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Biochim Biophys Acta
2001 Feb 16;15173:339-50. doi: 10.1016/s0167-4781(00)00281-5.
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New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis.
Watanabe Y
,
Kobayashi H
,
Suzuki K
,
Kotani K
,
Yoshizato K
.
Abstract
Xenopus larval keratin (XLK) was isolated by gel electrophoresis of proteins of tadpoleskin. Screening of an expression cDNA library of tail tissues by specific polyclonal antibodies against XLK produced XLK cDNA (xlk). Its complete nucleotide and predicted amino acid sequences revealed that XLK was a new member of type II keratin. Screening of a cDNA library of adult Xenopus skin using an oligonucleotide probe which had been designed from well-conserved N-terminal amino acid sequences of the rod domain of type I keratin produced two cDNAs, xak-a and xak-b, which were found to be new members of type I keratin gene. Northern blot analysis showed that xlk was expressed exclusively in the larval skin whereas xak-a and xak-b were expressed exclusively in the adult skin. Their expression level was regulated in a region- and metamorphic stage- dependent manner during larval skin development. mRNA in situ hybridization experiments identified the cells that expressed xlk, and xak-a and xak-b as larva- specific epidermal cells (skein cells and basal cells), and adult suprabasal epidermal cells, respectively. These three genes were found to be late responsive to thyroid hormone. Phylogenetic relationships of these keratins with known ones are discussed.
Fig. 1. Speci¢city of antibodies against XLK. Proteins of tailskin of tadpoles at stage 54 were separated by 2-D SDS^PAGE and subjected to CBB
staining (A) or Western blot analysis with antibodies against XLK (B). The proteins enclosed by a broken line in A are those reacted with the antibodies.
The speci¢city of the antibodies was also immunohistochemically examined on tissue sections prepared from back skin of tadpoles at stage 56 (C)
and stage 61 (D). The structure of tadpole back skin at stage 56 and 61 is presented in Fig. 6A1 and B1, respectively, wherein the skin sections were
stained with H p E. The epidermis at stage 61 showed a weak background signal. The location of the collagen lamella in C and D was determined by
observing the section in a bright ¢eld and is indicated by two broken lines. ap, apical cells ; sk, skein cells ; bs, basal cells ; cl, collagen lamella; bm,
basement membrane; ct-1, connective tissues beneath the collagen lamella; ct-2, connective tissues between the basement membrane and the collagen lamella;
gl, secretory glands. Bar in C,D =20 Wm.
Fig. 2. The deduced amino acid sequences of XLK, XAK-A, and XAK-B. Amino acid sequences of XLK (type II keratin) are compared with other
known Xenopus type II keratins, CK1(8) [19] and CK55(5/6) [20] (A). Likewise, sequences of XAK-A and -B (type I keratins) are compared with other
known Xenopus type I keratin, XL51 [18] (B). Gaps shown by dots were introduced to optimize the alignment. Identical amino acid residues in at least
two alignments are denoted with boldfaced letters. The K-helical tracts (C1A, C1B, and C2) and the linker segments (L1 and L12) are shown above the
sequences. Their assignment was done according to Franz and Franke [19]. The asterisk denotes the position of the proline residue in C1B segment of
XAK-A. The underline in A denotes the sequences determined for XLKs by protein microsequencing. GeneBank accession numbers for the nucleotide
sequences of XLK, XAK-A, and -B are AB045599, AB045600, and AB045601, respectively.
Fig. 3. Relatedness of the sequences of Xenopus keratins. (A) Type I
keratin. The sequences of C1A to L12 of the rod domain were compared
among reported Xenopus keratins. (B) Type II keratin. The sequences
of C1B to C2 of the rod domain were compared among reported
Xenopus keratins.
Fig. 4. Expression of xlk, xak-a, and xak-b during spontaneous metamorphosis. RNAs were extracted from the body (A) and tail (B) of tadpoles at different
metamorphic stages indicated. Ad in A represents RNAs of the adult body skin. These RNAs were electrophoretically separated on agarose gels
and were processed for Northern blot analysis. Northern blots were also done for rpL8 mRNA to estimate the amount of RNAs loaded on the gels.
Fig. 5. Expression of xlk, xak-a, and xak-b during T3-induced metamorphosis. RNAs were extracted from the body (A) and tail (B) of tadpoles at stage
54 treated with T3 for days indicated and were used for Northern blot analysis as in Fig. 4.
Fig. 6. In situ hybridization of mRNAs of xlk, xak-a, and xak-b for the skin of tadpoles and adults. Cross sections of the body skin were prepared
from tadpoles at stage 56 (A1^A4), stage 61 (B1^B4), and from adults (C1^C4). Histology of the skin was examined by HpE staining (A1,B1,C1). The
epidermis at stage 56 (A1) was composed of apical (ap), skein (sk), and basal cells (bs). The epidermis of stage 61 (B1) transformed to the adult epidermis
composed of granular (gr) and basal cells. The adult epidermis (C1) contained corni¢ed cells (c) in addition to granular and basal cells. The skin at
stage 56 (A1) contained the subepidermal connective tissue (ct-1), but did not develop the connective tissue (ct-2) between the basement membrane (bm)
and the collagen lamella (cl), but the skin at stage 61 (B1) and the adult (C1) formed it. In the HpE section of adults the collagen lamella is out of vision.
The sections were hybridized with antisense probes of xlk (A2,B2), xak-a (A3,B3,C3), and xak-b (A4,B4,C4). The results with sense probes were
shown only for xak-a in adult tissues (C2), because all of the sense probes gave a similar background stain for all the tissues tested as in C2. f, Fibroplasts;
gl, secretory glands. Bar =20 Wm.
