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• W2036712925 abstract "We examined the functional implication of nucleolin in the mouse first molar development. Both the nucleolin mRNA and protein expressions were demonstrated in the odontogenic epithelial cells in the early stage and in the inner enamel epithelial layer in the late stage. The expression pattern of nucleolin corresponded to the proliferating cells in the tooth germ, thus showing that nucleolin could possibly be related to cell proliferation. No in situ signal of nucleolin was found in the primary enamel knot (PEK). Furthermore, nucleolin protein was demonstrated in the PEK by immunohistochemistry. The existence of nucleolin protein in the PEK may possibly be related to the apoptosis in the PEK cells. An inhibition assay using the hemagglutinating virus of Japan-liposome containing nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) in cultured mouse mandibles at embryonic day (E) 11.0 showed a marked growth inhibition of tooth germ. Moreover, no developmental arrest was found in the cultured tooth germ at E15.0 treated with nucleolin AS S-ODN. Real time PCR was performed to examine the mRNA expression of nucleolin-related genes, and a significant reduction in the midkine mRNA expression was thus observed in the mouse mandible after being treated with nucleolin AS S-ODN. This inhibition assay indicated that nucleolin could thus be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the midkine expression. We examined the functional implication of nucleolin in the mouse first molar development. Both the nucleolin mRNA and protein expressions were demonstrated in the odontogenic epithelial cells in the early stage and in the inner enamel epithelial layer in the late stage. The expression pattern of nucleolin corresponded to the proliferating cells in the tooth germ, thus showing that nucleolin could possibly be related to cell proliferation. No in situ signal of nucleolin was found in the primary enamel knot (PEK). Furthermore, nucleolin protein was demonstrated in the PEK by immunohistochemistry. The existence of nucleolin protein in the PEK may possibly be related to the apoptosis in the PEK cells. An inhibition assay using the hemagglutinating virus of Japan-liposome containing nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) in cultured mouse mandibles at embryonic day (E) 11.0 showed a marked growth inhibition of tooth germ. Moreover, no developmental arrest was found in the cultured tooth germ at E15.0 treated with nucleolin AS S-ODN. Real time PCR was performed to examine the mRNA expression of nucleolin-related genes, and a significant reduction in the midkine mRNA expression was thus observed in the mouse mandible after being treated with nucleolin AS S-ODN. This inhibition assay indicated that nucleolin could thus be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the midkine expression. A multistep and complex process of the gene expressions are involved in the early stage of tooth development (1Pispa J. Mikkola M.L. Mustonen T. Thesleff I. Gene Expr. Patterns. 2003; 3: 675-679Crossref PubMed Scopus (69) Google Scholar). There have been many reports regarding the expression of various kinds of genes that are related to tooth morphogenesis (2Salazar-Ciudad I. Jernvall J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8116-8120Crossref PubMed Scopus (296) Google Scholar). However, the precise signaling pathway, which might be related with the initiation, growth, and differentiation of tooth germ, has not yet been fully disclosed. We previously performed cDNA subtraction between the mandibles of mice at embryonic day (E) 2The abbreviations used are: E, embryonic day; PEK, primary enamel knot; S-ODN, phosphorothioate oligodeoxynucleotide; AS, antisense; MK, midkine; CK, casein kinase; PP1, protein phosphatase; HVJ, hemagglutinating virus of Japan; TUNEL, deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling; DAPI, 4′,6-diamino-2-phenylindole; PBS, phosphate-buffered saline; fw, forward; rv, reverse; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AMGN, amelogenin; AMBN, ameloblastin; DSPP, dentin sialophosphoprotein; SE, sense; RS, random sequence. 10.5 and 12.0. Thirty five highly expressed positive clones were obtained from the E10.5 mandible by means of colony array screening. 47 highly expressed positive clones were also obtained from the E12.0 mandible (3Yamaza H. Matsuo K. Kiyoshima T. Shigemura N. Kobayashi I. Wada H. Akamime A. Sakai H. Int. J. Dev. Biol. 2001; 45: 675-680PubMed Google Scholar). We have already reported that the expressions of several genes among them were closely associated with the developing tooth germ (4Yamaza H. Matsuo K. Kobayashi I. Wada H. Kiyoshima T. Akhtar M. Ishibashi Y. Sakai T. Akamine A. Sakai H. Histochem. J. 2001; 33: 437-441Crossref PubMed Scopus (12) Google Scholar, 5Wada H. Kobayashi I. Yamaza H. Matsuo K. Kiyoshima T. Akhtar M. Sakai T. Koyano K. Sakai H. Histochem. J. 2002; 34: 105-109Crossref PubMed Scopus (18) Google Scholar, 6Akhter M. Kobayashi I. Kiyoshima T. Matsuo K. Yamaza H. Wada H. Honda J.Y. Ming X. Sakai H. Histochem. Cell Biol. 2005; 124: 207-213Crossref PubMed Scopus (31) Google Scholar). Nucleolin was one of the highly expressed genes in the E10.5 mandible. Nucleolin is a major nucleolar phosphoprotein that belongs to a large family of RNA-binding proteins (7Bugler B. Bourbon H. Lapeyre B. Wallace M.O. Chang J.H. Amalric F. Olson M.O. J. Biol. Chem. 1987; 262: 10922-10925Abstract Full Text PDF PubMed Google Scholar, 8Shaw P.J. Jordan E.G. Annu. Rev. Cell Dev. Biol. 1995; 11: 93-121Crossref PubMed Scopus (409) Google Scholar). Nucleolin is thought to play a role in the pre-rRNA transcription and ribosome assembly that is implicated in the early stage of preribosomal ribonucleoprotein assembly and processing (9Roger B. Moisand A. Amalric F. Bouvet P. Chromosoma (Berl.). 2003; 111: 399-407Crossref PubMed Scopus (53) Google Scholar, 10Borer R.A. Lehner C.F. Eppenberger H.M. Nigg E.A. Cell. 1989; 56: 379-390Abstract Full Text PDF PubMed Scopus (923) Google Scholar). The amount of nucleolin mRNA fluctuates in parallel with the status of DNA synthesis (11Konishi T. Karasaki Y. Nomoto M. Ohmori H. Shibata K. Abe T. Shimizu K. Itoh H. Higashi K. J. Biochem. (Tokyo). 1995; 117: 1170-1177Crossref PubMed Scopus (25) Google Scholar). The intact 110-kDa nucleolin molecule is the major species in actively dividing cells, and degraded forms are relatively abundant in nondividing cells (12Westmark C.J. Malter J.S. J. Biol. Chem. 2001; 276: 1119-1126Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition, nucleolin has been known to participate in the packaging and shuttling of the ribosome between the nucleus and cytoplasm (13Ginisty H. Serin G. Ghisolfi-Nieto L. Roqer B. Libante V. Amalric F. Bouvet P. J. Biol. Chem. 2000; 275: 18845-18850Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). There have also been numerous reports regarding the functional roles of nucleolin by forming large molecular complexes with other related factors, such as casein kinase (CK) II, c-Myb, midkine (MK), histone H1, nucleophosmin, p53, and protein phosphatase 1 (PP1) (14Tuteja R. Tuteja N. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 407-436Crossref PubMed Scopus (153) Google Scholar, 15Ying G.G. Proost P. van Damme J. Bruschi M. Introna M. Golay J. J. Biol. Chem. 2000; 275: 4152-4158Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Take M. Tsutsui J. Obama H. Ozawa M. Nakayama T. Maruyama I. Arima T. Muramatsu T. J. Biochem. (Tokyo). 1994; 116: 1063-1068Crossref PubMed Scopus (103) Google Scholar, 17Erard M.S. Belenguer P. Caizergues-Ferrer M. Pantalon Amalric F. Eur. J. Biochem. 1988; 175: 525-530Crossref PubMed Scopus (179) Google Scholar, 18Yang C. Maiguel D.A. Carrier F. Nucleic Acids Res. 2002; 30: 2251-2260Crossref PubMed Scopus (118) Google Scholar, 19Klibanov S.A. O'Hagan H.M. Ljungman M. J. Cell Sci. 2001; 114: 1867-1873Crossref PubMed Google Scholar, 20Kito S. Shimizu K. Okamura H. Yoshida K. Morimoto H. Fujita M. Morimoto Y. Ohba T. Haneji T. Biochem. Biophys. Res. Commun. 2003; 300: 950-956Crossref PubMed Scopus (30) Google Scholar), and then either directly or indirectly playing a role in the regulation of cell proliferation and growth, cytokinesis, replication, embryogenesis, and nucleogenesis (14Tuteja R. Tuteja N. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 407-436Crossref PubMed Scopus (153) Google Scholar, 21Huang Y. Shi H. Zhou H. Song X. Yuan S. Luo Y. Blood. 2006; 107: 3564-3571Crossref PubMed Scopus (160) Google Scholar, 22Wurm T. Chen H. Hodgson T. Britton P. Brooks G. Hiscox J.A. J. Virol. 2001; 75: 9345-9356Crossref PubMed Scopus (167) Google Scholar, 23Caizergues-Ferrer M. Mariottini P. Curie C. Lapeyre B. Gas N. Amalric F. Amaldi F. Genes Dev. 1989; 3: 324-333Crossref PubMed Scopus (71) Google Scholar, 24Bjerreqaard B. Wrenzychi C. Strejcek F. Laurincik J. Holm P. Ochs R.L. Rosenkranz C. Callesen H. Rath D. Niemann H. Maddox-Hyttel P. Biol. Reprod. 2004; 70: 867-876Crossref PubMed Scopus (23) Google Scholar). In addition to the many known functions of nucleolin, it may also function as a low affinity receptor of extracellular growth factor on the cell surface (25Said E.A. Krust B. Nisole S. Svab J. Briand J.P. Hovanessian A.G. J. Biol. Chem. 2002; 277: 37492-37502Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Here we examined the detailed in situ and immunohistochemical expression patterns of nucleolin in developing tooth germ. To further analyze the functional role of nucleolin, mandibles at E11.0 and tooth germs at E15.0 were cultured with nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) by using the hemagglutinating virus of Japan (HVJ)-liposome transfection method. We performed a histological analysis and real time PCR to estimate the effect of the nucleolin AS S-ODN on the formation of tooth germ and the mRNA expression of nucleolin-related gene in nucleolin AS S-ODN-treated tissue specimens, respectively. Nucleolin may be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the MK expression. Animals—Embryos of BALB/c mice at 10.5, 11.0, 12.0, 14.0, 15.0, 16.0, 17.0, and 18.0 days after gestation were used in this study. Adult BALB/c mice were obtained from Charles River Japan Inc. (Yokohama, Japan). All mice were bred in an air-conditioned clean room with a 12:12-h light-dark cycle and were provided standard laboratory food and water ab libitum. All manipulations of mice were performed in accordance with the guidelines of the Animal Center of Kyushu University. Female BALB/c mice (10–30 weeks) were caged together with male mice. After 3 h, successful insemination was determined based on the presence of a post-copulatory plug in the vagina, and the embryonic day was defined as E0 after such a plug was recognized. In Situ Hybridization—The details of the section preparation, the probe labeling, the specificity of the digoxigenin-labeled in situ RNA probes, and in situ hybridization methods have been shown in our previous studies (4Yamaza H. Matsuo K. Kobayashi I. Wada H. Kiyoshima T. Akhtar M. Ishibashi Y. Sakai T. Akamine A. Sakai H. Histochem. J. 2001; 33: 437-441Crossref PubMed Scopus (12) Google Scholar, 5Wada H. Kobayashi I. Yamaza H. Matsuo K. Kiyoshima T. Akhtar M. Sakai T. Koyano K. Sakai H. Histochem. J. 2002; 34: 105-109Crossref PubMed Scopus (18) Google Scholar, 6Akhter M. Kobayashi I. Kiyoshima T. Matsuo K. Yamaza H. Wada H. Honda J.Y. Ming X. Sakai H. Histochem. Cell Biol. 2005; 124: 207-213Crossref PubMed Scopus (31) Google Scholar). Three embryos at each embryonic day were removed from the pregnant mice under ether anesthesia. The removed embryos were fixed in 4% paraformaldehyde in diethyl pyrocarbonate-treated phosphate-buffered saline (PBS, pH 7.4) for 12 h at 4 °C and embedded in Tissue-Tek OCT (Miles Inc., Elkhart, IN). Serial cryosections were cut at a thickness of 8 μm and then were mounted on silane-coated glass slides for in situ hybridization. Murine nucleolin cDNA (GenBank™ accession number AK031606, nucleotides 536–1281) was subcloned into pGEM-3Z vector (Promega, Madison, WI) to synthesize both of the antisense and sense probes. Nucleolin sense probe was also applied to the tissue specimens as a control; however, no hybridization signal was detected (data not shown). Immunohistochemistry and Antibody—For immunohistochemistry, the preparation of serial cryosections was processed in the same way as for in situ hybridization. After the cryosections were dried at room temperature for 1 h, the sections were rinsed with PBS containing 0.1% Triton X-100 for 10 min. To block nonspecific immunoreaction, the sections were incubated with 5% chicken serum (Cosmo Bio Co., Ltd., Tokyo, Japan) and 5% donkey serum (Cosmo Bio) in PBS for 30 min, respectively. Next the sections were incubated with the primary antibody against nucleolin (goat polyclonal C23, Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:200 in PBS. The sections were washed four times with PBS for 10 min each and incubated with Alexa 568-labeled donkey anti-goat IgG (Invitrogen), diluted 1:2000 in PBS, for 2 h at room temperature. After rinsing with PBS five times in PBS, the sections were incubated with 4′,6-diamino-2-phenylindole (DAPI, 0.5 μg/ml) (Wako, Osaka, Japan) for 15 min at room temperature. For the negative control, the application of the primary antibody was omitted from the above described procedure. These sections were examined under a fluorescence microscopy Olympus IX71 (OLYMPUS, Tokyo, Japan), and immunofluorescent images were acquired using an Olympus DP70 camera. Double Staining for Nucleolin Immunohistochemistry and TUNEL—The tissue sections were prepared in the same method as mentioned above and double-stained by immunohistochemistry for nucleolin and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nickend labeling method (TUNEL) for apoptosis using the in situ apoptosis detection kit (Takara Bio Inc., Shiga, Japan). Briefly, the sections were treated with the permeabilization buffer for 5 min at 4 °C. To block nonspecific immunoreaction, the sections were preincubated with 5% chicken serum and 5% donkey serum (Cosmo Bio) in PBS for 30 min, respectively, and then incubated with the C23 antibody (Santa Cruz Biotechnology, diluted 1:200) for 90 min at room temperature. After washing in PBS, the sections were incubated with the mixture of reaction solution containing the terminal deoxynucleotidyltransferase and Alexa 568-labeled anti-goat IgG (Invitrogen) for 90 min at 37 °C. The sections were counterstained with DAPI. In a negative control incubated without the terminal deoxynucleotidyltransferase, no fluorescent products were observed. Quantitative Real Time PCR—Total RNA was isolated from the mandibles removed from E10.5 and E12.0 mouse embryos and 6-day cultured mandible explants using the SV total RNA isolation system (Promega). The cDNA was prepared by a reverse transcription reaction using the SuperScript III first strand synthesis system (Invitrogen) according to the manufacturer’s instructions. Real time PCR was performed in 10 μl of mixture consisting of 5 μl of SYBR Premix Ex Taq (Takara Bio Inc.) containing TaqDNA polymerase, oligonucleotide primers (0.2 μm each), and 1 μl of template cDNA. The amplification consisted of a two-step procedure as follows: denaturation at 95 °C for 10 s, and 40 cycles with denaturation at 95 °C for 5 s, and then annealing/elongation at 60 °C for 31 s by using ABI PRISM® 7000 sequence detection systems (Applied Biosystems, Foster City, CA). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. The primer sequences were designed using the Primer Express software version 1.0 (Applied Biosystems). The gene-specific forward (fw) and reverse (rv) primers for nucleolin and GAPDH were as follows: nucleolin fw, 5′-AAG CAG CAC CTG GAA AAC G-3′, and nucleolin rv, 5′-TCT GAG CCT TCT ACT TTC TGT TTC TTG-3′; GAPDH fw, 5′-GAA CAT CAT CCC TGC ATC CA-3′, and GAPDH rv, 5′-CCA GTG AGC TTC CCG TTC A-3′. Western Blot Analysis—A Western blot analysis for the nucleolin level was performed from the cytosolic fraction of the homogenate of E10.5 and E12.0 mandibles and 6-day cultured mandible explants. The tissue specimens were lysed in RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, pH 8.0, 0.1% SDS), supplemented with protease inhibitor mixture (50 μm), lactacystin (20 μm), β-glycerophosphate (25 mm), and sodium orthovanadate (1 mm). Protein samples (20 μg) were separated by 12% SDS-polyacrylamide gel and transferred to Immun-Blot® polyvinylidene difluoride membrane (Bio-Rad). The membrane was probed with antibody C23 (Santa Cruz Biotechnology) against nucleolin for 1 h at room temperature, and incubated for 1 h with secondary rabbit anti-mouse IgG conjugated with horseradish peroxidase (Amersham Biosciences). The membrane was developed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences). Emitted light was detected using a cooled CCD camera (LAS-1000, Fujifilm, Tokyo, Japan). Preparation of S-ODN—The HVJ-liposomes were prepared as described elsewhere (26Saeki Y. Kaneda Y. Cell Biology: A Laboratory Handbook. 1997; (Celis, J. E., ed) 2nd Ed., pp. , Academic Press, San Diego: 123-130Google Scholar, 27Kaneda Y. Uchida T. Kim J. Ishiura M. Okada Y. Exp. Cell Res. 1987; 173: 56-69Crossref PubMed Scopus (92) Google Scholar). In brief, egg yolk phosphatidylcholine (Sigma), cholesterol (Sigma), and bovine brain phosphatidylserine (Sigma) were each dissolved in chloroform, mixed in a weight ratio of 1:2:4.8, and then dried with a rotary evaporator (Eyela, Tokyo, Japan). Purified HVJ (kindly donated by Professor Katsuo Sueishi, Kyushu University) adjusted to 15,000 hemagglutinating units/ml was inactivated by ultraviolet light. Thereafter, 10 mg of liposomes mixture was fused with 1 ml of HVJ and 20 nmol of S-ODNs in 6 ml of balanced salt solution (140 mm NaCl, 5.4 mm KCl, 10 mm Tris-HCl, pH 7.5). HVJ-liposome complex was collected by centrifugation for use. The concentration of S-ODNs was adjusted to 0.2 mm by measuring absorbance of HVJ-liposome containing S-ODNs (27Kaneda Y. Uchida T. Kim J. Ishiura M. Okada Y. Exp. Cell Res. 1987; 173: 56-69Crossref PubMed Scopus (92) Google Scholar). Translation Arrest by Nucleolin AS S-ODN and Organ Culture—For the translation arrest of nucleolin, we designed AS S-ODN as 5′-CTC GGA AGA GCT CAG CGG AA-3′ corresponding to nucleotides 118–137 of murine nucleolin (GenBank™ accession number AK031606). The corresponding sense (SE) S-ODN (5′-TTC CGC TGA GCT CTT CCG AG-3′) and random sequence (RS) S-ODN (5′-AGT CGA CGC AAG TGC GCG AA-3′) were used for the control experiments. The protocol for the organ culture of the tooth germs was almost identical to that used in our previous studies (28Kobayashi I. Kiyoshima T. Wada H. Matsuo K. Nonaka K. Honda J.Y. Koyano K. Sakai H. Bone (NY). 2006; 38: 836-844Crossref PubMed Scopus (41) Google Scholar). Briefly, the mandibles and tooth germs of the lower first molar were dissected out from E11.0 and E15.0 embryos, respectively. These explants were supported by a filter (0.8 μm pore size, Millipore, MA) mounted on metal mesh, and then were incubated in the Fitton-Jackson’s modified BGJb medium (Invitrogen) supplemented with 5% fetal bovine serum (Filtron, Brooklyn, Australia), 100 μg/ml ascorbic acid (Invitrogen), and 100 unit/ml penicillin/streptomycin (Invitrogen) in a 5% CO2 atmosphere at 37 °C (29Slavkin H. Nuckolls G. Shum L. Methods Mol. Biol. 2000; 136: 45-54PubMed Google Scholar). The final concentration used in the culture medium included 1.25 μm for each ODN, 2.4 mg/ml for the HVJ-liposome complex, and 1 mm CaCl2. The culture media containing HVJ-liposome S-ODNs were changed every 24 h. Tissue Preparation and Histological Analysis of Cultured Explants—The cultured mandibles and tooth germs were fixed with 4% paraformaldehyde for 12 h at 4 °C at the 2nd, 4th, 5th, 6th, 7th, 8th, and 10th day after cultivation. At least three (up to 14) mandibles and tooth germs explants were used on each culture day for histological analysis. Five-μm-thick sections were cut in an antero-posterior direction, and hematoxylin and eosin double staining was then performed, and the sections were examined under light microscopy. Measurement of Nucleolin Suppression and Statistical Analysis—The effect of the AS S-ODNs on blocking the translation of nucleolin was confirmed by real time PCR and Western blot analysis. Each experiment was performed independently three times. The density for the blotting signals was measured using NIH Image software version 1.37. Unpaired Student’s t test was used to determine the significance of differences. Differences with a probability value of less than 0.05 were considered to be significant. Effects of Nucleolin Suppression on Gene Transcription—Real time PCR was performed to estimate the subsequent expression of selected genes after nucleolin transcription was arrested in 6-day cultured E11.0 mandibles. The following primers were used in the present study: MK fw, 5′-CTC TCC CAC AGG CCC AAG A-3′, and MK rv, 5′-AGG ACA GGC GTG ATT GAC AGA-3′; CKII fw, 5′-CAA TAT GAT GTC AGG GAT TTC TTC A-3′, and CKII rv, 5′-GTT GGC AGC GGC AAT CAC-3′; histone H1 fw, 5′-TGG GTG AGA ACG CCA ACT C-3′, and histone H1 rv, 5′-ACC CCT TTG GTT TGC TTG AGA-3′; nucleophosmin fw, 5′-GAA CAG GAG GCA GTT GTT TTC C-3′, amd nucleophosmin rv, 5′-GTC CAT ATC CAT CGA GTC TTC CA-3′; p53 fw, 5′-CAC TGC ATG GAC GAT CTG TTG-3′, and p53 rv, 5′-CAC TCG GAG GGC TTC ACT TG-3′; PP1 fw, 5′-GCT GGG AGT GAG CGT CTT CT-3′, and PP1 rv, 5′-TCC ACC ACG ATC TGA GTT ACG T-3′; c-Myb fw, 5′-ATG CCA AAT GGA GAA ATG TGT TC-3′, and c-Myb rv, 5′-TCT ATT GCC CCC TGA CAC AAG-3′. Quantitative real time PCR was also performed to estimate the expressions of amelogenin (AMGN), ameloblastin (AMBN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1) after nucleolin transcription was arrested in 6-day cultured E11.0 mandibles. The following primers were used in this study: AMGN fw, CCC CTG TCC CCA TTC TT, and AMGN rv, ACT TCT TCC CGC TTG GTC TTG; AMBN fw, TGC AGG AAG GAG AGC TGA TAG C, and AMBN rv, CAG GTG TTG GTG GGT TTT CG; DSPP fw, TGG GCA TGA CAG TTA CGA GTT C, and DSPP rv, TTT CGT CAC TTC CGT TAG ATT CG; DMP1 fw, TGC TCT CCC AGT TGC CAG AT, and DMP1 rv, GGT GAC CCA GCC AAA TCA TC. Different Expression of Nucleolin between E10.5 and E12.0—Real time PCR and Western blotting analyses were performed to estimate the different expression of nucleolin between the mandibles at E10.5 and at E12.0. The expression of nucleolin was 2.6-fold higher for mRNA (p < 0.01) and 1.8-fold higher for protein (p < 0.01) in the mandible at E10.5 than in the mandible at E12.0, and the difference was confirmed to be significant (Fig. 1, A–C). Expression of Nucleolin mRNA and Protein during Odontogenesis—Membrane hybridization was carried out to confirm the specificity of the digoxigenin-labeled in situ RNA probes. The details of the assay have been described in our previous study (6Akhter M. Kobayashi I. Kiyoshima T. Matsuo K. Yamaza H. Wada H. Honda J.Y. Ming X. Sakai H. Histochem. Cell Biol. 2005; 124: 207-213Crossref PubMed Scopus (31) Google Scholar). The binding activity of antisense nucleolin probe to sense nucleolin probe was inhibited when an excessive unlabeled sense or antisense probe was added to the reaction mixture (data not shown). The expression of nucleolin mRNA and protein in the lower first molar were analyzed by in situ hybridization (Fig. 2A) and immunohistochemical method (Fig. 2B) from the initiation stage (E10.5) to the bell stage (E18.0) in a series of serial sections. Other special terms in the developmental stages of tooth germ have been referred to in previous studies (3Yamaza H. Matsuo K. Kiyoshima T. Shigemura N. Kobayashi I. Wada H. Akamime A. Sakai H. Int. J. Dev. Biol. 2001; 45: 675-680PubMed Google Scholar, 4Yamaza H. Matsuo K. Kobayashi I. Wada H. Kiyoshima T. Akhtar M. Ishibashi Y. Sakai T. Akamine A. Sakai H. Histochem. J. 2001; 33: 437-441Crossref PubMed Scopus (12) Google Scholar, 5Wada H. Kobayashi I. Yamaza H. Matsuo K. Kiyoshima T. Akhtar M. Sakai T. Koyano K. Sakai H. Histochem. J. 2002; 34: 105-109Crossref PubMed Scopus (18) Google Scholar, 6Akhter M. Kobayashi I. Kiyoshima T. Matsuo K. Yamaza H. Wada H. Honda J.Y. Ming X. Sakai H. Histochem. Cell Biol. 2005; 124: 207-213Crossref PubMed Scopus (31) Google Scholar). At the initiation stage of E10.5, the expression of nucleolin mRNA was observed in the oral epithelium and mesenchyme at the site where the presumptive molar tooth germ was estimated to be formed. At E12.0, both nucleolin in situ signal and immunofluorescence were detected in the thickening oral epithelium. At the bud stage of E14.0, the expression of nucleolin mRNA and protein was mainly detected in the epithelial cells of the tooth bud. The immunolocalization of nucleolin was demonstrated in the whole enamel organ at the cap stage of E15.0, but no in situ signal was found in the primary enamel knot (PEK). The immunofluorescence reaction of nucleolin protein was observed in the cytoplasm and nucleus as a dot-like appearance (Fig. 2C). At the early bell stage of E16.0, in situ signal and immunofluorescence staining patterns were presented in the inner enamel epithelium, cervical loop, and in part of the outer enamel epithelial cells. The in situ signal and staining patterns of immunofluorescence were restricted in the inner enamel epithelial cells at the subsequent E17.0 and E18.0 (Fig. 2, A and B). The expression of mRNA thus generally coincided with that of protein. In particular, both mRNA and protein were concomitantly expressed in the early bud stage of E12.0 and the bell stage at E16.0–E18.0 during tooth germ development. However, several differences were also detected in the expression patterns between mRNA and protein. At the bud stage of E14.0, the expression intensity of protein in the dental papilla appeared stronger than that of mRNA. The expression of mRNA was absent in the PEK in the tooth germ at E15.0; meanwhile, a strong expression of immunoreaction was found in this structure. Double staining for nucleolin protein and TUNEL showed that the fluorescence images of nucleolin and apoptosis were not merged (Fig. 2D). Arrest of Nucleolin Translation by AS S-ODN—Based on the in situ hybridization and immunohistochemistry results for nucleolin, we examined the role of nucleolin in early tooth germ development and in tooth mineralization at the later stage using AS S-ODN in the cultured mandible at E11.0 and tooth germ at E15.0, respectively. Real time PCR was performed to examine the effect of nucleolin AS S-ODN on mouse E11.0 mandible explants cultured for 6 days by using HVJ-liposome transfection method. After performing a statistical analysis for the nucleolin expressions, more than 30% reduction of nucleolin mRNA expression (Fig. 3A) and 70% reduction of nucleolin protein (Fig. 3, C and D) were found in the nucleolin AS S-ODN-treated samples in comparison with control explants cultured with nucleolin SE or RS S-ODN (Fig. 3A). Regarding the toxicity assay, no significant difference of nucleolin mRNA expression was detected between control samples cultured without ODN and samples cultured with HVJ-liposome containing SE S-ODN, RS S-ODN, or HVJ-liposome only (Fig. 3B). Arrest of Tooth Germ Development by the Nucleolin AS S-ODN in E11.0 Mandible Culture—Preliminary experiments of organ culture for mandibles showed normal development of tooth germ to the cap stage by the 8th day of culture. However, no further development of the tooth germ was observed after the 8th day (supplemental Fig. 1A). The mandible explants removed from E11.0 mouse embryos were used and cultured with nucleolin SE S-ODN, RS S-ODN, or without ODNs as controls. After culturing for 8 days, the morphogenesis of the enamel organ could be detected, and most of enamel organs showed normal cap-like tooth germ (Fig. 4, A and B). However, no morphogenesis of the enamel organ was observed in the mandible treated with AS S-ODN (Fig. 4A). During the first 4 days of culture, the tooth germs in the E11.0 mandibles treated with AS S-ODN showed a normal bud-like structure as similar to the ODN-untreated tooth germs (Fig. 4A). At 4, 6, and 8 days of culture, the tooth germ in the ODN-untreated mandibles further developed (Fig. 4A), thus demonstrating features of the early and late cap stage. On the other hand, although the bud-like tooth germ treated with AS S-ODN had increased in size at day 6 (Fig. 4A), the epithelial buds were smaller than those treated without ODN (Fig. 4A). The epithelial bud at 8 days cultured and treated with AS S-ODN showed a similar size in comparison with 6 days of treatment with AS S-ODN and after 4 days without the ODN treatment (Fig. 4A). Almost all of the tooth germs in the mandible treated with AS S-ODN did not show differentiation into the early cap stage (p < 0.05; Table 1). However, some (15%) of the tooth bud developed an early cap stage similar to that in the control mandibles.TABLE 1The effects of AS S-ODN on the enamel organ development in cultured mandible Development of enamel organs at day 8 of culture was significantly inhibited.StagesUntreatedASaValues are p < 0.05SERSBudCapBudCapBudCapBudCapSample no.2 (14%)12 (86%)11 (85%)2 (15%)2 (25%)6 (75%)2 (33%)4 (67%)a Values are p < 0.05 Open table in a new tab No Effect of Nucleolin AS S-ODN on the Differentiation of Enamel Organ Cells in E15.0 Tooth Germ Culture—Tooth germs were removed micro-surgically from E" @default.
• W2036712925 created "2016-06-24" @default.
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