Highlights d Fibroblast-derived Prrx1 + cells are the main constituent of a frog limb blastema d Frog fibroblasts only undergo partial dedifferentiation due to intrinsic limitations d Adult chondrogenesis is distinct from the embryonic program

The salamander limb regenerates only the missing portion. Each limb segment can only form segments equivalent to- or more distal to their own identity, relying on a property termed, positional information. How positional information is encoded in limb cells has been unknown. By cell-type-specific chromatin profiling of upper arm, lower arm, and hand, we found segment-specific levels of histone H3K27me3 at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. This means that, in the hand segment, the permissive chromatin state of the hand homeoprotein gene HoxA13 engages with regeneration regulatory elements, bypassing the upper limb program.

Investigating cell lineage requires genetic tools that label cells in a temporal and tissue‐specific manner. The bacteriophage‐derived Cre‐ERT2/loxP system has been developed as a genetic tool for lineage tracing in many organisms. We recently reported a stable transgenic Xenopus line with a Cre‐ERT2/loxP system driven by the mouse Prrx1 (mPrrx1) enhancer to trace limb fibroblasts during the regeneration process (Prrx1:CreER line). Here we describe the detailed technological development and characterization of such line. Transgenic lines carrying a CAG promoter‐driven Cre‐ERT2/loxP system showed conditional labeling of muscle, epidermal, and interstitial cells in both the tadpole tail and the froglet leg upon 4‐hydroxytamoxifen (4OHT) treatment. We further improved the labeling efficiency in the Prrx1:CreER lines from 12.0% to 32.9% using the optimized 4OHT treatment regime. Careful histological examination showed that Prrx1:CreER lines also sparsely labeled cells in the brain, spinal cord, head dermis, and fibroblasts in the tail. This work provides the first demonstration of conditional, tissue‐specific cell labeling with the Cre‐ERT2/loxP system in stable transgenic Xenopus lines.

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