, 2010). Thus, modulation of
the proteomic composition of extracellular fluids together with the integration of cell autonomous determinants of self-renewal by the apical complex may ultimately provide unexpected ways to regulate stem cell behavior in health and SCH 900776 molecular weight disease. Time pregnant Sprague-Dawley, C57BL/6, and Swiss Webster dams were purchased from Charles River Laboratories and Taconic. Pals1loxP/loxP/NestinCre+/−, Pals1loxP/loxP/Emx1Cre+/−, Igf1RloxP/loxP/NestinCre+/−, and GFAP:Igf_1Tg mice were obtained from heterozygous breedings, and PtenloxP/+/Pals1loxP/+/Emx1Cre+/−, and PtenloxP/loxP/Pals1loxP/+/Emx1Cre+/− mice were obtained from PtenloxP/+/Pals1loxP/loxP/Emx1Cre+/− and PtenloxP/loxP/Emx1Cre−/− crosses ( Groszer et al., 2001, Kim et al., 2010,
Liu et al., 2009 and Ye et al., 2004). Igf2−/− and control CSF was collected from embryos obtained from homozygous breedings. Igf2−/− and control P8 brains were obtained from homozygous crosses or paternal heterozygotes mated with homozygous knockouts ( DeChiara et al., 1991). All animal experimentation was carried out under protocols approved see more by the IACUCs of Harvard Medical School, Children’s Hospital Boston, and UNC-Chapel Hill. The following antibodies were purchased: Ctip2, Igf2 (for EM), Tbr2 (Abcam); BrdU (AbD Serotec); Ki67 (Abnova); Vimentin 4A4 (Assay Designs); Pax6 (Developmental Studies Hybridoma Bank); β-catenin, Cdc42 (BD Biosciences); AKT, phospho-AKT, Igf1R, phospho-Igf1R, CC3, and phospho-S6rp (Cell Signaling); GLAST (Chemicon); Tuj1 (Covance); HRP conjugated
anti-Transferrin (Immunology Consultants Laboratory, Inc.); Igf2 (NAb; Millipore); Cux1, Igf2 (for WB) (Santa Cruz Biotechnology); and phospho-Histone H3 (Upstate). Tbr1 was a kind gift of R. Hevner. Embryonic rodent CSF, isolated as described (Zappaterra et al., 2007), was kept on ice during collection, centrifuged at 10,000 × g at 4°C for 10 min., and stored at −80°C. Human GBM and disease-free CSF samples were collected because by lumbar puncture from patients undergoing clinical evaluation. The 56 GBM samples tested were obtained from 21 individuals representing the full-range of disease progression. The samples used in analyses of highest and lowest CSF Igf2 concentration were obtained from distinct individuals. All research was approved by the IRBs at BIDMC and Children’s Hospital Boston. The telencephalic wall was dissected onto polycarbonate membranes (Whatman; 13 mm, 8.0 μm) and cultured for 24 hr as described in text. Artificial (A)CSF (NaCl 119 mM, KCl 2.5 mM, NaHCO3 26 mM, NaH2PO4 1 mM, glucose 11 mM, MgCl2 2 mM, CaCl2 2.8 mM) was supplemented with Igf2 (2 ng/ml; US Biologicals) as indicated. Igf2 NAb antibody was incubated with E17 CSF for 1 hr at 4°C. Explants were pulsed with BrdU for 30 min and fixed (60% methanol, 30% chloroform, and 10% acetic acid; 10 min). For in vivo BrdU labeling, pregnant dams were administered a 3 hr BrdU (60 mg/kg) pulse. Tissue was paraffin sectioned (5 μm). We thank A. Bonni, S. Gygi, R.