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First published online 25 May 2006
doi: 10.1242/dev.02419

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Non-cell-autonomous action of STAT3 in maintenance of neural precursor cells in the mouse neocortex

¹ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
² Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
³ Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

View larger version (20K): [in a new window]   Fig. 1. Inhibition of FGF2-dependent NPC maintenance by an inhibitor of JAK2. (A) Neuroepithelial cells prepared from E12.5 mouse neocortex were dissociated and cultured in suspension for 7 days with FGF2 (20 ng/ml) in the absence or presence of either the MEK inhibitor U0126 (10 µmol/l), the PI3K inhibitor LY294002 (20 µmol/l) or the JAK2 inhibitor AG490 (10 µmol/l). The number of generated neurospheres was then counted. *P<0.01. (B) Neuroepithelial cells prepared from E12.5 mouse neocortex were dissociated and cultured in suspension with FGF2 (20 ng/ml) for 2 days and in the additional absence or presence of U0126, LY294002 or AG490 for 1 day. The resulting primary neurospheres were dissociated and cultured in suspension for 7 days with FGF2 (20 ng/ml) and EGF (20 ng/ml) in the absence of inhibitors. The number of secondary neurospheres was then counted. *P<0.01.

View larger version (119K): [in a new window]   Fig. 2. Expression of STAT3 in the embryonic mouse brain. Brain sections from embryos at E13.5 (A-C,G,H,K-M,Q) or E16.5 (D-F,I,J,N-P,R) were immunostained with anti-STAT3 together with anti-nestin or anti-ßIII-tubulin (TuJ1), as indicated. STAT3 was expressed in nestin-positive cells (A-F,H,J), but not in cells positive for the neuronal marker TuJ1 (G,I). K and N show the phase-contrast micrographs of the sections. STAT3 was expressed in cells in the VZ and apical side of the IMZ at E13.5 (K-M,Q) and in the VZ, SVZ and the apical side of the IMZ at E16.5 (N-P,R). Q and R show magnification of cells expressing STAT3 (arrow) in the VZ and IMZ in L and in the VZ, SVZ and IMZ in O, respectively. In-situ hybridization analysis of the E13.5 mouse brain revealed that STAT3 mRNA was present in the neuroepithelium of the telencephalic plate (S). Dorsal side is up in A-J,S and left in K-R. Scale bars: 400 µm in A,S; 40 µm in G,K,N,Q,R.

View larger version (24K): [in a new window]   Fig. 3. Requirement of STAT3 for maintenance of FGF2-sensitive NPCs. (A) NPC cultures prepared from E12.5 mouse neocortex were incubated in the absence of FGF2 for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to STAT3, to the Tyr705-phosphorylated form of STAT3 (Anti-phospho STAT3) and to GAPDH (loading control). Reduction of STAT3 phosphorylation by FGF2 deprivation suggests FGF2-dependent phosphorylation of STAT3, although it may also reflect the reduction of sphere-forming NPCs by FGF2 deprivation. (B) Neuroepithelial cells prepared from the neocortex of E12.5 STAT3flox/flox mice were infected with a retrovirus encoding Cre recombinase (Cre) or with a control retrovirus (Control). After culture in the presence of FGF2 (20 ng/ml) for the indicated times, the cells were lysed and subjected to immunoblot analysis. (C) Neuroepithelial cells from STAT3flox/flox mice were infected and then assayed for the formation of primary neurospheres in the presence of FGF2 (20 ng/ml). *P<0.01. (D) Neuroepithelial cells prepared from the neocortex of E12.5 wild-type mice were infected with retroviruses encoding both STAT3-C and GFP (STAT3-C) or GFP alone (Control). They were subsequently assayed for the formation of secondary neurospheres. *P<0.01. (E-G,M) Neuroepithelial cells prepared from E12.5 wild-type mouse neocortex were infected and plated in the presence of FGF2 (20 ng/ml) for 2 days (E,F) or in the absence of FGF2 for 3 (G) or 4 (M) days. Then the cells were stained with Hoechst to identify apoptotic cells by counting cells exhibiting condensed and fragmented nuclei (F), or stained with anti-GFAP (G) or anti-ßIII-tubulin (TuJ1) antibody together with anti-GFP (M), or incorporated BrdU for 2 hours and stained with anti-BrdU and anti-GFP (E). *P<0.02. (H-L) Neuroepithelial cells prepared from E12.5 STAT3flox/flox mouse neocortex were infected and plated in the presence of FGF2 (20 ng/ml) for 2 days (H,I) or a lower concentration of FGF2 (2 ng/ml) for 3 (J) or 4 (K,L) days. Then the cells were analyzed as in E-G,M. Scale bar: 80 µm (K). *P<0.01.

View larger version (143K): [in a new window]   Fig. 4. (A) Requirement of STAT3 for inhibition of neurogenesis in vivo. Vectors encoding GFP alone (Control) or Cre recombinase plus GFP (Cre) were injected into the lateral ventricle of STAT3flox/flox mice at E14.5. After 2 days, the fate of the GFP-positive cells was examined by immunohistochemistry with anti-GFP, anti-ßIII-tubulin (TuJ1), anti-SOX2, anti-nestin, anti-Musashi and anti-MAP2, as indicated. The percentages of TuJ1-positive cells and SOX2-positive cells among total cells in the transfected VZ region (60 µm width from the ventricular surface) are shown. The boxed regions in A are shown enlarged in B. Arrows in B indicate TuJ1-positive cells and MAP2-positive cells co-expressed without GFP. Scale bars: 20 µm in A; 40 µm in B.

View larger version (16K): [in a new window]   Fig. 5. Inhibition of neurogenesis by STAT3 in a non-cell-autonomous manner. (A,B) NPC cultures were prepared from the neocortex of E12.5 STAT3flox/flox mice and infected with a retrovirus encoding GFP (pMX-GFP). Neuroepithelial cell cultures were prepared from STAT3flox/flox embryos and infected with retroviruses encoding CD8 alone (Control) or both CD8 and Cre recombinase (Cre). The infected NPCs (1x105 cells) were co-cultured for 4 days in the presence of low dose FGF2 (2 ng/ml) with the infected neuroepithelial cells (1x103 cells) and stained with anti-GFP and anti-CD8 (A), or anti-GFP and TuJ1 antibody (B). (A) GFP and TuJ1 fluorescence merged image are shown for typical field of co-culture experiments. Scale bar: 80 µm. (B) The percentage of clones containing only TuJ1-positive cells among GFP-positive clones was then determined by immunocytofluorescence analysis. *P<0.01. (C) NPC cultures prepared from the neocortex of E12.5 wild-type mice were infected with pMX-GFP and co-cultured with neuroepithelial cell cultures infected with pMX-CD8 (Control) or a retrovirus encoding both CD8 and STAT3-C (STAT3-C) as in B. *P<0.05.

View larger version (54K): [in a new window]   Fig. 6. Regulation of DLL1 expression by STAT3. (A-D) Brain sections of E16.5 mice were immunostained with anti-STAT3 and anti-DLL1, as indicated. DLL1 was detected in STAT3-expressing cells. Dorsal side is up in all panels. Scale bars: 400 µm in A; 80 µm in B. (E) NPC cultures prepared from the neocortex of E12.5 STAT3flox/flox mice were infected with retroviruses encoding GFP alone (Control) or both GFP and Cre-recombinase (Cre). After culture for 2 days, the cells were subjected to RT-PCR analysis for the indicated mRNAs. Data are expressed relative to the corresponding normalized value for control cells. *P<0.01. (F) NPC cultures infected and cultured for 2 days as in E were subjected to immunoblot analysis with the indicated antibodies. (G) Promoter regions of the mouse DLL1 genes that contain consensus sequences for STAT3 binding (white arrowheads and the nucleotides in boldface type). NPC cultures prepared from the neocortex of E12.5 mice were subjected to ChIP analysis with anti-STAT3 or control immunoglobulin G (IgG). The immunoprecipitates were analyzed by PCR with primers spanning the indicated regions (underlined) of mouse Dll1.

View larger version (11K): [in a new window]   Fig. 7. Requirement of Delta-Notch signaling for NPC maintenance by STAT3. (A) NPC cultures prepared from the neocortex of E12.5 mice were infected with a retroviral vector for DLL1 siRNA or a control siRNA. After culture for 3 days, cell lysates were subjected to immunoblot analysis with antibodies to DLL1 and GAPDH. (B) Neuroepithelial cells prepared from the neocortex of E12.5 mice were infected with a retrovirus for DLL1 siRNA or a control siRNA. They were also separately infected with a retrovirus for STAT3-C or a control retrovirus. The cells were then assayed for the formation of secondary neurospheres. *P<0.01. (C) Neuroepithelial cells prepared as in B were infected with a retrovirus encoding STAT3-C (STAT3-C) or a control retrovirus (pMX-GFP) and cultured to allow the formation of primary neurospheres for 2 days. The primary neurospheres were then incubated in the presence of the -secretase inhibitor L685,458 or of vehicle (dimethyl sulfoxide, DMSO) for 1 day. They were then dissociated and cultured to allow the formation of secondary neurospheres in the presence of FGF2 (20 ng/ml) and EGF (20 ng/ml) and in the absence of the inhibitor. *P<0.01.