Thyroid hormone (TH) binds TH receptor α (TRα) and β (TRβ) to induce amphibian metamorphosis. Whereas TH signaling has been well studied, functional differences between TRα and TRβ during this process have not been characterized. To understand how each TR contributes to metamorphosis, we generated TRα- and TRβ-knockout tadpoles of Xenopus tropicalis and examined developmental abnormalities, histology of the tail and intestine, and messenger RNA expression of genes encoding extracellular matrix-degrading enzymes. In TRβ-knockout tadpoles, tail regression was delayed significantly and a healthy notochord was observed even 5 days after the initiation of tail shortening (stage 62), whereas in the tails of wild-type and TRα-knockout tadpoles, the notochord disappeared after ∼1 day. The messenger RNA expression levels of genes encoding extracellular matrix-degrading enzymes (MMP2, MMP9TH, MMP13, MMP14, and FAPα) were obviously reduced in the tail tip of TRβ-knockout tadpoles, with the shortening tail. The reduction in olfactory nerve length and head narrowing by gill absorption were also affected. Hind limb growth and intestinal shortening were not compromised in TRβ-knockout tadpoles, whereas tail regression and olfactory nerve shortening appeared to proceed normally in TRα-knockout tadpoles, except for the precocious development of hind limbs. Our results demonstrated the distinct roles of TRα and TRβ in hind limb growth and tail regression, respectively.

When anuran tadpoles are treated with vitamin A after tail amputation, hindlimb-like structures can be generated instead of the lost tail part at the amputation site. This homeotic transformation was initially expected to be a key to understanding the body plan of vertebrates. Unfortunately, homeotic limb formation has been reproduced in only some Indian frog species and a European species, but not in experimental anurans such as Xenopus laevis or Rana catesbeiana. Consequently, this fascinating phenomenon has not been well analyzed, especially at the molecular level. In addition, the initial processes of ectopic limb development are also unclear because morphological changes in the early phases have not been analyzed in detail. In this study, we report the induction of homeotic transformation using Japanese brown frogs and present a detailed morphological analysis. Unexpectedly, the ectopic limbs developed not only at the ventral sites, but also at the dorsal sites of the tail regenerates of vitamin A-treated tadpoles. The relationship between position and axial orientation of ectopic limbs suggested the double duplication of positional value order along the rostral-caudal axis and the dorsal-ventral axis of the tail regenerates.

The urodele is capable of regenerating its limb by forming a blastema even in the adult. By contrast, the anuran, which is phylogenetically close to the urodele, loses this ability during metamorphosis and forms blastema-like tissues that develop only into a spike-like structure in the adult. In order to compare the molecular mechanism of the formation and maintenance of the blastema between the urodele and anuran, the genes encoding helix-loop-helix (HLH) type negative regulators of differentiation were characterized for both the Japanese newt, Cynops pyrrhogaster, and African clawed frog, Xenopus laevis. Cynops homologs of Id2, Id3, and HES1 and Xenopus Id2 were identified. To learn the roles of these genes in regeneration, their expression was examined. The expression of Id2 and Id3 was low in unamputated limbs, but was up-regulated in blastemas of both adult newt and Xenopus. Interestingly, transcripts of the two Id genes showed specific localizations in the blastema and the expression patterns were very similar in both species through the early to medium bud stage. Id2 was expressed predominantly in the blastemal epidermis, and Id3 was expressed equally in the blastemal epidermis and mesenchyme including cells in precartilage condensations. HES1 expression was up-regulated in the newt blastemal epidermis. It was thought that the up-regulation of these genes in the epidermis was related to the proliferation of the cells and that increased expression of these genes in the mesenchyme was related to the undifferentiated state of the blastemal cells. These results and considerations strongly suggested that the state of differentiation is similar in the early to medium bud blastema of both urodeles and anurans. The expression of Id3 remained high through to the digits stage in newts. In contrast, its expression in Xenopus decreased in spike-like regenerates, which correspond to palette-digits stage of newt regenerates. From these results, it was suggested that the blastema redifferentiates earlier in the frog than in the newt, and therefore the timing of redifferentiation of the cartilage is crucial for complete regeneration.

Tail resorption during anuran metamorphosis is perhaps the most dramatic tissue transformation that occurs during vertebrate development. Earlier studies in highly related anuran species Xenopus laevis and Xenopus tropicalis have shown that thyroid hormone (T3) receptor (TR) plays a necessary and sufficient role to mediate the causative effect of T3 on metamorphosis. Of the two known TR genes in vertebrates, TRα is highly expressed during both premetamorphosis and metamorphosis while TRβ expression is low in premetamorphic tadpoles but highly upregulated as a direct target gene of T3 during metamorphosis, suggesting potentially different functions during metamorphosis. Indeed, gene knockout studies have shown that knocking out TRα and TRβ has different effects on tadpole development. In particularly, homozygous TRβ knockout tadpoles become tailed frogs well after sibling wild type ones complete metamorphosis. Most noticeably, in TRβ-knockout tadpoles, an apparently normal notochord is present when the notochord in wildtype and TRα-knockout tadpoles disappears. Here, we have investigated how tail notochord resorption is regulated by TR. We show that TRβ is selectively very highly expressed in the notochord compared to TRα. We have also discovered differential regulation of several matrix metalloproteinases (MMPs), which are known to be upregulated by T3 and implicated to play a role in tissue resorption by degrading the extracellular matrix (ECM). In particular, MMP9-TH and MMP13 are extremely highly expressed in the notochord compared to the rest of the tail. In situ hybridization analyses show that these MMPs are expressed in the outer sheath cells and/or the connective tissue sheath surrounding the notochord. Our findings suggest that high levels of

A novel cytokeratin (CK) gene, xlk2, was cloned from a cDNA library prepared from regenerating limbs of Xenopus larvae. The deduced amino acid sequence indicated that its product, XLK2, is a 48 kDa type I (acidic) CK and has a high similarity to CK13, 15, and 19 with the highest homology (58%) to mouse CK15. The gene of xlk2 exclusively expressed in basal cells of the bi-layered larval epidermis, but not in other cells in larvae and not in other periods of life. Its expression was down-regulated during spontaneous and thyroid hormone-induced metamorphosis. The basal cells of the apical epidermal cap (AEC) formed on the regenerate of larval limbs terminated the expression of xlk2, whereas those of the adjacent normal epidermis continued to express it. The AEC-basal cells did not re-express the gene in the regenerate. In contrast, the basal cells of the tail regenerate also once terminated the expression of xlk2, but was able to re-express xlk2 later, supporting a notion that the "de-differentiated" basal cells of the tail epidermal regenerate re-differentiate into larval normal epidermal cells.