Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal

Abstract

Specialized cellular microenvironments, or 'niches', modulate stem cell properties, including cell number, self-renewal and fate decisions. In the adult brain, niches that maintain a source of neural stem cells (NSCs) and neural progenitor cells (NPCs) are the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus of the hippocampus. The size of the NSC population of the SVZ at any time is the result of several ongoing processes, including self-renewal, cell differentiation, and cell death. Maintaining the balance between NSCs and NPCs in the SVZ niche is critical to supply the brain with specific neural populations, both under normal conditions or after injury. A fundamental question relevant to both normal development and to cell-based repair strategies in the central nervous system is how the balance of different NSC and NPC populations is maintained in the niche. EGFR (epidermal growth factor receptor) and Notch signalling pathways have fundamental roles during development of multicellular organisms. In Drosophila and in Caenorhabditis elegans these pathways may have either cooperative or antagonistic functions. In the SVZ, Notch regulates NSC identity and self-renewal, whereas EGFR specifically affects NPC proliferation and migration. This suggests that interplay of these two pathways may maintain the balance between NSC and NPC numbers. Here we show that functional cell-cell interaction between NPCs and NSCs through EGFR and Notch signalling has a crucial role in maintaining the balance between these cell populations in the SVZ. Enhanced EGFR signalling in vivo results in the expansion of the NPC pool, and reduces NSC number and self-renewal. This occurs through a non-cell-autonomous mechanism involving EGFR-mediated regulation of Notch signalling. Our findings define a novel interaction between EGFR and Notch pathways in the adult SVZ, and thus provide a mechanism for NSC and NPC pool maintenance.

Figure 1. EGFR overexpression reduces NSC proliferation and self renewal in the adult mouse SVZ

(a) Decreased number of GFAP⁺BrdU⁺ and GFAP⁺Nestin⁺ NSCs, and increased number of NG2⁺ cells in the SVZ of the CNP-hEGFR mouse (*p<0.05). Means ± SEM. (b) Decreased percentage of GFAP⁺Nestin⁺LeX⁺ NSCs and increased percentage of GFAP⁺S100b⁺ astrocytes in the SVZ of the CNP-hEGFR mouse (**p<0.02). (c) Neurospheres from WT (c1) and CNP-hEGFR (c2) cells. (d) Reduced neurosphere numbers (d1) and size (d2) in cultures from CNP-hEGFR mice (*p<0.02; **p<0.001).

Figure 2. EGFR overexpression downregulates Notch signaling in the SVZ, and NICD overexpression rescues proliferation and self-renewal of SVZ NSCs

(a) Notch signaling is downregulated, but Numb is upregulated in the SVZ of CNP-hEGFR mice. (b) NICD, RBPjk, Hes1 are upregulated in the Wa2 SVZ, and Numb is downregulated. (c-d) High NICD levels were detected after in vivo electroporation by immunostaining. (e–f) GFAP-GFP⁺ neurosphere number (f1) and size (f2) increased after NICD electroporation. Means (n=3) ± SEM (*p<0.01). Scale bars = 100μm. (g) High b-gal expression levels in the SVZ after viral infection in WT (g1) and CNP-hEGFR (g2) ventricles. (h) Ad-NICD transduction increased NICD, but reduced Numb expression. (i) Ad-NICD infection increased neurosphere formation in CNP-hEGFR mice, but not in WT. Means (n=3) ± SEM (*p<0.02). Scale bars = 200μm.

Figure 3. EGFR-expressing NPCs regulate NSC properties through a non-autonomous cellular mechanism

(a) Increased BrdU⁺ cells and reduced GFAP⁺ cells in CNP-hEGFR mice (**p<0.002). AraC: Increased GFAP⁺ cells compared with NaCl in CNP-hEGFR mice (**p<0.002; *p<0.05). (b1) AraC upregulates Hes1 and RBPjk mRNAs and downregulates Numb. (b2) AraC upregulates NICD protein and downregulates Numb. (c) CNP-hEGFR mice: AraC increases NSC proliferation and self-renewal (*p<0.001). Means (n=3) ± SEM. (d) Reduced proliferation and self-renewal in WT NSCs cultured with CNP-hEGFR NPCs, as compared with WT cells. Ad-NICD overexpression rescued NSC proliferation. Means (n=3) ± SEM (*p<0.02). (e) Higher proliferation and self-renewal of NSCs cultured with NPCs of the Wa2 mouse, as compared with WT cells. Means (n=3) ± SEM (*p<0.05).

Figure 4. EGFR signaling reduces Notch1 expression through Numb

(a–d) Hes1-dsRED-NICD co-electroporation in GFAP-GFP and GFAP-GFP/CNP-hEGFR mice. More Hes1-dsRED⁺GFAP-GFP⁺ cells are observed in GFAP-GFP mouse. NICD increases GFAP-GFP⁺Hes1-dsRED⁺ cell number. (e) WT cells infected with: i) LacZ or NICD, or ii) GFP retrovirus (CLE-GFP) or EGFR-GFP. NICD upregulates NICD and Notch1, but downregulates Numb. EGFR reduces Notch1 and NICD, but increases Numb. (f) DAPT upregulates Numb. (g) Increased Numb/Notch1 immunoprecipitation correlates with enhanced degradation. (h) Ad-NICD reduces Notch1/Numb interaction and Notch1 degradation in CNP-hEGFR SVZ. (i) EGFR increases Numb and reduces NICD; PD168393 prevents these effects. (j) EGFR increases Numb/Notch immunoprecipitation, and Notch1 degradation. (k) hEGFR siRNAs decrease Numb/Notch1 interaction and Notch1 degradation in CNP-hEGFR cells. (l–o) Numb siRNA knockdown enhances Notch signaling in CNP-hEGFR NSCs. Scrab=scrambled siRNA. (l) Numb siRNA downregulates Numb and upregulates Notch1. (m) Numb siRNA decreases Numb/Notch interaction and Notch1 degradation. (n–o) Co-electroporation of Scrab or Numb siRNA, and Hes1-dsRED constructs. Numb siRNA increases the number of Hes1-dsRED⁺CAG-GFP⁺ cells. Scale bar = 50μm.