DEV02166 Supplementary Material

Files in this Data Supplement:

• Supplemental Table - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. The rGFAPp-eGFP retrovirus and FACS faithfully select GFAP⁺ cells. Neurospheres were infected with the rGFAPp-eGFP retrovirus and plated for differentiation for 7 days, with or without cytokines. (A,B) Differentiating cells were processed for immunocytochemistry. eGFP (green) driven by the 1.9 kb rat GFAP promoter is expressed by oligodendrocytes, as indicated by the arrows (B; red, bIII-Tubulin, neurons; blue, O4, oligodendrocytes).  (C-G) Live GFP fluorescent imaging to monitor the cell morphologies of the selected cells (green indicates eGFP-expressing cells). Untreated (control) cells had a mixture of morphologies.  Treatment with BMP4 induced a stellate morphology, whereas LIF treatment induced a bipolar/tripolar morphology.  Thus, findings with the GFP-selected cells recapitulated the findings from GFAP immunocytochemistry of cultured neurosphere cells exposed to cytokines (see Fig. 1). (H,I) FACS analysis for eGFP expression purifies GFAP-expressing cells. Dead cells are excluded by the gates in the forward-side scatter plot (H). GFP-expressing cells are selected according to high fluorescence. Scale bars: 20 mm for A,B; 50 mm for C-G.

• Supplemental Figure 2 -

  Fig. S2. Noggin decreases astrocyte markers in the postnatal hippocampus. Noggin was overexpressed in transgenic animals under the control of the neuron-specific enolase (NSE) promoter and processed for immunocytochemistry at postnatal day 15 (P15). Cell counts for the astrocyte markers GFAP and S100b were acquired at high magnification throughout the hippocampus. Noggin overexpression significantly decreased the total number of GFAP⁺ and S100b⁺ cells compared with wild-type littermates. *P< 0.01, Student’s t-test.

• Supplemental Figure 3 -

  Fig. S3. BMP signaling promotes exit of GFAP-expressing cells from the cell cycle in vivo. High magnification images demonstrate co-localization of Ki67 and GFAP in the postnatal SGZ. BMP overexpression decreases the number of GFAP⁺ cells that incorporate Ki67 (C), whereas BMP inhibition with noggin increases Ki67 incorporation into GFAP⁺ cells (A) compared with wild-type controls (B). SGZ, subgranular zone; GCL, granule cell layer; h, hilus; WT, wild type.

• Supplemental Figure 4 -

  Fig. S4. BMP signaling decreases vimentin expression in GFAP⁺ cells in vivo. Neural progenitor status was investigated in wild-type, NSE-noggin and NSE-BMP4 animals by processing P15 brains for immunofluorescence for GFAP (green) and for the neural progenitor marker, vimentin (red) in the hippocampus SGZ. (A-C,G-I) Some GFAP-expressing cells in the SGZ express vimentin and that number is increased when noggin is overexpressed. (D-F) Overexpression of BMP4 promotes the loss of vimentin in GFAP⁺ cells, demonstrating that BMP inhibition is necessary in this region to maintain progenitor cell traits. Blue color indicates Hoechst nuclear counterstain. GCL, granule cell layer; SGZ, subgranular zone; h, hilus; WT, wild type. Scale bar: 10 mm for A-I.

• Supplemental Figure 5 -

  Fig. S5. BMP inhibition maintains vimentin expression in GFAP⁺ cells in vivo. Neural progenitor status was investigated in wild-type, NSE-noggin and NSE-BMP4 animals by processing P15 brains for immunofluorescence for GFAP (green) and a neural progenitor marker, vimentin (red), in the hippocampus ML. (D-I) GFAP-expressing cells in the ML rarely co-express vimentin in wild-type or BMP4-overexpressing mice. (A-C) BMP inhibition in the ML increases vimentin expression in GFAP⁺ cells, demonstrating that BMP is necessary for astrocyte maturation in this region. Blue color indicates Hoechst nuclear counterstain. ML, molecular layer; WT, wild type. Scale bar: 10 mm for A-I.