Salamanders regenerate appendages via a progenitor pool called the blastema. The cellular mechanisms underlying regeneration of muscle have been much debated but have remained unclear. Here we applied Cre-loxP genetic fate mapping to skeletal muscle during limb regeneration in two salamander species, Notophthalmus viridescens (newt) and Ambystoma mexicanum (axolotl). Remarkably, we found that myofiber dedifferentiation is an integral part of limb regeneration in the newt, but not in axolotl. In the newt, myofiber fragmentation results in proliferating, PAX7(-) mononuclear cells in the blastema that give rise to the skeletal muscle in the new limb. In contrast, myofibers in axolotl do not generate proliferating cells, and do not contribute to newly regenerated muscle; instead, resident PAX7(+) cells provide the regeneration activity. Our results therefore show significant diversity in limb muscle regeneration mechanisms among salamanders and suggest that multiple strategies may be feasible for inducing regeneration in other species, including mammals.
The establishment of synaptic connections requires precise alignment of pre- and postsynaptic terminals. The glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 is enriched at pre- and postsynaptic compartments in hippocampal neurons, suggesting that it has a function in synapse formation. GDNF triggered trans-homophilic binding between GFRalpha1 molecules and cell adhesion between GFRalpha1-expressing cells. This represents the first example of a cell-cell interaction being mediated by a ligand-induced cell adhesion molecule (LICAM). In the presence of GDNF, ectopic GFRalpha1 induced localized presynaptic differentiation in hippocampal neurons, as visualized by clustering of vesicular proteins and neurotransmitter transporters, and by activity-dependent vesicle recycling. Presynaptic differentiation induced by GDNF was markedly reduced in neurons lacking GFRalpha1. Gdnf mutant mice showed reduced synaptic localization of presynaptic proteins and a marked decrease in the density of presynaptic puncta, indicating a role for GDNF signaling in hippocampal synaptogenesis in vivo. We propose that GFRalpha1 functions as a LICAM to establish precise synaptic contacts and induce presynaptic differentiation.
The axolotl (Mexican salamander, Ambystoma mexicanum) has become a very useful model organism for studying limb and spinal cord regeneration because of its high regenerative capacity. Here we present a protocol for successfully mating and breeding axolotls in the laboratory throughout the year, for metamorphosing axolotls by a single i.p. injection and for axolotl transgenesis using I-SceI meganuclease and the mini Tol2 transposon system. Tol2-mediated transgenesis provides different features and advantages compared with I-SceI-mediated transgenesis, and it can result in more than 30% of animals expressing the transgene throughout their bodies so that they can be directly used for experimentation. By using Tol2-mediated transgenesis, experiments can be performed within weeks (e.g., 5-6 weeks for obtaining 2-3-cm-long larvae) without the need to establish germline transgenic lines (which take 12-18 months). In addition, we describe here tamoxifen-induced Cre-mediated recombination in transgenic axolotls.
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