BMP signaling modulates astrocyte maturation <i>in</i><i>vitro</i>.

<p>A–C. Purified astrocytes were cultured for 3DIV in HbEGF (A), BMP5 (B), or FGF2 and GFAP expression examined by immunostaining. CellMask HCS staining was used to visualize all astrocytes. Images show individual channels from the same field. Scale bars are 100 µm. The percentage of astrocytes expressing GFAP at 3DIV was quantified using ImageJ (C). D–G. Purified astrocytes were cultured for 3DIV in HbEGF or BMP5 and the expression of AQP4 and S100ß examined by immunostaining. All cultures were also stained with GFAP. Scale bars are 50 µm. H–J. The proliferative capabilities of purified astrocytes in HbEGF, BMP5 and FGF2 were quantified <i>in</i><i>vitro</i> over 7 days using clonal analysis. Average clone size was calculated for both individual growth factors (H) and combinations of factors (I). Clonal analysis was also performed on astrocytes pre-treated with BMP5 for 7DIV to examine reversibility (J). K–L. Western blots for EGFR. Protein samples collected from acutely purified astrocytes at different postnatal time points confirm strong developmental downregulation of astrocyte EGFR (K). EGFR is also strongly downregulated in astrocytes cultured with BMP5 for 6DIV, even in the presence of other growth factors (L). Actin bands confirm equal protein loading. N ≥3 for each condition. Significance determined using one-way ANOVA with Tukey correction. Error bars represent SEM.