The Effects of Retinoids on Regenerating Axolotl Limbs

Abstract

The limbs of amphibians normally regenerate only those parts distal to the amputation plane, an observation that has been formalized as the rule of distal transformation. The basis for this rule is the inheritance by the blastema of a cellular memory of its position of origin, termed positional value. Recently, vitamin A and its derivatives (retinoids) have been found to proximalize positional value, causing duplication of stump structures in the regenerate. Positional values along the proximodistal (PD) axis are associated with a gradient of cell surface adhesivity, and retinoids have been shown to alter adhesivity and glycoconjugate composition of cell surfaces in cultured avian and mammalian cells. These two observations suggest a potential approach to a cellular and molecular analysis of positional value in regenerating limbs.

To use retinoids for this purpose, the morphological and histological effects of retinoids on axial pattern must first be thoroughly characterized. Within this context, the experiments of the present study were designed to accomplish the following: (1) to compare the efficacy of retinoic acid (RA), etretinate (ET), and arotinoid (AR) in evoking PD duplication; (2) to compare the effect of a given dose of these retinoids on two different PD levels of amputation; (3) to test whether retinoids can change positional value in the anteroposterior (AP) axis; (4) to examine retinoid-induced histological changes in regenerating limbs. The results show that retinoid-induced pattern duplication in the PD axis is dose-dependent, and that the efficacy of the three retinoids is in the order AR >>> RA >> ET. At a given dose of retinoid, more pattern was duplicated from a distal level (zeugopodium) than from a proximal level (stylopodium). In the AP axis, retinoid was found to posteriorize positional values but not anteriorize them. Therefore, retinoid-induced change of positional value is unidirectional in both the PD and AP axes. Histologically, retinoid treatment prolongs the period of dedifferentiation and blastema cell accumulation, and the extent of dedifferentiation of stump tissues is also increased. Blastema cell density is increased, suggesting that retinoid treatment may cause changes in extracellular matrix composition as well as cell surface properties.