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First published online March 4, 2005
doi: 10.1242/10.1242/dev.01693

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Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex

Ken-ichi Mizutani and Tetsuichiro Saito^*,

Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

View larger version (12K): [in a new window]   Fig. 1. Structure of a double-promoter vector, pCAG-ExNotchZ. The caNotch gene, which encodes the caNotch protein N-terminally tagged with the FLAG-peptide, is excised out at its flanking loxP sites by Cre recombinase to allow translation of ß-gal.

View larger version (83K): [in a new window]   Fig. 2. Electroporation of pCAG-ExZ, carrying EYFP and nlacZ, at two developmental stages. Coronal sections of the primary somatosensory area of P5 brains after electroporation at E13.5 (A-D,I-N), and E15.5 (E-H,O-Q) are shown. Green and blue signals show EYFP fluorescence (A,E), and immunostaining with an anti-ß-gal antibody (B,D,F,H,I,K,L,N,O,Q), respectively. Red signals show immunostaining with antibodies recognizing ER81 (C,D,J,K) and Lhx2 (G,H,M,N,P,Q). ß-Gal+ neurons of layer V, and of layers II and III, were positive for ER81 and Lhx2, respectively (arrowheads). ER81+ neurons expressed ER81 relatively uniformly at high levels, whereas 79.7±2.3% of ß-gal+/Lhx2+ neurons expressed Lhx2 at high levels. The ratio closely resembled that of strongly Lhx2-positive cells in the total population of Lhx2+ cells of layers II and II (78.6±2.7%). A-D, E-H, I-K, L-N, and O-Q each show the same section; D,H,K,N and Q are merged views. WM, white matter; LV, lateral ventricle. Scale bars: 200 µm for A-H; 20 µm for I-Q.

View larger version (58K): [in a new window]   Fig. 3. Double electroporation of DsRed and EYFP at different stages. P5 brains after single electroporation of DsRed (red) at E13.5 (A) or EYFP (green) at E15.5 (B), and after double electroporation of DsRed at E13.5 and EYFP at E15.5 (C,D) are shown. (D) Magnified view of upper-layer neurons, showing co-expression of DsRed and EYFP. Scale bars: 200 µm for A-C; 10 µm for D.

View larger version (86K): [in a new window]   Fig. 4. Effects of caNotch on cortical progenitors. pCAG-EYFP (A,B) or pCAG-ExNotchZ (C,D,F-J,L,M) was transfected at E13.5. (A-D,F,G) Embryonic brains were labeled at E14.5 for 2 hours (A-D) or 15 hours (F,G) with BrdU. Green and red signals show immunostaining with anti-GFP and anti-BrdU antibodies, respectively. (E) The ratio of BrdU+ cells in the total population of EYFP+ cells or EYFP+/CaNotch+ cells of 2-hour-labeled brains. Each ratio was calculated from three brains. (H-J) An E15.5 brain, 2 days after transfection. Green and red signals show EYFP fluorescence and immunostaining with the RC2 antibody, respectively. The ratio of RC2+ cells in the total population of EYFP+ cells was calculated from three brains. (K-M) TUNEL assay performed on the E15.5 brain. The areas of the transfected hemisphere (L,M) and the opposite non-transfected hemisphere (K) of an electroporated brain are shown. Green signals show EYFP fluorescence. Transfected green cells were not detected in the non-transfected hemisphere (K). A,B; C,D; F,G; H-J; and K-M each show the same section. B,D,G,J,K and M are merged views. CP, cortical plate; LV, lateral ventricle; VZ, ventricular zone. Scale bars: 50 µm for A-D,F,G; 20 µm for H-J; 100 µm for K-M.

View larger version (99K): [in a new window]   Fig. 5. Neurogenesis resumes after the removal of caNotch. P5 brains after single electroporation of pCAG-ExNotchZ at E13.5 (A,E-G), and after double electroporation of pCAG-ExNotchZ at E13.5 and pXCANCre at E15.5 (B-D,H-K). (E-G) Magnified view of an EYFP+ region close to the lateral ventricle (LV). (H-K) Magnified views of layers II and III. Green and blue signals show EYFP fluorescence (A,B,E,G,H), and immunostaining with the anti-ß-gal antibody (C,D,I,K), respectively. Red signals show immunostaining with antibodies recognizing FLAG (F-H) and Lhx2 (D,J,K). B-D, E-G, and I-K each show the same section; D,G,H and K are merged views. Note, EYFP+ neurons only signal green, no yellow colouration was observed even after the merge of two colors (H) because of a lack of FLAG signals. All ß-gal+ neurons of layers II and III were positive for Lhx2. 80.5±2.0% of ß-gal+/Lhx2+ neurons expressed Lhx2 at high levels. The ratio was almost identical to that of strongly Lhx2-positive cells in the total Lhx2+ population of layers II and III (79.6±2.45%). A similar distribution of cells was also confirmed in five brains by using EYFP fluorescence and X-gal staining (data not shown). Scale bars: 200 µm for A-D; 20 µm for E-H,I-K.

View larger version (23K): [in a new window]   Fig. 6. Apoptosis is not enhanced by caNotch and Cre. We performed TUNEL assays for the E17.5 brain after double electroporation of pCAG-ExNotchZ at E13.5 and pXCANCre at E15.5. The areas of the double-transfected hemisphere (B,C) and the opposite non-transfected hemisphere (A) of an electroporated brain are shown. Green signals show EYFP fluorescence. All three panels show the same section; A and C are merged views. Similarly, no increase of apoptosis was observed at E16.5 (data not shown). Scale bar: 200 µm.

View larger version (61K): [in a new window]   Fig. 7. Neurons of layers II and III are not born from caNotch+ cells before Cre transfection. P5 cortices after electroporation of pCAG-EYFP (A-D) or pCAG-ExNotchZ (E-H) at E13.5, BrdU labeling at E14.5 and electroporation of pXCANCre at E15.5, are shown. Red and green signals show immunostaining with the anti-BrdU and anti-GFP antibodies, respectively. Arrowheads and arrows indicate neurons that were heavily and lightly labeled with BrdU, respectively. C and G are merged views of the sections shown in A and B, and E and F, respectively. (D,H) Ratios of neurons that were heavily (h) and lightly (l) labeled with BrdU in the EYFP+ population. Each ratio was calculated from five brains. After EYFP transfection, layers II and III contained 17.4±1.6% heavily- and 49.8±2.9% lightly-labeled neurons, whereas layer IV contained 68.8±6.6% heavily- and 21.2±5.8% lightly-labeled neurons. EYFP/caNotch transfection showed 0.7±1.1% heavily and 14.0±1.3% lightly-labeled neurons in layers II and III. After EYFP/caNotch transfection, many EYFP+ cells were not labeled with BrdU in the upper layers (85.8±1.8%), presumably because the amount of BrdU in nuclei was reduced to below the detection limit after multiple cell divisions. Scale bars: 200 µm for A,E; 20 µm for B,C,F,G.

View larger version (51K): [in a new window]   Fig. 8. EYFP+/ß-gal+ neurons of layers II and III are born after Cre transfection. P5 cortices after double electroporation of pCAG-EYFP (A-D) or pCAG-ExNotchZ at E13.5 (E-H), and of pXCANCre at E15.5, and after BrdU labeling at E16.5, are shown. Red and green signals show immunostaining with the anti-BrdU and anti-GFP antibodies, respectively. Arrowheads indicate neurons that were heavily labeled with BrdU. C and G are merged views of the sections shown in A and B, and E and F, respectively. (D,H) Ratios of neurons that were heavily (h) and lightly (l) labeled with BrdU in the EYFP+ population. Ratios were calculated from 5 brains. After EYFP transfection, layers II and III contained 46.1±3.7% heavily- and 12.0±3.4% lightly-labeled neurons, whereas layer IV contained 2.9±0.1% heavily- and 1.3±1.1% lightly-labeled neurons. EYFP/caNotch transfection showed 48.1±3.5% heavily- and 8.2±2.3% lightly-labeled neurons in layers II and III. Scale bars: 200 µm for A,E; 20 µm for B,C,F,G.

View larger version (35K): [in a new window]   Fig. 9. caNotch+ progenitors lose the ability to generate low-layer neurons. Shown are P5 cortices after transplantation at E13.5 of progenitors that had been prepared after electroporation of pCAG-ExZ at E13.5 (A,E), after double electroporation of pCAG-ExZ at E13.5 and pXCANCre at E15.5 (B,F), or after double electroporation of pCAG-ExNotchZ at E13.5 and pXCANCre at E15.5 (C,D,G). Blue and red signals show immunostaining with antibodies recognizing ß-gal (A-D) and Lhx2 (D), respectively. C and D show the same section. D is a merged view. All ß-gal+ neurons were also positive for Lhx2. E15.5 progenitors single transfected with pCAG-ExZ at E13.5 showed the same results as did E15.5 progenitors double transfected with pCAG-ExZ at E13.5 and pXCANCre at E15.5 (data not shown), confirming that Cre did not affect the laminar fate of neurons. (E-G) Ratios of ß-gal+ neurons that were distributed to each layer of the cortex. Each ratio was calculated from 10 brains. Results were as follows. In E: layers II and III, 23.8±9.1%; layer IV, 32.7±5.1%; layer V, 40.1±8.7%; layer VI, 0.3±0.5%; and white matter (WM), 3.1±2.3%. In F: layers II and III, 97.1±3.5%; layer IV, 1.0±1.2%; layer V, 0.9±0.7%; and WM, 1.1±3.1%. In G: layers II and III, 89.7±7.4%; Layer IV, 4.3±3.5%; layer V, 2.0±4.2%; layer V1, 1.8±0.5%; and WM, 0.3±1.1%. Scale bar: 150 µm for A-D.