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Abstract
During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema−dorsal root ganglia (DRG) co‐culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for 5 days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function in injury and neurodevelopmental responses observed for mammalian DRG. We then cultured 5‐day DRG explants for an additional 5 days with or without co‐cultured blastema cells. On day 10, we identified 27 genes whose expression in cultured DRG was significantly affected by the presence or absence of blastema cells. Overall, our study established a DRG−blastema in vitro culture system and identified candidate genes for future investigations of axon regrowth, nerve−blastema signaling, and neural regulation of limb regeneration.
Highlights
Salamanders and humans have structurally homologous tetrapod limbs, but only in salamanders is this structure capable of regeneration
In addition to regenerating neurites, the morphology of dorsal root ganglia (DRG) appeared normal in histological sections (Fig. 1C and E). 4’,6-Diamidino-2phenylindole (DAPI) stained nuclei within both the DRG and the distal nerve trunk appeared normal with little evidence of pyknosis (Fig. 1E)
Cells within both the DRG and the nerve trunk were proliferating as evidenced by the presence of 5-ethynyl-2 -deoxyuridine (EdU) positive nuclei
Summary
Salamanders and humans have structurally homologous tetrapod limbs, but only in salamanders is this structure capable of regeneration. The blastema grows and eventually becomes a self-organizing structure that provides progenitor cells for regrowth and patterning of the missing limb (McCusker and Gardiner 2013). One of the earliest discoveries of an essential regulator of the process of blastema formation and subsequent limb regeneration was that severing the nerve supply either “retarded or entirely prevented” limb regeneration (Todd 1823). More than a century and a half later, researchers are still looking for the elusive factor(s) that the nerve provides in order to exert its effect on regeneration. Over the years many putative factors have been investigated, and with the availability of new techniques and genomic resources it is possible to identify specific gene regulatory networks associated with this phenomenon
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