Forelimb regeneration in thyroidectomized adult newts following organ culture and autografting of the thyroid glands

Summary Thyroidectomy and organ culture of adult newt thyroid glands three days prior to forelimb amputation was followed by autografting the glands subcutaneously into the animal's lower jaw region 9, 18 or 25 days postamputation (GC^{9, 18, 25} day series). This was an attempt, utilizing 515 animals, to elucidate further the role of the thyroids in regeneration. Amputated limbs of the thyroidectomized (Thx) and autografted muscle explant (MC = sham) cases underwent stumping or were significantly delayed in their regeneration rate and displayed abnormal morphogenesis compared with control regenerates. In the GC⁹ series newts, regenerates were identical to controls 45 days postamputation. However, regenerates of the GC¹⁸ series cases exhibited delayed and abnormal development at 45 days; but they were not as delayed and had fewer abnormalities than those cases in the Thx and MC groups. Results of the GC²⁵ series newts were similar to those of the Thx group. Within 5 days of autografting the thyroids, epidermal moulting resumed and long-term survival ensued. We conclude that normal limb regeneration in the adult newt is thyroid hormone(s) dependent, specifically the later stages of growth, differentiation and morphogenesis.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

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.

     

Carcinogens on Regeneration

A microcrystal (ca 5 μg) of N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) or 4-nitroquinoline-1-oxide (4NQO) was directly administered to the regeneration blastema on day 7 after amputation of a forelimb in the newt in order to analyze the effect of such potent carcinogenic substances on regeneration cells. Although neither MNNG nor 4NQO arrested regeneration completely, they caused great retardation of the regeneration cone formation followed by various abnormalities in the bony structures. Abnormal regenerants could be classified into the following four categories; (1) complete absence of both ulna and radius; (2) subregeneration or superregeneration of carpals and digits; (3) multiple disorganization of skeletal elements; (4) arrest of regeneration at the stage of regeneration cone. The polarity of regenerants developed after application of MNNG or 4NQO was very often shifted, during which the regeneration cone was always formed from the site where a microcrystal of the carcinogens was administered. The secondary regeneration initiated by reamputation of the regenerating limb, which had received the carcinogens at the early blastema stage, proceeded in the same way as observed in the case of a simple amputation. This suggested local and temporal effects of the carcinogens applied. Nevertheless, tumor formation has not induced in the newt limb so far. We can learn from these data that both MNNG and 4NQO only alter behaviour of the newt regeneration cells without excerting their carcinogenic effects on them, and that the newt cells are highly resistant and stable against the above-mentioned carcinogens.     

Growth and apoptosis during larval forelimb development and adult forelimb regeneration in the newt (<Emphasis Type="Italic">Notophthalmus viridescens</Emphasis>)

Many of the genes involved in the initial development of the limb in higher vertebrates are also expressed during regeneration of the limb in urodeles such as Notophthalmus viridescens. These similarities have led researchers to conclude that the regeneration process is a recapitulation of development, and that patterning of the regenerate mimics pattern formation in development. However, the developing limb and the regenerating limb do not look similar. In developing urodele forelimbs, digits appear sequentially as outgrowths from the limb palette. In regeneration, all the digits appear at once. In this work, we address the issue of whether regeneration and development are similar by examining growth and apoptosis patterns. In contrast to higher vertebrates, forelimb development in the newt, N. viridescens, does not use interdigital apoptosis as the method of digit separation. During adult forelimb regeneration, apoptosis seems to play an important role in wound healing and again during cartilage to bone turnover in the advanced digits and radius/ulna. However, similar to forelimb development, demarcation of the digits in adult forelimb regeneration does not involve interdigital apoptosis. Outgrowth, rather than regression of the interdigital mesenchyme, leads to the individualization of forelimb digits in both newt development and 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.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated,nerve‐dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF‐like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine‐rich domain (CRD‐NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD‐NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve‐dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve‐dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 150–158, 2000     

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.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Amphibian Limb Regeneration and its Relation to Nerves

Much circumstantial evidence points to a neurotrophic influencein amphibian limb regeneration. Although fine-structural observationsof nerves in regenerating limbs have indicated the possibilitythat neurosecretory vesicles accumulate distally in these axons,there is no clear-cut demonstration available that these organellesare neurotropic. Evidence is accumulating that the neural influencein newt limb regeneration is transneuronal. There is also evidencethat trophic substances other than those found in the nerveitself may be involved in the supporting limb regeneration.The characterization of the neurotrophic substance is considereda central task for students of regeneration in the future.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated, nerve-dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD-NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response.     

Regeneration and supernumerary limb formation under sparsely innervated conditions

Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0–5; 1 with 5–10; and 1 with 20⁺ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent. Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0–5; 5 with 5–10; 7 with 10–19; and 1 with 20⁺ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.     

Nvbeta-actin and NvGAPDH as normalization factors for gene expression analysis in limb regenerates and cultured blastema cells of the adult newt, Notophthalmus viridescens

The red-spotted newt has the ability to fully regenerate complex structures by creating a pool of dedifferentiated cells that arise in response to tissue injury. An understanding of the mechanisms involved in the regenerative ability of the newt is limited by a lack of characterized assays. This deficiency includes the cloning and validation of housekeeping genes for normalizing gene expression data. We describe the cloning, characterization and real-time quantitative PCR evaluation of the normalization potential of the newt homologues of cytoplasmic beta-actin and GAPDH during newt limb regeneration and within the blastemal B1H1 cell line. Nvbeta-actin demonstrates a heterogeneous expression during limb regeneration and may be associated with differentiation state. The level of Nvbeta-actin expression in B1H1 cultures under conditions of myogenesis and serum resupplementation varies with the treatment. NvGAPDH is ubiquitously expressed during limb regeneration and within B1H1 cultures and does not demonstrate overall variations in expression levels. Thus, NvGAPDH is a more appropriate normalization factor in gene expression analyses during limb regeneration and treatments of B1H1 cultures.     

The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

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.     

Increased slow transport in axons of regenerating newt limbs after a nerve conditioning lesion

We have previously shown that a nerve conditioning lesion (CL) made 2 weeks prior to amputation results in an earlier onset of limb regeneration in newts. Studies in fish and mammals demonstrate that when a CL precedes a nerve testing lesion, slow component b (SCb) of axonal transport is increased compared to axons that had not received a CL. We wanted to know whether the earlier initiation of limb regeneration after a CL was associated with an increase in SCb transport. The transport of [35S]methionine labeled SCb proteins was measured by using SDS-PAGE, fluorography, and scintillation counting. The rate of transport and quantity of SCb proteins was determined at 7, 14, 21, and 28 days after injection of [35S]methionine into the motor columns of normal; single lesioned (i.e., transection axotomy, amputation axotomy, or sham CL followed by amputation); and double-lesioned limb axons (i.e., nerve transection CL followed 2 weeks later by amputation axotomy). The rate of SCb transport in axons of unamputated newt limbs was 0.19 mm/day. There was an increase in the amount of labeled SCb proteins transported in axons regenerating as the result of a single lesion but no acceleration in the rate of SCb transport, which was 0.21 mm/day in axons that received a sham CL followed by limb amputation. The rate of SCb transport doubled (0.40 mm/day) and the amount of labeled SCb proteins being transported was increased when amputation was preceded by a CL. This study demonstrates that the earlier onset of limb regrowth, seen when amputation follows a CL, is associated with an increased transport of SCb proteins. This suggests that limb regeneration is, in part, regulated by axonal regrowth. We propose that the blastema requires a minimum quantity of innervation before progressing to the next stage of limb regeneration, and that the transport of SCb proteins determines when that quantity will be available.     

A comparison of beta-endorphin levels in regenerating and nonregenerating vertebrates

The mean plasma level of beta-endorphin in the newt, Notophthalmus viridescens, as measured by radioimmunoassay, is significantly higher than the circulating levels of beta-endorphin in the anuran, Rana pipiens, and in mammals (humans and mice). The newt beta-endorphin level is found to be 6-fold greater than mammalian levels and three times greater than the levels observed in Rana pipiens. The high levels are maintained in the newt throughout limb regeneration.     

Co-operative Bmp- and Fgf-signaling inputs convert skin wound healing to limb formation in urodele amphibians

Urodele amphibians have remarkable organ regeneration capability, and their limb regeneration capability has been investigated as a representative phenomenon. In the early 19th century, nerves were reported to be an essential tissue for the successful induction of limb regeneration. Nerve substances that function in the induction of limb regeneration responses have long been sought. A new experimental system called the accessory limb model (ALM) has been established to identify the nerve factors. Skin wounding in urodele amphibians results in skin wound healing but never in limb induction. However, nerve deviation to the wounded skin induces limb formation in ALM. Thus, nerves can be considered to have the ability to transform skin wound healing to limb formation. In the present study, co-operative Bmp and Fgf application, instead of nerve deviation, to wounded skin transformed skin wound healing to limb formation in two urodele amphibians, axolotl (Ambystoma mexicanum) and newt (Pleurodeles waltl). Our findings demonstrate that defined factors can induce homeotic transformation in postembryonic bodies of urodele amphibians. The combination of Bmp and Fgf(s) may contribute to the development of novel treatments for organ regeneration.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: An organ culture model for the urodele retinal regeneration

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented “neuron‐like cells” with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU‐labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron‐specific proteins; HPC‐1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite‐like processes. Numerous lightly pigmented cells with neuron‐like morphology showed HPC‐1 immunoreactivity. Fibroblast growth factor‐2 (FGF‐2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron‐like cells and HPC‐1‐like immunoreactive cells in a dose‐dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue‐intrinsic factors responsible for newt retinal regeneration. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 209–220, 2002; DOI 10.1002/neu.10031     

Expression profiles of elastase1 (NvElastaseI) and secretory leukocyte protease inhibitor (NvSLPI) during forelimb regeneration in adult Notophthalmus viridescens suggest a role in epithelial remodeling and delamination

Extracellular proteases and their inhibitors may regulate a number of important processes involved in forelimb regeneration in the adult newt, including epithelial remodeling, breakdown of extracellular matrix, and dedifferentiation. We have identified a newt homologue of human ElastaseI (NvElastaseI) and its potential inhibitor, SLPI (NvSLPI), and evaluated their spatial and temporal expression during limb regeneration. NvElastaseI is upregulated early in regeneration and is associated with subdermal and wound epithelial cells, suggesting an involvement in wound healing and the generation of the wound epithelium. Up until 15 days post-amputation, NvElastaseI is also scattered throughout the developing blastema and may have a role in the dedifferentiation of stump tissues. NvSLPI is found at the interface between the intact skin and the wound epithelium, and may limit NvElastaseI activity. NvSLPI is also expressed in dermal glands, and is likely involved in anti-microbial activity or function. Quite apart from regeneration, complementary patterns of expression of NvElastaseI and NvSLPI are associated with newt epithelial sloughing.     

Adoptive immunotherapy of a mouse colon carcinoma with recombinant interleukin-2 alone or combined with lymphokine-activated killer cells or tumor-immune lymphocytes

Summary We have used a BALB/c colonic adenocarcinoma (C-26) to evaluate the therapeutic potential of recombinant interleukin-2 (rIL-2) at high and low dosages in combination with or without lymphokine-activated killers (LAK) or tumor-specific, immune lymphocytes in either an adjuvant spontaneous or an artificial metastasis system. Most (80%) of the mice that underwent s.c. C-26 tumor excision were shown to die of spontaneous metastasis with lung involvement by 1–4 months after excision. Postsurgical systemic treatment with low-dose rIL-2 (3 × 10⁴ U/day, i.p.) increased the survival rate to 31% as compared to 21% (not significant) in excised controls while administration of high-dose rIL-2 (8 × 10⁴ U/day) led to 53% survival (P <0.01). Both LAK cells and C-26-tumor-immune lymphocytes given during rIL-2 treatment significantly increased the effects of rIL-2 at the low but not at the high-dose, with tumor-immune effectors resulting in the highest percentage (63%) of cures. When mice bearing 3-day artificial lung metastases of C-26 cells were treated with low- or high-dose rIL-2, in combination with or without LAK or tumor-immune lymphocytes, a highly significant reduction or abrogation of the number of lung foci was observed with all treatments, including those involving or tumor-immune lymphocytes alone. Assessment of survival benefit in these mice, however, showed survival prolongation, with 20% cures achieved by low-dose rIL-2 alone and up to 65% cures by LAK in combination with low-dose rIL-2. In this system of artificial metastasis high-dose rIL-2 alone increased the survival time but failed to cure the animals, and the addition of LAK was ineffective whereas that of tumor-immune lymphocytes led to 80% cure. These results suggest that tumorimmune lymphocytes are more effective than LAK when combined with rIL-2 and that caution is necessary in extrapolating findings obtained in artificial metastasis models.