Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

Authors

  • K. Suzuki,

    1. Laboratory of Developmental Biology, Department of Biological Science, Graduate School of Science
      Hiroshima University, 1-3-1, Kagamiyama
      Higashihiroshima, 739-8526, Japan
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  • K. Sato,

    1. Laboratory for Amphibian Biology
      Department of Biological Science
      Graduate School of Science
      Hiroshima University
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  • K. Katsu,

    1. Laboratory for Amphibian Biology
      Department of Biological Science
      Graduate School of Science
      Hiroshima University
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  • H. Hayashita,

    1. Laboratory of Developmental Biology, Department of Biological Science, Graduate School of Science
      Hiroshima University, 1-3-1, Kagamiyama
      Higashihiroshima, 739-8526, Japan
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  • D. Bach Kristensen,

    1. Yoshizato MorphoMatrix Project
      Exploratory Research for Advanced Technology,
      Japan Science and Technology Corporation
    2. Hiroshima Tissue Regeneration Project, Hiroshima Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Corporation, Hiroshima Prefectural Institute of Industrial Science and Technology, Japan
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  • K. Yoshizato

    Corresponding author
    1. Laboratory of Developmental Biology, Department of Biological Science, Graduate School of Science
      Hiroshima University, 1-3-1, Kagamiyama
      Higashihiroshima, 739-8526, Japan
    2. Yoshizato MorphoMatrix Project
      Exploratory Research for Advanced Technology,
      Japan Science and Technology Corporation
    3. Laboratory for Amphibian Biology
      Department of Biological Science
      Graduate School of Science
      Hiroshima University
    4. Hiroshima Tissue Regeneration Project, Hiroshima Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Corporation, Hiroshima Prefectural Institute of Industrial Science and Technology, Japan
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✉ Tel: + 81 – 824 – 24 – 7440
Fax: + 81 – 824 – 24 – 1492
e-mail: kyoshiz@hiroshima-u.ac.jp

Abstract

Abstract The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.

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