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. 2013 May 10;8(5):e62959. doi: 10.1371/journal.pone.0062959

Figure 6. Loss of Notch signaling accelerates neuronal differentiation.

Figure 6

Time-course analysis of Pax6 (A) and Sox1 (B) mRNA in CSL+/− and CSL−/− ES cell neural differentiations reveal normal neural induction but a rapid depletion of neuroepithelial progenitors in CSL−/− differentiations. Immunohistochemistry for Pax6, Tuj1 (C) and Sox2 (D) confirm that these markers are induced lost beginning at day 5 in CSL−/− differentiations. Note that oval structures with neural stem cells surrounding a central lumen (yellow arrows) can be found in both CSL+/− and CSL−/− cultures at day 5. (E,F) A larger number of neurospheres are derived from CSL+/− cultures after 7 days of monolayer differentiation than from CSL−/− differentiations, quantified in (F). In addition, CSL−/− spheres display an aberrant appearance with cells bulging out (boxed region in E). At day 8 (G) the neural stem cell marker Sox3 and the radial glia marker and Notch target gene BLBP are absent in CSL−/− differentiations. BLBP mRNA (H) is not detected in CSL−/− cultures over 6 days of differentiation. Finally, CSL−/− differentiations (I) lose Nestin expression and differentiate into Tuj1+ neurons more rapidly than CSL+/− differentiations, this is statistically significant (J) from day 3 of differentiation, as seen in this quantification of the number of colonies in CSL+/− and CSL−/− differentiations containing TuJ1 positive cells at day 2 and day 3. Scalebar in (C) (confocal, 5 μm section, 20×) and (D) (confocal, 2 μm section, 40×) is 100 μm, scalebar in (G) (confocal, 2 μm section, 20×) is 50 μm. Panels in (I) were taken on a fluorescence microscope at 10×. Graphs depict means from three experiments performed in triplicate, error bars indicate standard deviation: ***significant difference at p<0.001; **significant difference at p<0.01; *significant difference at p<0.05.