Tetsuya Endo

Shh expression in developing and regenerating limb buds of Xenopus laevis

The zone of polarizing activity (ZPA) is a specialized region involved in the antero‐posterior (A‐P) axis formation in chick and mouse limb buds. The existence of ZPA in the posterior margin is suggested in Xenopus hindlimb buds because 180° rotation of the distal limb tip induces the supernumerary limb. In this study, we investigated the expression of Sonic hedgehog (shh), a molecular marker for ZPA, in Xenopus developing limb buds and regenerating blastemas by whole‐mount in situ hybridization. Although shh was expressed in the posterior margin of the limb bud like in chicks, its expression domain did not correspond to the ZPA map of Xenopus hindlimb buds. shh expression was distant from the ZPA at stage 53 in particular. To clarify the difference between the shh expression domain and ZPA, we examined shh expression in 180° rotated limb buds. As a result, ectopic shh expression was newly induced in the proximal region to its original expression domain. These results suggest that ZPA is accompanied by shh expression as in chick limb buds. Furthermore we examined shh expression in regenerating blastemas. shh was reexpressed in the posterior margin of the blastema. This result supports the possibility that ZPA also exists in the regenerating blastema. Dev. Dyn. 209:227–232, 1997.

The molecular basis of amphibian limb regeneration: integrating the old with the new.

Is regeneration close to revealing its secrets? Rapid advances in technology and genomic information, coupled with several useful models to dissect regeneration, suggest that we soon may be in a position to encourage regeneration and enhanced repair processes in humans.

Characterization of Xenopus Digits and Regenerated Limbs of the Froglet

Xenopus has 4 and 5 digits in a forelimb and hindlimb, respectively. It is thought that their limbs and digits develop in Xenopus by mechanisms that are almost conserved from amphibians to higher vertebrates. This is supported by some molecular evidence. The 5′hoxd genes are convenient marker genes for characterizing digits in the chick and mouse. The anteriormost digit is characterized by being hoxd13‐positive and hoxd12 (hoxd11)‐negative in the chick and mouse. In this study, we revealed that the anteriormost digit of the Xenopus forelimb is hoxd13‐positive and hoxd11‐positive, that is, a more posterior character than digit I. The order of formation of digit cartilages also suggested that Xenopus forelimb digit identity is II to V, not I to IV. We have also been interested in the relationship between digit identity and shh. The anteriormost digit develops in a shh‐independent way. A limb treated with cyclopamine (a shh inhibitor) has a gene expression pattern (hoxd11‐negative) similar to that in shh‐deficient mice, suggesting that a hindlimb treated with cyclopamine has a digit I character. However, a Xenopus froglet regenerate (spike), which lacks shh expression during its regeneration process, does not have such an expression pattern, being hoxd11‐positive. We investigated hoxd11 transcriptions in blastemas that formed in the anteriormost and posteriormost digits, and we found that the blastemas have different hoxd11 expression levels. These findings suggest that the froglet limb blastema does not have a mere digit I character in spite of shh defectiveness and that the froglet limb blastema recognizes its positional differences along the anterior‐posterior axis. Developmental Dynamics 235:3316–3326, 2006.

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.

Pattern formation in dissociated limb bud mesenchyme in vitro and in vivo

A fundamental process in limb bud development is the formation of position‐dependent cartilage pattern. Cells of the distal mesenchyme maintain positional values as the expression pattern of transcription factors, for example, hox genes, which induce position‐related cell differentiation and cell surface differences. Cultured, dissociated limb bud mesenchymal cells segregate from each other, and eventually form cartilage nodules. This sorting out is position‐dependent, not cell‐type dependent, suggesting that the positional values may be involved. Positional valves were found to be retained in limb bud recombinants. In the chick system, the expression of HoxA13 and HoxD12 was present in the distal half of stage 20 recombinants, whereas these markers were expressed throughout the stages 25 recombinants. In the Xenopus system, multiple digit formation was introduced in limb recombinants, and a position‐related relationship between regeneration potency and the multiple digit formation could be established. This determination of multiple digit formation with different stages of limb mesenchyme may be useful in understanding mechanisms of the loss of vertebrate limb regeneration potency.

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

Abstract 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.

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.

The Accessory Limb Model: An Alternative Experimental System of Limb Regeneration

Accessory limb model (ALM) was developed as an experimental model and functional assay for limb regeneration. The ALM provides several ways to identify pathways and test for signaling molecules that regulate limb regeneration. Here, we summarize the history of the ALM and describe the specific details involved in inducing ectopic blastemas and limbs from a skin wound on the side of the arm.

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

Abstract 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.