DEV004895 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Diagram illustrating the strategy for generating transgenic mice. (A) Generation of NG2DsRedBAC and NG2creBAC transgenes. Diagram 1 shows the structure of the mouse NG2 gene (not to scale). Exons are indicated as black blocks. The regions used for 5′ and 3′ homology arms are indicated. Diagram 2 shows the structure of the NG2DsRedBAC-RecA shuttle vector that was used to modify the BAC DNA in order to insert the DsRed cDNA and polyadenylation sequence into the BAC clone that contained the NG2 gene in the center. Diagram 3 shows the structure of the modified BAC DNA that was used for microinjection into fertilized oocytes. pA signal, polyadenylation signal; ATG, translation initiation codon of the NG2 gene; ts ori, temperature-sensitive origin of replication. (B) Strategy for fate mapping of NG2 glia using mice that are double transgenic for NG2creBAC (top, mouse line 1) and cre reporter Z/EG (mouse line 2, middle). In the double transgenic mice (bottom, double tg line), Cre that is activated in NG2 cells will permanently excise the lacZ transcription unit, including its associated polyadenylation signal, thereby permanently activating transcription of EGFP under the universally active CA promoter (cytomegalovirus enhancer/chicken β-actin promoter).

• Supplemental Figure S2 -

  Fig. S2. Fluorescence-activated cell sorting of DsRed+ cells. (A-D) Plots of cells from FACS. (A,C) Forward scatter (x-axis; FSC-H) and side scatter (y-axis; SSC-H) distribution of cells from P3 wt (A) and tg (C) brains. Polygon (R2) indicates the population of cells selected for sorting. Analysis of 200,000 events. (B,D) Red fluorescence intensity (x-axis; FL2-H) of 200,000 cells from P3 wt (B) and tg (D) brains. Horizontal lines in D indicate gates M1, M2 and M3 used to collect cells in a pilot study. The M3 gate represents the gate with the highest stringency, including only cells with DsRed fluorescence above 1×10². The M3 gate yielded 100% DsRed+ cells when analyzed 4 hours after plating and was used for all the experiments. (E) Graph representing the percentage of cells immunoreactive for NG2, PDGFRα, O1, GFAP or βIII-tubulin 4 hours, 4 days, and 11 days after sorting. Cells were maintained in NBM with B27 supplements with PDGF AA during the first 4 days. Values are expressed as mean ± s.d.

• Supplemental Figure S3 -

  Fig. S3. Antigenic expression of sorted cells 4 hours after sorting. (A-C) Living cells were immunolabeled with anti-NG2 antibody. (D-F) Living cells were immunolabeled with anti-PDGFRα antibody. (G-I) Living cells were immunolabeled with anti-O1 antibody. In each row the images are of the same field: (A,D,G) DAPI, (B,E,H) DsRed fluorescence, and immunolabeling with (C) rabbit anti-NG2 antibody, (F) rabbit anti-PDGF Rα antibody, and (I) mouse O1 antibody. Secondary anti-rabbit or anti-mouse antibodies were conjugated with Alexa Fluor 488 (anti-rabbit) or FITC (anti-mouse IgM). Images were acquired on a Leica DMR epifluorescence microscope. Scale bar: 25 µm.

• Supplemental Figure S4 -

  Fig. S4. Immunolabeling of sorted cells after 4, 7 or 11 aays in vitro (div). (A-C) Cells were labeled after 4 div using antibodies to NG2 (A), PDGFRα (B), or O1 (C). Red represents DsRed fluorescence. (D) Double immunolabeling for NG2 (red) and O1 (green) at 11 div showing a cluster of differentiated oligodendrocytes. (E-G) Double immunolabeling for NG2 (red) and GFAP (green) after 7 div (E) or 11 div (F,G). (E) Type 1 and type 2 astrocyte-like cells both co-express GFAP and NG2. (F shows the different intensities of GFAP and NG2 immunoreactivity in colabeled cells. G shows the appearance of GFAP+ cells that are NG2-negative by 11 div. (H,I) Double immunolabeling for PDGF Rβ (green) and GFAP (red) at 11 div. PDGFRβ is detected in pericytes in unsorted P3 wt cultures (I) but not in cultures of sorted cells (H). (J,K) Immunolabeling for βIII-tubulin (green) after 11 div showing no βIII-tubulin+ cells among the sorted cells cultured for the first 4 days in NSC medium in the presence of EGF and FGF2. (K) Positive control for βIII tubulin staining showing many βIII-tubulin+ neurons in P5 wt mouse cerebellar culture. Blue in all the panels represents nuclear labeling with DAPI. Images were acquired on a Leica DMR epifluorescence microscope. Scale bars: 25 µm.