AMPHIBIAN LIMB REGENERATION
219
stages of the regeneration process—from tissue dedifferentiation, through
blastemal cell proliferation, to tissue ^differentiation in the regenerate.
Holtzer (1961), using labeled antimyosin techniques, has followed mononucleate muscle cells, containing solubilized myosin, until the seventh or
eighth day after amputation when blastemal cells no longer bind antibody (although they do so again on the twelfth or thirteenth day). In
these experiments, from 40 to 80% of the early blastemal cells are reported to contain the myosin label—an indication of how high a proportion of the blastemal cells may be derived specifically from muscle.
Nevertheless, these experiments, due to the absence of label for about 4
to 5 days, are unable to provide us with unequivocal answers to the
possibility of metaplasia in these regenerates.
Numerous experiments have indicated that of all the tissues normally
found in the limb stump, the skeleton is the only one the absence of
which does not interefere with typical regeneration (reviewed by Goss,
1961). Removal of the humerus (Bischler, 1926, and others) does not
prevent the formation of a new humerus in the regenerate. Similar evidence is not available for muscle, connective tissue, or skin due to the
great difficulty of excluding all of these tissues from the limb stump. The
problem, however, has been approached in a variety of ingenious ways.
Rose and Rose (1965), for example, labeled only the epidermal cells of
the limbs of adult newts with
3
H-thymidine and, after amputation, followed these labeled cells through as many as five generations—a program
made possible only by the great care they used in their techniques. They
report that labeled nuclei (3 to 5 grains) are found in muscle and cartilage of the regenerates which, they conclude, would indicate that metaplasia had occurred. Steen (1967), however, points out that, in axolotl
regeneration experiments in which he implanted thymidine3
!! labeled
triploid tissues into diploid limb stumps, transfer was routinely detected
(i.e., in radioactive diploid cells) at a level sufficient to give 3 to 5 grains
over a transfer cell. He suggests that the results reported by Rose and
Rose (1965) might also be a consequence of label transfer, a possibility
testable by the double label technique. A number of other investigations
have provided data at variance with the conclusion that epidermis may
serve as a source for blastemal cells in urodele limb regeneration. Hay
and Fischman (1961), by injecting thymidine3
H into newts before
amputation, labeled only the epidermis and failed to find any labeled
cells subsequently in the blastema. Riddiford (1960) transplanted regenerate epidermis labeled with thymidine3
H to unlabeled regenerating
limbs and also failed to detect any labeled cells in the blastema. O'Steen
219
stages of the regeneration process—from tissue dedifferentiation, through
blastemal cell proliferation, to tissue ^differentiation in the regenerate.
Holtzer (1961), using labeled antimyosin techniques, has followed mononucleate muscle cells, containing solubilized myosin, until the seventh or
eighth day after amputation when blastemal cells no longer bind antibody (although they do so again on the twelfth or thirteenth day). In
these experiments, from 40 to 80% of the early blastemal cells are reported to contain the myosin label—an indication of how high a proportion of the blastemal cells may be derived specifically from muscle.
Nevertheless, these experiments, due to the absence of label for about 4
to 5 days, are unable to provide us with unequivocal answers to the
possibility of metaplasia in these regenerates.
Numerous experiments have indicated that of all the tissues normally
found in the limb stump, the skeleton is the only one the absence of
which does not interefere with typical regeneration (reviewed by Goss,
1961). Removal of the humerus (Bischler, 1926, and others) does not
prevent the formation of a new humerus in the regenerate. Similar evidence is not available for muscle, connective tissue, or skin due to the
great difficulty of excluding all of these tissues from the limb stump. The
problem, however, has been approached in a variety of ingenious ways.
Rose and Rose (1965), for example, labeled only the epidermal cells of
the limbs of adult newts with
3
H-thymidine and, after amputation, followed these labeled cells through as many as five generations—a program
made possible only by the great care they used in their techniques. They
report that labeled nuclei (3 to 5 grains) are found in muscle and cartilage of the regenerates which, they conclude, would indicate that metaplasia had occurred. Steen (1967), however, points out that, in axolotl
regeneration experiments in which he implanted thymidine3
!! labeled
triploid tissues into diploid limb stumps, transfer was routinely detected
(i.e., in radioactive diploid cells) at a level sufficient to give 3 to 5 grains
over a transfer cell. He suggests that the results reported by Rose and
Rose (1965) might also be a consequence of label transfer, a possibility
testable by the double label technique. A number of other investigations
have provided data at variance with the conclusion that epidermis may
serve as a source for blastemal cells in urodele limb regeneration. Hay
and Fischman (1961), by injecting thymidine3
H into newts before
amputation, labeled only the epidermis and failed to find any labeled
cells subsequently in the blastema. Riddiford (1960) transplanted regenerate epidermis labeled with thymidine3
H to unlabeled regenerating
limbs and also failed to detect any labeled cells in the blastema. O'Steen