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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.
Fig. 2. Expression of xlk2 during development and metamorphosis. mRNAs were quantified by RT-PCR. The developmental stages are indicated at the top of the panel. (A) Expression in embryos and tadpoles. Total RNA was extracted from the whole body of embryos and tadpoles at the indicated stages. No products were observed when RT-PCR was done without reverse-transcriptase. (B) Expression in the epidermis of the developing tadpoles. Total RNA was extracted from the epidermis of head and trunk (body), hind limb (limb) and tail of tadpole at the indicated stages.
Fig. 3. Spatial expression of xlk2 in developing hind limbs. (A) Whole mount in situ hybridization was performed on developing hind limbs of tadpoles at the indicated stages with the antisense probe. The figures represent views of the dorsal side of the limb. (B) A control experiment of A using the sense probe. (C) A section of the limb at the stage 55 shown in A. The arrow indicates the dorsoventral direction. The arrowheads point to the regions where xlk2 was not expressed. (D) Magnification of the region enclosed by the rectangle in C. (E) A serial section of D was stained with hematoxylin. The insets in D and E show magnified figures of the areas enclosed by rectangles. a, the apical layer; b, the basal layer; c, collagen layer; and m; mesenchyme. The magnification of B and E is identical to A and D, respectively. Scale bars = 1 mm in A, and 100 μm in C and D.
Fig. 5. Spatial expression of xlk2 during regeneration of hind limbs and tails. [I] Limb regeneration. Limbs of tadpoles at stage 54 were amputated and were subjected to whole mount in situ hybridization for xlk2 mRNA at 3 days post-amputation with the antisense (A) and the sense probe (B). Three limbs are shown. The closed arrowheads point to the amputation sites. The rightlimb in A is sectioned and shown in C. A serial section of C was stained with hematoxylin (D). The magnification of B and D is identical to that of A and C, respectively. Scale bars = 1 mm in A, and 200 μm in C. [II] Tail regeneration. Normal tails of tadpoles at stage 50 were subjected to whole mount in situ hybridization for xlk2 mRNA. A representative photograph is shown in A. Tails of tadpoles at stage 49–50 were amputated and subjected to whole mount in situ hybridization for xlk2 mRNA at 3 days post-amputation with the antisense (B) and the sense probes (C). Three tails are shown. There were non-specific signals in the notochord of tails and the mesenchyme of regenerates. The closed arrowheads point to the amputation sites and the open arrowhead in the left sample in C points to the notochord. The lefttail in B is sectioned and shown in D. A serial section of D was stained with hematoxylin (E). Similarly, whole mount in situ hybridization was performed at 5 days post-amputation with the antisense (F) and the sense probes (G). The dotted line of the left sample in F indicates the regenerate. A longitudinal section was made from a proximal and distal region of the lefttail shown in F and is shown in H and J, respectively. Serial sections of H and J were stained with hematoxylin and are shown in I and K, respectively. The magnification of B, C, F, and G is identical to that of A. The magnification of sections of D, E, and H–K is identical and its scale bar is shown in D. Scale bars = 1 mm in A, and 100 μm in D.
Fig. 6. Immunohistochemistry on regenerating limbs using monoclonal antibody AE1. Limbs of tadpoles at stage 54 were amputated and were subjected to immunohistochemistry at 3 days post-amputation. The arrowheads point to the amputation sites. Scale bar = 200 μm.
Fig. 4. Expression of xlk2 in the epidermis during T3-induced metamorphosis. Tadpoles were reared in water without (T3−) or with T3 (T3+). RT-PCR was done using total RNA as a template extracted from the epidermis of the body and the tail. The Arabic numerals at the top of the gel indicate the periods (days) of the hormone treatment. The expression level of the hormone-untreated tadpoles did not significantly change for 3 days.
