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Cadherin-related Neuronal Receptors in Incisor Development

¹ Department of Preventive Dentistry,
² Dental Pharmacology, and
³ Pediatric Dentistry, Nagasaki University School of Dentistry, Nagasaki 852-8588, Japan;
⁴ CREST, KOKORO Biology Group, Laboratories of Intedgrated Biology, Graduated School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita 565-0871, Japan;

View larger version (50K): [in a new window]   Figure 1. Isolation of CNRs by Differential Display Method. (A) (a) Schematic diagram of the differential display method using three different stages of ameloblasts: secretory (S), early-maturation (EM), and late-maturation (LM) stages. Panels (b) and (c) show differential displays with reverse primers oligo(dT)15A and oligo(dT)15G, respectively. Arrows represent CNR1 (b) and CNR5 (c) cDNA identified by band sequencing. (B) Schematic structures of cadherins and CNRs. Bold lines under CNRs represent conserved regions in the CNR family. S, signal peptide; EC, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain. A comparison of rat, mouse, and human C-terminal protein sequences is also shown. The nucleic acid and amino acid sequences for these clones were deposited in GenBank (accession number AB045585 for CNR1 and AB045586 for CNR5). (C) Tissue-specific expression of CNR mRNA in adult (upper) and P1 rats (lower) was analyzed by RT-PCR. Primers from the conserved regions were used. CNRs were expressed in the brain and incisor but not in other tissues. (D) mRNA expression was quantified by real-time PCR in three stages of ameloblasts. The relative percentage in each stage is shown in relation to the highest value as 100%. Statistical analysis was performed with the use of InStat3 software (GraphPad Software, San Diego, CA, USA). All results were expressed as mean ± SE, and P < 0.05 was used for significance. S, secretory stage; EM, early-maturation stage; LM, late-maturation stage.

View larger version (79K): [in a new window]   Figure 2. In situ hybridization of CNR1 and CNR5 mRNA in the rat incisor. (A) Whole stages of ameloblasts and odontoblasts. Arrows indicate the points of the signal that disappeared in ameloblasts. (B) Tissues were hybridized with CNR5 antisense (a,d,g,j), CNR1 antisense (b,e,h,k), and CNR5 sense cRNA probes (c,f,i,l). Bulbous portion of the odontogenic epithelium (a,b,c); secretory stage (d,e,f); early-maturation stage (g,h,i); and late-maturation stage (j,k,l). Magnification: x40. am, ameloblasts; od, odontoblasts. (C) High magnification of CNR5 mRNA expression. CNR5 antisense (a, c, e) and sense cRNA probes (b, d, f); secretory stage (a, b); early-maturation stage (c, d); and late-maturation stage (e, f). Magnification: x200. am, ameloblasts; pc, papillary cell layer. Scale bars: 50 µm (Bl); 20 µm (Cf).

View larger version (66K): [in a new window]   Figure 3. Immunolocalization of CNRs in rat incisor (red). (A) A monoclonal antibody that reacts with all CNR isoforms was used. The bulbous portion of the odontogenic epithelium and mesenchyme (a), secretory stage (b), early-maturation stage (c), and late-maturation stage of ameloblasts (d). Magnification: x40. (B) High magnification of the early-maturation stage of ameloblasts and odontoblasts (a); second antibody only (b). Magnification: x200. am, ameloblasts; pc, papillary cell layer; od, odontoblasts; de, dentin. Scale bars: 50 µm (Ad); 20 µm (Bb).

View larger version (23K): [in a new window]   Figure 4. CNR1 binding to dental epithelial and mesenchymal cells. (A) Schematic structure of CNR1 and tagged CNR1 fusion proteins and their cell-binding activity. Recombinant proteins were incubated with cells, and bound proteins were analyzed by staining with anti-Hisx6 or anti-HA antibody, with FITC-conjugated secondary antibody. Activity of CNR binding to dental epithelial cells and mesenchymal cells is shown as + (positive-binding cells are more than 30%) or - (positive-binding cells are 0%). The percentage of positive-binding cells is calculated as 100 x [number of FITC-positive cells] / [number of Hochest-positive cells]. S, signal peptide; EC, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain; Hisx6, polyhistidine; HA, hemaglutinin. (B) Visualized image of EC1 binding to dental epithelial or mesenchymal cells. EC1-His was incubated with cells, and bound proteins were visualized by FITC-conjugated secondary antibody. Purple signal was Hochest staining for the cell nucleus. (C) Only recombinant protein containing EC1 (lane3) bound to the dental epithelial cells, not S-His or EC1-His (lanes 1 and 2). EC1-HA was used to compete with EC1-His for cell binding at equal (lane 4) or 10-fold concentrations (lane 5). Bound CNR1 proteins were detected by Western blotting with anti-His antibody (upper) and anti-HA antibody (lower).