Transcription activator-like effector nucleases (TALENs) are attractive and powerful molecular tools for targeted gene disruption because of their simple design and quick assembly. To evaluate the utility of TALENs in genome editing in Xenopus tropicalis, we prepared nine pairs of TALENs for the tyrosinase, noggin and MMP-9TH genes. All of the TALENs had some activity in a single-strand annealing assay using a cultured frog cell line, suggesting double-stranded DNA cleavage activity by the TALENs at their target site. The injection of mRNAs encoding TALENs into fertilized X. tropicalis embryos resulted in Cel-1 cleavage of the PCR fragment containing the target site amplified from embryo genomic DNA, indicating that a mutation in the target gene had occurred during embryogenesis. These mutations were confirmed by the sequencing of clones derived from the PCR fragments of genomic DNA. Patches of vitiligo were observed in tadpoles raised from fertilized eggs that had been injected with mRNAs of TALENs for the tyrosinase gene. TALENs containing the repeat variable di-residue (RVD) NN appeared to show more activity than TALENs containing RVD NK, although both RVD NN and NK preferentially associate with a G nucleotide.

Amphibian metamorphosis has historically attracted a good deal of scientific attention owing to its dramatic nature and easy observability. However, the genetic mechanisms of amphibian metamorphosis have not been thoroughly examined using modern techniques such as gene cloning, DNA sequencing, polymerase chain reaction or genomic editing. Here, we review the current state of knowledge regarding molecular mechanisms underlying tadpole tail resorption.

Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within three days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval-skin-specific antigens, Ouro proteins. We generated ouro-knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1- and ouro2-knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis revealed that mutant frogs lacked splenic CD8+ T cells, which play a major role in cytotoxic reaction. Furthermore, T cell-dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which demonstrates that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.

Tail regression in amphibian tadpoles during metamorphosis is one of the most dynamic morphological changes in animal development and is induced by thyroid hormone (TH). It has been proposed that tail resorption is driven by immunological rejection in Xenopus laevis, based on experimental evidence showing that larval skin grafts become atrophic on syngenic recipient adult frogs. This led to the hypothesis that tail regression is induced by an immunological rejection against larval skin-specific antigens called Ouro proteins. However, our group has demonstrated that ouro-knockout tadpoles undergo normal metamorphosis, including tail resorption in Xenopus tropicalis, which indicates that the expression of ouro genes is not necessary for tail regression. In the present study, we showed that an inhibitor of TH synthesis promotes the survival of larval tail skin grafts on syngenic adult Xenopus tropicalis frogs. The levels of endogenous THs in adult frogs were also comparable to those in metamorphosing tadpoles of Xenopus laevis with a regressing tail, and TH induced the regression of tadpole tail tips of Xenopus tropicalis in organ culture. Taken together, these results strongly suggest that endogenous THs in the recipient adult frog induce the degeneration of syngenic tail skin grafts.