Spatially unique Shh-Fgf8 distribution in regenerating limb guarantees consistent limb morphogenesis in different limb sizes and contributes to axolotl specific digit patterning.

Limb regeneration in Ambystoma mexicanum occurs in various sizes of fields and can recreate consistent limb morphology. It was not known what mechanism supports such stable limb morphogenesis regardless of size. Limb regeneration in urodele amphibians has been basically considered to recapitulate the limb developmental processes. Many molecules in the limb developmental processes are conserved with other tetrapods. SHH and FGF8 play important roles in the morphogenesis of limbs among them. Focusing on these two factors, we investigated the detailed expression pattern of Shh and Fgf8 in the various sizes of blastema in axolotl limb regeneration. Fgf8 is expressed in the anterior side of a blastema and Shh is expressed in the posterior side. These are maintained in a mutually dependent manner. We also clarified that the size of Shh and Fgf8 expression domains were scaled as the size of the blastemas increased. However, it was found that the secretion and working range of SHH were kept constant. We also found that the consistent SHH secretion range contributed to promoting cell proliferation and the first digital cartilage differentiation near the Shh expression domain. This would be a reasonable system to guarantees constant limb morphogenesis regardless of the blastema size. We also showed that the Shh-Fgf8 expression domain was shifted posteriorly as the digital differentiation progressed. Consistently, slowing the timing of blocking Shh signaling resulted in morphological defects that could be observed in only posterior digits. The revealed posteriorly shifting Shh-Fgf8 domain might explain urodele specific digit formation, in which digits are added posteriorly. ### Competing Interest Statement The authors have declared no competing interest.