In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.

Skeletal callus formation is a nerve-independent regenerative response to limb amputation in mice and Xenopus.

To clarify the mechanism of limb regeneration that differs between mammals (non-regenerative) and amphibians (regenerative), responses to limb amputation and the accessory limb inducible surgery (accessory limb model, ALM) were compared between mice and Xenopus, focusing on the events leading to blastema formation. In both animals, cartilaginous calluses were formed around the cut edge of bones after limb amputation. They not only are morphologically similar but show other similarities, such as growth driven by undifferentiated cell proliferation and macrophage-dependent and nerve-independent induction. It appears that amputation callus formation is a common nerve-independent regenerative response in mice and Xenopus. In contrast, the ALM revealed that the wound epithelium (WE) in Xenopus was innervated by many regenerating axons when a severed nerve ending was placed underneath it, whereas only a few axons were found within the WE in mice. Since nerves are involved in induction of the regeneration-permissive WE in amphibians, whether or not nerves can interact with the WE might be one of the key processes separating successful nerve-dependent blastema formation in Xenopus and failure in mice.

Growth and differentiation of a long bone in limb development, repair and regeneration.

Repair from traumatic bone fracture is a complex process that includes mechanisms of bone development and bone homeostasis. Thus, elucidation of the cellular/molecular basis of bone formation in skeletal development would provide valuable information on fracture repair and would lead to successful skeletal regeneration after limb amputation, which never occurs in mammals. Elucidation of the basis of epimorphic limb regeneration in amphibians would also provide insights into skeletal regeneration in mammals, since the epimorphic regeneration enables an amputated limb to re-develop the three-dimensional structure of bones. In the processes of bone development, repair and regeneration, growth of the bone is achieved through several events including not only cell proliferation but also aggregation of mesenchymal cells, enlargement of cells, deposition and accumulation of extracellular matrix, and bone remodeling.

Ectopic blastema induction by nerve deviation and skin wounding: a new regeneration model in Xenopus laevis.

Recently, the accessory limb model (ALM) has become an alternative study system for limb regeneration studies in axolotls instead of using an amputated limb. ALM progresses limb regeneration study in axolotls because of its advantages. To apply and/or to compare knowledge in axolotl ALM studies to other vertebrates is a conceivable next step. First, Xenopus laevis, an anuran amphibian, was investigated. A Xenopus frog has hypomorphic regeneration ability. Its regeneration ability has been considered intermediate between that of non-regenerative higher vertebrates and regenerative urodele amphibians. Here, we successfully induced an accessory blastema in Xenopus by skin wounding and rerouting of brachial nerve bundles to the wound site, which is the regular ALM surgery. The induced Xenopus ALM blastemas have limited regenerative potential compared with axolotl ALM blastemas. Comparison of ALM blastemas from species with different regenerative potentials may facilitate the identification of the novel expression programs necessary for the formation of cartilage and other tissues during limb regeneration.

Skeletogenesis in Xenopus tropicalis: characteristic bone development in an anuran amphibian.

In mammals and birds, most of the skeletal bones develop via endochondral ossification. Chondrocytes in the cartilaginous anlagen undergo processes of maturation such as hypertrophy, calcification and apoptosis. Concomitantly, osteoblasts are recruited to replace the cartilage scaffold gradually with bone matrix and become osteocytes in the trabecular bones. Throughout the successive development of bones, several gene products have been identified as being the components of the molecular mechanism regulating bone development. Transcription factor SOX9 plays essential roles during developmental steps from undifferentiated mesenchymal cells to proliferating chondrocytes, meanwhile, it inhibits transition of proliferating chondrocytes to hypertrophy. Other transcription factors RUNX2 and OSTERIX are critical in osteoblast differentiation, and RUNX2 is also essential for chondrocyte maturation such as hypertrophy and matrix mineralization. GDF5, a protein belonging to the transforming growth factor beta superfamily, is involved in joint formation and chondrogenesis. The limb skeleton of one of the ancestral tetrapod, anuran amphibians also develops through cartilaginous anlagen to bones, but their skeletogenesis has some unique characteristics compared with that of mammals and birds. Anuran amphibians develop and grow with less bone trabeculae and poor epiphyseal growth plates, and its endochondral ossification was found to be a delayed process. In order to address the characteristic skeletal development of anuran amphibians, we cloned Xenopus tropicalis RUNX2 (Xt-runx2), OSTERIX (Xt-osterix) and GDF5 (Xt-gdf5) homologue, and observed expression patterns together with Xt-sox9. In X. tropicalis limbs, histological observation and section in situ hybridization analysis suggest that Xt-SOX9 is involved in chondrogenesis, Xt-RUNX2 and Xt-OSTERIX are involved in osteogenesis, and Xt-GDF5 is involved in joint formation. In the cartilaginous anlagen, Xt-runx2 expression was found in perichondrium and immature chondrocytes as seen in other vertebrates. However, Xt-runx2 expression in enlarged chondrocytes was weak and dissimilar to common hypertrophic chondrocytes. These observations suggest that weak Xt-runx2 expression in maturing chondrocytes affects characteristic bone development in X. tropicalis long bones.