Fig 1.
Cells do not proliferate during follicular morphogenesis of Ciona intestinalis (A-C), Molgula citrina (D-F), and Phallusia mammillata (G).
Oocytes at different stages of differentiation (S1: stage 1 oocyte; ES2: early stage 2 oocyte; LS2: late stage 2 oocyte; S3: stage 3 oocyte) were collected and incubated overnight with 50μM BrdU, then fixed and stained with FITC-phalloidin (green pseudo-color), DAPI (blue pseudo-color) and anti-BrdU antibody (red pseudo-color). Note that in these oocytes, the follicular system is not fully achieved (as shown by phalloidin staining) and that no follicular cell was found BrdU positive. Note also that in areas devoid of follicular cells (A-C) blue DAPI-positive staining is indicative of the presence of numerous test cells. (H-I): confocal images of spheroid COS cells at two growing stages triple labeled with FITC-phalloïdin (green pseudo-color), TRITC-secondary antibodies directed against anti-BrdU antibodies (red pseudo-color) and DAPI (blue pseudo-color). BrdU positive cells appear in magenta pseudo-color and served as positive control for BrdU incorporation experiments. Bars (from A to I respectively) = 20, 30, 40, 30, 25, 40, 50, 40 and 60μm.
Fig 2.
Cells do not undergo apoptosis during follicular morphogenesis of Ciona intestinalis (A-F), Ascidiella aspersa (G), Phallusia mammillata H,I) and Styela clava (J-L).
(A,B,C,E,F): confocal images of oocytes, triple labeled with FITC-phalloïdin (green pseudo-color), TRITC-TUNEL (red pseudocolor) and DAPI (blue pseudo-color) after dislocation of Ciona intestinalis gonads. (D): actin staining with FITC-phalloïdin of another plane at the same stage of C in order to better visualize the accretion process. Note the elongated, fibroblast-like shape of follicular cells spread onto young oocytes (A). (G): Accretion process just before the terminal stage of Ascidiella aspersa oocyte maturation. (H, I): first stages of accretion process in Phallusia mammillata. (J-L): Accretion process in Styela clava from first stage to mature oocyte. S1: stage 1 oocyte; ES2: early stage 2 oocyte; S3: stage 3 oocyte; S4: stage 4 mature oocyte; arrowhead: germinal vesicle. Note that in areas devoid of follicular cells (E, K) blue DAPI-positive staining is indicative of the presence of test cells. Bar (from A to L respectively) = 5, 10, 15, 15, 20, 25, 25, 16, 16, 25, 25 and 22μm. Note that no apoptotic cell was detected in A-L, except the artifactual refringent bodies in F.
Fig 3.
Localization of EdU positive cells in the gonad of Ciona intestinalis.
A-B: double fluorescence of single confocal sections (bleu pseudocolor: DAPI staining; red pseudo-color: EdU labeling). C-D: double fluorescence, as in A-B, of stacked confocal sections (C) and (D) phase contrast image of the same field. E-F: mixed double fluorescence of single confocal sections, as in A-B, and phase contrast images of oocytes at various stages of maturation devoid of EdU labeling. Note that clustered EdU positive cells (magenta pseudo-color resulting from DAPI and EDU merged staining) were only detected at proximity of the gonad wall (C-D) but never in contact with oocytes whatever their stage of maturation. S1: stage 1 oocyte; ES2: early stage 2 oocyte; LS2: late stage 2 oocyte; S3: stage 3 oocyte; arrowhead: germinal vesicle. Bar = 50 μm.
Table 1.
List of parameters used to run computer simulations of epithelial morphogenesis.
Fig 4.
Topological organization of non-proliferative epithelial follicular cells in ascidians.
Cell membranes were labeled with FITC-WGA in fixed oocytes (left panels). The percentage of cell shapes (the shape of one cell being directly deduced from the number of its neighboring cells as indicated in the abscissa of histograms) was manually determined for each indicated species. All epithelia are exclusively organized with hexagons, pentagons and heptagons. n: number of experiments. All standard errors were lower than 0.09. Bars = 35μm.
Fig 5.
Topological organization is significantly modified in proliferative epithelia.
The polygonal cell frequency was determined, as in Fig 4, for flat (A) or spherical (B) COS cell monolayers and Drosophila melanogaster wing discs (C-E). Unless otherwise indicated, actin is color-coded red, nuclei blue. Arrows point to TUNEL-positive COS cells (green), and arrowheads to mitotic cells. Wing discs, wild type (C), expressed green-coded GFP-nubgal4 empty driver (D) or GFP-nubgal4-dIAP (E). In inserts (C-E), TUNEL-positive cells are color-coded red. n: number of experiments. All standard errors were lower than 0.07. Bars = 10μm (except C, D, E left panels: 100μm).
Fig 6.
Hyper-proliferation does not impact the topological features of proliferative epithelia.
A (wild type) and B (PH mutant) of Drosophila eye discs double labeled for actin (red pseudo-color) and nucleus (blue pseudo-color). The polygonal cell frequency (C) was determined, as in Fig 4, for wild type (dark grey) or PH mutant (light grey). n: number of experiments. All standard errors were lower than 0.08. Bars = 100 μm (left panel) and 10 μm (right panels).
Fig 7.
Simulation of the accretion scenario.
Four successive steps of epithelial morphogenesis are represented with cell membrane contours in green and cytoplasmic grains in red; the process begins with 15 cells contacting the support, followed by cell growth and the successive addition of cells, up to a limit of 60 (as in Ciona intestinalis), that eventually assemble as hexagons and pentagons and entirely cover the sphere surface.
Fig 8.
Simulations of epithelial morphogenesis in non-proliferative and proliferative context.
Left pictures represent numerical objects at 99% coverage of the sphere surface (color-coded as in Fig 7, with blue lines indicating the axis of division). Apoptotic cells presented a twisted membrane and a loss of internal grains. Respective right skeleton pictures illustrate the 3D perspective. A-B: accretion-driven morphogenesis at two different organization rates (A, low rate; B, high rate, see also Table 1). C-E: morphogenesis in a proliferative context (7% mitotic cells at any time) with different mitosis/apoptosis ratios: 1/3 (C); 1/5 (D); no apoptosis (E). The number of cells covering the surface of the sphere is indicated in each case with the standard deviation from n number of simulations. All standard errors for histograms were lower than 0.09.