expression is layer-specific in perinatal neocortex and tectum. In the embryonic telencephalon, Shh is expressed within the mantle of the medial ganglionic eminence, the preoptic area and the amygdala [35,36,37]. Shh secreted from differentiated cells in the cortex can affect Gli1-positive, cycling precursor cells located at a distance; in addition, Shh can also be produced by the precursors themselves [18,38,39]. Genetic loss-of-function and knock-in studies wherein Gli genes have been ablated and then reintro- duced have shown that the Shh-Gli pathway controls the growth and dorsal-ventral patterning of brain structures by regulating proliferation of neural stem cells through EGF signaling [40,41,42,43,44]. Shh signaling also plays a role in inducing apoptosis. Signaling via Ptch, a 12–transmembrane domain receptor of Shh, induces caspase-mediated apoptosis in neuroepithelial cells. The intracel- lular domain of Ptch harbors a cleavage site for caspase 3; cleavage at this site by caspase 3 exposes the proapoptotic domain of the receptor. In vitro treatment of neuroepithelial cells with recombi- nant Shh blocks Ptch-induced cell death [45,46]. Here we demonstrate that: (i) PIC negatively regulates NPC proliferation by inhibiting Shh signaling, (ii) PIC induces apoptosis in association with inhibition of Ras-ERK signaling and elevated expression of Fas, and (iii) that these effects depend on TLR3 activation. Results Phenotypic analysis of EGF-responsive primary cortical neurospheres Neurospheres were generated from dissociated cerebral cortex obtained from Wild-type (WT) or TLR3 knockout (TLR32/2) C57BL/6 mouse embryos at GD14. The cellular composition of neurospheres was surveyed by FACS after culture for 1 or 7 days in proliferation medium supplemented with EGF (20 ng/ml). After 1 day of culture, 65.2 6 1.8% of WT cells expressed GFAP, a marker for astrocytes; 22.6 6 3.0% expressed tubulin-b-III, a marker for young postmitotic neurons; 4.8 6 1.5% expressed nestin, a marker for neuroepithelial stem cells (Fig. 1a; values represent mean 6 SEM) [47,48,49]. After 7 days, the percentage of GFAP+ and tubulin-b-III+ cells in WT neurospheres decreased to 11.8 6 0.6% and 7.5% 6 1.2%, respectively, whereas the percentage of nestin+ precursors increased to 55.9 6 2.0% (Fig. 1a; values represent mean 6 SEM). We observed similar percentages of cells in day 7 neurosphere cultures derived from TLR3 knockout mice (TLR32/2). After 7 days of culture, 63.062.0% of TLR32/2 neurosphere cells expressed nestin; 12.662.0% expressed GFAP; and 8.461.0% expressed tubulin-b- III, suggesting that neurospheres derived from TLR32/2 mice are equally responsive to EGF (Fig. 1a; values represent mean 6 SEM). To further characterize the phenotype of nestin+ NPC, day 7 WT and TLR32/2 neurosphere cells were analyzed by FACS for expression of PAX6 and TBR2, two transcription factors expressed in vivo by NPC during mid- and late cortical neurogenesis [50,51,52]. 46.2 6 2.8% of WT and 62.3 6 2.0% of TLR32/2 precursor cells co-expressed nestin and PAX6; 2.2 6 0.07% WT and 2.6 6 0.08% of TLR32/2 cells co-expressed nestin and TBR2. PAX6 is a radial glial (RG) marker; thus, these results indicate that the majority of the nestin+ neurosphere- derived cells in these dorsal telencephalon cultures are self- renewing NPC (Fig. 1b; values represent mean 6 SEM). In WT neurosphere cells, intracellular expression of TLR3 was observed (in nestin+, PAX6+, and TBR2+ NPC populations (Fig. 1c). TLR3 Regulates Neural Stem Cell Proliferation The neurosphere culture model contains cells at various stages of differentiation [8,9]. Whereas nestin-positive precursor cells are typically located toward the outside of the sphere, GFAP-positive cells are found in the center. Tubulin-b-III-positive neurons are less common and tend to be evenly distributed [53]. In our experiments, neurospheres were detected after one week of culture of WT and TLR32/2 GD14 cortices (Fig. S1). The number of nestin-positive cells was decreased by 50% in secondary versus primary WT neurospheres (Fig. S2) and decreased continuously with subsequent passages (data not shown) [54]. Thus, our experiments were performed with primary neurospheres wherein nestin expression was maximal. When dissociated and transferred to differentiating conditions for 7 days, WT primary neurospheres lost their spherical shape and flattened to form monolayers (Fig. S3a). FACS analysis using cell lineage-specific antisera showed that WT neurospheres were multipotent and capable of giving rise to neurons, astrocytes, and oligodendrocytes. During differentia- tion, WT cells expressed GFAP, tubulin-b-III, and O4, markers characteristic for astrocytes, neurons and oligodendrocyte popu- lations, respectively (Fig. S3b–f). PIC negatively regulates neurosphere-derived NPC proliferation in a TLR3-dependent manner To assess the direct role of TLR3 signaling on NPC proliferation, we used primary cortical neurosphere cultures obtained from GD14 WT and TLR3-deficient (TLR32/2) embryos. Neurospheres maintained in proliferation media for 7 days were treated with the TLR3 ligand, PIC (50 mg/ml), for 24 hours; BrdU was added for the final 12 hours of the culture. Proliferative responses of neurosphere-derived cells were assessed by flow cytometric measurement of labeled BrdU. Our results indicate that BrdU was incorporated into the majority of nestin+ cells, and that PIC exposure was associated with a reduced percentage of BrdU+nestin+ precursor cells in WT neurosphere cultures as compared to PBS-treated WT control cultures (n = 12; Mann-Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 2a, b). PIC treatment resulted in a concomitant increase in the percentage of BrdU-negative GFAP+ and Tubulin- b-III+ subpopulations in the WT neurospheres (n = 12; Mann- Whitney U, p = 0.04; relative to PBS-treated WT control group; Fig. 2c, e). No significant differences were observed in expression levels of GFAP and Tubulin-b-III markers in WT neurospheres (n = 12; Mann-Whitney U, p = 0.29; Fig. 2d). Neurosphere cultures derived from TLR32/2 mice were used to assess the role of TLR3 signaling in the PIC-mediated inhibition of NPC proliferation. No differences were observed in the percentage of BrdU+nestin+ precursor cells in PIC-treated TLR32/2 neuro- spheres as compared to PBS-treated TLR32/2 control cultures (n = 12; Mann-Whitney U, p = 0.26; Fig. 2a, b). Taken together, these results implicate TLR3 signaling in PIC-induced impairment of proliferation of EGF-responsive NPC in cortical neurospheres. PIC decreases neurosphere-derived neural precursor cell proliferation by inhibiting Shh signaling Shh signaling regulates the proliferation of EGF-responsive precursor cells in the developing mouse neocortex [41]. To test whether Shh signaling is active in the EGF-responsive precursor cells found in neurospheres, we examined the expression of Shh pathway receptors and targets in 7-day cultures from GD14 embryos. FACS analysis revealed that WT and TLR32/2 nestin+ neurosphere cells expressed the membrane-associated Shh recep- tor, Ptch (Fig. 3a). No change in the percentage of nestin+ Ptch+ cells was observed with PIC treatment. 11.0–15.0% of nestin+ cells PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26766