Figure 3

E-protein mRNA expression is enriched in germinal regions of the postnatal and adult forebrain and is increased during NSC differentiation in vitro and in vivo . (A) ISH of E2-2, E2A, and HEB revealed its enhanced expression in the SVZ (dashed zone at P60), the RMS, and the dentate gyrus, when compared to non-neurogenic regions (i.e., thalamus, cortex) at P6 as well as P60. (B) Inverted greyscale quantification of A . (C) E2-2, E2A, and HEB transcripts were highly expressed in the lateral SVZ compared to the striatum (dashed zone circling both regions) (0.18 ± 0.03 vs. 0.5 ± 0.06, 0.02 ± 0.004 vs. 0.46 ± 0.08, 0.003 ± 0.0004 vs. 0.07 ± 0.01, respectively). (D) Upon differentiation, E-protein mRNA levels were upregulated in NS5 cells (two-way ANOVA followed by Dunnett’s post hoc test). Differentiation progress was confirmed by upregulation of doublecortin (Dcx) transcripts, an early neuronal marker, and normalized to GAPDH and shown as a percentage vs. D0. (E) E-protein transcription, in particular E2-2, was enhanced in more mature PSA-NCAM⁺ MAC sorted neuroblasts compared to earlier Prominin-1⁺ (or CD133⁺) NSCs/progenitors (100 ± 32.0 vs. 687.8 ± 226.4, 100 ± 9.9 vs. 145.4 ± 15.4, 100 ± 12.1 vs. 144.0 ± 17.4, respectively). Cell sorting efficiency was confirmed by enrichment of Dcx (100 ± 34.0 vs. 1291.3 ± 438.6) and CD24 (100 ± 17.8 vs. 270.1 ± 48.1) transcripts in neuroblasts, while the generic NSC transcripts GFAP (100 ± 20.4 vs. 22.1 ± 4.5) and Id1 (100 ± 6.5 vs. 72.8 ± 4.7) were enriched within Prominin-1⁺ sorted cells. P values: *P <0.05; **P <0.01; ***P <0.001. All quantifications were normalized to control conditions (D, E).