The regulation of the tooth morphogenesis via actin reorganization.

<p><b>(A)</b> The localizations of p-cofilin (green, left upper), cofilin (green, left lower), and F-actin (white, right) were detected by immunohistochemistry. The G0/G1 phase cells (red, center) are visualized with a Fucci probe. The lingual side is on the left in all panels. <b>(B)</b> Estimations of the p-cofilin/cofilin ratios in parts of the tooth germ epithelium (EK, enamel knot; DL, dental lamina; B and L, buccal side and lingual sides of the growing apex of the epithelium). The relative amounts of cofilin and p-cofilin in the regions of the epithelium were determined by immunoblotting (left). The intensities of the bands were calculated and are indicated in the bar graphs (right). <b>(C)</b> Measurements of actin dynamics in the epithelium using fluorescence recovery after photobleaching (FRAP). The right graph illustrates the best-fit curves of the normalized fluorescence intensity during the FRAP assay. The spots indicate the half-recovery times. <b>(D)</b> Gene expression of upstream molecules that regulate cofilin activity in the E14.5 tooth germ. The lingual side is on the left in all panels. The scale bars represent 100 μm. <b>(E)</b> Inhibition of cell proliferation by cofilin phosphorylation in a WST-8 assay. The results are presented as the mean ± s.d. of triplicate experiments. *<i>P</i> < 0.01, analyzed by <i>t</i>-test. LIMK WT, wild-type LIM-kinase (LIMK); LIMK D460A, dominant negative LIMK mutant; Rac V12, dominant active mutant of Rac1. <b>(F)</b> Inhibition of cell migration by cofilin phosphorylation in a wound healing assay. The bars indicate the migration distances of the epithelial cells. The results are presented as the mean ± s.d. of triplicate experiments. *<i>P</i> < 0.01, analyzed by <i>t</i>-test. <b>(G)</b> Schematic summarizing the observed results in this study.</p

Coordinated behaviors of the epithelial cells during tooth morphogenesis.

<p><b>(A)</b> Time-lapse sequences of the dental lamina (DL), enamel knot (EK) and growing apex of the epithelium (GAE) regions showing cell motility. The cells randomly expressed transgenes (YFP-Actin; green). The red, yellow and blue dots indicate individual cells in each region. The arrowhead indicates a membrane protrusion extending from the cell in the GAE. The scale bars represent 5 μm. <b>(B)</b> Distribution and measurement of mitotic spindle orientation in the developing tooth germ. The locations and orientations of the mitotic spindles of the dividing cells are shown in the wire frames. The mean distributions of the mitotic spindle angles (<i>θ</i>) in stellate reticulum (SR; upper) and growing apex of the epithelium (GAE; lower) are shown in the pie chart graph. The results are shown as the mean ± s.d. of three samples (<i>n</i> = 557, 627 and 626 cells [SR], <i>n</i> = 263, 183 and 234 cells [GAE]). *<i>P</i> < 0.01, * * 0.001 < <i>P</i> < 0.005, analyzed with <i>t</i>-tests. The scale bars represent 100 μm.</p

The quantitative kinetic analysis of tooth morphogenesis.

<p><b>(A)</b> Trajectories of epithelial cells over 20 hours (h) are shown in fluorescent images (upper panel) and wire frames (lower panel) at each time point of the long-term live image. The contours of the epithelium before and after 20 hours are shown in blue and grey wire frames, respectively. The growth-arrested regions are shown in red wire frame. <b>(B)</b> Trajectories of epithelial cells in parts (EK, enamel knot; DL, dental lamina; GAE, growing apex of epithelium) of the tooth germ epithelium during tooth development. <b>(C)</b> The changes in the relative positions of cells in parts of the tooth germ epithelium. The red and gray spots indicate the center cells and surrounding cells, respectively. <b>(D)</b> Measurements of the relative distances of the epithelial cells in each region. The numbers of spots for the calculations in each region were as follow: <i>n</i> = 17 cells (DL), <i>n</i> = 16 cells (EK), <i>n</i> = 14 cells (GAE). The error bars indicate the standard errors. <b>(E)</b> Deformation analysis of the epithelial tissue for 5–10 hours. The upper and lower panels show the spatial patterns of the volume growth rate and anisotropic tissue stretching, respectively. In the lower panels, the colors indicate the degree of anisotropy, and the arrows indicate the major axes of tissue stretching. The numbers of spots used to estimate the deformation map for each time intervals were as follows: <i>n</i> = 425 cells (25–30 hours), <i>n</i> = 552 cells (50–55 hours), <i>n</i> = 532 cells (75–80 hours), <i>n</i> = 615 cells (100–110 hours). The scale bars represent 100 μm.</p

Tooth morphogenesis and spatial-temporal cell proliferation.

<p><b>(A)</b> Frontal sections of mandibular molar tooth germ derived from Fucci mice at embryonic day (E) 13.5–18.5. The lingual side is on the left in all panels. The scale bars represent 100 μm. <b>(B)</b> Three-dimensional volume rendering images of the molar epithelium shown from the jaw side. The scale bars represent 100 μm. <b>(C)</b> Three-dimensional reconstructions of frontal sections of Fucci tooth germ epithelia showing the cell proliferation pattern. The scale bars represent 100 μm. <b>(D)</b> Length and width measurements of each part of the molar tooth germ. The results are provided as the mean ± s.d. of 6 samples (E14), 6 samples (E15), 8 samples (E16), 7 samples (E18) and 4 samples (E20). The measurement parts are indicated in the left figure. The scale bars represent 100 μm. <b>(E)</b> <i>Ex vivo</i> imaging of four phases during tooth germ epithelium morphogenesis. Schematic (upper panels) and captured live images (lower panels) are provided. The lingual side is on the left in all panels. Red indicates the growth-arrested regions. DL, dental lamina; EK, enamel knot; GAE, growing apex of epithelium. The scale bars represent 100 μm.</p

Downregulation of Carbonic Anhydrase IX Promotes <em>Col10a1</em> Expression in Chondrocytes

<div><p>Carbonic anhydrase (CA) IX is a transmembrane isozyme of CAs that catalyzes reversible hydration of CO₂. While it is known that CA IX is distributed in human embryonic chondrocytes, its role in chondrocyte differentiation has not been reported. In the present study, we found that <i>Car9</i> mRNA and CA IX were expressed in proliferating but not hypertrophic chondrocytes. Next, we examined the role of CA IX in the expression of marker genes of chondrocyte differentiation <i>in vitro</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#s1" target="_blank">Introduction</a> of <i>Car9</i> siRNA to mouse primary chondrocytes obtained from costal cartilage induced the mRNA expressions of <i>Col10a1</i>, the gene for type X collagen α-1 chain, and <i>Epas1</i>, the gene for hypoxia-responsible factor-2α (HIF-2α), both of which are known to be characteristically expressed in hypertrophic chondrocytes. On the other hand, forced expression of CA IX had no effect of the proliferation of chondrocytes or the transcription of <i>Col10a1</i> and <i>Epas1</i>, while the transcription of <i>Col2a1</i> and <i>Acan</i> were up-regulated. Although HIF-2α has been reported to be a potent activator of <i>Col10a1</i> transcription, <i>Epas1</i> siRNA did not suppress <i>Car9</i> siRNA-induced increment in <i>Col10a1</i> expression, indicating that down-regulation of CA IX induces the expression of <i>Col10a1</i> in chondrocytes in a HIF-2α-independent manner. On the other hand, cellular cAMP content was lowered by <i>Car9</i> siRNA. Furthermore, the expression of <i>Col10a1</i> mRNA after <i>Car9</i> silencing was augmented by an inhibitor of protein kinase A, and suppressed by an inhibitor for phosphodiesterase as well as a brominated analog of cAMP. While these results suggest a possible involvement of cAMP-dependent pathway, at least in part, in induction of <i>Col10a1</i> expression by down-regulation of <i>Car9</i>, more detailed study is required to clarify the role of CA IX in regulation of <i>Col10a1</i> expression in chondrocytes.</p> </div

Effects of <i>Car9</i> siRNA on the expressions of transcription factors related to chondrocyte differentiation in primary chondrocytes cultured under a hypoxic condition.

<p><i>Car9</i> siRNA (+) or control siRNA (-) was introduced into mouse primary chondrocytes under a hypoxic condition (5% CO₂ and 95% N₂). The cells were additionally cultured for 48 hours under the same condition. The expressions of <i>Car9</i>, <i>Col10a1</i>, <i>Epas1</i>, <i>Sox5</i>, <i>Sox6</i>, and <i>Sox9</i> were quantitatively analyzed by real-time RT-PCR. The expression level of each gene was normalized to that of <i>Gapdh</i>. Data are expressed as the mean ± SD (n = 4) in the fold change by introduction of <i>Car9</i> siRNA. <i>P</i>-values determined by two-tailed Mann-Whitney U-test are indicated.</p

Localization of CA IX in epiphyseal cartilage.

<p>Frozen sections of lower limbs excised from 1-day-old postnatal ddY mice were analyzed for immunolocalization of type II collagen (<b>A</b>, <b>D</b>, <b>G</b>, <b>J</b>), type X collagen (<b>B</b>, <b>E</b>, <b>H</b>, <b>K</b>), and CA IX (<b>C</b>, <b>F</b>, <b>I</b>, <b>L</b>). Photographs show the entire epiphysis with lower magnification (<b>A</b>–<b>C</b>), regions containing stationary to round-shaped proliferating chondrocytes (<b>D</b>–<b>F</b>), columnar chondrocytes proliferating to pre-hypertrophic chondrocytes (<b>G</b>–<b>I</b>), and hypertrophic chondrocytes (<b>J</b>–<b>L</b>) at higher magnifications. The regions magnified (<b>D</b>–<b>L</b>) were shown in the photographs of the entire epiphysis (<b>A</b>–<b>C</b>). Bars are 0.2 mm (A–C) and 0.1 mm (D–L).</p

Effects of <i>Car9</i> siRNA on chondrocyte proliferation and expressions of marker genes of chondrocyte differentiation.

<p><i>Car9</i> siRNA or control siRNA was introduced into primary chondrocytes isolated from the rib cages of 1-day-old postnatal ddY mice. <b>A.</b> The expression of <i>Car9</i> mRNA was analyzed by real-time RT-PCR at 48 hours after introduction of <i>Car9</i> siRNA or control siRNA, with the level normalized to that of <i>Gapdh</i>. Data are expressed as the mean ± SD (n = 4) for fold changes caused by introduction of <i>Car9</i> siRNA. <i>P</i>-value obtained by two-tailed Mann-Whitney <i>U</i>-test (n = 4) indicated that the <i>Car9</i> siRNA used in this study significantly lowered the expression level of <i>Car9</i> mRNA (α = 0.05). <b>B.</b> Expression of CA IX protein in chondrocytes was detected by immunocytochemical staining at 48 hours after introduction of control siRNA (left 2 panels) or <i>Car9</i> siRNA (right 2 panels). Lower 2 panels show the results obtained without primary antibody against CA IX. Bar, 50 µm. <b>C. </b><i>Car9</i> siRNA (unfilled square) or control siRNA (filled square) was introduced to chondrocytes on day 0. Proliferation of chondrocytes was assessed spectrophotometrically using CellTiter 96^® Aqueous One Solution. Data are expressed as the mean ± SD (n = 4). At each time point, Mann-Whitney <i>U</i>-test with Bonferroni correction was performed to evaluate the difference between control siRNA- and <i>Car9</i> siRNA-introduced cells. No significant difference between the values was indicated at any time point (α = 0.01). <b>D.</b> At 48 hours after introduction of siRNAs, expressions of mRNAs for <i>Car9</i>, <i>Col2a1</i>, <i>Col10a1</i>, <i>Vegfa</i>, and <i>Mmp13</i> were analyzed by RT-PCR. <b>E-G.</b> The expressions of <i>Col2a1</i> (<b>E</b>), <i>Acan</i> (<b>F</b>), and <i>Col10a1</i> (<b>G</b>) were quantitatively analyzed by real-time RT-PCR. The expression level of each gene was normalized to that of <i>Gapdh</i>. Data are expressed by boxplots (n = 4) in the fold change by introduction of <i>Car9</i> siRNA (the sample maximum, the upper quartile, the median, the lower quartile, and the minimum observation). <i>P</i>-values determined by two-tailed Mann-Whitney U-test are indicated. <b>H.</b> At 4 days after introduction of <i>Car9</i> or control siRNA, chondrocyte cultures were stained with Alcian blue. Alcian blue bound to the cell matrix was extracted and determined spectrophotometrically (n = 4). A <i>P</i>-value determined by two-tailed Mann-Whitney <i>U</i>-test is indicated. Typical photographs are shown above the columns.</p

Effects of forced expression of CA IX on chondrocyte proliferation and mRNA expression of <i>Col2a1</i>, <i>Acan</i>, <i>Col10a1</i>, and <i>Epas1</i>.

<p>Primary mouse chondrocytes were transfected with a CA IX-expression plasmid (+) or its control plasmid (-). A. At 48 hours after transfection, the expression of CA IX (58kDa) was assessed by western blot analysis using anti-mouse CA IX antibody. CA IX band is indicated by an arrowhead. B. Proliferation of chondrocytes introduced with CA IX-expression (filled square) and control (unfilled circle) plasmids was assessed spectrophotometrically using CellTiter 96^® Aqueous One Solution. Data are expressed as the mean ± SD (n = 4). At each time point, Mann-Whitney <i>U</i>-test with Bonferroni correction was performed to evaluate the difference between control plasmid- and CAIX-expression plasmid-transfected cells. No significant difference between the values was indicated at any time point (α = 0.01). C-F. The expressions of <i>Col2a1</i> (C), <i>Acan</i> (D), <i>Col10a1</i> (E), and <i>Epas1</i> (F) were quantitatively analyzed by real-time RT-PCR, with the expression level of each normalized to that of <i>Gapdh</i>. Data are expressed by boxplots (n = 6) for fold changes caused by introduction of <i>Car9</i> siRNA (the sample maximum, the upper quartile, the median, the lower quartile, and the minimum observation). <i>P</i>-values determined by two-tailed Mann-Whitney <i>U</i>-test are indicated.</p

Schematic representation of possible roles of CA IX in regulation of <i>Col10a1</i> expression in chondrocytes.

<p>Blue and red lines represent pathways already known and those indicated in this study, respectively. Arrows with equilateral-triangular heads and T-shaped bars show facilitation and suppression of the pathways, respectively. HIF-1α induces the expression of <i>Car9</i> mRNA and hence that of CA IX protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Kaluz1" target="_blank">[8]</a>. It is known that cAMP-dependent pathways inhibit hypertrophic differentiation of chondrocytes including the expression of <i>Col10a1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Tintut1" target="_blank">[26]</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Chung1" target="_blank">[29]</a>. On the other hand, it is reported that HIF-2α, encoded by <i>Epas1</i> gene, transactivates the <i>Col10a1</i> gene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Saito1" target="_blank">[14]</a>. We propose that CA IX suppresses the expression of <i>Col10a1</i> mRNA partly via a cAMP-dependent manner based on the following observations. <i>Col10a1</i> expression was induced by introduction of <i>Car9</i> siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g003" target="_blank">Figure 3G</a>). The cAMP level was lowered by <i>Car9</i> siRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8B</a>). Inhibition of PKA by H89 augmented the expression of <i>Col10a1</i> induced by <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8A</a>). In addition, inhibition of PDE by IBMX and activation of PKA by Br-cAMP suppressed the expression of <i>Col10a1</i> expression induced by <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figures 8A and 8D</a>). While <i>Car9</i> siRNA also enhanced the expression of <i>Epas1</i> mRNA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g004" target="_blank">Figure 4E</a>), HIF-2α does not mediate the <i>Col10a1</i> induction by <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g007" target="_blank">Figure 7C</a>). It is partly because <i>Car9</i> siRNA lowers HIF-2α protein via an unknown mechanism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g007" target="_blank">Figure 7D</a>). <i>Epas1</i> siRNA rather enhanced the expression of <i>Col10a1</i> mRNA by an unknown mechanism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g007" target="_blank">Figure 7C</a>). It is reported that Src kinase mediates the expression of <i>Epas1</i> and <i>Col10a1 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Bursell1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone.0056984-Sato1" target="_blank">[36]</a>. In this study, Src kinase inhibitor I lowered the induction of <i>Epas1</i> and <i>Col10a1</i> expressions after <i>Car9</i> silencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8A</a>), which may indicate a possibility that CA IX suppresses the Src kinase-mediated pathways. Augmentation of <i>Epas1</i> expression in the presence of the inhibitors of p38 MAPK (SB202190) and PI3K (LY294002) suggests a possible inhibition of <i>Epas1</i> expression by these kinases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056984#pone-0056984-g008" target="_blank">Figure 8A</a>).</p