A chromatin code for limb segment identity in axolotl limb regeneration.

The salamander limb correctly regenerates missing limb segments because connective tissue cells have segment-specific identities, termed "positional information". How positional information is molecularly encoded at the chromatin level has been unknown. Here, we performed genome-wide chromatin profiling in mature and regenerating axolotl limb connective tissue cells. We find segment-specific levels of histone H3K27me3 as the major positional mark, especially 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. In the hand, the permissive chromatin state of the homeoprotein gene HoxA13 engages with the regeneration program bypassing the upper limb program. Comparison of regeneration regulatory elements with those found in other regenerative animals identified a core shared set of transcription factors, supporting an ancient, conserved regeneration program.

Artificial Insemination in Axolotl.

In axolotls (Ambystoma mexicanum), fertilization takes place internally. After courtship, the male axolotl deposits spermatophores, which the female takes up into her cloaca in order to fertilize eggs internally. The success of axolotl breedings is subject to several poorly understood factors including age, pairing, and genotype. In some cases, individuals are unable to breed naturally despite having significant scientific value. Assisted reproductive technologies represent one approach to maintaining stocks of such individuals, as well as supplementing natural breedings of laboratory stocks.Here, we describe a protocol for artificial insemination--an assisted reproductive technology in which sperm is extracted from a male and transferred into the female cloaca, thus mimicking natural fertilization in axolotls. We believe that this simple method can be applied to other salamander species that have internal fertilization and also help restore endangered wild populations.

Inducible and tissue-specific cell labeling in Cre-ERT2 transgenic Xenopus lines.

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.

Fibroblast dedifferentiation as a determinant of successful regeneration.

Limb regeneration, while observed lifelong in salamanders, is restricted in post-metamorphic Xenopus laevis frogs. Whether this loss is due to systemic factors or an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that connective tissue (CT) cells form the post-metamorphic frog blastema, as in the case of axolotls. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells dedifferentiate and integrate to form lineages, including cartilage. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT, but not to the developing cartilage. Furthermore, using single-cell RNA-seq analysis we find that embryonic and adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration.