Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.

Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.

This study aimed at characterizing the Sonic hedgehog (

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.

The regeneration of structures in adult animals depends on a mechanism for coupling the acute response to tissue injury or removal with the local activation of plasticity in residual differentiated cells or stem cells. Many potentially relevant signals are generated after injury, and the nature of this mechanism has not been elucidated for any instance of regeneration. Lens regeneration in adult vertebrates always occurs at the pupillary margin of the dorsal iris, where pigmented epithelial cells (PEC) reenter the cell cycle and transdifferentiate into the lens, but the basis of this striking preference for the dorsal margin over the ventral is unknown. In this study, we report that a critical early event after lentectomy in the newt is the transient and selective activation of thrombin at the dorsal margin. The thrombin activity was blocked with two different irreversible inhibitors and was shown to be strictly required for cell cycle reentry at this location. The axolotl, a related urodele species, can regenerate its limb, but not its lens, and thrombin is activated in the former context, but not the latter. Our results indicate that selective activation of thrombin is the pivotal signal linking tissue injury to the initiation of vertebrate regeneration.