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

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

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

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.

Symmetrical vascularization patterns in normal and retinoic acid treated limb regenerates of the axolotl, Ambystoma mexicanum

1998 ◽  
Vol 76 (9) ◽  
pp. 1795-1796 ◽  
Author(s):  
Steven R Scadding ◽  
Andrew Burns
Keyword(s):  
Retinoic Acid ◽  
Pattern Formation ◽  
Vascular System ◽  
Limb Regeneration ◽  
Ambystoma Mexicanum ◽  
Limb Pattern ◽  
Regeneration Blastema

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.

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

1999 ◽  
Vol 77 (11) ◽  
pp. 1835-1837 ◽  
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.

Use of retinoids to analyze the cellular basis of positional memory in regenerating amphibian limbs

1987 ◽  
Vol 65 (8) ◽  
pp. 750-761 ◽  
Author(s):  
David L. Stocum ◽  
Karen Crawford
Keyword(s):  
Retinoic Acid ◽  
Molecular Basis ◽  
Limb Regeneration ◽  
Molecular Property ◽  
Cell Recognition ◽  
Dorsoventral Axis ◽  
Modifying Agent ◽  
A Cell ◽  
Regeneration Blastema

Cells of the amphibian limb regeneration blastema inherit memories of their level of origin (positional memory) along the limb axes. These memories serve as boundaries of what is to be regenerated, thus preventing regeneration of any but the missing structures. Because of its importance in determining the boundaries of regenerate pattern, it is essential to understand the cellular and molecular basis of positional memory. One approach to this problem is to look for position-related differences in a cell or molecular property along a limb axis and then show, using an agent that modifies regenerate pattern, that the cell or molecular property and the pattern are coordinately modified. We have done this using retinoic acid (RA) as a pattern-modifying agent and an in vivo assay that detects position-related differences in a cell recognition–affinity property along the proximodistal (PD) axis of the regenerating axolotl limb. RA proximalizes positional memory in the PD axis, posteriorizes it in the anteroposterior axis, and ventralizes it in the dorsoventral axis. The level-specific PD cell recognition–affinity property is proximalized by RA, indicating that this property and positional memory are causally related. The effects of RA on positional memory may be mediated through a cellular RA-binding protein (CRABP), since the concentration of unbound (apo) CRABP molecules is highest during early stages of regeneration when the proximalizing effects of RA are greatest.

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

1988 ◽  
Vol 66 (9) ◽  
pp. 2065-2070 ◽  
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.

Retinoic acid coordinately proximalizes regenerate pattern and blastema differential affinity in axolotl limbs

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 687-698 ◽  
Author(s):  
K. Crawford ◽  
D.L. Stocum
Keyword(s):  
Retinoic Acid ◽  
Dorsal Surface ◽  
Blastema Cell ◽  
Specific Affinity ◽  
Cell Affinity ◽  
Differential Affinity ◽  
Regeneration Blastema

An assay that detects position-related differences in affinity of axolotl regeneration blastema cells in vivo was used to test whether retinoic acid, which proximalizes regenerate pattern, simultaneously proximalizes blastema cell affinity. The assay involved autografting or homografting late bud forelimb blastomas derived from the wrist, elbow or midupper arm levels to the dorsal surface of the blastema-stump junction of an ipsilateral, medium-bud-stage hindlimb regenerating from the midthigh level. The grafted blastemas consistently displaced to their corresponding levels on the proximodistal axis of the host regenerate, indicating the existence of level-specific differences in blastema cell affinity. Retinoic acid proximalized the pattern of donor forelimb regenerates to the level of the girdle and abolished their displacement behaviour on untreated host hindlimbs. Conversely, untreated forelimb donor blastemas displaced distally to their corresponding levels on host ankle regenerates, that had been proximalized to the level of the girdle by retinoic acid. These results indicate that positional memory in regenerating limbs is directly related to blastema cell affinity, and that very similar or identical sets of level-specific affinity properties are shared by forelimb and hindlimb cells.

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