Figure 2: EGFL7 affected SVZ-derived neurospheres.

(a) Scheme of neurosphere assay. (b) Ectopic EGFL7 expression increased the amount of small neurospheres (19.7±3.31 versus 1.54±1.34% in AdMock; n=100; P<0.01), while (c) EGFL7^−/− spheres displayed an increased size (35.3±2.98 versus 20.15±5.23% in WT; n=100; P<0.01). (d) SILAC-based mass spectrometric proteome analyses of EGFL7^−/− and WT neurospheres was performed after 2 and 5 d in culture. WT spheres upregulated proteins involved in neuronal signalling, for example, glutamate receptors or synaptic proteins at d5, suggesting differentiation in vitro. However, EGFL7^−/− spheres displayed limited upregulation of these proteins but instead, retained an expression profile at d5 that was comparable to WT and knockout (KO) spheres at d2 in culture with high levels of riboproteins and metabolic enzymes. This indicates that EGFL7^−/− spheres displayed delayed differentiation but sustained proliferation in vitro resulting in larger spheres. (e) Cell cycle analysis revealed an increased amount of cells in the G₂/M phase (9.08±0.62 versus 6.91±0.61% in WT; n=3; P<0.05) at the expense of cells in G₀/G₁ phase (65.36±2.40 versus 69.76±0.80% in WT; n=3; P<0.05). (f) FACS sorting of qNSCs, aNSCs, TAPs and NBs from the SVZ of EGFL7^−/− and WT mice was performed using the following markers: qNSCs (GLAST⁺/CD133⁺/EGFR⁻), aNSCs (GLAST⁺/CD133⁺/EGFR⁺), TAPs (CD133⁻/EGFR⁺) and NBs (CD24⁺). Subsequently, an in vitro neurosphere formation assay yielded an increased amount of spheres derived from the aNSC population (32.3±5.1 versus 21.7±3.7% in WT; n=3; P<0.05). (g–r) Time-lapse video microscopy analysis of primary NSC/NPC cultures. (g–p) Individual time points of the experiment. (q) Post hoc cell identification by IF. Scale bar, 50 μm. (r) A typical differentiation tree of EGFL7^−/− aNSCs displays initial cell divisions (red arrows) and differentiation but subsequently, re-entering of cell cycle (yellow arrows) and further differentiation, which is not observed in WT. Statistical analysis was performed by Student’s t-test/Mann–Whitney U-test. For all experiments data, error bars indicate s.d.