The use of retinoids and an in vivo "affinophoresis" assay to analyze the cellular basis of positional memory in regenerating axolotl limbs

117 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.Amphibian limb regeneration blastema cells normally regenerate only those parts distal to the amputation plane, and have been shown to inherit an expressed memory of their position of origin, termed positional value, by developing autonomously when grafted away from their normal limb site, and by inducing intercalary regeneration from more proximal limb stumps. Recently, retinoids (vitamin A and its derivatives) have been shown to predictably modify positional memory in regenerating amphibian limbs resulting in PD duplications of stump structures within the regenerate. This discovery creates a means by which the cellular and molecular basis of pattern formation can be investigated. This study describes an in vivo assay that was devised to detect level-specific differences in cell affinity along the proximodistal (PD) axis of regenerating axolotl limbs, and several experiments employing retinoic acid (RA) designed to correlate these differences with positional memory.In the assay, forelimb late bud wrist, elbow or mid-upper arm regeneration blastemas were autografted to the dorsal blastema-stump junction of mid-thigh medium bud blastemas. The regenerates formed by the donor blastemas were subsequently found at those levels of the host regenerate corresponding to the levels of origin of the graft: ankle, knee and mid-thigh respectively. This result suggests that blastema cells possess level-specific cell affinity properties that allow them to recognize and associate with cells of corresponding host levels.To correlate this level-specific property with positional memory, RA treatment was combined with the in vivo assay and grafting in three experiments: (1) homografting retinoic acid treated wrist, elbow or mid-upper arm blastemas to the untreated mid-thigh blastema hosts; (2) the reciprocal experiment, homografting untreated wrist, elbow or mid-upper arm blastemas to RA-treated ankle level hosts; and (3) homografting blastemas, derived from distal levels of RA-treated animals, to the stump surface of more proximal level hosts. The results of all three experiments indicate that the level-specific cellular properties which mediate non-neighbor recognition and initiate intercalary regeneration and in vivo sorting are coordinately proximalized with positional memory by RA. These results suggest that the same set of cell surface molecules may mediate these different responses of regenerate cells to pattern discontinuities.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD