Neocortex / NFIA / Sox9 Supplementary Figure 1. Expression of, NFIA, and Sox9 in the mouse neocortex at. The lower panels are higher magnification views of the oxed area. Arrowheads indicate triple-positive cells. Scale ars, 5 and 25 μm in the main panel and higher magnification images, respectively. 5 μm
Forerain Higher-magnification Forerain Higher-magnification / S1β Sep Sep / FoxJ1 Sep Sep c GFP / FoxJ1 / Hoechst Sep GFP / FoxJ1 / Hoechst Sep Supplementary Figure 2. Expression of in ependymal cells. (a) Coexpression of and either S1β or FoxJ1 in the and adult forerain. Right panels of each pair are higher magnification views of the oxed areas. (, c) Expression plasmids for GFP alone (control) or for oth GFP and were injected into the lateral ventricle of the E15.5 mouse forerain in utero and were introduced into the dorsolateral region of the neocortex () and the medial dorsoventral oundary of rains (c) y electroporation. The rain was isolated at and sujected to immunostaining for FoxJ1 and GFP., lateral ventricle; Sep, septum. Arrowheads indicate doule-positive cells (a, c). Scale ars, 5 μm (a, c), 1 μm (), and 25 μm (higher magnification images in a).
Neocortex Neocortex / HuC/D P3 / NeuN a / NeuN / NeuN Supplementary Figure 3. Transient expression of in NeuN+ neurons and HuC/D+ immature neurons. (a) Coexpression of and NeuN in the P3,, and adult neocortex. () Coexpression of and HuC/D in the neocortex. Arrowheads indicate doule-positive cells. Scale ars, 5 μm.
GFP / Sox9 GFP / GFAP GFP / TuJ1 8 6 4 2 TuJ1 GFAP O4 Sox9 c d e GFP / EdU GFP / cl-casp3 EdU + cells among GFP + cells (%) 4 3 2 1 NS cl-casp3 + cells among GFP + cells (%) 2. 1.5 1..5. NS Supplementary Figure 4. Effects of on the proliferation, survival, and differentiation of NPCs in vitro. (a, ) NPCs derived from E16.5 mouse forerain were infected with retroviruses encoding GFP alone (control) or oth GFP and. Two days after infection, the cells were induced to differentiate for 6 days and then stained for TuJ1, GFAP, O4, Sox9, and GFP (a). Arrows indicate marker + /GFP + cells. The percentages of marker + cells among total GFP + cells were determined as means ± s.d. (n = 3) (). (c e) E16.5 NPCs infected with control or retroviruses were laeled with EdU for 2 h in the presence of FGF2 and EGF. The cells were stained for EdU and cleaved caspase 3 (cl-casp3) 1 day after plating (c). Arrows indicate EdU + /GFP + cells or cleaved caspase 3 + /GFP + cells. The percentages of EdU + (d) or cleaved caspase 3 + (e) cells among GFP + cells were determined as means ± s.d. (n = 3). P <.1 versus the corresponding control value; NS, non-significant; P =.45 (d) and.53 (e) versus corresponding control value. Scale ars, 5 μm.
GFP / TuJ1 / GFAP GFP / GalC / TuJ1 GFP / GalC / GFAP NA NO AO 1 8 6 4 2 N NA A c 1 8 6 4 2 N NO O d 1 8 6 4 2 A AO O e Cell numer / clone 14 12 1 8 6 4 2 NS f 1 (sh-luc) sh- #1 g 1 (sh-luc) sh- #1 h 1 (sh-luc) sh- #1 i 14 8 6 4 2 N NA A NS 8 6 4 2 N NS NO O 8 6 4 2 A AO O Cell numer / clone 12 1 8 6 4 2 NS sh- (sh-luc) #1 Supplementary Figure 5. Clonal analysis of -overexpressing and knockdown cells. (a i) The control, -overexpressing or knockdown cells were plated at a clonal density and cultured with FGF2 for 3 days and without FGF2 for 4 days. The percentages of neuron clones (N) which contain TuJ1 + cells and do not contain GFAP + cells, neuron and astrocyte clones (NA) which contain oth TuJ1 + cells and GFAP + cells, or astrocyte clones (A) which contain GFAP + cells and do not contain TuJ1 + cells among total clones were quantified (, f). The percentages of neuron clones (N) which contain TuJ1 + cells and do not contain GalC + cells, neuron and oligodendrocyte clones (NO) which contain oth TuJ1 + cells and GalC + cells, or oligodendrocyte clones (O) which contain GalC + cells and do not contain TuJ1 + cells among total clones were quantified (c, g). The percentages of astrocyte clones (A) which contain GFAP + cells and do not contain GalC + cells, astrocyte and oligodendrocyte clones (AO) which contain oth GFAP + cells and GalC + cells, or oligodendrocyte clones (O) which contain GalC + cells and do not contain GFAP + cells among total clones were quantified (d, h). The cell numer per clone was also determined (e, i). Data are means ± s.d. (n = 3). P <.1, P <.5 versus corresponding control value; NS, non-significant; P =.92 (e),.19 (f),.15 (g) and.75 (i) versus corresponding control value. Scale ar, 5 μm.
VZ/SVZ GFP / Hoechst VZ/SVZ Forerain E18.5 GFP / Hoechst VZ/SVZ Supplementary Figure 6. The distriution pattern of -overexpressing cells around the VZ/SVZ and in the neocortex. (a, ) Expression plasmids for GFP alone (control) or for oth GFP and were injected into the lateral ventricle of E15.5 mouse forerain in utero and were introduced into the dorsolateral region of the neocortex y electroporation. The rains were isolated at (a) or E18.5 () and sujected to immunostaining for GFP., lateral ventricle; SVZ, suventricular zone; VZ, ventricular zone. Scale ars, 75 μm (a) and 1 μm ().
1 8 6 4 2 TuJ1 # E11.5 NPC culture GFAP + sh-sox9 #1 # O4 1 8 6 4 2 TuJ1 # E11.5 NPC culture GFAP + sh-nfia #1 # O4 # Supplementary Figure 7. Effects of knockdown of Sox9 or NFIA on the promotion of astrocyte differentiation y. (a, ) E11.5 NPCs were infected with control,, or plus either sh-sox9 #1 (a) or sh-nfia #1 () retroviruses, cultured without FGF2 and EGF for 6 days, and sujected to immunostaining for TuJ1, GFAP, O4, and GFP. The percentages of marker + cells among total GFP + cells were determined as means ± s.d. (n = 3). P <.1 versus corresponding control value; #P <.1 versus value for alone.
1 8 6 4 2 TuJ1 GFAP NICD CNTF NICD + CNTF NICD + sh- #1 NICD + CNTF + sh- #1 Supplementary Figure 8. Effect of knockdown on astrocyte differentiation induced y NICD and CNTF. E16.5 NPCs were infected with control, NICD, or NICD plus sh- #1 retroviruses, cultured without or with CNTF for 6 days, and then stained for TuJ1, GFAP, and GFP for determination of the percentages of marker + cells among total GFP + cells (means ± s.d., n = 3). P <.1 versus corresponding control value.
4. E11.5 NPC culture GFAP promoter-luc Luciferase activity (fold) 3. 2. 1..1.3.5.7.7 STAT3-C (μg) Supplementary Figure 9. Effect of on the activity of the Gfap promoter. Luciferase reporter assay of relative Gfap promoter activity in NPCs transfected with the indicated amounts of expression plasmids for or STAT3-C (positive control). Data are means ± s.d. (n = 3). P <.1 versus corresponding control.
Astrocytic genes Aldh1L1 Aldoc CNTFR FGFR3 GFAP GLAST GLT-1 gp13 NFIA NFIX S1β Sox9 STAT1 STAT3 Fold change (/).95.85 1.5 1. 1.29.94 1..87.82.98 1.55.87.97.98 Downregulated neuronal genes Brn2 (Pou3f2) Brn4 (Pou3f4) NFIB Sox4 Sox11 Fold change (/).56.78.72.7.64 Supplementary Figure 1. Microarray analysis of -overexpressing NPCs. (a, ) E11.5 NPCs were infected with control or viruses. The virus-infected NPCs were isolated 3 days after infection and sujected to microarray analysis. Fold changes in the expression level of the indicated astrocytic (a) and neuronal () genes in -overexpressing NPCs relative to control are shown.
FLAG- FLAG- NFIA Sox9 NFIA IP: FLAG- Sox9 IP: FLAG- FLAG- FLAG- NFIA WCL Sox9 WCL FLAG- FLAG- Supplementary Figure 11. interacts with NFIA. (a, ) 293T cells were transfected with plasmids for FLAG-tagged, Sox9, or NFIA, as indicated. The cell lysates were sujected to co-immunoprecipitation and western lot analysis. IP, immunoprecipitation; WCL, whole cell lysate.
Ventral forerain Hippocampus Spinal cord GM Spinal cord WM / Aldh1L1 P14 CA1 P14 P14 P14 Ventral forerain Hippocampus Spinal cord GM Spinal cord WM CA1 / GFAP c Ventral forerain Hippocampus Spinal cord GM Spinal cord WM CA1 / S1β Supplementary Figure 12. Expression of in the CNS regions other than neocortex. (a) Doule staining for and Aldh1L1-GFP in the P14 ventral forerain, hippocampus, and spinal cord. (, c) Doule staining for and either GFAP () or S1β (c) in the adult ventral forerain, hippocampus, and spinal cord. CA1, hippocampal CA1 sector; GM, gray matter; WM, white matter. Arrowheads indicate doule-positive cells. Scale ars, 5 μm.
E11.5 NPC culture E11.5 NPC culture GFP / GalC GFP / GFAP GFP / TuJ1 8 6 4 2 TuJ1 GFAP GalC Supplementary Figure 13. Effects of on the differentiation of spinal cord NPCs in vitro. (a, ) NPCs derived from E11.5 mouse spinal cord were infected with retroviruses encoding GFP alone (control) or oth GFP and. Two days after infection, the cells were induced to differentiate for 6 days and then stained for TuJ1, GFAP, GalC, and GFP (a). Arrows indicate marker + /GFP + cells. The percentages of marker + cells among total GFP + cells were determined as means ± s.d. (n = 3) (). P <.1 versus the corresponding control value. Scale ar, 5 μm.
E11.5 NPC culture 1.2 c Sox1 mrna / GAPDH mrna 1..8.6.4.2 GFP / Sox1 Sox1 + cells among GFP + cells (%) 15 1 5 d. Forerain Higher-magnification e Day 1 Day 2 GFP / Sox1 / Hoechst f Fold enrichment 6 5 4 3 2 P3 SVZ E11.5 NPC culture, mouse IgG, TY1 A TY1-, mouse IgG TY1-, TY1 A Sox1 + cells among GFP + cells (%) 15 1 5 1-639 ~ -6259-2183 ~ -2133-523 ~ -473-231 ~ -181 Sox1 gene region (p) -11 ~ +43 +32 ~ +37 Supplementary Figure 14. suppresses oligodendrocyte differentiation. (a) Quantitative RT-PCR analysis of relative Sox1 mrna aundance in -overexpressing and control NPCs. Data are means ± s.d. (n = 3). (, c) E16.5 NPCs were infected with retroviruses encoding GFP alone (control) or oth GFP and. Two days after infection, the cells were induced to differentiate for 1 or 2 days and then stained for Sox1 and GFP (). The percentages of Sox1 + cells among total GFP + cells were determined as means ± s.d. (n = 3) (c). (d, e) Expression plasmids for GFP alone (control) or for oth GFP and were injected into the lateral ventricle of the E15.5 mouse forerain in utero and were introduced into the lateral dorsoventral oundary of rains y electroporation. The rain was isolated at P3 and sujected to immunostaining for Sox1 and GFP (d). The percentages of Sox1 + cells among total GFP + cells were determined as means ± s.d. (n = 1) (e). (f) ChIP analysis of inding to the Sox1 gene region in NPCs. Six different regions of the Sox1 locus were tested in control cells and cells expressing TY1-tagged. Data are expressed as fold enrichment relative to the corresponding value for control cells and normal mouse immunogloulin G (IgG). Data are means ± s.d. (n = 3). SVZ, suventricular zone; p, ase pair; A, antiody. The oxed region in d is shown at higher magnification in the right-most panel. Arrows indicate Sox1 + /GFP + cells (, d). Scale ars, 5 μm (d) and 25 μm ( and higher magnification image in d). P <.1 versus the corresponding control value.
Forerain / Sox1 / Olig2 Neocortex / Ki67 / Olig2 P3 P3 CC SVZ Supplementary Figure 15. + /Olig2 + cells are astrocyte precursors in the developing neocortex. (a) + /Olig2 + /Sox1 cells (arrowheads) and /Olig2 + /Sox1 + cells (arrows) in the P3 neocortex. () + /Olig2 + /Ki67 + cells (arrowheads) and /Olig2 + /Ki67 + cells (arrows) in the P3 neocortex. CC, corpus callosum;, lateral ventricle; SVZ, suventricular zone. The lower panels are higher magnification views of the oxed areas. Scale ars, 5 μm or 25 μm (higher magnification images).
8 Zt16 mrna / GAPDH mrna 2 16 12 8 4 Zt16 6 4 2 TuJ1 GFAP O4 sh- #1 Zt16 sh- #1 + Zt16 Nestin ## c d 8 Zt45 mrna / GAPDH mrna 1 8 6 4 2 Zt45 6 4 2 TuJ1 GFAP sh- #1 Zt45 sh- #1 + Zt45 O4 Supplementary Figure 16. Zt16 and Zt45 fail to rescue the knockdown phenotypes in NPC cultures. (a, c) E16.5 NPCs were infected with retroviruses encoding GFP alone (control) or GFP plus either Zt16 (a) or Zt45 (c). The expression level of Zt16 (a) or Zt45 (c) mrna was determined y quantitative RT-PCR analysis. Data are expressed relative to the control value and are means ± s.d. (n = 3). (, d) E16.5 NPCs were infected with retroviruses for control, sh- #1, Zt16, or sh- #1 plus Zt16 (), or for control, sh- #1, Zt45, or sh- #1 plus Zt45 (d). The cells were induced to differentiate for 6 days, after which the cells were immunostained for TuJ1, GFAP, O4, nestin, and GFP. The percentages of marker + cells among total GFP + cells were quantified as means ± s.d. (n = 3). P <.1, P <.5 versus corresponding control value; ##P <.5 versus value for sh- #1 alone.