Animals exhibit extreme diversity in regenerative ability. This likely reflects different, lineage-specific selective pressures in their evolutionary histories, but how specific molecular features of regenerative programs help solve species-specific challenges has not been examined in detail. Here we discover that, in the highly-regenerative axolotl salamander, a conserved, body-wide stem cell activation response triggered in response to limb removal primes undisturbed limbs for regeneration upon subsequent amputation. This response should be particularly useful to salamanders, which frequently lose limbs in response to cannibalism. We further demonstrate the body-wide response requires both peripheral nervous system input at these distant sites and mTOR signaling. We defined gene expression changes within the nerves and nearby tissues, harboring responsive stem cells, leading to identification of candidate genetic pathways influencing distant stem cell activation following amputation. Functional experimentation confirmed a requirement for adrenergic signaling in amputation-induced activation of distant stem cells. These findings reveal a direct link between systemic cellular activation responses to local tissue damage and overall regenerative ability. Similar systemic activation responses to tissue removal have been observed in animals with widely differing regenerative abilities (e.g., planaria to mice), suggesting that it is the responses downstream of these signals, likely sculpted by differing evolutionary pressures, that ultimately distinguish regenerators from non-regenerators.

INTRODUCTIONAs discussed in detail at this conference, natural and synthetic analogs of vitamin A (retinoids) have been demonstrated to be capable of inhibiting the development of chemically induced carcinomas in a variety of laboratory animals and at a variety of anatomical sites.'-4 Furthermore, dietary deficiency in experimental animals has been shown to enhance susceptibility to chemical carcinogenesiss, and there is recent suggestive evidence from epidemiological studies in human populations that groups ingesting low levels of vitamin A are at increased risk of lung6 and bladder7 cancer. Because of the importance of these observations, we have studied the effects of retinoids in the C3H/lOT1/2 CL8 cell line of mouse embryo fibroblasts,a which is readily transformable by many chemicalg and physical agents10 and in which transformation can be readily quan-titated9. In this line transformation is expressed as a lack of post-confluence inhibition of growth and transformed foci appear as piled up regions of overlapping cells amid a highly regular monolayer of growth-inhibited nontransformed cells. That about 90% of these morphologically transformed foci are malignant can be demonstrated by injection into immunosuppressed syngeneic C3H mice.9 In many studies the behavior of these cells to carcinogens has been shown to reflect in vivo events This paper will discuss our recent studies on the cell biology of retinoid action, structure/activity requirements for activity of retinoids in the 10T112 cells, and the effects of retinoids on the cell membrane. METHODS Cells and Culture ConditionsA transformable line of mouse embryo fibroblasts designated C3H/lOT1/2 CL8 (10T1/2)8 and neoplastically transformed lines derived from the parent cell line by treatment with chemical carcinogens were used throughout this study." Cultures were passaged weekly in basal medium Eagle's (BME) supplemented with 10% heat-inactivated fetal calf serum (HIFCS) without antibiotics, and were