Origin of the blastema in regenerating limbs of the newt Triturus viridescens: An autoradiographic study using tritiated thymidine to follow cell proliferation and migration

Abstract

The present study has utilized autoradiography to detect incorporation of tritiated thymidine by the cells of the regenerating Triturus viridescens limb during blastema formation and to follow the subsequent migration of cells labeled by the isotope. New information was obtained on sites of DNA synthesis during blastema formation and the role of the internal tissues and epithelium was re-evaluated by tracing, for the first time, the actual fate of labeled cells during normal regeneration. Three series of experiments were performed. In the first, regenerating limbs 1–28 days post amputation were fixed the same day that the animals were injected with tritiated thymidine. This experiment revealed that DNA synthesis begins 4–5 days after amputation in the dedifferentiating muscle, endomysium, epimysium, periosteum, nerve sheaths, and loose connective tissue of the stump for a distance ∼ 1 mm proximal to the amputation surface. The number of cells in the inner tissues synthesizing DNA increases rapidly 10–20 days post amputation. The epidermis which migrates over the wound surface ceases to synthesize DNA within about 2 days. Epithelium proximal to the amputation surface, however, reaches a high level of DNA synthesis 8 days after amputation, and its cells migrate distally to increase the size of the apical cap 10–15 days post amputation. During blastema formation, no more than 2% of the cells in the apical cap incorporate thymidine, these rather feebly. After the blastema is established and the regenerate has begun to elongate, the apical cap thins out and its cells begin to synthesize DNA again. In the second experimental series, limbs were treated with tritiated thymidine during regeneration (5, 10, and 15 days post amputation) and representative limbs were fixed at daily intervals after treatment. The dedifferentiating inner cells incorporated a large amount of thymidine on the day of injection, whereas the apical epithelium did not. Almost all the blastema cells that appeared subsequently were labeled, and it can be concluded that they were derived from the dedifferentiating internal tissues. In the third experimental series, animals were injected with tritiated thymidine before their limbs were amputated. Epidermis is the only limb tissue that labels under these circumstances. Blood cells are labeled in their sites of origin, the liver and spleen. After amputation the labeled epidermis migrates over the wound surface and forms an apical cap which remains well labeled throughout blastema formation. The isotope becomes diluted in that area of proliferating proximal epithelium shown in Series I to be actively synthesizing new DNA. Blood cells labeled at the time of injection extravasate into the wounded limb tissues, but disappear from the limb 12–15 days post amputation The blastema cells which form in these limbs that contained labeled blood and apical epithelium are not labeled. In the discussion, the concept that the morphological and physiological changes which take place in dedifferentiating tissues make possible a phase of active cellular proliferation, is emphasized. The apical epidermal cap does not exhibit the DNA synthesis or any of the other essential features of dedifferentiating cells and, when this epidermis is labeled by appropriate treatment, no transformation of epithelial cells into blastema cells can be demonstrated. Indeed, the cells of the apical cap give evidence of being more highly differentiated than epidermis elsewhere.