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First published online 1 August 2006
doi: 10.1242/jcs.03079

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Cell and fibronectin dynamics during branching morphogenesis

Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370, USA

View larger version (56K): [in a new window]   Fig. 1. Epithelial cells in intact salivary glands undergo dynamic movements. (A) Schematic diagram of an intact E13 SMG with epithelium (e) and surrounding mesenchyme (m); the box indicates the region imaged by time-lapse microscopy in panel B. (B) Epithelial buds with surrounding mesenchyme (top) imaged in supplementary material Movie 1 at t=0. The white dashed box indicates the region shown magnified below from individual frames of Movie 1. Two cells that move towards the right are outlined (red and cyan). (C) Schematic diagram indicating the site of Ad-GFP microinjection into epithelium; the box indicates the epithelial region shown in D. (D) First frame from a time-lapse movie showing brightfield-imaged (left) and GFP-labeled cells (right), revealing substantial apparent epithelial cell movements (supplementary material Movie 2). (E) Immunostaining of the microinjected gland after time-lapse analysis with the epithelial marker E-cadherin (red) indicated that the injected GFP-expressing cells (green) are epithelial (arrowheads). Time is indicated in hours:minutes. Bars, 20 µm.

View larger version (83K): [in a new window]   Fig. 2. Mesenchyme-free epithelial cells move relative to each other. (A) Schematic diagram indicating the region of isolated E12 SMG epithelial cells analyzed by time-lapse microscopy in supplementary material Movie 3. (B) First frame of time-lapse Movie 3 with the basement membrane marked by a white dashed line. The yellow dashed box indicates the region examined at higher magnification in C and D. (C) Three frames from time-lapse Movie 3 with cells analyzed in D marked with arrowheads. (D) Frames from C were converted to a binary image (dark green overlay), and cell centroids were calculated automatically (yellow points). The distance between the two cell centroids (cyan dashed line) increased over time, indicating that the cells were migrating: 25.6 µm (0:00), 29.1 µm (0:24), and 38.6 µm (0:48). The velocity varied between frames: 8.7 µm/hour and 23.7 µm/hour. Bars, 20 µm. Time, hours:minutes.

View larger version (120K): [in a new window]   Fig. 3. Epithelial cells show dynamic cell movements that differ in distinct regions of the gland. (A) Still frames from time-lapse supplementary material Movie 4 of E13 SMG epithelia. The frames are single confocal sections through the center of the gland with epithelial cells labeled by GFP (green) and fibronectin labeled with Alexa Fluor-647-FN (red). (B) Individual cell movements were manually tracked in 3D; an example bud track is shown in white overlaid on an XY projection with XZ (top) and XY (left) projections. (C) Representative tracks of migrating cells from the bud interior (black) or duct interior (green) compared to bud cells that had contacted the basement membrane (BM, purple) displayed as a 3D rose plot (X, Y, and Z axis) with the origin of each track at 0, 0, 0. Cell tracks were quantified for velocity (T/t), displacement (D), total distance traveled (T), and meandering index (D/T) and displayed as bar graphs: mean ± s.e.m. On average, cells in buds traveled at higher velocities and for longer distances than duct cells, and cells that contacted the basement membrane traveled even faster and further than cells in the interior of buds. Cells were tracked for 5 hours; n=44 (bud), 29 (duct) and 45 (BM), averaged from six experiments. The Wilcoxon signed-rank test was used to calculate statistical significance of differences, *P<0.001, **P<0.0001 compared with bud cells. (D) After time-lapse imaging, the epithelia were fixed and stained with anti-E-cadherin antibody: E-cadherin (blue), GFP (green), and FN (red). All GFP-labeled cells (green) were inside of the basement membrane demarcated by FN, and all expressed E-cadherin. Bars, 50 µm (A), 10 µm (D).

View larger version (35K): [in a new window]   Fig. 4. Cell migration by salivary epithelial cells is not affected by inhibiting cell proliferation. E12 SMG epithelial rudiments were labeled with GFP and Alexa Fluor-647-FN and treated with 0.5 mM hydroxyurea (supplementary material Movie 5). (A) Cells were tracked manually, and 10 representative tracks for each treatment are displayed as 3D rose plots. (B) To measure inhibition of cell proliferation after 24 hours in culture, SMGs were pulse-labeled with BrdU for 3 hours. The mitotic index was calculated as the ratio of total BrdU pixels to total SMG area. Velocity, total distance traveled in 5 hours, displacement and meandering index were calculated as an average of all tracks, and displayed as bar graphs comparing control (black) with hydroxyurea (blue), mean ± s.e.m. Cells were tracked for 3 hours; n=20, averaged from two experiments. Values displayed represent mean ± s.e.m. The Wilcoxon signed-rank test was applied to compare cell tracks of inhibitor-treated SMGs with untreated SMGs, *P<0.05 compared with untreated SMGs.

View larger version (63K): [in a new window]   Fig. 5. Cell migration is severely reduced in day 1 postnatal (P1) salivary glands. SMGs from P1 mice were labeled with GFP and Alexa Fluor-647-FN and imaged by confocal microscopy (supplementary material Movie 6). (A) Randomly chosen bud cells were tracked for 5 hours, and migration tracks are displayed as a 3D rose plot, n=10. (B) Frames from Movie 6 showing cells (green) overlaid on a DIC image and surrounded by FN (red). Prior to tracking, epithelial cells were confirmed to be in an acinus surrounded by FN (red). (C) After time-lapse imaging, SMGs were stained with E-cadherin antibody (blue) to reveal epithelial cell boundaries. GFP-labeled cells (green) expressing E-cadherin (blue) were surrounded by FN (red). Bars, 10 µm.

View larger version (80K): [in a new window]   Fig. 6. FN at the base of clefts translocates into the bud whereas newer FN assembles behind older FN. (A) E12 SMGs were labeled with Alexa Fluor-647-FN-containing media for 6 hours and imaged by time-lapse confocal microscopy. After 1 hour 48 minutes of imaging, medium was removed and replaced with fresh medium, and imaging was continued (supplementary material Movie 7). The initiation points of two clefts are labeled with open arrowheads. The bases or bottoms of each of these clefts move inward into the interior of the gland as indicated by filled arrowheads. (B) To image FN displacement, SMGs were incubated with Alexa Fluor-488-FN (green) for 12 hours, washed, and then treated with Alexa Fluor-647-FN (red) for two additional hours. The earliest FN (green) was concentrated at the bottom and lower sides of the cleft, whereas the FN added later (red) assembled in the bud basement membrane and the upper portion of the clefts. (C) Higher-magnification images of clefts at 16 hours from time-lapse images in (A) showing concentration of FN in a wedge-like pattern. Bars, 100 µm.

View larger version (29K): [in a new window]   Fig. 7. Working model for roles of cell migration and fibronectin translocation in branching morphogenesis. Cells engage in active migration, providing a highly plastic tissue that is susceptible to local matrix signals. Three representative motile cells have been colored in yellow, blue and cyan. As cleft formation begins, FN is located at the cleft initiation site. Cleft formation proceeds towards the center of the gland with continued synthesis and assembly of FN fibrils by neighboring epithelial cells and dynamic inward progression of an aggregate or wedge of FN between mobile epithelial cells. FN washout experiments show that older FN (green) is translocated deep into the gland at the base of deepening clefts, whereas new FN (red) assembly occurs behind it. We propose that the dynamic inward translocation of FN as a wedge, followed by later assembly behind it, provides the missing mechanism for precise local deposition of FN synthesized by a broad zone of cells surrounding developing clefts. The local wedge of FN would mediate the previously described conversion of E-cadherin cell-cell adhesions to integrin-mediated cell-matrix adhesions, thereby promoting the separation of actively jostling cells to form the cleft during branching morphogenesis.