The effect of hypophysectomy upon cholinesterase activity in the forelimbs of regenerating adult Notophthalmus news

In order to study endocrine influence upon cholinesterase activity during regeneration, adult newts were hypophysectomized either prior to limb transection or during regeneration. Homogenates of limb tissues were assayed for cholinesterase activity during each stage of regeneration.In animals with pituitaries intact, cholinesterase activity in regenerating limb tissues decreases soon after amputation, and then it rises to the level of activity in intact limbs of normal animals, during the period of differentiation. In hypophysectomized newts there seems to be no alteration of this basic pattern of activity, but removal of the pituitary does result in more elevated levels of enzymatic activity. In the intact forelimbs of control newts undergoing regeneration, cholinesterase activity greatly increases as the other transected limb begins to regenerate but it returns to normal as regeneration progresses. If these animals are hypophysectomized, no such increase is observed during the early stages of regeneration. Rather, there is an initial decrease in cholinesterase activity that is followed by an increase in such activity.These data are compatible with the hypothesis that the pituitary modulates cholinesterase activity in the limb tissues of adult newts.     

An investigation of the prolactin-thyroxine synergism in newt limb regeneration

The use of hormone replacement to support limb regeneration in hypophysectomized newts has been the subject of many investigations. Growth hormone, as well as prolactin (PL) in combination with exogenously supplied thyroxine, have all been shown to he effective. However, the bovine growth hormone used to support limb regeneration was contaminated by prolactin and thyroidstimulating hormone (TSH). The present investigation evaluates the significance of (1) prolactin contamination and (2) endogenous thyroxine synthesis resulting from TSH contamination on limb regeneration in hypophysectomized newts. The effect of supplying exogenous thyroxine was also evaluated. Our studies showed that when hypophysectomized newts were injected with contamination levels of PL and TSH, regeneration occurred, suggesting that the newt's thyroid synthesized sufficient thyroxine to support a prolactin-thyroxine synergism. The endogenous thyroxine was synthesized by thyroid glands that were indistinguishable from those of saline-injected, hypophysectomized controls.     

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.     

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.     

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.     

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.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

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.     

Effects of growth hormone and nutrient on limb regeneration in hypophysectomized adult newts

The effect of hypophysectomy, growth hormone (GH) and an amino acid-glucose mixture on the regenerative ability of the hypophysectomized Triturus pyrrhogaster yielded the following results:

• 1 The survival time of hypophysectomized newts can be prolonged substantially by the sulfamide application.
• 2 Although the limb regeneration in the hypophysectomized newt is retarded as compared with that of the pituitary intact control, it finally completes morphogenetic process under such conditions of prolonged survival.
• 3 The injection of 100 μg of GH restored the speed of regeneration of pituitary-deprived limbs to almost a normal level.
• 4 Injections of the amino acid-glucose mixture also promoted the limb regeneration in hypophysectomized newts. However, initial delay in regeneration to the time of bud appearance was not restored by the nutrients.

     

Regenerative ability of double-half and half upper arms in the newt, Notophthalmus viridescens

The upper arms of adult newts (Notophthalmus viridescens) were surgically manipulated to create double-half dorsal, double-half ventral, double-half anterior, and double-half posterior upper arms, and longitudinal half-dorsal, half-ventral, half-anterior, and half-posterior upper arms. Amputation through the double-half upper arms usually failed to elicit normal distal regeneration, despite the fact that an apparently normal regeneration blastema was initially formed. Instead, regeneration in these cases was limited to the formation of a variable number of small cartilage elements. On the basis of these results it is concluded that a complete limb circumference is required for distal transformation in newts, in addition to the well-established requirements for a wound epidermis, adequate innervation and dedifferentiation leading to blastema formation. A model for the sequential generation of new parts of the limb pattern during distal transformation from a complete circumference is presented. This model can also account for the occurrence of normal early stages of regeneration in double-half upper arms. Half upper arms which were amputated immediately were shown to develop single, complete regenerates. If amputation of half upper arms was delayed three or more weeks to permit complete wound healing, a supernumerary limb from the lateral wound surface sometimes developed in addition to a complete, single limb from the distal amputation surface.     

An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: immunological relationship of MT1 antigen with tenascin.

Several well-characterized extracellular matrix (ECM) components have been localized to the amphibian limb regenerate, but the identification and characterization of novel ECM molecules have received little attention. Here we describe, using mAb MT1 and immunocytochemistry, an ECM molecule expressed during limb regeneration and limb development. In limb stumps, mAb MT1 reactivity was restricted to tendons, myotendinous junctions, granules in the basal layers of epidermis, periosteum (newts) and perichondrium (axolotls). In regenerating limbs, reactivity in the distal limb stump was first detected 5 days and 1 day after amputation of newt and axolotl limbs, respectively. In both species, mAb MT1 recognized what appeared to be an abundant blastema matrix antigen, localized in both thin and thick cords between and sometimes closely associated with blastema cells. Reactivity was generally uniform throughout the blastema except for a particularly thick layer that was present immediately beneath the wound epithelium. During redifferentiation stages, mAb MT1 reactivity persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and connective tissue differentiation. During the entire period of embryonic limb development, mAb MT1 reactivity was seen in the ECM of the mesenchyme and in a layer beneath the limb bud ectoderm, similar to its distribution during regeneration. Considerable mAb MT1 reactivity was also associated with the developing somites. The reactivity of mAb MT1 in blastema and limb bud was similar if not identical to that of a polyclonal Ab against tenascin (pAbTN), a large, extracellular matrix glycoprotein implicated in growth control, inductive interactions, and other developmental events. This pAbTN effectively competed against mAb MT1 binding on blastema sections. In immunoblots, both mAb MT1 and pAbTN recognized a very high molecular weight (approximately Mr 1000 x 10(3)) protein in blastema extracts of both newts and axolotls. mAb MT1 immunoprecipitated a protein of Mr 1000K size which reacted to both mAb MT1 and pAbTN in immunoblots. These data show that tenascin is in the matrix of the urodele blastema and limb bud, and suggest that mAb MT1 identifies urodele tenascin.     

Changes in RNA and protein synthesis in the neural retina during lens regeneration in newts

Since neural retina stimulates regeneration of a lens from the dorsal iris in newts, RNA and protein synthesis in the neural retina was investigated during this process. Incorporation of 3H-uridine and 3H-leucine using liquid scintillation counting was employed to compare RNA and protein synthesis in the neural retina from sham-operated control eyes with that in eyes during lens regeneration. An initial increase in 3H-uridine uptake was seen one to three days after lentectomy. This was followed by greater incorporation of 3H-leucine, indicating increased protein synthesis between 5 to 15 days after lens removal. A decrease in 3H-uridine uptake was also seen at 5 to 12 days after lentectomy. After 20 days both the RNA and protein synthesis returned to the normal level. Since the increase in protein synthesis is preceded by an increase in RNA synthesis, the two processes might be related. The results indicate significant changes in the synthesis of macromolecules by the neural retina following lentectomy. These may be indirectly related to the production of the neural retinal factor with stimulates lens differentiation.     

Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

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.     

The irradiated epidermis inhibits newt limb regeneration by preventing blastema growth. A histological study

It has been established that X-ray irradiation localized to a forelimb or entire irradiation of premetamorphic Pleurodeles larvae prevented limb regeneration. Transplantation of non-irradiated skin, dermis or muscle to limb stumps of locally irradiated newts was sufficient to allow a blastema to develop. Transplantation of the same tissues to limb stumps of entirely irradiated newts yielded different results with the different graft types. Skin graft allowed a normal blastema to be established but dermis or muscle grafts did not. In order to define more precisely the role played by the epidermis in the establishment of a blastema, and in the growth of a regenerate, different combinations of limb tissues, either irradiated or not, were carried out at the level of amputated limb stumps. At four different times (8-10 days; 13-15 days; 20-23 days; 30 days or more) after amputation the stumps were examined in histological longitudinal sections to study the first events of regeneration, that is dedifferentiation and growth. Dedifferentiation occurred in both normal and irradiated tissues of mesodermal origin. The healthy mesenchymal cells began dividing and formed a growing blastema only when associated with a non-irradiated epidermis. Healthy mesenchymal cells covered with an irradiated epidermis exhibited a few mitoses after dedifferentiation, but the mitotic figures became rarer and rarer until the animals died. The lack of dense accumulation of blastemal cells in such limb stumps suggested that the healthy epidermis allows the mesenchymal cells to divide actively to constitute a growing blastema. Hence, X-ray irradiation seems to be responsible for the loss of such an epidermal mitogenic influence on the underlying mesenchymal cells.     

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.     

Skin flaps inhibit both the current of injury at the amputation surface and regeneration of that limb in newts

For over two decades, we have been investigating a strong (ca. 20-100 microA/cm2), outwardly directed electric current driven through the limb stump for the first few days following amputation in regenerating salamanders. This current is driven through the stump in a proximal/distal direction by the amiloride-sensitive transcutaneous voltage of the intact skin of the stump. Limb regeneration can be manipulated by several technique that manipulate this physiology, demonstrating that the ionic current is necessary, but not sufficient, for normal regeneration of the amphibian limb. Here, we demonstrate that a full thickness graft of skin covering the forelimb stump of newts strikingly inhibits the regeneration of the limb, and that this procedure is also highly correlated to a suppression of peak outwardly directed stump currents in those animals that fail to regenerate.     

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens

 A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration. Received: 6 November 1998 / Accepted: 12 December 1998     

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.