Radioautographic studies of regeneration in the common newt. II. Regeneration of the forelimb

After amputation of the forelimb in urodele amphibians, a blastema develops in the regenerating appendage from cells contributed by the tissues in the immediate vicinity of the injury. The blastema is composed of morphologically dediff erentiated cells with only a reduced amount of cytoplasm surrounding each nucleus. During later stages of regeneration a fully differentiated limb develops from this blastema. The phenomenon of regeneration has suggested many fundamental problems (review, Nicholas, ' 5 5 ) , but one of the most important is related to the specific origin of the cells within the regeneration blastema. The origin of these cells might be, in the case of limbs, the internal tissues, such as cartilage, muscle, and connective tissue, or the epidermis, or both sources. There are, therefore, several schools of thought concerning the origin of blastema cells. Godlewski ('28) first advanced the hypothesis that epidermal cells from the thickened apical cap migrated into the blastema in large numbers. Rose ('48) and Rose et al. ('55) reintroduced this hypothesis. Hay ('52), using tissues with modified chromosome numbers, showed that cells in the blastema of amphibian larvae came in part both from internal tissues and from epidermis. Results from the work of others (e.g., Butler, '33; Thornton, '38a, '38b, '42; Forsyth, '46; and Chalkley, '54, '59) indicated that the principle source of blastema cells was the internal tissues of the regenerating limbs, such as fibroblasts, and dissociating muscle and cartilage. Although the methods previously used have contributed to a better understanding of regenerative processes and have stimulated additional related research, some of them have not proven completely satisfactory. They provided evidence for the origin of blastema cells, but did not mark the cells so that they might be followed during later stages of regeneration. Radioautography using tritiated thymidine is a method of labeling desoxyribonucleic: acid (DNA) in the nuclei of cells preparing to divide. Using this technique, the reactive nuclei may be traced for varying lengths of time. The period of observation for such a nucleus depends upon several factors, such as the dilution of the labeled DNA and the life span of the cell. After each cell division, the labeled DNA originally in a single nucleus is distributed to the two daughter nuclei. Following several divisions, the diluted DNA of reactive nuclei may not be defined on radioautographs. Death, phagocytosis, and desquamation of reactive cells also reduces the population of labeled cells. The use of radioautographic techniques for studying regenerating amphibian tissues was previously proposed during an investigation of the normal replacement of cells in tissues in the common newt (O'Steen and Walker, '60). Results from this study indicated that intraperitoneal injections of tritiated thymidine in the adult newt heavily labeled the epidermis of the appendages without labeling tissues such as bone, cartilage, muscle, and associated connective tissues. Therefore, if only epidermal cells were labeled prior to amputating the forelimb, the behavior of the epidermis in relation to the formation