Recent studies suggest that maintenance of the differentiated state requires continuous regulation. Limb regeneration in urodele amphibians provides a context in which to address this issue, as limb regeneration may involve the dedifferentiation of multinucleate myotubes to yield mononucleate blastemal cells, which then proliferate and contribute to regenerate tissues. To evaluate this possibility, cultured newt limb myotubes were selectively microinjected with the lineage tracer rhodamine-dextran and introduced into regenerating limbs. In culture, such labeled myotubes were stable after 6-8 weeks, and transfer of the tracer to mononucleate cells was not observed. In contrast, after implantation of labeled myotubes under the wound epidermis of limb blastemas, strongly labeled mononucleate cells were observed after 1 week. These cells could be double-labeled with the cytoplasmic lineage tracer and [3H]thymidine that had been incorporated into the nuclei of implanted myotubes. The number of labeled mononucleate cells increased significantly by 2-3 weeks after implantation, indicating that these cells proliferated. Although the fate of these cells at later times was uncertain, we provide evidence consistent with their subsequent differentiation. These results demonstrate reversal in the mononucleate-to-multinucleate transition of vertebrate myogenesis.Following the isolation of several myogenic genes (1-4), muscle has become a key system for investigating the control and maintenance of the differentiated state. The fusion of mononucleate myoblasts to form multinucleate, postmitotic syncytial myotubes is one of the most striking examples of terminal differentiation. In understanding the mechanisms that underlie the stability ofdifferentiation, the reversal ofthe process, termed dedifferentiation, or a switch to another lineage, referred to as transdifferentiation (5), are of particular interest. In a recent study it has been demonstrated that expression of simian virus 40 large tumor antigen can induce nuclei in cultured mouse myotubes to reenter the cell cycle and, furthermore, that this observation and the normal cell cycle arrest involved in myogenesis may reflect changes in activation of the retinoblastoma gene product (6). Although these experiments suggest the possibility of reversing myogenic differentiation, cytokinesis of myotubes to yield mononucleate progeny was not reported.One natural context in which dedifferentiation may occur is during limb regeneration of urodele amphibians such as the newt and axolotl. Blastemal cells, the mesenchymal progenitors of the regenerate, arise locally at the amputation plane and may derive in part from dedifferentiation of stump tissues such as cartilage (7) and muscle (8-10), although the contribution of multinucleate myofibers remains uncertain (11,12). To assess the reversibility of the mononucleate-to-multinucleate transition in urodeles, we induced myotube formation in cultured newt limb cells and selectively labeled such myotubes by microinjection of a nont...
The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.
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