that discontinuous growth hormone therapy is sufficient to initiate and maintain the conducive environment for limb regeneration to advanced stages in the hypophysectomized newt. While initiating this process, prolactin and thyroxine therapy on a discontinuous regime does not maintain regeneration. The direct and indirect role of growth hormone in supporting limb regeneration in normal and hypophysectomized newts is discussed.     

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.

     

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.     

Cell interactions and regeneration control

This paper is a review of the main findings of our laboratory on the control of regeneration by cell interactions. These include results related to the role of both cell contact and local soluble factors in regeneration of the legs of insects and newts and of the parapodia and segments of nereis. The pattern of these structures is considered to be defined by positional information distributed as longitudinal and transverse positional value sequences carried by epidermal (insect) or mesenchymal (newt) cells. By associating tissues to create transverse and longitudinal discontinuities in these sequences, single or multiple regenerating structures were obtained. These structures are formed by the intercalation of cells characterized by intermediate positional values which fill the gap between the tissues in contact. Positional information may also be changed during regeneration by the nerve cord in nereis and retinoids in the newts. We describe additional cases where morphogenesis occurs without any overt discontinuity in positional information, such as from a locally injured or non-injured insect trochanter, or after deflection of nerves in nereis and newt. Regeneration following an amputation may be considered as a special case of intercalary regeneration, the first stage being the juxtaposition of normally non-contiguous cells resulting in a longitudinal or/and a transverse gap. We also report studies on local factors produced by nerves and the blastema during newt limb regeneration. The nerve factor is necessary for the division of blastemal cells. After denervation, mesenchyme differentiates in an abnormal way. The mitogenic signal from the nerves is mediated by the PKC pathway. Its production is enhanced by regeneration of cut nerve fibers. The blastema also produces growth factors. We show that the epidermal cap and mesenchyme contain acidic FGF-like factor, and that the proliferating mesenchyme stimulates nerve fibers to regrow into the blastema.     

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.