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Figure 3. In situ hybridization of xak-c mRNA on skin of tadpoles and adults. Histologic sections were prepared from tadpoles at stage 56 (A,B), 57 (C,D), 61 (E,F), and adults (G,H) and used for in situ hybridization of xak-c mRNA (A,C,E,G) and hematoxylin and eosin staining (B,D,F,H). The epidermis at stage 56 contained apical cells (ap), skein cells (sk), and small basal cells (bs). The skin at stage 57 showed drastic changes in its connective tissue (ct-1) compartment. Fibroblasts (f) increased their number and invaded into collagen layers (cl). Basal cells also increased their number and became larger in size than at stage 56. The epidermis at stage 61 did not contain apical cells or skein cells, and basal cells started to get into the path of the terminal differentiation to spinous and granular (gr) cells. New connective tissues (ct-2) were formed between the basement membrane and collagen layers. Secretory glands (gl) were developed. The adult skin produced the cornified layer (c) and well-developed glands. Sense probes did not show any appreciable stains in in situ hybridization experiments. Identical in situ experiments were performed four times, and similar results were obtained reproducibly. Scale bar = 20 mu m in A (applies to A-H).
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Figure 2. Expression of xak-c mRNA during spontaneous and 3,5,3′-triiodothyronine (T3) -induced metamorphosis. A: RNAs were isolated from the body and the tail of tadpoles at the indicated metamorphic stages and used as templates for reverse transcriptase-polymerase chain reaction (RT-PCR) of xak-c together with xlk, xak-b, and rpL8. The rpL8 gene was used as an internal control (Shi and Liang, 1994). Identical RT-PCR experiments were repeated four times, and similar results were reproducibly obtained. B: Tadpoles at stage 54 were treated with 5 nM T3. RNAs were isolated from the body and the tail of tadpoles at the indicated days and used as templates for RT-PCR as in A. Identical RT-PCR experiments were repeated four times, and similar results were obtained reproducibly.
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Figure 4. Immunohistochemistry of XAK-C and XAK-B. Sections of skin tissues were prepared from tadpoles at stage 56 (St. 56, A), 58 (B,C), and 61 (D), and adults (G) and subjected to immunohistochemistry for XAK-C (A,B,D,G) and XAK-B (C,E,H). Red and green color represent stains of XAK-C and XAK-B, respectively. F and I are the merged images of D and E, and G and H, respectively. There was no XAK-C signal at stage 56. Basal cells at stage 58 were XAK-C positive, but no cells were positive for XAK-B. Basal cells of the skin at stage 61 and of adults were stained with antiAK-C antisera. XAK-B signals were detected in differentiated cells seen on both sections. Immunohistochemistry experiments were performed four times, and similar staining patterns were obtained reproducibly. Scale bar = 20 μm in I (applies to A).
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Figure 6. Transfection of the adult skin with XS2.8k/pEGFP. Skin pieces were isolated from adult Xenopus and were transfected with XS2.8k/pEGFP (A), CMV/pEGFP (B), and pEGFP-1 (null, C) by using a gene gun. The tissues were cultured for 48 hr and observed through a fluorescence-dissecting microscope. Identical gene gun experiments were carried out three times, and similar results were obtained reproducibly. Scale bar = 1 mm.
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Figure 7. Keratinocyte-specific induction of the promoter activity of the 5′-flanking region of xak-c. Xenopus adult keratinocytes were isolated, cultured for 72 hr, and fixed. The cells were stained with Hoechst 33342 and then with antisera against XAK-C and XAK-B. The immunosignals were visualized by anti-rabbit immunoglobulin (Ig) G Alexa 594 (red) and anti-rat IgG Alexa 488 (green) for antibodies against XAK-C and XAK-B, respectively. A: Bright-field, B: fluorescent field for Hoechst, XAK-C, and XAK-B, respectively. E: SH2.8k/pLUC was introduced into each of Xenopus keratinocytes, human keratinocytes, Xenopus A6 kidney cell line, and Swiss 3T3 cells. The transfected cells were cultured for 48 hr, and their luciferase activity was measured. The results are shown as the average with SEM of at least three independent experiments. *P < 0.01 and **P < 0.001 indicates the statistical level at a 99 and 99.9% significance between pLUC and SH2.8k/pLUC, respectively. Scale bar = 50 μm in A (applies to A).
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Figure 8. Effects of 3,5,3′-triiodothyronine (T3) on promoter activity of the 5′-flanking region of xak-c. Pieces of body skin were dissected from tadpoles at stage 56/57 tadpoles (A) and adults (B), transfected with SH2.8k/pLUC, and were cultured for 72 hr with or without 10 nM T3. As a control experiment, null-vector (pLUC) was similarly transfected to the tissues. The luciferase activity was determined for these skin tissues, and are shown as the average with SEM of at least three independent experiments. *P < 0.001, statistically significant level at 99.9% between with and without T3. C:Xenopus laevis adults were treated with 5 nM T3 for up to 7 days. RNAs were isolated from the skin at the indicated days and used as templates for reverse transcriptase-polymerase chain reaction of xak-c and rpL8.
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xak-b (adult keratin XAK-B ) gene expression in Xenopus laevis adult frogs, as assayed by immunofluorescence (green channel). Coronal sections of skin, superficial up.
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xak-c (adult keratin XAK-C ) gene expression in Xenopus laevis adult frogs, as assayed by immunofluorescence (red channel). Coronal sections of skin, superficial up.
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