PsyR, a transcriptional regulator in quorum sensing system, binds lux box-like sequence in psyI promoter without AHL quorum sensing molecule and activates psyI transcription with AHL in Pseudomonas syringae pv. tabaci 6605

Quorum sensing (QS) is a mechanism for bacterial cell-cell communication using QS signals. N-acyl-homoserine lactones (AHLs), QS signals in Pseudomonas syringae pv. tabaci (Pta) 6605, are synthesized by an AHL synthase (PsyI) and recognized by the cognate transcription factor PsyR. To reveal the role of PsyR in virulence, we generated a psyR mutant and complemented strains of Pta 6605 and found that the psyR mutant is remarkably reduced in AHL production and ability to cause disease and propagate in host tobacco leaves. The phenotypes of complemented strains were restored to that of the wild type (WT). Because the psyR mutant lost nearly all AHL production, we investigated the function of PsyR in the transcription of psyI and production of AHL. Electrophoretic mobility shift assays suggested that the recombinant PsyR protein binds the promoter region of psyI but not psyR without AHL. The addition of AHL did not significantly affect this binding. The binding core sequence of this region was identified as a 20-bp lux box-like sequence. To reveal the function of PsyR and AHL on psyI transcription, we constructed a psyI promoter::lacZYA chimeric reporter gene, and inserted it into the WT and psyI mutant of Pta 6605. beta-galactosidase activity increased in a bacterial density-dependent manner in the WT and also in a psyI mutant after the addition of exogenous AHL. These results indicate that the solo PsyR binds the lux box in the psyI promoter and activates transcription in the concomitant presence of AHL

Coordination of Cellular Dynamics Contributes to Tooth Epithelium Deformations.

The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses

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

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

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