1. Volume 27, Issue 9, Pages 1381-1386 (May 2017)
Cell-Cycle-Coupled Oscillations in Apical Polarity and Intercellular Contact Maintain Order in Embryonic Epithelia 
Katerina Ragkousi, Kendra Marr, Sean McKinney, Lacey Ellington, Matthew C. Gibson 
Current Biology 
Volume 27, Issue 9, Pages (May 2017)
DOI: /j.cub
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2. Figure 1
The Nematostella vectensis Embryo Forms an Epithelial Monolayer and Compacts Early and Periodically during Development
(A and B) Sum projection (A) and single-plane view (B) of embryos at 0 hours post-fertilization (hpf) (1-cell), 2.5–3 hpf (4-cell), 3–3.5 hpf (16-cell), 4.5 hpf, 5.5 hpf, and 7 hpf fixed and stained for F-actin. Monolayer organization is manifested at the 16-cell stage.
(C) Selected frames from a time-lapse movie (Movie S1) of a developing transgenic embryo with Par6-GFP expression (F2 mat > Par6-GFP/+). Par6-GFP is enriched at the four-cell contacts at 0 min (2.5 hpf), gradually reduces at 6 min, and is completely absent during cytokinesis at 12 min. Par6-GFP enrichment is recovered at the cell contacts of the compacted embryo at 24 min. A second cycle follows soon after (30 min–48 min).
(D and E) Single-plane image showing Par6-GFP enriched at the cell surface of one blastomere from embryo shown in (C), and cartoon (E) of the same blastomere illustrating how Par6-GFP intensity levels were measured in (F).
(F) Plot of Par6-GFP intensity levels at the cell-cell contacts (green) from three blastomeres and the cell surface (blue) from five blastomeres (left y axis) of the same embryo. Compaction efficiency (orange) is plotted on the right y axis and was determined as described in Figure S1C. Gray bars indicate cytokinesis intervals.
Error bars indicate SD. Scale bars, 35 μm. See also Figure S1 and Movie S1.
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3. Figure 2
Dividing Cells of Nematostella Embryonic Epithelia Lose Par3-GFP but Maintain α-Catenin-GFP and Lgl-GFP at Their Junctions
(A) Dividing cells of embryos expressing injected mRNA of Par3-GFP (green) and H2B-RFP (magenta) that label apical junctions and chromatin, respectively, show loss of GFP signal by metaphase and recovery of signal by interphase. Two sequential cycles (interphase-metaphase-interphase) are shown from the same embryo (Movie S1; see also Figures S2A and S2D).
(B) Dividing cells of embryos expressing injected mRNA of α-catenin-GFP (green) that labels apical junctions and H2B-RFP (magenta) show mildly reduced GFP signal at metaphase cell contacts (see also Figure S2B).
(C) Dividing cells of embryos expressing injected mRNA of Lgl-GFP (green) that labels baso-lateral junctions and H2B-RFP and PH-mCherry (magenta for both) that labels chromatin and cell membrane, respectively, show retained GFP signal at metaphase cell contacts (see also Figures S2C and S2D). All images are selected time-lapse frames from sum projections of live embryos.
(D) Boxplot of protein retention levels for Par3-GFP (blue; n = 48 cells), α-catenin-GFP (red; n = 64 cells), and Lgl-GFP (green: n = 62 cells). Retention is defined as the ratio of GFP intensity at metaphase divided by GFP intensity at interphase. Broken line indicates that metaphase and interphase GFP levels are equal. Data points were collected from three independent experiments as described in the Supplemental Experimental Procedures. Differences between groups were found to be statistically significant (Wilcoxon Mann-Whitney rank-sum test; more information is given in the Supplemental Experimental Procedures).
Scale bars, 10 μm. See also Supplemental Experimental Procedures, Figure S2, and Movie S1.
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4. Figure 3
Embryonic De-compaction Is Coupled to Mitotic Cell Rounding, and Its Prolonged Extension Perturbs Development
(A) Selected time-lapse frames show that blastomeres of the transgenic Par6-GFP (F2 mat > Par6-GFP/+) and the Par3-GFP (green)/H2B-RFP (magenta)-expressing embryo round up but do not complete cytokinesis when grown in the presence of the Aurora kinase inhibitor (Movie S2). White arrows indicate endo-replicated chromatin.
(B) Plots of Par6-GFP dynamics in embryos grown in the presence of two separate cell-cycle inhibitors. Error bars indicate SD. Additional plots from different embryos are shown in Figures S3A and S3B.
(C–F) In (C) and (D), selected frames are shown from time-lapse movies of developing embryos (F2 mat > Par6-GFP/+) in sea water (control) or Con A-supplemented sea water (shown in C); corresponding compaction dynamics of each embryo plotted in (E) or injected with either wild-type Moesin as a control or constitutively active MoesinT562E (shown in D); and corresponding compaction dynamics of each embryo plotted in (F) (see also Figures S4I and S4J and Movie S3). Cell-cycle phases were identified with co-injection of H2B-RFP (not shown). Stars indicate cells that divide asynchronously with respect to neighbors. In (E) and (F), the red dotted line indicates complete compaction, and the blue dotted line indicates complete de-compaction, as evaluated from normal embryos. Bars show cytokinesis intervals.
(G) Embryos were treated with either sea water (control) or sea water supplemented with Con A at the four-cell stage (3 hours post-fertilization [hpf]) and then moved to sea water 8 hr later. Control embryos maintain a monolayer at 5 hpf (segmented yellow line), and 90% gastrulate normally (n = 19 embryos), while Con A-treated embryos lose the monolayer organization within 2 hr after Con A treatment, and only 6% gastrulate normally (n = 35 embryos).
(H) Embryos injected with either wild-type Moesin or MoesinT562E were fixed during early (5.5hpf) and post-gastrulation (25.5hpf) stages. 76% Moesin-injected embryos maintain a monolayer (n = 75 embryos), and 74% gastrulate normally (n = 63 embryos). 12% MoesinT562E-injected embryos maintain monolayer organization (n = 70 embryos), and 30% gastrulate normally (n = 61 embryos). 88% of un-injected controls (data not shown) developed a monolayer (n = 72 embryos), and 88% gastrulated successfully (n = 97 embryos).
Scale bars, 35 μm. Data in (G) and (H) were collected from two independent experiments. See also Figures S3 and S4 and Movies S2 and S3.
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5. Figure 4
Dividing Cells of Drosophila Wing Disc Epithelia Lose Par3-GFP from Apical Junctions
Selected time-lapse frames show protein levels in dividing cells (indicated by a star in all images) at interphase 1 prior to division, at mitosis, and in daughter cells (indicated by a square point in all images) at interphase 2. Scale bars, 2 μm. See also Supplemental Experimental Procedures and Movie S4.
(A) Par3-GFP levels in a dividing cell.
(B–D) Par3-GFP (B), E-cadherin-GFP (C), and ATPα-GFP (D) levels in a dividing cell of a mosaic clone. (For protein levels in mosaic clones, see also Movie S4.) All images are sum projections (4–6 μm thick) of cells from wing disc epithelia.
(E and F) Cartoon (E) depicting the construction of mosaic cell clones in the fly wing disc and the measurement of GFP intensity levels as shown in a boxplot (F) for Par3-GFP (n = 18 cells from nine wing discs), E-cadherin-GFP (n = 14 cells from five wing discs), and ATPα-GFP (n = 20 cells from four wing discs) at junctions shared with either GFP-marked (blue) or un-marked neighboring cells (yellow). Retention levels were calculated as described in Figure 2 and in Supplemental Experimental Procedures. Three asterisks indicate differences between groups found to be statistically significant (Wilcoxon Mann-Whitney rank-sum test; more information in Supplemental Experimental Procedures).
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