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First published online November 7, 2008
doi: 10.1242/10.1242/dev.024570

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Selection of differentiating cells by different levels of delta-like 1 among neural precursor cells in the developing mouse telencephalon

¹ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
² Department of Immunology, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.

View larger version (115K): [in this window] [in a new window]   Fig. 1. Expression of Dll1 in the developing mouse telencephalon. (A,B,E-T) Brain sections from ICR mouse embryos at E13.5 (A,E-T) or E16.5 (B) were immunostained with anti-Dll1 alone (A,B) or together with anti-Pcna (E-H), anti-nestin (I-L), anti-Sox2 (M-P) or anti-β III-tubulin (TuJ1) (Q-T). (C,D) Dll1 mRNA in sections of E13.5 ICR mouse brain detected by in situ hybridization. Higher magnifications of the boxed region in C,G,K,O and S are shown in D,H,L,P and T, respectively. NCX, neocortex. Scale bars: 200 µm in A-C; 20 µm in D-T.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Dll1 expression and Notch1 activation are segregated into distinct cells in the VZ of the developing mouse telencephalon. (A-H) Brain sections from ICR mouse embryos at E13.5 were immunostained with anti-active Notch1 (actN1) and anti-Dll1 and TO-PRO-3 (nuclear staining). Higher magnifications of the boxed regions in C and G are shown in D and H, respectively. Arrowheads in D,H indicate Dll1-positive actN1-negative cells in the VZ. (I) The proportions of active Notch1-positive cells among Dll1-negative or Dll1-positive cells in the VZ of the neocortex were determined by immunohistochemical analysis. Data are the mean ± s.e.m. of values from three sections of each brain, and similar results were obtained from six independent brains. *P<0.0005. NCX, neocortex; GE, ganglionic eminence. Scale bars: 20 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Dll1 overexpression in a small proportion of NPCs induces neuronal differentiation in vitro. Primary NPCs were prepared from (A-D) 3-day sphere cultures [3 days in vitro (3 DIV)] or (E,F) 12-day sphere cultures (fourth passage, 12 DIV) of the neocortical cells of E12.5 ICR mice. The NPCs were infected with a retrovirus encoding GFP (pMX-GFP, control), or both GFP and Dll1 (pMX-Dll1-IG), at a low titer and subjected to clonal analysis (see Materials and methods). (A) A representative GFP-positive clone stained with anti-GFP, TuJ1 and Hoechst. After incubation for 2 days in the presence of a low dose of human FGF2 (2 ng/ml) (B-D) or in the absence of FGF2 (E,F), cells were stained with anti-GFP and TuJ1 or anti-GFP and anti-Gfap. The percentage of clones containing only TuJ1-positive (B,E) or only Gfap-positive (D,F) cells among GFP-positive clones was then determined by immunocytochemical analysis. The percentage of TuJ1-positive cells among GFP-positive cells was also determined by immunocytochemistry (C). *P<0.02, **P<0.01, ***P<0.0001. Scale bar: 50 µm in A.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Dll1 overexpression induces neuronal differentiation in a non-cell-autonomous manner. (A) Primary NPCs were prepared from 3-day sphere cultures (3 DIV) of the neocortical cells of E12.5 ICR mice, infected with a retrovirus encoding GFP (pMX-GFP, control), or both GFP and Dll1 (pMX-Dll1-IG), at a high titer and analyzed as in Fig. 3C. (B) Primary NPCs were prepared from 3-day sphere cultures (3 DIV) of the neocortical cells of E12.5 ICR mice and plated at different cell densities (0.26x, 0.52x and 1.04x105 cells/cm2). These cells were infected with a retrovirus encoding GFP (pMX-GFP, control), or both GFP and Dll1 (pMX-Dll1-IG), at a low titer and analyzed as in Fig. 3B (clonal analysis). *P<0.05. (C) Primary NPCs were prepared from 3-day sphere cultures (3 DIV) of the neocortical cells of E12.5 ICR mice and infected with a retrovirus encoding GFP (pMX-GFP, control), or both GFP and the delta-like 1 intracellular domain (pMX-DICD-IG), at a low titer and analyzed as in Fig. 3B (clonal analysis).

View larger version (51K): [in this window] [in a new window]   Fig. 5. Dll1 overexpression in a small number of NPCs in the developing mouse neocortex induces neuronal differentiation. Retroviruses encoding GFP alone (pMX-GFP, control), or both GFP and Dll1 (pMX-Dll1-IG), were injected into the lateral ventricle of ICR mice at E12.5. Under these conditions, only a small proportion of cortical NPCs were infected. Brains were excised 2 days later and examined. (A,B) The location of the GFP-positive cells was determined by immunohistochemistry with anti-GFP and TO-PRO-3 (nuclear staining). Typical results are shown. (C) Quantification of the distribution of GFP-positive cells in the neocortex. Data are the mean±s.e.m. of values from eight sections of each brain compared between littermates. Similar results were obtained from seven independent littermates. (D) Brain sections were stained with anti-GFP and TuJ1, and the percentage of TuJ1-positive cells among GFP-positive cells in the whole neocortex was determined. Data are the mean±s.e.m. of values from eight sections of each brain compared between littermates. Similar results were obtained from three independent brains. *P<0.01, **P<0.0001. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars: 200 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 6. Dll1 deletion in a large number of NPCs in the developing mouse telencephalon induces premature neurogenesis. The Dll1 gene was ablated in the majority of NPCs by crossing Dll1 floxed with nestin-Cre mice. (A-Z) Immunoreactivity was compared between Dll1-intact mice (control, these mice were littermates of Dll1 conditional KO mice of genotype Dll1flox/flox or Dll1flox/wt without Cre) (A,C,E,E',G,I,K,M,O,Q,S,U,W,Y) and Dll1 conditional KO (cKO) mice (genotype was Dll1flox/flox with nestin-Cre) (B,D,F,F',H,J,L,N,P,R,T,V,X,Z). Brain sections from each mouse at E11.5 (A-H,E',F',K,L,O,P,S,T,W,X) or E13.5 (I,J,M,N,Q,R,U,V,Y,Z) were immunostained with anti-Dll1 (A-D), anti-active Notch1 (actN1) (E-F'), anti-β III-tubulin (TuJ1) (G-J), anti-Sox2 (K-N), anti-BrdU (O-R), anti-phosphorylated histone H3 (pH3) (S-V) or anti-Tbr2 (W-Z). TO-PRO-3 was used for nuclear staining (E',F',O-Z). Dorsal side is up in all panels. NCX, neocortex; GE, ganglionic eminence. Scale bars: 100 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 7. Dll1 deletion in a small number of NPCs in the developing mouse neocortex suppresses neuronal differentiation. Retroviruses encoding GFP alone (pMX-SV40-GFP, control), or both GFP and Cre recombinase (pMX-Cre-SV40-GFP), were injected into the lateral ventricle of Dll1flox/flox mice at E12.5. Under these conditions, only a small proportion of cortical NPCs were infected. Brains were excised 3 days later and examined. (A,B) The location of the GFP-positive cells was determined by immunohistochemistry with anti-GFP and TO-PRO-3 (nuclear staining). Typical results are shown. (C) Quantification of the distribution of GFP-positive cells in the neocortex. Data are the mean±s.e.m. of values from eight sections of each brain compared between littermates. Similar results were obtained from five independent littermates. (D) Brain sections were stained with anti-GFP and anti-Pax6, and the percentage of Pax6-positive cells among GFP-positive cells in the whole neocortex was determined. Data are the mean±s.e.m. of values from six sections of each brain compared between littermates. Similar results were obtained from three independent brains. *P<0.005, **P<0.0005. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars: 200 µm.

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