Analysis of fibronectin expression during tooth development.

<p>A. Immunofluorescence analysis of fibronectin expression using an anti-fibronectin antibody in sagittal sections from 6-week-old mouse incisors. The incisors were separated into the apical bud (AB), presecretory (PS), secretory (S), early maturation (EM), and late maturation (LM) stages. B. Immunofluorescence and immunohistochemical results are shown at a higher magnification. Fibronectin was expressed in the basal lamina at the S stage and in the papillary layer at the LM stage. C. Fibronectin mRNA was detected using <i>in situ</i> hybridization in the basal lamina, pre-odontoblasts, pre-ameloblasts from PS to S (PS-S) and LM ameloblasts, with lower expression seen during the EM stage. D. The expression levels of fibronectin and amelogenin were investigated by real-time PCR using PS, S, EM, and LM dental epithelial cells separated from the incisor. E. The expression of fibronectin in dental epithelial (DE) and dental mesenchymal (DM) cells isolated from the molars of P1 and P7 mice (n = 5) was investigated by real-time PCR. Amelogenin mRNA expression increased in DE cells, while fibronectin mRNA expression decreased with differentiation. iee, inner enamel epithelium; dp, dental pulp; si, stratum intermedia; am, ameloblasts; pam, pre-ameloblasts; od, odontoblasts; pod, pre-odontoblasts; pl, papillary layer.</p

Sagittal sections of lower incisors from a 6-week-old WT and <i>Itgβ1</i>CKO mouse.

<p>A. The lower incisors of a wild type (WT) and a <i>Itgβ1</i> conditional knockout (CKO) mouse were stained with hematoxylin and eosin (H-E) and an anti-dentin sialoprotein (DSP) antibody. The white dotted arrow indicates the starting point of dentin formation from DSP-positive odontoblasts, and the blue arrow indicates the starting point of enamel formation from ameloblasts. Dentin (white dotted arrow) and enamel-formative regions (blue arrow) of the <i>Itgβ1</i>CKO-mouse incisors were separated by a longer distance as compared with those from the WT mice. B. High magnification of the area enclosed by the dotted box (a, amelogenesis origin of WT; b, dentinogenesis origin of <i>Itgβ1</i>CKO; c, amelogenesis origin of <i>Itgβ1</i>CKO) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121667#pone.0121667.g005" target="_blank">Fig. 5A</a> (DAPI staining). C. Ameloblast cell and nuclear heights are indicated by the red and blue arrows, respectively. The ratios of cells to nuclear heights are shown in the graph. The heights of the nuclei of undifferentiated ameloblasts did not change in the dentinogenesis region, and the ameloblasts became polarized and matured in the amelogenesis area. *P < 0.01. am, ameloblasts; od, odontoblasts.</p

Dental epithelial cell adhesion assay.

<p>A. SF2 rat dental epithelial cells were cultured in plates coated with fibronectin, collagen I, mouse laminin 111 and 211, and human laminin 511/521 in the presence and absence of RGD or RAD peptides. Plates were stained with crystal violet. The relative number of adherent cells was evaluated using spectrophotometric analysis. B. SF2 cells were cultured in plates coated with fibronectin or laminin 511/521 in the presence and absence of IgGs, RGD peptide, and anti-β1, -α6, or -β4 antibodies. Cell adherence to fibronectin was inhibited by the RGD peptide and the anti-β1 integrin antibody, and adhesion of cells to plates coated with laminin 511/521 was inhibited in the presence of the RGD peptide, as well as anti-integrin α6 or anti-integrin β4 antibodies. *P < 0.01; **P < 0.05 (n = 6).</p

A proposed model in which Med1 ablation alters epithelial cell fate.

<p>Med1 regulates Notch signaling by activating Notch1 target genes (upper diagram). Med1 maintains the niche architecture containing Sox2-expressing dental epithelial stem cells (DE-SC) (blue). Enamel is formed as dental epithelia differentiate in control incisors partly due to Notch signaling. In contrast, DE-SCs fail to commit to the dental lineage when Med1 is ablated (lower diagram). Instead, Sox2-expressing cells (blue) extend into the differentiating zones. Med1 deficient cells are exposed to extracellular calcium through the blood vessels (red-dotted circles) adjacent to the papillary layers (yellow). Med1 deficient cells are differentiated into epidermal cells (light green) and hair keratinocyte-like cells (green) and produce mature hair shafts (black) in the Med1 KO incisors (inserted picture).</p

Gene expression profiling predicted changes in Notch and calcium signaling upon Med1 deletion.

<p>(A) Strategy of microarray analyses. CL and Mat tissues were dissected from Med1 KO and CON mice at 4 wk and 10 wk. Array 1 calculated fold changes in CL/Mat in control mice revealed genes that are enriched in CL compared to the Mat stage, which are DE-SC signature candidates. Array 2 calculated fold changes in KO/CON at CL shows genes up-or down-regulated in Med1 KO compared with CON. Arrays were performed on RNA samples from Med1 KO and CON. (B) These analyses revealed a list of DE-SC signatures affected in the CL of Med1 KO in comparison with gene pools identified through other stem cells of ESC or HF-SC. Green letters show down-regulation, and the red letters indicate up-regulation. Fold increases and other details are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099991#pone.0099991.s007" target="_blank">Table S4</a> (10 wk). (C, D) The IPA pathway analysis predicted that Notch1 (C) and calcium (D) are potential upstream regulators to cause these changes in Med1 KO. The sub-cellular location and the up- and down-regulation of genes are shown by red and green, respectively. (E) The protein expressions of Notch1 at 4 wk are shown in CL of Med1 KO and CON.</p

Expression of collagen IV, laminin β1γ1, and ameloblastin in incisors from WT and <i>Itgβ1</i>CKO mice.

<p>A. The lower incisor of a wild type (WT) mouse was analyzed using immunostaining with anti-collagen IV and anti-laminin β1γ1 antibodies. Laminin β1γ1 was expressed in the basal membrane, papillary layer, and immature ameloblasts. B, C. The lower incisors of WT and <i>Itgβ1</i> conditional knockout (CKO) mouse were immunostained by anti-ameloblastin and anti-laminin β1γ1 antibodies. Ameloblastin was detected in the incisor of a WT but not a <i>Itgβ1</i>CKO mouse. Laminin β1γ1 was detected in <i>Itgβ1</i>CKO (asterisk) but not WT incisors. iee, inner enamel epithelium; am, ameloblasts; od, odontoblasts.</p

Phenotype of incisors from <i>Itgβ1</i>CKO mice.

<p><i>Itgβ1</i> was deleted from cells that expressed cytokeratin 14. A. Southern blot analysis of genomic DNA from P1 molars using a probe for exon 3. Most of the region containing exon 3 was removed by Cre recombinase under the control of the <i>K4</i> promoter in the dental epithelium. B. Expression levels of β1 integrin mRNA and protein in P1 molar tooth germs. C. Immunohistolocalization of β1 integrin in E16.5 molar tooth germs. D. Frontal view of a 8-week-old wild type (WT) and homozygous <i>Itgβ1</i> conditional knockout (CKO) mouse. Many vitiligo patches are visible in the image of the lower incisor from an <i>Itgβ1</i>CKO mouse. E. The lower incisors were observed under higher magnification and the total size of the yellow areas was measured. Yellow areas were markedly decreased in the incisors from <i>Itgβ1</i>CKO mice (n = 5). F. Sagittal sections of lower incisors from a 6-week-old WT and <i>Itgβ1</i>CKO mouse were stained with hematoxylin and eosin (H-E). High magnifications of the areas enclosed by the dashed box in the left panel. Cysts were formed in the late maturation (LM) stage. G. Immunostaining using an anti-collagen IV antibody. Nuclei were stained using DAPI. The image shows an irregular and multilayer arrangement of ameloblasts in the incisor from an <i>Itgβ1</i>CKO mouse. The dotted line shows the border of enamel and ameloblasts. *P < 0.01. iee, inner enamel epithelium; dp, dental pulp; alv, alveolar bone; en, enamel; de, dentin; am, ameloblasts; pl, papillary layer.</p

Phosphorylation of ERK1/2 after stimulation of neurotrophic factor NT-4 in the presence of fibronectin.

<p>A. Western blot analysis was performed using anti-ERK1/2 and phospho-ERK1/2 antibodies. Cell lysates were prepared after stimulation of NT-4 at 0, 5, and 15 minutes with or without fibronectin or an siRNA for β1 integrin. B. Quantitative data of ERK1/2 phosphorylation.</p

Real-time PCR analysis of expression of mRNAs encoding ameloblast markers in primary cultures of ameloblasts.

<p>A. Wild type (WT) ameloblasts were cultured with RGD or RAD peptide for 0 to 72 hours. The expression of ameloblastin was delayed by RGD but not by RAD. B. WT and <i>Itgβ1</i>CKO ameloblasts were cultured for 0 to 72 hours. The expression of ameloblastin was delayed in <i>Itgβ1</i>CKO ameloblasts as compared with WT. C. Analysis of the expression of mRNAs encoding ameloblast markers in WT ameloblasts cultured in the presence of an anti-β1 integrin antibody. The expression of all ameloblast markers was decreased. D. WT ameloblasts were transfected with fibronectin siRNA for 0 to 72 hours. The inhibition of fibronectin expression delayed the expression of ameloblastin. *P < 0.01; **P < 0.05 (n = 5).</p

Med1 ablation prevents dental fate but drives epidermal fate.

<p>The IPA pathway analysis on 4(A) The expressions of dental genes were decreased in KO at the Mat stage. Instead, hair genes (B) and epidermal genes (C) increased in KO at the Sec and Mat stages. The full list of altered genes with fold changes and detailed information is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099991#pone.0099991.s004" target="_blank">Tables S1</a>, 2 and 3. Green letters indicate down-regulation and red letters indicate up-regulation. The mRNA levels of representative genes for dental (A, b Klk4), hair (B, b Krt71), and epidermal (C, b Lor) are confirmed by QPCR. The average and SD of relative expressions (% of GAPDH) in CL, Sec and Mat at 4 wk are shown. The numbers show the value of the relative expression, which are too low to represent using bars. The statistically significant increases/decreases in Med1 KO (closed bars) compared to KO (open bars) at each stage are shown by asterisks (n = 3, <i>p</i><0.05). (D) Immuno-staining of epidermal keratin Krt1 (left) and hair keratin Krt71 (right) (Krt1 or Krt71 green, Notch1 red, DAPI blue) in the Med1 KO and CON at 4 wk. Equivalent sections stained by HE are presented on the right side of each image.</p