Expression of Regeneration-Associated Antigens in Normal and Retinoid-Treated Regenerating Limbs of Ambystoma mexicanum. (limb regeneration/blastema/retinoid/monoclonal antibody)

The purpose of this investigation was to determine whether there were any asymmetries in the vascularization of the limb-regeneration blastema in the axolotl, Ambystoma mexicanum, that might be related to pattern formation, and to determine if retinoic acid could modify the vascular patterns of the blastema. We used acrylic casts of the vascular system of the limbs to assess the pattern of vascularization. We observed a very regular symmetrical arrangement of capillaries in the limb-regeneration blastema that did not appear to be modified by doses of retinoic acid sufficient to modify the limb pattern.

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A monoclonal antibody stains myogenic cells in regenerating newt muscle

Development ◽

10.1242/dev.101.2.267 ◽

1987 ◽

Vol 101 (2) ◽

pp. 267-277 ◽

Cited By ~ 1

Author(s):

K.J. Griffin ◽

D.M. Fekete ◽

B.M. Carlson

Keyword(s):

Monoclonal Antibody ◽

Muscle Regeneration ◽

Tissue Repair ◽

Limb Regeneration ◽

Alternative Interpretation ◽

Muscle Fibres ◽

Muscle Repair ◽

Contralateral Limb ◽

Minced Muscle ◽

Regeneration Blastema

Monoclonal antibodies have been used to study minced muscle regeneration in the adult newt, Notophthalmus viridescens. The contralateral limb was amputated and the immunostaining patterns in the regenerating blastema were compared with the minced tissue in sectioned material. Staining with a myofibre-specific antibody, called 12/101 (Kintner & Brockes, 1984), showed that myofibre degeneration was complete by 8–10 days after mincing, with myogenesis commencing 2 days later. Another monoclonal antibody, called 22/18, previously shown to label a subset of cells in the regeneration blastema of the newt (Kintner & Brockes, 1984, 1985), was found also to recognize a population of cells in regenerating minced muscle. At 6 days after mincing, the number of 22/18-positive (22/18+) cells was low but by days 12–16, during the period of myogenesis, their number had increased to become a major population within the minced tissue. A small number of the 22/18+ cells could be double labelled with 12/101 at this time. Prior to this, there was a phase in which 12/101 staining had disappeared from the mince. Cells immunoreactive with both antibodies after this phase confirm that at least some of the 22/18+ cells are myogenic. The number of 22/18+ cells decreased as muscle repair and maturation progressed. These results show that 22/18 is not specifically associated with blastemal cells but is a more general marker for regenerating systems in the newt. They further suggest an alternative interpretation of the double-labelled cells used by Kintner & Brockes (1984) as evidence for myofibre dedifferentiation in limb regeneration. Instead, we propose that such cells represent new myogenesis occurring by tissue repair of locally damaged muscle fibres.

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Gap junctions in the limb regeneration blastema of the axolotl, Ambystoma mexicanum, are not distributed uniformly and are regulated by retinoic acid

Canadian Journal of Zoology ◽

10.1139/cjz-77-6-902 ◽

1999 ◽

Vol 77 (6) ◽

pp. 902-909

Author(s):

Leigh-Anne D. Miller ◽

Melissa L. Farquhar ◽

John S. Greenwood ◽

Steven R. Scadding

Keyword(s):

Retinoic Acid ◽

Gap Junctions ◽

Limb Regeneration ◽

Ambystoma Mexicanum ◽

Regeneration Blastema

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Gap junctions in the limb regeneration blastema of the axolotl, Ambystoma mexicanum, are not distributed uniformly and are regulated by retinoic acid

Canadian Journal of Zoology ◽

10.1139/z99-045 ◽

1999 ◽

Vol 77 (6) ◽

pp. 902-909

Author(s):

Leigh-Anne D Miller ◽

Melissa L Farquhar ◽

John S Greenwood ◽

Steven R Scadding

Keyword(s):

Retinoic Acid ◽

Gap Junctions ◽

Limb Regeneration ◽

Ambystoma Mexicanum ◽

Limb Bud ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Gap Junctional ◽

Regeneration Blastema

Gap junctions are thought to play a role in pattern formation during limb development and regeneration by controlling the movement of small regulatory molecules between cells. An anteroposterior gradient of gap junctional communication that is higher posteriorly has been reported in the developing chick limb bud. In both the developing chick limb bud and the amphibian regenerating limb, an anteroposterior retinoic acid gradient is present, and this is also higher posteriorly. On the basis of these observations, we decided to examine the role of gap junctional communication in the regenerating amphibian limb. Gap junctions were observed in both the axolotl, Ambystoma mexicanum, limb regeneration blastema and cardiac tissue (as a positive control), using immunohistochemical labelling and laser scanning confocal microscopy. The scrape-loading/dye transfer technique for tracing the movement of a gap junction permeable dye, Lucifer yellow, showed that in blastemal epidermis there were nonuniform distributions of gap junctions in both the dorsoventral and anteroposterior axes of the blastema. Retinoic acid was found to increase gap junctional permeability in blastemal epidermis 48 h after injection and in blastemal mesenchyme 76 h after injection. The potential role of gap junctions during pattern formation in limb regeneration is discussed based on these results.

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Vitamin A modification of the positional information of blastema cells during limb regeneration in the axolotl Ambystoma mexicanum

Canadian Journal of Zoology ◽

10.1139/z88-304 ◽

1988 ◽

Vol 66 (9) ◽

pp. 2065-2070 ◽

Cited By ~ 2

Author(s):

Steven R. Scadding

Keyword(s):

Vitamin A ◽

Limb Regeneration ◽

Normal Structure ◽

Positional Information ◽

Ambystoma Mexicanum ◽

Limb Amputation ◽

Temporary Change ◽

Cellular Changes ◽

Regeneration Blastema

Vitamin A is known to cause proximodistal duplication of parts of the limb during limb regeneration in amphibians. The objective of this study was to investigate the nature and location of the cellular changes induced by vitamin A when it causes this duplication in the axolotl Ambystoma mexicanum. When axolotls were treated with retinol palmitate by immersion for 14 days before limb amputation, proximodistal duplications were still observed in subsequent regenerates of limbs amputated after vitamin A treatment was discontinued. This observation suggests that some characteristic of the cells is changed by vitamin A and that exogenous vitamin A need not be present while the limb is regenerating. When a limb that was induced to undergo proximodistal duplication by vitamin A was reamputated 49 days later through the original mid radius–ulna amputation plane, it regenerated a limb of normal structure. A regeneration blastema transplanted from a vitamin A treated axolotl to an untreated axolotl regenerated on the host limb stump, producing a limb with proximodistal duplication; this indicates that the blastema cells underwent some change by the early to mid cone stage, which was expressed later when the blastema redifferentiated into a new limb. Conversely, when an untreated blastema was transplanted onto a vitamin A treated axolotl from which the forelimb blastema had been removed, proximodistal duplications developed. This result is interpreted to mean that the stump cells, although morphologically of the radius–ulna level, were proximalized by the prior vitamin A treatment and still displayed proximal positional values, leading to intercalation of missing proximodistal structures. These results are consistent with the hypothesis that vitamin A brings about a temporary change in the positional information of the limb stump and blastema cells, and that when the vitamin A treatment is discontinued, there is a gradual return to normal positional values over a period of several weeks.

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Cell cycle controls and the role of nerves and the regenerate epithelium in urodele forelimb regeneration: possible modifications of basic concepts

Biochemistry and Cell Biology ◽

10.1139/o87-097 ◽

1987 ◽

Vol 65 (8) ◽

pp. 739-749 ◽

Cited By ~ 17

Author(s):

Roy A. Tassava ◽

David J. Goldhamer ◽

Bruce L. Tomlinson

Keyword(s):

Cell Cycle ◽

Monoclonal Antibody ◽

Limb Regeneration ◽

Blastema Formation ◽

Basic Concepts ◽

Regenerate Epithelium ◽

Early Wound ◽

Skin Epidermis ◽

Wound Epithelium

Data from pulse and continuous labeling with [3H]thymidine and from studies with monoclonal antibody WE3 have led to the modification of existing models and established concepts pertinent to understanding limb regeneration. Not all cells of the adult newt blastema are randomly distributed and actively progressing through the cell cycle. Instead, many cells are in a position that we have designated transient quiescence (TQ) and are not actively cycling. We postulate that cells regularly leave the TQ population and enter the actively cycling population and vice versa. The size of the TQ population may be at least partly determined by the quantity of limb innervation. Larval Ambystoma may have only a small or nonexisting TQ, thus accounting for their rapid rate of regeneration. Examination of reactivity of monoclonal antibody WE3 suggests that the early wound epithelium, which is derived from skin epidermis, is later replaced by cells from skin glands concomitant with blastema formation. WE3 provides a useful tool to further investigate the regenerate epithelium.

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Citral, an inhibitor of retinoic acid synthesis, modifies pattern formation during limb regeneration in the axolotl Ambystoma mexicanum

Canadian Journal of Zoology ◽

10.1139/z99-147 ◽

1999 ◽

Vol 77 (11) ◽

pp. 1835-1837 ◽

Cited By ~ 8

Author(s):

Steven R Scadding

Keyword(s):

Retinoic Acid ◽

Pattern Formation ◽

Limb Regeneration ◽

Acid Synthesis ◽

Ambystoma Mexicanum

While the effects of exogenous retinoids on amphibian limb regeneration have been studied extensively, the role of endogenous retinoids is not clear. Hence, I wished to investigate the role of endogenous retinoic acid during axolotl limb regeneration. Citral is a known inhibitor of retinoic acid synthesis. Thus, I treated regenerating limbs of the larval axolotl Ambystoma mexicanum with citral. The result of this inhibition of retinoic acid synthesis was that limb regeneration became extremely irregular and hypomorphic, with serious pattern defects, or was inhibited altogether. I conclude that endogenous retinoic acid plays an important role in pattern formation during limb regeneration.

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The influence of aminopterin on limb regeneration in Ambystoma mexicanum

Development ◽

10.1242/dev.16.1.143 ◽

1966 ◽

Vol 16 (1) ◽

pp. 143-158

Author(s):

D. O. E. Gebhardt ◽

J. Faber

Keyword(s):

Toluidine Blue ◽

Growth Retardation ◽

Limb Regeneration ◽

Ambystoma Mexicanum ◽

Ontogenetic Development ◽

Antimitotic Agent ◽

Chemical Substances ◽

Histamine Formation ◽

Number Of Authors

During the last twenty-five years a number of authors have studied the influence of chemical substances on limb regeneration in amphibians. Examples of compounds which have been tested so far are: (1) the antimitotic agent, colchicine (Thornton, 1943); (2) the salt, beryllium nitrate (Thornton, 1949, 1950, 1951); (3) the carcinogens, dibenzanthracene and methylcholanthrene (Karczmar & Berg, 1952; Ruben & Balls, 1964); (4) the lathyrus factor, β- aminopropionitrile (Chang, Witschi & Ponseti, 1955); (5) the hormone, thyroxine (Hay, 1956); (6) atropine and other neuropharmacological drugs (Singer, Davis & Scheuing, 1960); (7) the metachromatic dye, toluidine blue (Csaba, Bierbauer & Törö, 1961); and (8) semicarbazide, an inhibitor of histamine formation (Deck & Shapiro, 1963). Most of these substances caused growth retardation as well as malformations of the limb regenerates. A number of other investigators have studied the effects of chemicals on the ontogenetic development of the amphibian limb.

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