| Budget Amount *help |
¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Budding tunicates possess a remarkable potential of tissue renewal. Interestingly, this renewal is not accomplished by undifferentiated stem cell but by differentiated somatic cells. As soon as budding or regeneration begins, pigmented epithelial cells (atrial epithelium) undergo dedifferentiation, enter cell division cycle and take undifferentiated shape. This kind of morphogenesis that accompanies dedifferentiation and/or transdifferentiation is also known in Cnidaria and Annelida. In contrast with vertebrate stem cell system, we call this as 'primitive' stem cell system. The goal of our project is to disclose structure and function of proteins that regulate cell growth and differentiation of multipotent cells in budding tunicates.
First we have looked for genes that are expressed specifically at budding phase by the method of subtraction, differential display and EST analysis. Secondly, spatio-temporal expression of those genes has been examined by means of in situ hybridization and
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immunohistochemistry. Thirdly, in order to elucidate gene functions, recombinant proteins and mutant proteins have been produced and assayed in vitro using tunicate cell lines.
In this project, we have cloned eight kinds of budding-specific genes {retinoid X receptor (RXR), aldehyde dehydrogenase (ALDH), serine protease (TRAMP), serine protease inhibitor (SERPIN), lectin (TC14), reverse transcriptase (RT), cytochrome b5 (Cb5), mitochondrial 16S ribosomal RNA (MTlrRNA)}. ALDH is expressed in the epidermis very early during budding or regeneration. It plays a role in biosynthesis of retinoic acid. Then, retinoic acid induces production and excretion of TRAMP and SERPIN that are dedifferentiation factors of the multipotent cells in budding tunicate. TC14 counteracts the dedifferentiation factors, and its activity is canceled with ease by inhibition sugar such as galactose. Cb5 is known to plays a role in fatty acid biosynthesis. In budding tunicates, fatty acids show cell growth-promoting activity. RT is expressed in the multipotent epithelium. As it resembles fruit fly retroposon, gypsy, RT may play a role in rejuvenation of multipotent, but differentiated cells in budding tunicates. Less
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