TLR3 Regulates Neural Stem Cell Proliferation Figure 6. PIC induces apoptosis in primary cortical neurospheres. Primary neurospheres were obtained from cerebral cortex cells isolated from WT and TLR32/2 embryos at GD14. Neurospheres were cultured for 7 days in the presence of EGF (20 ng/ml) and treated with PBS or PIC for 24 hours. (a, b) Representative histograms showing Annexin V-positive cells in PBS- or PIC-treated WT or TLR32/2 neurospheres. Numbers in gates represent the percentages of Annexin V+ cells. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (c) Box plot showing the percentages of Annexin V+ cells in PBS- and PIC-treated neurospheres derived from WT and TLR32/2 embryos. Results are expressed as percentages of total cells (*, p,0.05 relative to PBS control group; Mann-Whitney U test). Height of box plot shows inter-quartile range; horizontal line, median. Due to the low numbers involved in the flow cytometry experiments, the box plot upper and lower interquartile ranges also represent the maximum and minimum scores. White bars: PBS-treated mice, hatched bars: PIC-treated mice. doi:10.1371/journal.pone.0026766.g006 followed by generation of neurons through asymmetric divisions [72]. During this critical period of central nervous system development, aberrant TLR3 activation may have profound implications for NPC proliferation, leading to deficits in telencephalon morphogenesis. These findings provide a new mechanistic framework for under- standing the pathogenesis of neurodevelopmental disorders associat- ed with gestational and perinatal infections. Materials and Methods Mice Ethics Statement. All mice were handled in accordance with the Association for Assessment and Accreditation of Laboratory Animals Care international guidelines, with the approval of the Institutional Animal Care and Use Committee at Columbia University under approval ID AC-AAAA4827. The Columbia University IACUC reviewed and approved this study under ID AC-AAAA4827. Wild-type (WT) pregnant C57BL/6 mice at gestational day (GD) 14 were obtained from Harlan Laboratories (Dublin, VA). Congenic TLR3 deleted mice (TLR32/2) on a C57BL/6 background were obtained by repeated breeding of heterozygous B6;129S1-Tlrtm1 Flv/J TLR3 knockout mice (Jackson Laboratories, Bar Harbor, ME) with WT C57BL/6 for ten generations. Heterozygous mice, carrying the TLR3 deletion were identified at each generation by PCR analysis of total genomic DNA using the following primer sets: NEO_F CTTGGGTGGAGAGGC- TATTC, NEO_R AGGTGAGATGACAGGAGATC, TLR3_F PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26766