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