Forty-eight hours following forced expression of a tagged-CyclinD2 version, cells expressing GFP (green) and the transgenic protein detected with an anti-Tag-V5 antibody (blue) are observed in the ventricular zone and differentiating field

Detection of the cells in S phase visualized following a 30 minute pulse of BrdU (red). Detection of the phospho-histone H3 (P-H3) on a cross-section of the spinal cord 48 h after overexpression of CyclinD2 (e,f, red) or D1 (g-i, blue). (h,i) Magnifications of the cell in anaphase shown in (g) (arrowhead). (a-f) Maximum projections of eight optical sections acquired at 5 μm Z steps; (g-i) single optical sections.<p><b>Copyright information:</b></p><p>Taken from "Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord"</p><p>http://www.neuraldevelopment.com/content/3/1/4</p><p>Neural Development 2008;3():4-4.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2265710.</p><p></p

Immunodetection of Sox2, Olig2, Pax6, and Pax7, showing that transgenic cells (green) co-express markers of the ventricular zone (yellow cells) only when located within that zone

For each marker, a total of at least 400 transgenic cells were analyzed from 5 independent 40 μm sections from 4 transgenic embryos. Each image represents the maximum projections of 8 optical sections acquired at 5 μm Z steps. (c,d,g,h,k,l,o,p) High magnifications of the zones framed by dashed lines on the adjacent sections.<p><b>Copyright information:</b></p><p>Taken from "Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord"</p><p>http://www.neuraldevelopment.com/content/3/1/4</p><p>Neural Development 2008;3():4-4.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2265710.</p><p></p

Schematic representation showing the age of the embryo at the time of electroporation and the different times after electroporation at which the embryos were fixed to evaluate the phenotype

For each experimental condition, 23 to 48 sections from 2 to 3 electroporated embryos were analyzed and the percentage of sections displaying ectopic P-H3 cells on the transgenic side determined. The corresponding values are reported for each experimental condition. Cross-sections of the spinal cord showing the phenotype 24 h after electroporation at E1.5 (b,c) and E2.5 (d), respectively. Tuj1 (b) marks the differentiating neurons. The arrows in (c,d) mark mitotic cells in ectopic positions. (b-d) Images obtained with an epifluorescent microscope.<p><b>Copyright information:</b></p><p>Taken from "Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord"</p><p>http://www.neuraldevelopment.com/content/3/1/4</p><p>Neural Development 2008;3():4-4.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2265710.</p><p></p

Transverse sections through the spinal cord at the brachial level showing the axonal tracts

eGFP+ axons are found in motor nerves, in the ventral commissure and in the sensory nerves, that is, corresponding to the tracts originating from the different locations where transgenic cells are detected. Immunodetection of BEN/SC1/DM-GRASP (red) reveals an enlarged ventral root on the electroporated side compared to the contralateral control side (underlined with dashed lines in (d)). (a,d) Images obtained with an epifluorescent microscope. Co-immunodetection of BEN/SC1/DM-GRASP (red) and P-H3 (blue) 72 h after electroporation with CyclinD1 reveals the presence of mitotic cells displaying an axon. (e-f) Single optical sections; (f) high magnification of the cell marked with an arrow in (e) – the GFP channel is off. Single optical sections showing eGFP and Islet1/2 (red) 72 h after CyclinD1 electroporation. The population of motor neurons is increased on the transgenic side. Single optical sections showing eGFP, Islet1/2 (red) and BrdU (blue). (j) A high magnification of (i). Seventy-two hours following forced expression of a tagged version of CyclinD2, cells expressing GFP (green) still express CyclinD2 visualized with an anti-Tag-V5 antibody (red). The TagV5 is clearly detected in the differentiating field. (k,l) Maximal projections.<p><b>Copyright information:</b></p><p>Taken from "Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord"</p><p>http://www.neuraldevelopment.com/content/3/1/4</p><p>Neural Development 2008;3():4-4.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2265710.</p><p></p

Co-immunodetection of Islet1/2 (a-i, green) or MNR2 (j,k, green) with P-H3 (red)

(a,b) Maximum projections; the electroporated side is on the left. (c) Single optical section showing a high magnification view at the level of the cell marked with an arrow in (a,b). (d-g) Orthogonal sections along the cell marked with an arrow in (c). (h-k) Single optical sections taken with a 63× objective. Dotted lines mark the limit of the mitotic transgenic cell. Co-immunodetection of BrdU (red) and Islet1/2 (green). All the pictures are single optical sections. (n-q) Orthogonal projections along the cell marked with an arrow in (m).<p><b>Copyright information:</b></p><p>Taken from "Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord"</p><p>http://www.neuraldevelopment.com/content/3/1/4</p><p>Neural Development 2008;3():4-4.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2265710.</p><p></p

Analysis of CyclinD1 (a) and D2 (b) expression on cross-sections of the neural tube of 3- to 3

5-day-old chicken neural tube. Co-detection of the transcripts CyclinD1 or D2 (dark blue) with the protein Nkx2.2 (brown). hybridization performed on serial sections showing CyclinD1 (e), Olig2 in combination with anti-BrdU immunostaining (f), CyclinD2 (g), and Irx3 (h). Note that both CyclinD1 and D2 are present in the dorsal aspect of the ventricular zone (a,b). A small dorsal domain expresses CyclinD1 at a lower level (a, asterisk). In the ventral part of the spinal cord, distinct domains (1, 2, 3) of ventral progenitors express distinct CyclinDs, the pMN domain expressing mainly CyclinD1. Transcripts encoding CyclinD1 seem more homogenously distributed along the apico-basal axis than those encoding CyclinD2, which are reinforced on the basal side (b).<p><b>Copyright information:</b></p><p>Taken from "Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord"</p><p>http://www.neuraldevelopment.com/content/3/1/4</p><p>Neural Development 2008;3():4-4.</p><p>Published online 13 Feb 2008</p><p>PMCID:PMC2265710.</p><p></p

Mechanical Stress Impairs Mitosis Progression in Multi-Cellular Tumor Spheroids

<div><p>Growing solid tumors are subjected to mechanical stress that influences their growth rate and development. However, little is known about its effects on tumor cell biology. To explore this issue, we investigated the impact of mechanical confinement on cell proliferation in MultiCellular Tumor Spheroids (MCTS), a 3D culture model that recapitulates the microenvironment, proliferative gradient, and cell-cell interactions of a tumor. Dedicated polydimethylsiloxane (PDMS) microdevices were designed to spatially restrict MCTS growth. In this confined environment, spheroids are likely to experience mechanical stress as indicated by their modified cell morphology and density and by their relaxation upon removal from the microdevice. We show that the proliferation gradient within mechanically confined spheroids is different in comparison to MCTS grown in suspension. Furthermore, we demonstrate that a population of cells within the body of mechanically confined MCTS is arrested at mitosis. Cell morphology analysis reveals that this mitotic arrest is not caused by impaired cell rounding, but rather that confinement negatively affects bipolar spindle assembly. All together these results suggest that mechanical stress induced by progressive confinement of growing spheroids could impair mitotic progression. This study paves the way to future research to better understand the tumor cell response to mechanical cues similar to those encountered during in vivo tumor development.</p></div

Mechanically confined growth impairs the regionalization of mitotic cells in MCTS.

<p>(A) Transmitted light images of a control MCTS and of a MCTS grown in a PDMS microdevice for 6 days. (B) Upper panels: Detection by immunofluorescence of mitotic cells (anti-phosphorylated Histone H3 antibody, pH3; in green) in cryosections of a control MCTS and a mechanically confined MCTS. The orientation of mechanically confined MCTS cryosections is parallel to the bottom of the channel (middle, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080447#pone-0080447-g001" target="_blank">Fig 1C</a>) and perpendicular to the bottom of the channel (right, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080447#pone-0080447-g001" target="_blank">Fig 1C</a>). Nuclei are stained with DAPI (blue). Lower panels: mean fluorescence intensity of pH3 staining in 8 cryosections from 6 control MCTS (3 independent experiments), 11 parallel cryosections from 11 mechanically confined MCTS (4 independent experiments) and 8 perpendicular cryosections from 6 mechanically confined MCTS (3 independent experiments). Dashed lines represent the walls of the PDMS channel. White lines indicate the width of the area where mitotic cells are localized (scale bar, 100 µm). (C) Percentages of mitotic cells (pH3-positive cells) in the peripheral (P) and the central (C) areas of control MCTS (n = 14 areas analyzed, from 7 MCTS from 3 experiments) and in the peripheral (P) and central (C) areas and the tips (Tips) of confined MCTS (n = 29 areas analyzed, from 12 MCTS from 6 experiments). The bars correspond to the mean ± SEM.</p

Growth-associated external mechanical stress leads to accumulation of cells arrested in mitosis.

<p>(A) Upper panel: Immunodetection of EdU incorporation (green) and mitotic cells (pH3-positive, red) in a cryosection from MCTS grown in PDMS microdevices for 6 days. Nuclei are stained using DAPI (blue). Lower panel: High-contrast image of the immunodetection of EdU incorporation (white) (scale bar, 100 µm). (B) Analysis of EdU incorporation in mitotic cells. Images correspond to magnifications of the regions indicated by the white squares in A from the tip (top panels) and the body (bottom panels) of a mechanically confined MCTS. The white arrow indicates a pH3- positive cell that is not EdU-positive. This cell is next to a pH3-positive/EdU-positive cell. (C) Percentage of pH3-positive/EdU-negative cells in the body (589 mitotic cells from 48 cryosections) and in the tips (331 mitotic cells from 95 cryosections) of mechanically confined MCTS (20 MCTS from 4 independent experiments) and in control (CTL) MCTS (358 mitotic cells from 34 cryosections from 15 MCTS from 4 independent experiment). Bars correspond to the mean ± SEM. (D) Map showing the localization of pH3-positive/EdU-negative cells in 8 cryosections from 8 mechanically confined MCTS from 4 independent experiments. The white line represents the outline of the MCTS and the red dots the localization of the pH3-positive/EdU-negative cells. The dashed lines indicate the microdevice PDMS walls.</p

Mechanical confinement does not impair mitotic cell rounding within MCTS.

<p>(A) Cryosections of a control MCTS and a mechanically confined MTCS (6 days in the PDMS device), stained for DNA (blue) and E-Cadherin (green) (scale bar, 10 µm). The cell outlines are drawn manually (green dashed line) to extract the area and the circularity of cells. (B) Area values of interphase cells (blue) and mitotic cells (orange) in control MCTS and in the body and tips of mechanically confined MCTS (6 days in the PDMS microdevice). Lines correspond to the mean ± SD. (C) Circularity values of interphase cells (blue) and mitotic cells (orange) in control MTCS and in the body and tips of mechanically confined MCTS (6 days in the PDMS microdevice). The error bars represent the mean ± SD. For control MCTS, 167 interphase cells and 175 mitotic cells were analyzed from 8 MCTS from 2 experiments. For confined MCTS, 307 interphase cells and 125 mitotic cells were analyzed in the body, and 146 interphase cells and 91 mitotic cells were analyzed in the tip, both from 10 MCTS from 4 experiments. (D) Cryosections of a mechanically confined MCTS stained for DNA (blue), E-Cadherin (grey), EdU (green) and pH3 (red) (scale bar, 10 µm). (E) Area (left panel) and circularity (right panel) values of pH3-positive mitotic cells in the body of mechanically confined MCTS relative to EdU incorporation (EdU+/pH3+: 79 cells analyzed, EdU−/pH3+: 43 cells analyzed, from 3 MCTS from 2 independent experiments). The lines correspond to the mean ± SD.</p