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.     

Blastema cell proliferation in vitro: effects of limb amputation on the mitogenic activity of spinal cord extracts

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts of non-amputated animals in a dose-dependent manner up to 60 micrograms protein/ml of culture medium; at this concentration the mitotic index is increased 4-fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.     

Effects of a Carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine on Blastema Cells and Blastema Formation in Newt Limb Regeneration

The effects of a potent, carcinogen, N -methyl- N' -nitro- N -nitrosoguanidine (MNNG) on blastema formation and blastema cells were studied in the adult newt by means of scanning electron microscopy. By administration of MNNG to the blastema of the limbs the formation of the basement lamella, which lies between the epithelium and the mesenchyme, was effectively inhibited at least for a month. Basement lamella in treated limbs is formed 30 or 40 days after carcinogen administration. MNNG altered significantly cell surface morphology and cell motility of blastema cells isolated in vitro. Membrane's specializations such as the formation of filopodia were inhibited. Motility of the treated cells was much reduced compared to the control. The role of these alterations in the carcinogen-induced abnormal regeneration is discussed.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Strand breaks of mammalian mitochondrial DNA induced by carcinogens.

Closed circular mitochondrial DNA in mammalian cells was degradated to the open circular form by exposure of the cells to the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline 1-oxide (4NQO). MNNG caused more strand scission of mitochondrial DNA than 4NQO at the same concentration. The action of the carcinogens on mitochondrial DNA did not parallel that with nuclear DNA which was damaged by 4NQO more markedly than by MNNG. Mitochondrial DNA damaged by carcinogens was not repaired during 4-20 h of post-treatment incubation of the cells. Incorporation of labeled thymidine into the closed circular mitochondrial DNA, decreased by the treatment of cells with carcinogens, recovered during post-treatment incubation.     

Glial growth factor and nerve-dependent proliferation in the regeneration blastema of Urodele amphibians

After amputation of a limb from Urodele amphibians, division of the blastemal cells (the progenitor cells of the regenerate) depends on one or more unidentified growth factors provided by the nerve supply. Here we show that glial growth factor (GGF), a mitogenic protein previously purified from the bovine pituitary, is present in newt nervous system extracts. It is also detectable in extracts of the forelimb regeneration blastema, and its level there decreases after denervation. We have previously shown that blastemal cells dependent on the nerve for division are marked by a monoclonal antibody called 22/18. When denervated blastemas are cultured in the presence of partially purified GGF from newt brain, or pure GGF from the bovine pituitary, the thymidine labeling index of blastemal cells that are 22/18-positive is increased as much as sevenfold. These data indicate that GGF plays a role in nerve-dependent proliferation in the blastema.     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.     

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.     

New regulators of vertebrate appendage regeneration

Appendage regeneration is a complex and fascinating biological process exhibited in vertebrates by urodele amphibians and teleost fish. A current focus in the field is to identify new molecules that control formation and function of the regeneration blastema, a mass of proliferative mesenchyme that emerges after limb or fin amputation and serves as progenitor tissue for lost structures. Two studies published recently have illuminated new molecular regulators of blastemal proliferation. After amputation of a newt limb, the nerve sheath releases nAG, a blastemal mitogen that facilitates regeneration. In amputated zebrafish fins, regeneration is optimized through depletion of the microRNA miR-133, a mechanism that requires Fgf signaling. These discoveries establish research avenues that may impact the regenerative capacity of mammalian tissues.     

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.     

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     

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.     

Changes in the Distribution of Fibronectin During Limb Regeneration in Newts Using Immunocytochemistry

The distribution of fibronectin in regenerating newt limbs was studied using immunocytochemistry. At appropriate intervals after the initial amputation at the elbow (10–30 days), animals were reamputated at the shoulder and processed for light microscopy. The peroxidase-antiperoxidase technique was used to localize affinity-purified antibodies to fibronectin in limb tissues. At the amputation site, fibronectin was associated with basal laminae and connective tissues adjacent to dedifferentiating limb tissues destined to form the regeneration blastema. Accumulation and growth of the blastema was accompanied by the apparent de novo synthesis of fibronectin, where it appeared randomly in the interstitium between blastemal cells. The onset of chondrogenesis was characterized by a central condensation of prechondroblasts that formed the cartilage anlagen. Fibronectin formed an amorphous network between presumptive chondroblasts. As the mature cartilage phenotype was expressed and chondrocytes became isolated in lacunae, fibronectin was greatly reduced and then disappeared. The extracellular matrix surrounding undifferentiated blastemal cells still contained fibronectin. Fibronectin was also found in high concentrations between differentiating myoblasts. A condensation of fibronectin was also observed beneath the epidermis at the distal limb tip at the onset of digit formation. These observations are consistent with the hypothesis that fibronectin may play a key role in the morphogenetic events that result in the spatial organization and subsequent differentiation of cells during pattern formation in the regenerating limb.     

Treatment of brachial nerves with colchicine inhibits limb regeneration in the newt Notophthalmus viridescens

In urodele amphibians, limb regeneration is dependent on innervation and is blocked by the administration of colchicine. The objective of this experiment was to determine if colchicine blocks limb regeneration by a direct action on the blastema cells or by an indirect action on the nerves, specifically, if colchicine treatment of the brachial nerves would inhibit limb regeneration in the newt Notophthalmus viridescens. Colchicine was applied to the nerves by implanting a colchicine-loaded silastin block adjacent to the brachial nerves of an amputated newt limb. With appropriate dose levels of colchicine, limb regeneration was completely inhibited. Contralateral control limbs, carrying unloaded silastin blocks, and control limbs with colchicine-loaded blocks implanted equidistant from the blastema, but not adjacent to the brachial nerves, regenerated normally. Thus, the results indicate that the colchicine inhibition of limb regeneration is mediated by colchicine effects on the nerves. The possible mechanism of colchicine action on nerves may involve either wallerian degeneration, or inhibition of axoplasmic transport, or both.     

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.     

Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration

Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the 'aneurogenic' limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).     

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.