TY - JOUR
AU - Mouillet-Richard, Sophie
AB - Abstract The prion protein is infamous for its involvement in a group of neurodegenerative diseases known as Transmissible Spongiform Encephalopathies. In the longstanding quest to decipher the physiological function of its cellular isoform, PrPC, the discovery of its participation to the self-renewal of hematopoietic and neural stem cells has cast a new spotlight on its potential role in stem cell biology. However, still little is known on the cellular and molecular mechanisms at play. Here, by combining in vitro and in vivo murine models of PrPC depletion, we establish that PrPC deficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that the absence of PrPC in a neuroepithelial cell line or in primary neurospheres is associated with drastically reduced expression of Notch ligands and receptors, resulting in decreased levels of Notch target genes. Similar alterations of the Notch pathway are recovered in the neuroepithelium of Prnp−/− embryos during a developmental window encompassing neural tube closure. In addition, in line with Notch defects, our data show that the absence of PrPC results in altered expression of Nestin and Olig2 as well as N-cadherin distribution. We further provide evidence that PrPC controls the expression of the epidermal growth factor receptor (EGFR) downstream from Notch. Finally, we unveil a negative feedback action of EGFR on both Notch and PrPC. As a whole, our study delineates a molecular scenario through which PrPC takes part to the self-renewal of neural stem and progenitor cells. Prion protein, Notch, Neural stem cells, Epidermal growth factor receptor, Cadherins Significance Statement The cellular prion protein PrPC, participates to hematopoietic and neural stem cell self-renewal. By combining in vitro and in vivo models of PrPC depletion, we establish that PrPC deficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that Notch signaling fine-tunes neuroepithelial cell architecture by strengthening N-cadherin-mediated cell-cell contacts. Downstream from Notch, PrPC positively regulates the expression of epidermal growth factor receptor, whose activation exerts a negative feedback control on Notch and PrPC. Overall, our findings unveil a new facet of the contribution of PrPC to neural stem cell homeostasis. Introduction The cellular prion protein PrPC is a highly conserved, ubiquitous glycoprotein that is mainly studied for its involvement in a group of neurodegenerative disorders known as Transmissible Spongiform Encephalopathies [1]. It is mostly found at the cell surface, where it may interact with a great diversity of partners, from extracellular matrix components to soluble ligands such as the extracellular chaperone stress-inducible 1 (STI-1) protein [2]. Over the past decade, PrPC has emerged as a key actor of stem cell proliferation and/or self renewal [3, 4]. It was first identified as a cell-surface marker for hematopoietic stem cells (HSCs), and shown to sustain the long-term repopulating activity of HSCs [5]. It was also found to positively regulate neural precursor cell proliferation, in accordance with its abundant expression in the developing and adult central nervous system (CNS) [6]. In line with this, the interaction of PrPC with STI-1 was shown to enhance the self-renewal of neural progenitors [7]. PrPC has also been depicted as a marker for mammary stem cells [8] and cardioprogenitors [9]. Further, we [10] and others [11] have shown that PrPC is upregulated following the cell fate restriction of pluripotent embryonic stem (ES) or carcinoma (EC) cells toward the neuronal lineage. Finally, we recently documented an enrichment of PrPC at the base of the primary cilium in stem and progenitor cells from the CNS and cardiovascular system of developing embryos [12]. Collectively, these data support the notion that PrPC is involved in stem cell homeostasis, but its specific contribution to stem cell proliferation, maintenance in an undifferentiated state, choice of cell fate or ability to enter a differentiation program remains incompletely understood. The present work aims at delineating the contribution of PrPC to neuroepithelial stem cell homeostasis by combining a synergy of in vitro and in vivo analyses. Our in vitro experiments exploit the EC-derived murine 1C11 neuroepithelial cell line [13], endogenously expressing PrPC [10, 14], and its stably PrPC-depleted derivatives (PrPKD−1C11 cells) [15], as well as primary neurospheres from WT and Prnp−/− mice [16]. In 1C11 cells, shRNA-mediated silencing of PrPC severely affects the Hedgehog pathway [12], which plays an overriding role in neural stem cells [17]. Here, we found that PrPC silencing drastically impacts on Notch signaling, a critical pathway involved in stem cell maintenance [18], by hindering the expression of the Notch ligands Jagged1 and Jagged2 as well as that of Notch receptors. The in vivo relevance of these results is substantiated by our analyses on Prnp−/− mice embryos revealing defects in the Notch pathway slightly after the onset of PrPC expression in the neural folds. Furthermore, our data highlight various outcomes of PrPC depletion downstream from Notch, including alterations in neural stem cell markers, cell architecture and, most notably, expression of the epidermal growth factor receptor (EGFR), whose activation exerts a negative feedback action on the Notch pathway as well as PrPC expression. Materials and Methods Animals Animal experiments were carried out in strict accordance with the recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioral Research with Animals (Directive 86/609EC) and all efforts were made to minimize suffering. Experiments were approved by the Local Ethics Committee of Jouy-en-Josas (Comethea, Permit number 12-034). E8.5 to E10.5 FVB/N (WT) and FVB/N Prnp−/− [19] mouse embryos from, respectively, WT × WT and Prnp−/− × Prnp−/− crossings were dissected in ice cold PBS and immediately frozen in liquid nitrogen for RNA analysis or fixed for immunofluorescence analyses. Cell Culture and Treatments 1C11 cells and their PrP-KD counterparts were grown as in [15]. For cells grown on Jagged1, culture dishes were precoated with recombinant Jagged1-Fc (R&D systems (Minneapolis, MN, USA), www.rndsystems.com) at 3 µg/cm2 or control IgG1-Fc in PBS. Mouse neurospheres were obtained by whole brain dissection of E14 embryo from two lines from the same 129/ola strain: wild-type (WT) and Prnp−/− mice homozygous for a targeted null mutation in the Prnp gene [20]. Cells were grown as previously described in [16], that is in the presence of 20 ng/ml EGF (R&D systems) and 20 ng/ml basic Fibroblast Growth Factor (bFGF) (Eurobio, Courtaboeuf, France, www.eurobio.fr). Immunofluorescence, Western Analysis, and Quantitative Real-Time PCR Detailed methods are presented in Supporting Information Materials and Methods. Statistics The results are reported as the means ± SEM. The unpaired Student's t test and the paired Wilcoxon's test were used for comparisons. A p value < .05 was considered significant. Results PrPC Depletion in Neural Stem/Progenitor Cells Cancels the Expression of Notch Pathway Effectors and Switches Off Notch Signaling To assess the contribution of PrPC to neural stem cell homeostasis, we first analyzed the impact of the absence of PrPC on the status of the Notch pathway, which plays a critical role in neural stem cell maintenance [18]. Activation of Notch transmembrane receptors induces the proteolytic cleavage of the Notch protein and nuclear translocation of its intracellular domain (Notch ICD, NICD), which interacts with the DNA-binding protein CBF1 to induce the transcription of target genes [18]. We first exploited the 1C11 cell line, in which we had previously documented a nuclear staining with anti-Notch polyclonal antibodies, indicating an activation of the Notch pathway [13]. The Notch nuclear staining was consistently observed in 1C11 cells, but was not recovered in their PrP-KD counterparts (Supporting Information Fig. S1), indicating that PrPC knockdown in 1C11 cells switches off the Notch pathway. In line with this, the level of transcripts encoding the Notch target gene Hes1 in PrP-KD cells was only 30% of that found in the parental 1C11 cells, as measured through quantitative PCR (qRT-PCR) (Fig. 1A). We went on to analyze the expression of mRNAs encoding the Notch receptors and their ligands Jagged1 (Jag1) and Jagged2 (Jag2) in 1C11 cells and their PrP-KD derivatives. We monitored strong reductions in the mRNA levels of Jag1 and Jag2 (16% and 22% vs. control, respectively) (Fig. 1B) as well as Notch1 (20% vs. control) (Fig. 1C) when PrPC was knocked down in 1C11 cells. The amount of transcripts encoding Notch2 were slightly but significantly lower in PrPKD−1C11 cells versus their parental 1C11 cells, while Notch3 transcripts were upregulated by 40% (Fig. 1C). Notch4 mRNAs were barely detected, irrespective of PrPC expression (data not shown). Figure 1 Open in new tabDownload slide PrPC depletion compromises Notch signaling in neural stem/progenitor cells. (A): The Notch target gene Hes1 is downregulated in PrPKD−1C11 cells as measured in quantitative PCR (qRT-PCR). Further qRT-PCR analysis of effectors of the Notch pathway in 1C11 cells and their PrP-KD derivatives indicates that PrPC silencing drastically reduces Jag1 and Jag2 expression (B). Notch1 level is strongly decreased and Notch2 slightly reduced in PrP-depleted 1C11 cells while Notch3 level is upregulated (C). Neurospheres from Prnp−/− embryos exhibit reduced Hes1 (D), Jag1, Jag2 (E) as well as Notch1 and Notch3 (F) mRNAs as compared with WT controls. The protein levels of Jagged1 (G, I) and Notch1 (H, J) measured through Western blot and normalized to α-tub (G, H) or actin (I, J) are drastically reduced in PrPKD−1C11 cells and Prnp−/− NSC versus their PrPC-expressing counterparts. Data are representative of a set of n = 3 independent experiments. Results are expressed as means ± SEM. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: α-tub, α-tubulin; Jag1, jagged1; Jag2, jagged2; NSC, neurospheres, WT, wild type. Figure 1 Open in new tabDownload slide PrPC depletion compromises Notch signaling in neural stem/progenitor cells. (A): The Notch target gene Hes1 is downregulated in PrPKD−1C11 cells as measured in quantitative PCR (qRT-PCR). Further qRT-PCR analysis of effectors of the Notch pathway in 1C11 cells and their PrP-KD derivatives indicates that PrPC silencing drastically reduces Jag1 and Jag2 expression (B). Notch1 level is strongly decreased and Notch2 slightly reduced in PrP-depleted 1C11 cells while Notch3 level is upregulated (C). Neurospheres from Prnp−/− embryos exhibit reduced Hes1 (D), Jag1, Jag2 (E) as well as Notch1 and Notch3 (F) mRNAs as compared with WT controls. The protein levels of Jagged1 (G, I) and Notch1 (H, J) measured through Western blot and normalized to α-tub (G, H) or actin (I, J) are drastically reduced in PrPKD−1C11 cells and Prnp−/− NSC versus their PrPC-expressing counterparts. Data are representative of a set of n = 3 independent experiments. Results are expressed as means ± SEM. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: α-tub, α-tubulin; Jag1, jagged1; Jag2, jagged2; NSC, neurospheres, WT, wild type. To potentially extend these findings to another paradigm of PrPC depletion, we compared the status of the Notch pathway in neurospheres (NSC) derived from Prnp−/− versus WT embryos [16]. Of note, we first confirmed the negative impact of PrPC depletion on the activity of the Notch pathway since Hes1 mRNA was significantly decreased (66% vs. control) in Prnp−/− NSC (Fig. 1D). Similarly to PrPKD−1C11 cells, Prnp−/− NSC also exhibited reduced levels of Jag1 and Jag2 mRNAs (35% and 19% vs. control, respectively), (Fig. 1E). In this case, however, we found a milder reduction in the transcripts encoding Notch1, together with a strong decrease in Notch3 mRNAs (19% vs. control) (Fig. 1F). At the protein level, we confirmed the downregulation of Jagged1 (16% and 58% vs. control) (Fig. 1G, 1I) in both PrPC-depleted 1C11 cells and NSC, respectively, while the expression of Jagged2 could not be examined in Western blot due to the lack of appropriate antibody. We further monitored reduced protein expression levels of Notch1 (21% and 27% vs. control) (Fig. 1H, 1J) in PrPKD−1C11 cells and Prnp−/− NSC, respectively. Altogether, these observations indicate that the absence of PrPC in 1C11 cells or NSC blunts the expression of Jagged1 and Jagged2 as well as Notch1 and/or Notch3 and obliterates Notch signaling, as mirrored by the reduction in Hes1 mRNA. PrPC Depletion In Vivo Is Associated with Defects in Notch Signaling at Early Embryonic Stages To assess the in vivo relevance of our in vitro findings, we analyzed the expression of Jag1, Jag2, Notch1, Notch2, and Notch3 through qRT-PCR in Prnp−/− versus WT mice at early embryonic stages. We also selected two Notch target genes for analysis: Hes5 and the brain lipid binding protein (Blbp), whose expression in the developing nervous system correlates with Notch activity and defines neural stem and progenitor cells in vivo [21, 22]. We chose to start our analyses at embryonic day (E) 8.5, just after the detection of Prnp expression in the neural folds (E8.0) [23]. Prnp−/− E8.5 embryos exhibited similar level of Jag1 but strongly reduced level of Jag2 compared to their WT counterparts (46% vs. WT) (Supporting Information Fig. S2A). In contrast, PrPC invalidation was associated with mild but significant increases in Notch1 and Notch2 mRNAs (130% and 117% vs. WT, respectively) (Supporting Information Fig. S2B). This may reflect Notch expression in tissues other than the developing nervous system [24]. The marked decrease in Blbp transcripts in Prnp−/− embryos (18% vs. WT) (Supporting Information Fig. S2C) is, however, consistent with an alteration of Notch signaling in the developing nervous system [22]. We next sought to refine our study focusing on the developing CNS by performing analyses on dissected neural tube samples, which can be prepared from E9 onwards. In E9 neural tubes, we confirmed an alteration of the Notch pathway in a PrP-null context with a significant decrease in the expression of Jag2 and most notably Hes5 and Blbp (81%, 75% and 33% vs. WT, respectively) (Supporting Information Fig. S2D, S2F). There was also a trend toward reduced Notch1 level in the absence of PrPC although not statistically significant (Supporting Information Fig. S2E). In Prnp−/− neural tubes corresponding to E9.5, that is, at the completion of cranial fold and caudal neuropore closure, the alteration of the Notch pathway was more prominent with significant reductions in Jag1, Jag2, Notch1 and Notch3 mRNAs (83%, 71%, 75% and 80% vs. WT, respectively) (Fig. 2A, 2B). To the opposite, Notch2 expression was very slightly but significantly increased (112% vs. WT) (Fig. 2B). The decrease in the mRNA levels of Hes5 and Blbp was further amplified at this stage (62% and 29% vs. WT, respectively) (Fig. 2C), revealing defective Notch signaling in the absence of PrPC. In neural tubes from E10.5 embryos, most of these changes vanished or even reversed, with slight but significant increases in Jag1 and Notch1 levels (Fig. 2F, 2G). In contrast, the decrease in Blbp expression persisted, although less pronounced than in E9.5 neural tubes (56% vs. WT) (Fig. 2H). Figure 2 Open in new tabDownload slide The Notch pathway is affected in neural tubes from embryos lacking PrPC, together with the expression of NSC and cell fate specification markers. Quantitative PCR analysis of the expression of Jag1, Jag2 (A, F), Notch1, Notch2, Notch3 (B, G), the Notch target genes Hes5 and Blbp (C, H), the neural stem cell markers Sox2, Nestin, and Pax6 (D, I), the motor neuron progenitor marker Olig2 and the oligodendrocyte progenitor marker PDGFRα (E, J) was performed in neural tube-enriched samples from Prnp−/− early embryos and WT controls at E9.5 and E10.5. In neural tubes from E9.5 Prnp−/− embryos (n = 5), reduced mRNA levels are observed for Jag1 and Jag2 (A), Notch1 and Notch3 (B), Hes5 and Blbp (C) as compared with neural tubes from WT controls. These changes are accompanied by reduced levels of Sox2 and Nestin (D). In neural tubes from E10.5 Prnp−/− embryos (n = 6), Jag1 (F) and Notch1 (G) levels are slightly increased while Blbp (H) and Nestin (I) level remains lower than in neural tubes from WT embryos. At this stage, Olig2 transcripts are significantly increased in Prnp-ablated neural tubes (J). Results are expressed as means ± SEM. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: Jag1, jagged1; Jag2, jagged2; NSC, neurospheres; WT, wild type. Figure 2 Open in new tabDownload slide The Notch pathway is affected in neural tubes from embryos lacking PrPC, together with the expression of NSC and cell fate specification markers. Quantitative PCR analysis of the expression of Jag1, Jag2 (A, F), Notch1, Notch2, Notch3 (B, G), the Notch target genes Hes5 and Blbp (C, H), the neural stem cell markers Sox2, Nestin, and Pax6 (D, I), the motor neuron progenitor marker Olig2 and the oligodendrocyte progenitor marker PDGFRα (E, J) was performed in neural tube-enriched samples from Prnp−/− early embryos and WT controls at E9.5 and E10.5. In neural tubes from E9.5 Prnp−/− embryos (n = 5), reduced mRNA levels are observed for Jag1 and Jag2 (A), Notch1 and Notch3 (B), Hes5 and Blbp (C) as compared with neural tubes from WT controls. These changes are accompanied by reduced levels of Sox2 and Nestin (D). In neural tubes from E10.5 Prnp−/− embryos (n = 6), Jag1 (F) and Notch1 (G) levels are slightly increased while Blbp (H) and Nestin (I) level remains lower than in neural tubes from WT embryos. At this stage, Olig2 transcripts are significantly increased in Prnp-ablated neural tubes (J). Results are expressed as means ± SEM. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: Jag1, jagged1; Jag2, jagged2; NSC, neurospheres; WT, wild type. As a whole, these results indicate that, as observed in vitro, the absence of PrPC in vivo is associated with a downregulation of the Notch pathway starting around E8.5. Importantly, this alteration is observed within a narrow time-window only and is the most apparent in neural tubes from E9.5 embryos, that is, at a stage corresponding to full neural tube closure, when the neuroepithelium is highly proliferative [25]. Further, the decrease in transcripts encoding Hes5 and BLBP, two markers of neuroepithelial and radial glial progenitors [21, 22], mirrors a reduction in the pool of neural stem/progenitor cells in the absence of PrPC. Prnp−/− Early Embryos Show Alterations in Neural Stem Cell Identity Markers In view of the well-established control exerted by the Notch pathway on neural cell fate, we went on to analyze the expression of the stem cell markers Sox2, Nestin and Pax6 in neural tubes from Prnp−/− versus WT embryos. At E9.5, both Sox2 and Nestin mRNAs were reduced in the absence of PrPC (Fig. 2D). The decrease in Nestin expression was still apparent at E10.5 (Fig. 2I), while no significant difference was observed for Pax6 at either stage (Fig. 2D, 2I). Finally, it is now acknowledged that Notch activity in the neural tube contributes to the specification of the most ventral so-called p3 domain and that defective Notch signaling is associated with an expansion of the adjacent, less ventral domain known as pMN (which gives rise to motor neurons and is marked by the expression of Olig2) at the expense of the p3 domain, while increased Notch signaling has opposite effects [26]. In line with this, NICD was shown to negatively regulate the expression of Olig2 in glioma stem cells [27]. In accordance with the above alterations in the Notch pathway in neural tubes from Prnp-ablated mouse embryos, we monitored a twofold increase in the expression of Olig2 in Prnp−/− versus WT neural tubes at E10.5 (Fig. 2J). In contrast, transcripts encoding PDGFRα, a marker of oligodendrocyte progenitor cells [28], were insensitive to Prnp ablation (Fig. 2J). We went on to examine the pattern of Nestin distribution in the neural tubes from E9.5 and E10.5 Prnp−/− versus WT embryos by immunostaining of transverse sections of the neural tube. In WT embryos, Nestin exhibits a regular staining of fibers extending from the ventricular zone to the mantle zone (Fig. 3A, left panels). At E9.5, Nestin staining appeared less organized in Prnp−/− embryos, especially across the neural tube and near the mantle zone in the presumptive hindbrain region (Fig. 3A, top right panels) as well as in the trunk region (Fig. 3A, bottom right panels). These differences were mitigated at E10.5 (Supporting Information Fig. S3, bottom panels). We then compared the distribution of the proliferation marker Ki67 between Prnp−/− and WT embryos. At E9.5, Ki67 staining was less regular in Prnp−/− versus WT embryos (Supporting Information Fig. S4). At E10.5, this effect was more pronounced, notably in the ventral part of the neural tube, with regions devoid of Ki67+ cells (Fig. 3B, compare right versus left panels, see arrow). These observations suggest reduced neural stem cell proliferation in the absence of PrPC. Finally, we found a slight but significant (123%, p < .05, Wilcoxon's test) extension of the Olig2-positive zone in the mid-trunk region of Prnp−/− versus WT embryos at E10.5 (Fig. 3C), which is no longer visible at E11.5 (data not shown). Figure 3 Open in new tabDownload slide Prnp−/− embryos exhibit altered Nestin distribution, as well as Ki67 and Olig2 staining in vivo. (A): Transverse sections of the neuroepithelium at the level of the presumptive hindbrain (top) or mid-trunk region (bottom) from WT (left) and Prnp−/− (right) mouse embryos at E9.5 costained for Nestin (green) and DAPI (blue) (scale bar 20 μm). (B): Transverse sections of the neuroepithelium at the level of the trunk region from WT (left) and Prnp−/− (right) mouse embryos at E10.5 costained for Ki67 (red) and nuclear marker DAPI (blue) (scale bar: 20 μm). (C): Transverse sections of the neuroepithelium at the level of the trunk region from WT (left) and Prnp−/− (right) mouse embryos at E10.5 co-stained for Olig2 (red) and DAPI (blue) (scale bar: 20 μm). Images are representative of a set of n = 3 to 6 Prnp−/− embryos and n = 3 to 6 WT embryos. Figure 3 Open in new tabDownload slide Prnp−/− embryos exhibit altered Nestin distribution, as well as Ki67 and Olig2 staining in vivo. (A): Transverse sections of the neuroepithelium at the level of the presumptive hindbrain (top) or mid-trunk region (bottom) from WT (left) and Prnp−/− (right) mouse embryos at E9.5 costained for Nestin (green) and DAPI (blue) (scale bar 20 μm). (B): Transverse sections of the neuroepithelium at the level of the trunk region from WT (left) and Prnp−/− (right) mouse embryos at E10.5 costained for Ki67 (red) and nuclear marker DAPI (blue) (scale bar: 20 μm). (C): Transverse sections of the neuroepithelium at the level of the trunk region from WT (left) and Prnp−/− (right) mouse embryos at E10.5 co-stained for Olig2 (red) and DAPI (blue) (scale bar: 20 μm). Images are representative of a set of n = 3 to 6 Prnp−/− embryos and n = 3 to 6 WT embryos. Thus, the defects in Notch signaling recorded in early Prnp−/− embryos are accompanied by changes in the expression of defined neural stem identity markers, with apparent—albeit subtle—alterations in neural tube patterning. PrPC Depletion in 1C11 Cells Affects N-Cadherin Dependent Cell-Cell Contacts In the developing CNS, neural stem cells and their progenies form a highly structured epithelial tissue, whose architecture is notably regulated through N-cadherin-based intercellular contacts [29]. N-cadherin contributes to the regulation of the balance between self-renewal and differentiation [29] and the disruption of adherens junction integrity is associated with premature differentiation of neural stem cells [30]. To get insight into the impact of PrPC knockdown on the architecture of 1C11 cells, we carried out immunofluorescence experiments to assess the distribution of N-cadherin as well as β-catenin, which associates with the intracellular domain of cadherins at the adherens junction. 1C11 cells form an epithelium and both N-cadherin and β-catenin were evenly distributed at the membrane, lining cell-cell contacts (Fig. 4A). In contrast, these contacts were strongly reduced in PrPC-depleted cells. In line with this, both N-cadherin and β-catenin membrane stainings were markedly affected (Fig. 4A). Similar alterations were found in 1C11 cells 48h after transfection with two distinct PrPC-targeting siRNAs (Supporting Information Fig. S5–S7). Changes monitored in PrPKD−1C11 cells correlated with an increase in N-cadherin mRNA (N-cad) (184% vs. control) (Supporting Information Fig. S8A) and protein (158% vs. control) (Fig. 4B) levels in PrPC depleted cells. Finally, while the expression of β-catenin mRNA (ß-cat) was not statistically different (Supporting Information Fig. S8A), we monitored a significant decrease at the protein level in PrPKD−1C11 cells (60% vs. control) (Fig. 4B). This observation is consistent with reduced stabilization of β-catenin due to decreased N-cadherin-mediated cell adhesion [31]. Figure 4 Open in new tabDownload slide PrPC depletion affects N-cadherin distribution in vitro and in vivo. (A): PrPC-deficient 1C11 cells exhibit drastic changes in N-cadherin and β-catenin staining associated with disrupted cell-cell contacts, as compared to 1C11 cells. Scale bar = 20 µm. (B): Western blot analysis of PrPKD−1C11 cell extracts indicates increased N-cadherin and decreased β-catenin protein expression. Protein levels were normalized to α-tub. Data are representative of n = 3 independent experiments. (C): Transverse section of the neuroepithelium at the level of the presumptive caudal trunk region from wild type (WT) (left) and Prnp−/− (right) mouse embryos at E9.5 costained for N-cadherin (green) and DAPI (blue) (scale bar: 10 μm). (D, E): Relative distribution of N-cadherin staining intensity from the lumen to the pial surface of the neural tube in WT (D) and Prnp−/− (E) embryos. Images are representative of n = 3 embryos of each genotype. Abbreviations: N-cad, N-cadherin; β-cat, β-catenin; α-tub, α-tubulin. Figure 4 Open in new tabDownload slide PrPC depletion affects N-cadherin distribution in vitro and in vivo. (A): PrPC-deficient 1C11 cells exhibit drastic changes in N-cadherin and β-catenin staining associated with disrupted cell-cell contacts, as compared to 1C11 cells. Scale bar = 20 µm. (B): Western blot analysis of PrPKD−1C11 cell extracts indicates increased N-cadherin and decreased β-catenin protein expression. Protein levels were normalized to α-tub. Data are representative of n = 3 independent experiments. (C): Transverse section of the neuroepithelium at the level of the presumptive caudal trunk region from wild type (WT) (left) and Prnp−/− (right) mouse embryos at E9.5 costained for N-cadherin (green) and DAPI (blue) (scale bar: 10 μm). (D, E): Relative distribution of N-cadherin staining intensity from the lumen to the pial surface of the neural tube in WT (D) and Prnp−/− (E) embryos. Images are representative of n = 3 embryos of each genotype. Abbreviations: N-cad, N-cadherin; β-cat, β-catenin; α-tub, α-tubulin. Altogether, these data indicate that PrPC deficiency in the 1C11 neuroprogenitor promotes a drastic reduction of intercellular contacts mediated by N-cadherin, as well as a decrease of β-catenin level. Prnp−/− Early Embryos Exhibit N-Cadherin Patterning Alterations In view of the alterations of N-cadherin expression and staining in PrPKD−1C11 cells, we then compared its expression in neural tubes from WT and Prnp−/− embryos at E9, E9.5 and E10.5. Whatever the developmental stage analyzed, we found similar N-cad mRNA levels in Prnp−/− and WT mice (Supporting Information Fig. S8B). Next, we examined the pattern of N-cadherin in Prnp−/− and WT embryos at E8.5 and E9.5 by immunostaining of transverse sections of the neural tube in the presumptive hindbrain and mid-trunk regions. At E8.5 in the presumptive mid-trunk region, while the pattern of N-cadherin staining in WT embryos was mainly localized to the apical face of the neuroepithelium (i.e. the ventricular zone, where neural stem cells reside), it appeared to extend toward the external part of the neuroepithelium (i.e. the mantle zone) in Prnp−/− embryos (Supporting Information Fig. S9). At E9.5, the difference in N-cadherin staining persisted in a fraction (n = 2 out of 3) of the Prnp−/− embryos, most notably in the caudal trunk region (Fig. 4C). When we measured the relative distribution of N-cadherin staining along the thickness of the neural tube in the caudal trunk region, from the apical face to the mantle zone, we found that the profile of N-cadherin distribution was very similar among WT embryos, with a peak in intensity in the apical-most part of the neural tube (Fig. 4D). While this peak in intensity is still present in the apical-most part of the neural tube in Prnp−/− embryos, we observed that 2 out of 3 Prnp−/− embryos exhibited shifted N-cadherin distribution toward the mantle zone (Fig. 4E). These data argue for subtle but consistent alterations in N-cadherin distribution in the neural tube of Prnp−/− embryos, which, interestingly, are reminiscent of those observed in the spinal cord after transfection with a dominant negative form of N-cadherin [30]. Intact Jagged-Notch Mediated Signaling is Necessary for the Maintenance of Cell-Cell Contacts Having shown that PrPC depletion disrupts cell architecture and affects Notch signaling, we probed the occurrence of a causal relationship between the two sets of alterations. To this purpose, we sought to restore the Jagged-Notch cascade in PrPKD−1C11 cells by growing the cells on dishes coated with Jagged1 for 24 hours. With Jagged1, we observed a robust (369% vs. control) increase in transcripts encoding the Notch target gene Hes1, indicating that the Notch pathway is indeed turned on (Fig. 5C). In these conditions, Hes1 mRNAs reached similar levels as those monitored in the parental 1C11 cells (data not shown). This treatment actually boosted the expression of Jag1, Jag2 and Notch1 (216%, 179%, and 175% vs. controls, respectively) (Fig. 5A, 5B), highlighting a positive autoregulatory loop induced by Jagged1, as reported in the literature [32, 33]. We next examined N-cadherin expression and found no variation at the mRNA level (Fig. 5D), but an increased protein level (138% vs. control) in PrPKD−1C11 cells exposed to Jagged1 (Fig. 5E). In immunofluorescence experiments, we observed a strengthening of cellular contacts, which were regularly stained with anti-N-cadherin antibodies, suggesting the establishment of N-cadherin-dependent cell-cell interactions in PrPC-depleted cells upon rescue of the Notch pathway (Fig. 5F). We finally analyzed the distribution of β-catenin, which is expected to be retained at the plasma membrane upon cadherin-mediated adhesion. Antibodies against β-catenin indeed yielded an even submembrane staining in Jagged-treated PrPKD−1C11 cells (Fig. 5F). Altogether, these data indicate that the restoration of an active Notch pathway in PrP-deficient cells rescues N-cadherin-mediated cell adhesion. Figure 5 Open in new tabDownload slide Restoring an active Notch pathway in PrPKD−1C11 cells induces N-cadherin dependent cell contacts. (A-C) PrPKD−1C11 cells were grown on Jagged1-coated dishes and mRNAs encoding the Notch ligands Jag1 and Jag2 (A), receptors Notch1, Notch2, Notch3 (B) and target gene Hes1 (C) were measured through quantitative PCR (qRT-PCR). (D) qRT-PCR analysis of N-cad expression in PrPKD−1C11 cells grown on Jagged1-coated dishes shows similar level as in control PrPKD−1C11 cells. (E) Western blot analysis of N-cadherin expression in PrPKD−1C11 cells grown on Jagged1-coated dishes reveals higher level as compared with control PrPKD−1C11 cells. Protein levels were normalized to α-tub. (F) PrPKD−1C11 cells grown on Jagged1-coated dishes were stained with antibodies against N-cadherin or β-catenin. Scale bar = 20 µm. Results in (A-E) are expressed as means ± SEM. of n = 3 independent experiments. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: Jag1, Jagged1; Jag2, Jagged2; N-cad, N-cadherin; α-tub, α-tubulin. Figure 5 Open in new tabDownload slide Restoring an active Notch pathway in PrPKD−1C11 cells induces N-cadherin dependent cell contacts. (A-C) PrPKD−1C11 cells were grown on Jagged1-coated dishes and mRNAs encoding the Notch ligands Jag1 and Jag2 (A), receptors Notch1, Notch2, Notch3 (B) and target gene Hes1 (C) were measured through quantitative PCR (qRT-PCR). (D) qRT-PCR analysis of N-cad expression in PrPKD−1C11 cells grown on Jagged1-coated dishes shows similar level as in control PrPKD−1C11 cells. (E) Western blot analysis of N-cadherin expression in PrPKD−1C11 cells grown on Jagged1-coated dishes reveals higher level as compared with control PrPKD−1C11 cells. Protein levels were normalized to α-tub. (F) PrPKD−1C11 cells grown on Jagged1-coated dishes were stained with antibodies against N-cadherin or β-catenin. Scale bar = 20 µm. Results in (A-E) are expressed as means ± SEM. of n = 3 independent experiments. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: Jag1, Jagged1; Jag2, Jagged2; N-cad, N-cadherin; α-tub, α-tubulin. PrPC Controls the Expression of EGFR via Notch We finally sought to assess the impact of PrPC depletion on a third protagonist, EGFR, which is known to modulate the proliferation, migration and fate of neural progenitors during embryonic development [34, 35]. EGFR was identified as a direct Notch target gene in adult neural stem cells [36], and its interaction with the Notch pathway regulates adult neural stem cell self-renewal [37]. We found a decrease in the expression of EGFR in PrPKD−1C11 cells as compared to their parental 1C11 cells at both the mRNA and protein levels (57% and 55% vs. control) (Fig. 6A, 6B). A similar impact of PrPC depletion on EGFR expression was observed in Prnp−/− NSC (Fig. 6C, 6D), as well as in vivo in neural tubes samples from Prnp−/− mice at E9 and E9.5 (Fig. 6E). The decrease in Egfr transcripts persisted at E10.5, with levels reaching only 55% of control (Fig. 6E). Thus, the downregulation of Egfr monitored in the developing CNS in Prnp−/− animals correlated with the alteration of Notch activity (Fig. 2). Figure 6 Open in new tabDownload slide PrPC positively controls EGFR expression via the Notch pathway. (A-D): Quantitative PCR (qPCR) and Western blot analyses indicate that EGFR mRNA (A, C) and protein (B, D) levels are reduced in PrPKD−1C11 cells versus their parental 1C11 cells (A, B) as well as in Prnp−/− versus WT NSC (C, D). (E): qPCR analysis of Egfr expression performed in neural tube-enriched samples from Prnp−/− early embryos and WT controls at E9, E9.5 and E10.5 reveals reduced levels at all stages in the absence of PrPC (n = 3-6 samples each). (F, G): Quantitative PCR and Western blot analyses indicate that EGFR mRNA (F) and protein (G) levels are enhanced in PrPKD−1C11 cells grown on Jagged1-coated dishes as compared to untreated PrPKD−1C11 cells. Protein levels were normalized to α-tub (B, G) or actin (D). Data shown on (A-D) and (F, G) are representative of a set of n = 3 independent experiments. Results are expressed as means ± SEM. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: EGFR, epidermal growth factor receptor; NSC, neurospheres; WT, wild type; α-tub, α-tubulin. Figure 6 Open in new tabDownload slide PrPC positively controls EGFR expression via the Notch pathway. (A-D): Quantitative PCR (qPCR) and Western blot analyses indicate that EGFR mRNA (A, C) and protein (B, D) levels are reduced in PrPKD−1C11 cells versus their parental 1C11 cells (A, B) as well as in Prnp−/− versus WT NSC (C, D). (E): qPCR analysis of Egfr expression performed in neural tube-enriched samples from Prnp−/− early embryos and WT controls at E9, E9.5 and E10.5 reveals reduced levels at all stages in the absence of PrPC (n = 3-6 samples each). (F, G): Quantitative PCR and Western blot analyses indicate that EGFR mRNA (F) and protein (G) levels are enhanced in PrPKD−1C11 cells grown on Jagged1-coated dishes as compared to untreated PrPKD−1C11 cells. Protein levels were normalized to α-tub (B, G) or actin (D). Data shown on (A-D) and (F, G) are representative of a set of n = 3 independent experiments. Results are expressed as means ± SEM. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: EGFR, epidermal growth factor receptor; NSC, neurospheres; WT, wild type; α-tub, α-tubulin. Next, we found an upregulation of EGFR mRNA (167% vs. control) and protein (237% vs. control) in PrPKD−1C11 cells exposed to Jagged1 (Fig. 6F, 6G). Altogether, these data indicate that the absence of PrPC in vitro and in vivo affects the expression of EGFR and provide evidence for an involvement of the Notch pathway in this regulation. EGFR Exerts a Reciprocal Negative Control on Notch and PrPC In our last set of experiments, we sought to delineate how EGFR may participate to the stem cell properties of 1C11 cells, in relation with PrPC. To this purpose, 1C11 cells were exposed to EGF (10 ng/ml, 24 hours) and further analyzed for the status of the Notch pathway as well as for PrPC expression through qRT-PCR. We found a clear-cut negative effect of EGF on the Notch pathway, with significant reductions in the expression of Jag1 (59% vs. control) (Fig. 7A), Notch1 and Notch3 (76% and 68% vs. control, respectively) (Fig. 7B) as well as Hes1 and Blbp (76% and 31% vs. control, respectively) (Fig. 7C). There was also a trend toward reduced Jag2 mRNAs, although not statistically significant (Fig. 7A). In contrast, Notch2 mRNAs were unaffected by EGF (Fig. 7B). Interestingly, EGF also promoted a reduction in the expression of the PrPC-encoding Prnp transcripts (76% vs. control) (Fig. 7D). The EGF-dependent downregulation of PrPC was confirmed at the protein level (76% vs. control) (Fig. 7E). Thus, EGFR activation appears to act as a brake on both the Notch pathway and PrPC expression. Figure 7 Open in new tabDownload slide EGFR exerts a negative feedback action on Notch signaling and PrPC expression. (A-D): 1C11 cells were exposed to EGF (10 ng/ml, 24 hours) and mRNAs encoding the Notch ligands Jag1 and Jag2 (A), receptors Notch1, Notch2, Notch3 (B) and target gene Hes1 and Blbp (C), as well as Prnp (D) were measured through quantitative real-time PCR (qRT-PCR). (E): Western blot analysis of PrPC expression in cell extracts from 1C11 cells exposed or not to EGF (10 ng/ml, 24 hours). Middle panel shows full-length PrPC deglycosylated with PNGaseF-treatment. Protein levels were normalized to α-tub. Data shown are representative of a set of n = 3 independent experiments. (F-J): 1C11 cells were grown on Jagged1-coated dishes and concomitantly exposed or not to EGF (10 ng/ml, 24 hours). Jag1, Jag2 (F), Notch1, Notch2, Notch3 (G) Hes1, Blbp (H), Egfr (I) and Prnp (J) mRNAs were measured through qRT-PCR. Results are expressed as means ± SEM. of n = 4 independent experiments. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: EGFR, epidermal growth factor receptor; Jag1, jagged1; Jag2, jagged2; α-tub, α-tubulin. Figure 7 Open in new tabDownload slide EGFR exerts a negative feedback action on Notch signaling and PrPC expression. (A-D): 1C11 cells were exposed to EGF (10 ng/ml, 24 hours) and mRNAs encoding the Notch ligands Jag1 and Jag2 (A), receptors Notch1, Notch2, Notch3 (B) and target gene Hes1 and Blbp (C), as well as Prnp (D) were measured through quantitative real-time PCR (qRT-PCR). (E): Western blot analysis of PrPC expression in cell extracts from 1C11 cells exposed or not to EGF (10 ng/ml, 24 hours). Middle panel shows full-length PrPC deglycosylated with PNGaseF-treatment. Protein levels were normalized to α-tub. Data shown are representative of a set of n = 3 independent experiments. (F-J): 1C11 cells were grown on Jagged1-coated dishes and concomitantly exposed or not to EGF (10 ng/ml, 24 hours). Jag1, Jag2 (F), Notch1, Notch2, Notch3 (G) Hes1, Blbp (H), Egfr (I) and Prnp (J) mRNAs were measured through qRT-PCR. Results are expressed as means ± SEM. of n = 4 independent experiments. *, p < .05; **, p < .01 versus control (Student's t test). Abbreviations: EGFR, epidermal growth factor receptor; Jag1, jagged1; Jag2, jagged2; α-tub, α-tubulin. Then, to mimic the neurogenic stem cell niche [38], we sought to boost the Notch pathway by growing 1C11 cells on Jagged1-coated dishes. As observed with PrPKD−1C11 cells, this treatment promoted a strong increase in the expression of various key components of the Notch pathway itself: Jag1 and Jag2 (290% and 280% vs. controls, respectively) (Fig. 7F), Notch1 and Notch3 (225% and 220% vs. controls, respectively) (Fig. 7G), Hes1 and Blbp (225% and 860% vs. controls, respectively) (Fig. 7H). In line with data obtained with PrPKD−1C11 cells upon restoration of an active Notch pathway, Egfr mRNA level was also upregulated in Jagged1-treated 1C11 cells (195% vs. control) (Fig. 7I). Of note, the expression of Prnp was also modestly but significantly increased in these conditions (129% vs. control) (Fig. 7J). Finally, in view of the crosstalk interactions between Notch and EGFR signaling in various paradigms [39, 40] and most notably in the regulation of adult neural stem cell self-renewal [37], we examined the impact of combined exposure of 1C11 cells to Jagged1 and EGF on the same target genes. Interestingly, EGF mitigated some of the effects of Jagged1, by counteracting the raise in expression of Jag2, Blbp and Prnp (Fig. 7F, 7H, 7J). This suggests that EGFR activation partly opposes the action of the Notch pathway, notably in the control of the stemness marker BLBP. We may thus conclude from these data that, EGFR, which is a target of the PrPC-Notch signaling axis, exerts a negative feedback action on both PrPC and Notch when it is activated. Thus, the induction of EGFR expression downstream from PrPC appears to poise cells to respond to an EGF signal, which antagonizes stem cell maintenance and likely favors the conversion into a more mature neuronal progenitor state. Discussion Although Prnp−/− mice develop without overt abnormalities [41], they have been instrumental in unveiling an unexpected involvement of PrPC in the self-renewal of various stem/progenitor cells, most notably along the hematopoietic and neural lineages [5, 6]. Despite recent progress regarding the contribution of this protein to stem cell homeostasis [4], and notably to Hedgehog signaling [12], the cellular and molecular mechanisms at play are yet to be fully understood. Here, by combining in vitro and in vivo experiments, we uncover a critical role of PrPC in the control of Notch signaling in neural progenitors, which in turn influences neural stem cell markers, N-cadherin-mediated cell contacts and EGFR expression. Our study further elaborates on an intricate interplay between Notch and EGFR signaling, as well as PrPC expression itself. Uncovering the PrPC-Notch connection was facilitated by our in vitro models, the 1C11 neuronal progenitor and its PrP-KD derivatives [15], and primary neurospheres from WT and Prnp−/− mouse embryos. The defect in Notch signaling in PrP-depleted cells is evidenced by the prominent reduction in the expression of the Notch target gene Hes1. From a mechanistic point of view, our data reveal that the absence of PrPC is associated with a dramatic decrease in the expression of the Notch ligands Jagged1 and Jagged2 as well as that of the Notch1/Notch3 receptors. From these cellular observations, screening early Prnp−/− embryos allowed us to delineate a narrow time-window when defects in the Notch pathway can be revealed under PrPC ablation. Our analyses at various embryonic stages encompassing neural tube closure indicate that these defects initiate at E8.5-E9 and are most apparent at E9.5. This time-window corresponds to that of alterations in Hedgehog signaling and tubulin post-translational modifications that we recently uncovered in Prnp−/− embryos [12]. At these stages, PrPC is abundantly expressed in the ventricular zone of the neural tube [12], where neural stem cells reside. As in vitro (PrPKD−1C11 cells and neurospheres from Prnp−/− embryos), alterations monitored in the neural tubes of Prnp−/− embryos include reductions in the mRNA expression of both Jag1 and Jag2, and their receptors Notch1 and Notch3. The milder and temporally-restricted defects detected in neural tubes from Prnp−/− embryos, in contrast to our in vitro observations, might be accounted for by the coexistence of diverse cell types, whose relative number may vary depending on the developmental stage and the dorso-ventral and rostro-caudal axes. Another likely explanation would be the occurrence of dynamic mechanisms of adaptation in vivo, as recently described in the case of Hedgehog signaling [42]. Importantly, that the ablation of PrPC compromises Notch signaling in vivo is firmly established by the strong reduction in the mRNA levels of the two Notch target genes Hes5 and Blbp, starting from E8.5-E9. Because these genes specify neuroepithelial and radial glial cells in the developing CNS [21, 22], these data argue that the lack of PrPC transiently affects the pool of early neural stem/progenitor cells and causes premature commitment. This conclusion is supported by the altered pattern of Ki67 in Prnp−/− embryos. The reduction in the expression of Nestin in neural tubes from Prnp−/− embryos at E9.5 and E10.5, together with an increase in Olig2 expression at the latter stage, further corroborates this view. These changes at the mRNA level are accompanied by a partly disorganized pattern of Nestin staining at E9.5 and a subtle extension of the Olig2-positive domain at E10.5 in the absence of PrPC. Overall, these data are in line with the positive regulation of Nestin expression by Notch [43] as well as with the recent demonstration that Notch activity influences dorso-ventral patterning of the neural tube by negatively regulating the expression of Olig2 [26]. A second contribution of this work is the demonstration that PrPC depletion is associated with a disruption of N-cadherin-based cell-cell contacts. This alteration of cell architecture arises, at least partly, from defects in Notch signaling. Indeed, PrPKD−1C11 cells exposed to Jagged1 restored N-cadherin-dependent intercellular contacts. These data fit in with the reported defects in apico-basal polarity of the neural tube in early embryos following blockade of the Notch pathway [44] and with the notion that N-cadherin patterning is affected when apico-basal polarity is disrupted [45]. In line with our in vitro data and in agreement with the transient reduction in Notch signaling in the developing CNS of animals lacking PrPC (Fig. 2), we observed subtle defects in N-cadherin patterning in the neuroepithelium of Prnp−/− embryos. These alterations notably include shifted distribution from the apical face of the neuroepithelium toward the mantle zone. Because N-cadherin-mediated contacts participate to the maintenance of neuroepithelial progenitors in the ventricular zone [30], such defects further argue that PrPC deficiency impacts on the proper expansion of the neural stem cell pool. In addition, in view of the link between cadherin-dependent contacts and β-catenin signaling [31], these data suggest that the lack of PrPC affects the pool of β-catenin in neural stem and/or progenitor cells of the neuroepithelium, which may in turn impact on their proliferation/differentiation balance. Altogether, these findings add to the complex interplay between PrPC and cadherins, since PrPC was previously shown to contribute to the recruitment of E-cadherin at cell-cell contacts [46, 47] or to the trafficking of N-cadherin in differentiating neurons [48]. Our data finally posit EGFR as a third protagonist whose expression is affected by the absence of PrPC. Reductions in EGFR expression were observed both in vitro in PrPKD−1C11 cells and neurospheres from Prnp−/− embryos, as well as in vivo in Prnp−/− embryos. Thus, beyond impinging on EGFR activity as previously described [47, 49], PrPC also contributes to the regulation of Egfr transcripts. In line with [36], we showed that EGFR expression is positively controlled by Notch signaling. Finally, upon EGF exposure, both Notch signaling and PrPC (mRNA and protein) levels were toned down, suggesting that the induction of EGFR downstream from the PrPC-Notch network poises cells to respond to an EGFR-activating stimulatory signal that will conversely dampen the PrPC-Notch axis. In this respect, it is noteworthy that EGFR signaling contributes to the recruitment of neural progenitors during development [34], or after injury [50]. According to our results, this process may involve a negative feedback on Notch and PrPC. Altogether, our study allows to propose a molecular cascade linking the various actors under the control of PrPC (Supporting Information Fig. S10): (1) PrPC positively regulates the expression of Notch ligands and that of the Notch receptors and its ablation compromises Notch signaling; (2) proper Notch signaling supports N-cadherin-mediated cell-cell contacts and (3) exerts a positive feedback action on PrPC; (4) downstream from PrPC, Notch signaling additionally upregulates the expression of EGFR, which, when activated, (5) negatively regulates both PrPC and Notch. Hence, our data strengthen the view that PrPC, the Notch pathway and EGFR belong to a gene regulatory network, whose imbalance may shift toward “more stemness” (higher Notch activity induces higher PrPC expression and vice-versa) or “less stemness” (EGFR activation dampens Notch signaling and PrPC expression). In view of the established roles of these various actors in the self-renewal of neuroprogenitors [30, 37, 51], this global cascade ultimately supports the involvement of PrPC in neural stem cell maintenance. In vivo, the changes observed in embryos lacking PrPC may altogether favor a precocious differentiation of neural stem/progenitor cells (see above). The subtle and transient defects occurring in the developing CNS of Prnp−/− mice could actually account for the behavioral and cognitive deficits recorded in mice lacking PrPC [52], in line with the proposed neurodevelopmental origin of psychiatric disorders [53]. Another hypothesis that would be worth considering is that the absence of PrPC disturbs the homeostasis of adult neural stem cells, since Notch signaling controls neural stem cell maintenance in the adult brain as well [54]. Incidentally, the Notch pathway appears to be also affected by the lack of PrPC after birth as the expression of Jag2 mRNA fails to increase in the hippocampus of Prnp−/− mice during postnatal development, while Jag1 mRNA level is higher in the same brain region of adult Prnp−/− mice versus their WT counterparts [55]. From a pathological point of view, the latter observations added to our findings may have relevance as to the disturbed cell fate of adult neural stem cells upon prion infection [56]. Conclusion In summary, our work demonstrates that PrPC contributes to the regulation of a major developmental signaling pathway, namely Notch, and thereby supports the maintenance of neural stem cell architecture as well as EGFR expression. Beyond neural stem/progenitor cells, the molecular scenario brought to light in the present study may more broadly account for the contribution of PrPC to the self-renewal of diverse types of stem cells within their niche. Since the overall protagonists shown here to be under the control of PrPC sustain the self-renewal of HSCs in their bone marrow niche [57–59], our results may notably accommodate the requirement of PrPC for the long-term maintenance of HSCs [5]. Acknowledgments We thank Dr. C. Brou (Institut Pasteur, Paris) for the kind gift of Notch antibody, Céline Urien (INRA-Jouy) and the MIMA2 platform (INRA-Jouy) for access to confocal microscopy and Johan Castille for mouse breeding. This work was supported by funds from ARC (Grant SFI2011205489) and INSERM to S.M.-R. S.M.-L. and T.Z.H. were supported by fellowships from DIM StemPole (Region Ile de France) and Fondation pour la Recherche Médicale, respectively. The J.-M.T laboratory was supported by grants from the Spanish Plan Nacional de I+D+I (RTA2012-00004 and AGL2012-37988-C04). S.H. is currently affiliated to CNRS UMR7592, Jacques Monod Institute, Paris Cedex 13, France; J.H.-R. is currently affiliated to Centre de Recherche du CHU de Québec, Université Laval, Québec, Québec, Canada. Author Contributions S.M.L.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript; S.H., T.Z.H., J.H.R., B.P., and C.T.R.: Collection and assembly of data, data analysis and interpretation and final approval of the manuscript; A.V.D.: Provision of study material; J.M.T.: Provision of study material and final approval of the manuscript; V.B., J.L.V., and J.M.L.: Data analysis and interpretation and final approval of the manuscript; S.M.R.: Conception and design, financial support, data analysis and interpretation, manuscript writing and final approval of the manuscript. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. 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[PMC][10.1016/j.celrep.2013.07.048] [24012753] Google Scholar Crossref Search ADS PubMed WorldCat Author notes S.H. and T.Z.H. contributed equally to this article. © 2016 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The Cellular Prion Protein Controls Notch Signaling in Neural Stem/Progenitor Cells
JO - Stem Cells
DO - 10.1002/stem.2501
DA - 2017-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-cellular-prion-protein-controls-notch-signaling-in-neural-stem-74fxOJFXci
SP - 754
EP - 765
VL - 35
IS - 3
DP - DeepDyve
ER -