| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
The present study has been designed to analyze the role of signal molecules during the morphogenesis of gustatory papillae and taste buds of rats and mice. For this purpose, immunofluorescence laser-scanning microscopy was employed to examine the localization of antibodies for several kinds of signal molecules containing growth factors. As markers for the differentiation of lingual epithelium, keratin subunits (K) were also detected by immunofluorescence laser-scanning microscopy. Furthermore, preliminary experiment of organ culture of the developing tongue of fetuses has been carried out in CO_2 incubator to clarify the effect of signal molecules on the morphogenesis of gustatory papillae and taste buds.
The results obtained as follows ;
1. Fetuses were removed on E13, E15, E17 and E19 from pregnant female rats and mice after they had been killed by an intraperitoneal overdose of sodium pentobarbital (200mg/kg body weight), and then their tongues were prepared for examination of fluores
… More
cence immunohistochemistry. Tongues were also removed postnatally from rats and mice just after birth (P0), on postnatal day 7 (P7), on postnatal day 14 (P14), and on postnatal day 21 (P21). In relation with the morphogenesis of lingual papillae and taste buds, monoclonal antibodies specific for K13, K14, K18, Egf, EgfR, Kgf (Fgf-7), Fgf-4, -5, FgfR-2, -3, Shh, Bmp-3, -4, -5, -6, -7, Tgfβ1, TgfβR and Bdnf were monitored by immunofluorescence staining with streptavidin-Alexa Fluor 488, 546 or 633 (Molecular Probes). The preparations were observed under confocal laser-scanning microscope. Ultrathin sections of the same specimens mentioned above have been immuno-electron microscopically observing until now.
2. In situ hybridization for m-RNA probe of EgfR, Kgf (Fgf-7), Fgf-4, -5, FgfR-2, -3, Shh, Bmp-3, -4, -5, -6, -7, Tgfβ1, TgfβR have been carried out, and the results are comparing to those of fluorescence immunohistochemistry to analyze the relationship between morphogenesis of lingual papillae and appearance of signal molecules. Preliminary experiments for in situ hybridization by the transmission electron microscopy using protein A-colloidal gold method have been preparing.
3. Organ culture of the tongue of fetal rodents has been attempted by CO_2 incubator to establish the experimental method for testing of the role of signal molecules in vivo.
4. Data obtained by fluorescence immunohistochemistry under the confocal laser-scanning microscopy have been analyzed considering the relationship between the morphogenesis of gustatory and non-gustatory papillae and the appearance of signal molecules containing growth factors. Some of them were already published in scientific journals, and another data are now in press and preparing for publication. Less
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