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 size fields and can recreate consistent limb morphology. The mechanism that supports such stable limb morphogenesis regardless of size is unknown. Sonic hedgehog (SHH) and fibroblasts growth factor 8 (FGF8) play important roles in anteroposterior limb patterning, similar to other tetrapods. Focusing on these two factors, we investigated the detailed expression pattern and function of Shh and Fgf8 in various blastema sizes in axolotl limb regeneration. We measured and functionally analyzed the expression domains of Shh and Fgf8 in regenerating limb blastema of various sizes, and found that, although the position and size of the Shh+ and Fgf8+ domains varied depending on the size of the blastema, the secretion of SHH was maintained at a relatively fixed working distance, regardless of blastema size. This stable secretory distance of SHH resulted in the formation of an active proliferative zone (aPZ) in the vicinity of SHH, regardless of blastema size. The aPZ was under the mitogenic influence of SHH and FGF8, resulting in high cell density in the aPZ. We also examined the impact of the aPZ on digit formation. We found that the first digit formation occurs in the aPZ. Next, the aPZ gradually shifts posteriorly as digits develop, which contributes to new digit formation at the site of the shifted aPZ. We also found that the exogenously formed aPZ caused extra digit formation even after the completion of autopod morphogenesis. Our findings suggest that the variable Shh-Fgf8 positioning in various blastema sizes causes various positioning of the aPZ, and that the aPZ leads to digit formation. The mechanism we propose here accounts for stable digit morphogenesis regardless of blastema sizes and urodele-specific digit formation. One-Sentence SummaryA unique SHH-FGF8 spatial interaction compensates for robust limb morphogenesis in various limb sizes.