consequences of Sonic Hedgehog (Shh) secretion from the notochord during spinal cord development [7,8]. Shh is also expressed in dorsal structures throughout the developing CNS, including the cerebellum, tectum and forebrain [9,10,11,12]. Within the neocortex, Komada and colleagues have identified domains of Shh expression in the proliferative zones and post- mitotic neurons of the neocortex from E13.5–E15.5. Loss of function studies utilizing conditional Shh and Smoothened (Smo) alleles indicate that Hh signaling acts by regulating the cell cycle of VZ stem and progenitor cells and in the absence of an active pathway there is significantly reduced neurogenesis, patterning defects and a smaller dorsal telencephalon at E18.5 [12]. Consistent with these findings, gain of function studies in vitro have indicated that Hh signaling is mitogenic for both tectal and neocortical progenitors [11,12]. Notch signaling plays a central role in the maintenance of neural stem cell phenotype [13,14,15]. During embryogenesis, Notch signaling promotes neural stem cell proliferation in the forebrain [14] by inhibiting neuronal fate and ablation of Notch signaling leads to premature differentiation of glial cells to neurons [16]. In addition, Notch signaling is essential for the maintenance of stem cell characteristics, possibly through the canonical Rbpj- mediated Notch signaling cascade [17]. Anthony et al have demonstrated that Blbp is a direct target of Notch signaling in radial glial cells thereby providing a direct link between active Notch signaling and the maintenance of the radial glial cell scaffold [18]. Despite its critical role in the maintenance of radial glial cells and the development of the mammalian neocortex, little is known to date about the interaction of Notch signaling pathway with other key developmental pathways in the spatiotemporal generation of the neocortex. On the basis of published data, both the Notch and Hedgehog signaling pathways act concomitantly on dorsal forebrain development. In particular, both in vitro and in vivo studies addressing either Notch or Hh signaling suggest that the activity of both pathways is vital for the correct regulation of the stem cell niche, and the subsequent formation of the cortical layers [11,12,14,17,18,19,20,21]. What is not known is whether these two pathways are capable of interacting to control neurogenesis. To address this issue we directly examined the interaction between Notch and Hedgehog signaling in the neocortex. Since the function of Hedgehog signaling in the neocortex appears to be the regulation of the size of stem/progenitor pool and loss of function studies lead to substantive patterning defects. As a general approach to defining Hh/Notch interaction, we used a gain of function mutation as our start point. We activated the Hedgehog pathway in neuroepithelial cells through the use of a conditional Ptc1 (Ptc1Lox/Lox) allele [22] in combination with a Nestin- promoter-driven Cre-recombinase mouse model (NestinCre) [23]. Ptc1 is the Hedgehog receptor and a negative regulator of Hedgehog signaling whilst Nestin is expressed in neuroepithelial cells from E10.5 into adult life but down regulated in differentiated cells derived from the neuroepithelium. Loss of Ptc1 from neuroepithelial cells creates a cell autonomous activation of the Hedgehog pathway. The Ptc1Lox/Lox;NestinCre embryos die at E15.5, therefore, the studies presented here were carried out on E14.5 or earlier developmental stages. To perturb Notch signaling we utilized a conditional allele of Rbpj (RbpjLox/Lox) [24], a common effector required for the activity of all four mammalian Notch receptors. When inactivated under the control of NestinCre, loss of Rbpj function results in premature differentiation of the stem cells of the dorsal forebrain [25]. Here we demonstrate that autonomous Hh signal activation in VZ progenitors leads to an increase in the symmetrical divisions of radial glial cells. The Control of Neural Stem Cells Ptc1Lox/Lox;NestinCre mice have an expansion of the VZ progenitors and improper patterning. Furthermore, Notch signaling targets Hes1 and Blbp which were upregulated in the Ptc1Lox/Lox;NestinCre VZ suggesting that Hedgehog and Notch signaling may directly interact in the determination of radial glial cell fate in these animals. We tested this suggestion and determined that loss of Rbpj restored the number of neurogenic divisions to wild type levels in the Ptc1Lox/Lox;RbpjLox/Lox;NestinCre neocortex. We used ex vivo neurosphere based assays to define the proliferative compart- ment and demonstrate that in vivo activation of Hh signaling dramatically expands the number of stem and progenitor cells in the neocortex but loss of Rbpj attenuates this expansion to wild type levels. Overall, our results demonstrate that Hh signaling can co-operate with Notch signaling in the maintenance and expansion of neocortical stem cells. Materials and Methods Mice with CNS-specific deletions of Ptc1 were obtained by breeding animals carrying the conditional Ptc1 (Ptc1Lox/Lox) [22] or Rbpj allele (RbpjLox/Lox) [24] with Nestin-Cre (NestinCre) transgenic mice [23] and genotyping confirmation was carried out as outlined. All experimentation involving animals was approved by University of Queensland animal ethics committee and conformed to relevant ethical guidelines. Pregnant females were injected intraperitoneally with 0.1 ml/g (volume/body weight) of BrdU labelling reagent (Zymed, San Francisco, CA) for 2, 24, 48 or 72 hours prior to sacrificing. Histology, immunohistochemistry, in situ hybridisation and cell cycle analysis Embryos were removed, fixed in 4% buffered paraformalde- hyde overnight at 4uC, paraffin embedded and cut in 6 mm thick sections. Sections were stained with haematoxylin and eosin or stained for Nissl substance using standard histological techniques. Non-radioactive in situ hybridisation was performed using digoxigenin (DIG)-labelled cRNA probes as described [26]. The Ptc1 probe was designed towards the C-terminal end of the stop codon, so that it detects Ptc1 transcript. For immunohistochem- istry, antigen retrieval of deparaffinised wax tissue sections was performed by boiling in antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) for 5 minutes. Sections were blocked in 5–10% horse serum, 2% BSA and 0.2% Triton-X in PBS prior to primary antibody incubation over night at 4uC as previously described [26]. Antibodies used were mouse anti-Nestin (1:200, Chemicon, Temecula, CA), rabbit polyclonal anti-Tbr2 (1:1000, Chemicon) and mouse anti-MAP2 (1:500, Sigma Aldrich, St. Louis, MO). Slides were incubated with secondary antibodies for 1 hour at room temperature. Immunofluorescence was performed on coronal sections incubated with goat anti-Sox2 (1:200, R&D Systems, Minneapolis, MN, USA); mouse anti- Nestin (1:200, Chemicon, Temecula, CA) and rabbit anti-bIII Tubulin or TuJ1 (1:300, Covance, Princeton, NJ). For immuno- fluorescence, a DAPI (4, 6-diamidino-2-phenylindole, Sigma Aldrich, St Louis, MO, USA, 1:10000) counterstain was performed for 5 minutes prior to mounting with Fluorescence Mounting Media (Dako, Carpentaria, CA, USA). Fluorescent secondary antibodies used were anti-rabbit Alexa Fluor 488 (1:250, Invitrogen), anti-rabbit Alexa Fluor 555 (1:250, Invitro- gen), anti-mouse Alexa Fluor 488 (1:250, Invitrogen) and anti-goat Cy3 (1:250, Abacus ALS Pty Ltd, Brisbane, Australia). Fluores- cence images were obtained by using a laser confocal microscope (LSM Meta, Carl Zeiss). All images were taken in the medial neocortical region. PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e14680