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. 2017 Apr 15;31(8):757–773. doi: 10.1101/gad.293027.116

Figure 3.

Figure 3.

FOXG1/SOX2 drives reacquisition of NS cell identity in post-mitotic astrocytes. (A) Schematic of the experimental strategy used to test dedifferentiation. Cells at clonal density (10 cells per square millimeter) were treated with 10 ng/mL BMP4 for 24 h and then switched to EGF/FGF-2 medium with or without transgene induction by Dox treatment. (B) EdU staining shows that no rapidly cycling cells remain after 24 h of BMP4 treatment. Twenty-four hours after plating in EGF/FGF-2 or BMP4, a 24-h pulse of EdU was administered in medium containing EGF/FGF-2. Representative images of EdU staining and quantification of the percentage of EdU-positive cells are shown for each condition. Mean ± SD. n = 2 independent experiments. Bar, 100 µm. (C) Transgene dose determines the extent of colony formation after 10 d in EGF/FGF-2. n = 3 independent experiments. Tumor-initiating IENS cells retained colony-forming ability after BMP treatment and served as a positive control, while ANS4 cells served as a negative control. Below are shown example 10-cm dishes for FS3 (no Dox), FS3 plus 1000 ng/mL Dox, and IENS treated with BMP4 for 24 h and returned to EGF/FGF-2 for 10 d. FS3 cells form colonies efficiently on transgene induction. (D) ICC for FS3 cells showing Gfap and Nestin protein levels after 24 h in EGF/FGF-2, 24 h in BMP4, return to EGF/FGF-2 for 10 d without Dox, and return to EGF/FGF-2 for 10 d with Dox. (E) Heat map of the most differentially expressed transcripts across RNA sequencing (RNA-seq) libraries at various time points during dedifferentiation; biological replicates are shown for each condition, with variability at early stages due to the low absolute numbers of cells that dedifferentiate. (F) FS3 cells retain astrocytic and neuronal differentiation potential after long-term expansion (∼30 d), as shown by ICC for Gfap and Tuj1. (G) Mouse primary astrocytes were derived from a postnatal day 3 (P3) mouse cortex, and the FOXG1/SOX2-inducible transgene was introduced by lipofection. Following the described colony-forming assay, colonies were scored 2 wk following restoration of EGF/FGF-2. (H) A working model: In the presence of mitogens, FOXG1/SOX2 acts to restrict differentiation commitment and drive proliferation.