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First published online 21 November 2007
doi: 10.1242/dev.009951

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Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex

¹ GSF-National Research Institute for Environment and Health, Institute for Stem Cell Research, Ingolstädter Landstr. 1, 85764 Neuherberg/Munich, Germany.
² Physiological Genomics, University of Munich, Schillerstr. 46, 80639 Munich, Germany.

View larger version (76K): [in this window] [in a new window]   Fig. 1. Par-complex proteins are reduced at the ventricular surface during cortical neurogenesis. (A-R) Coronal section through E12 (A-C,G-I,M-O) and E16 (D-F,J-L,P-R) mouse cerebral cortices immunolabeled for Par3 (A-F, red), Par6 (G-L, red) or aPKC (M-R, red), pan-cadherin (green) and stained with DAPI (blue). Note the high immunoreactivity for Par3, Par6 and aPKC in the apical domain of the VZ at E12 and its reduction at later developmental stages. Scale bar: 25 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 2. Decreased proliferation of cortical progenitors after Par3 knockdown. (A-D) Fluorescence micrographs depicting a two-cell clone generated from an E12 mouse cortical progenitor after Par3 shRNA transduction, immunolabeled for GFP (green), MAP2 (red) and Tbr1 (blue). Note that both GFP-positive cells express the neuronal markers MAP2 or Tbr1 (arrows). (E) Bar chart representing the frequency of control and Par3b shRNA-transduced cell clones according to the size (number of cells per clone, x-axis). Asterisks indicate statistically significant differences between control (n=80 clones) and Par3 shRNAs (Par3a, n=40; Par3b, n=87; Par3c, n=57 clones) for each clone size (*, P<0.05; **, P<0.01; Tukey's multiple comparison test). Note the lack of clones larger than 10 cells after Par3a shRNA-transduction. (F,G) Quantification of the mean number of GFP-positive cells per clone (F) and the proportion of neurons (G) generated after control or Par3 shRNA transduction (*, P<0.05; **, P<0.01; ***, P<0.001; Tukey's multiple comparison test). Scale bar: 25 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 3. Par3 loss-of-function increases the number of deep-layer neurons in vivo. (A-E) Fluorescence micrographs of E18 cortices injected with control (A,B) and Par3b shRNA (C,D) (6 days after injection) immunolabeled for GFP (green) and MAP2 (red) and stained with DAPI (blue). Note the GFP-positive neurons in both upper and deep cortical layers of a control-injected mouse (A,B). By contrast, a single GFP-positive neuron is found in the deep layers of a Par3b shRNA-injected mouse (C,D). (E) Representative section of E18 cortex indicating the different layers analyzed (VZ, SVZ, IZ and CP). (F,G) Histograms representing the proportion of GFP-positive cells in the cortex layers depicted in E (F) and GFP-positive neurons in the upper and deep cortical plate (G) of both control (n=3, 554 cells) and Par3b shRNA (n=3, 318 cells) -injected brains. Note the significant increase in neurons in the deep layers after Par3b shRNA transduction in comparison to the control (*, P<0.05; unpaired t-test). (H,I) Par3b shRNA-transduced neuron (arrow) colabeled for GFP (green), MAP2 (blue) and Tbr1 (red). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars: 40 µm in A,B; 50 µm in C,D; 100 µm in H; 25 µm in I.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Increased clone size upon overexpression of mouse Par3 in vitro. (A-D) GFP-labeled clone in E12 cortex dissociated-cell cultures after Par3 transduction and 7-days culture. Immunolabeled for GFP (A and D, green), nestin (C and D, blue) and MAP2 (B and D, red). Note the high number of nestin-positive MAP2-negative cells. (E-G) Bar charts representing the size (E,F) and neuronal content (G) of clones generated from E12 cortical cells after transduction with control or Par3-containing retroviral vectors. Note the significant increase in the average clone size (F; ***, P<0.001; unpaired t-test), as well as in the frequency of clones containing 20 or more cells (E; *, P<0.05; unpaired t-test) after Par3 transduction (n=132) in comparison to the control (n=170). The proportion of neurons amongst GFP+ transduced cells was also significantly reduced after Par3 gain-of-function (G; **, P<0.01; unpaired t-test). Scale bar: 50 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Increased clone size upon overexpression of mouse Par6 in vitro. (A-F) Bar charts representing the composition of clones generated from E12 (A-C) or E14 (D-F) cortical cells transduced with either control or Par6-containing plasmids after 7 days in vitro (7div). Par6 overexpression increased the mean number of cells per clone (A and D; *, P<0.05; unpaired t-test) as well as the frequency of large clones (C and F; *, P<0.05; unpaired t-test) in both E12 (control, n=150; Par6, n=150) and E14 (control, n=80; Par6, n=96) cell cultures, with a stronger effect in the E14 cells (compare A-C and D-F). The proportion of neurons within clones was also significantly reduced in both sets of experiments (B and D; *, P<0.05; unpaired t-test).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Lineage analysis by time-lapse video microscopy of control and Par6-transduced progenitors. (A-G) Examples of lineage trees from control (A-D) and Par6-transduced (E-G) cells obtained from time-lapse images taken every 2 minutes over a period of 5 days. Horizontal grid lines indicate every 12 hours of the experiment. Colored stars indicate cell divisions generating two progenitors (green), two postmitotic cells (black) or one progenitor and one postmitotic cell (red). Most cells leaving the cell cycle more than 36 hours before the end of the movies were identified in subsequent immunolabelings as MAP2-positive neurons (N) (see also Fig. S2 in the supplementary material). Horizontal lines indicate the time point of cell division. The length of the horizontal line is arbitrary and has no significance. The lengths of the vertical lines depicts the time elapsed between two cell divisions, i.e. the cell cycle length. (H) Quantification of symmetric progenitor, symmetric terminal and asymmetric cell divisions in the first, second, third and fourth cell divisions. Note the higher frequency of symmetric progenitor cell divisions in the lineages derived from Par6-transduced cells (n=56) in comparison to controls (n=68).

View larger version (57K): [in this window] [in a new window]   Fig. 7. Par6 overexpression increases the number of Pax6+ progenitors. (A-I) Examples of two-cell clones (pairs) obtained from E12 mouse cortical cells transduced with control (A-F) or Par6 (G-I) cDNA and immunolabeled for GFP (green) and Pax6 (red) after 2 days in vitro (2div). White arrows point to Pax6/GFP double-positive cells and black arrows to GFP-positive Pax6 negative cells. (J) Bar chart depicting the frequency of pairs (control, n=84; Par6, n=59) containing two Pax6-positive cells (black bar), only one Pax6-positive cell (gray), or only Pax6-negative cells (white). (K) Bar chart representing the same quantification as in J but for Tbr2 (control, n=61; Par6, n=54). Note the increased proportion of pairs containing two Pax6-positive cells after Par6 overexpression. By contrast, the frequency of pairs containing two Tbr2-positive cells was significantly reduced after Par6 transduction. Scale bar: 25 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 8. Par6 overexpression in vivo increases the number of Pax6+ VZ progenitors. (A,B) Coronal sections of E16 cerebral cortices injected with either control (A) or Par6-containing (B) retroviruses at E13, immunolabeled for GFP (green) and stained with DAPI (blue). (C) Quantification of GFP-positive cells in the dorsal telencephalon of control (n=4, 1642 cells) and Par6 (n=3, 3568 cells) -injected brains. Note the increased proportion of GFP-positive cells in the VZ of Par6-injected animals (***, P<0.001; unpaired t-test), whereas the proportion of GFP-positive cells in the cortical plate and intermediate zone was reduced (*, P<0.05; unpaired t-test). (D-G) Higher-power micrographs of coronal sections of E16 cortices injected with Par6-containing retroviruses and double labeled as indicated. Insets are higher magnifications of the boxed areas. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; LV, lateral ventricle; GE, ganglionic eminences. Scale bars: 100 µm in A,B; 50 µm in D-F; 75 µm in G.