In contrast, after transfection of Cre, only 1.15% ± 0.29% of transfected cells were BLBP+ ( Figures S3A–S3C). This result suggests that

MEK plays a cell-autonomous role in regulating glial progenitor specification. Our results strongly indicated that MEK is required for gliogenesis; however, it was unclear whether overactivation of MEK was sufficient to drive gliogenesis. We tested this by introducing pCAG-caMek1-EGFP into E15.5 WT radial progenitors by IUE and quantifying the proportion of transfected cells that express BLBP at E19.5. In EGFP transfected cortices, 5.68% ± 0.94% of transfected radial progenitors became astrocyte precursors. In caMek1-transfected cortices, the proportion of radial progenitors that became astrocyte precursors increased robustly click here to 24.78% ± 2.07% (Figures 3A–3C). In additional experiments, we assessed the ability of caMek1 to convert radial progenitors into mature astrocytes by allowing mice electroporated at E15.5 to survive until P60. Normally, electroporation of EGFP at E15.5 results in robust Veliparib clinical trial labeling

of neurons in layer II-III with virtually no astrocytes labeled. In striking contrast, transfection of caMek1 results in multiple mature astrocytes labeled in every section (Figures 3D and 3E). The astrocyte identity is confirmed by Acsbg1 immunostaining (Figures S3D and S3E). Overall, approximately 50% of labeled cells were astrocytes. Intriguingly, labeled neurons occupied a very superficial position in layer II consistent with overactive MEK stimulating accelerated

progression of radial progenitor development GPX6 (see below). Taken together, these data suggest that the regulation of glial progenitor specification by MEK is cell autonomous and demonstrate that MEK is both necessary and instructive for glial specification. To assess the mechanisms by which MEK regulates glial progenitor specification, we examined the response of Mek-deleted cortical cells to a gliogenic stimulus in vitro. Cytokine signaling through the LIFRβ/gp130-JAK-STAT pathway is a critical stimulus for astrocyte differentiation. We therefore induced astrocyte differentiation with CNTF (100 ng/ml) in WT or Mek1,2\Nes E17.5 cortical cultures. In WT cultures without CNTF, 4% of the cells differentiated into GFAP+ astrocytes after 5 days. Adding CNTF increased astrocyte differentiation as expected to 15%. In mutant cultures, only 1% of cells differentiated into astrocytes without CNTF stimulation, which increased to only 2% after stimulation with CNTF ( Figures 4A and 4B). Confirming the cell counting, western blotting showed that the level of GFAP protein in Mek1,2\Nes samples was only 20% of controls, while levels of the neuronal marker βIII-tubulin were comparable to controls ( Figures 4C and 4D). The STAT3 Tyrosine 705 site is directly phosphorylated by the upstream kinase JAK following CNTF stimulation (Liu et al., 1998).