Lineage tracing of Shh+ floor plate cells and dynamics of dorsal-ventral gene expression in the regenerating axolotl spinal cord

Both development and regeneration depend on signalling centres, which are sources of locally secreted tissue-patterning molecules. As many signalling centres are decommissioned before the end of embryogenesis, a fundamental question is how signalling centres can be re-induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration. The floor plate is an archetypal vertebrate signalling centre that secretes Shh ligand and patterns neural progenitor cells during embryogenesis. Unlike mammals, axolotls continue to express floor plate genes (including Shh) and downstream dorsal-ventral patterning genes in their spinal cord throughout life, including at steady state. The parsimonious hypothesis that Shh+ cells give rise to functional floor plate cells for regeneration had not been tested. Using HCR in situ hybridisation and mathematical modelling, we first quantitated the behaviours of dorsal-ventral spinal cord domains, identifying significant increases in gene expression level and floor plate size during regeneration. Next, we established a transgenic axolotl to specifically label and fate map Shh+ cells in vivo. We found that labelled Shh+ cells gave rise to regeneration floor plate, and not to other neural progenitor domains, after tail amputation. Thus, despite changes in domain size and downstream patterning gene expression, Shh+ cells retain their floor plate identity during regeneration, acting as a stable cellular source for this regeneration signalling centre in the axolotl spinal cord.