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

Abstract

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.