Figure 2

Antigenic properties of radial glial as well as non-radial glial cells and confirmation of differentiation blockade by dnE47 overexpression. (A, B), At 2 days post electroporation (dpe), 40% of the labeled cells showed morphological criteria of radial glial cells (RGCs, i.e., bipolar processes, apical connection to lateral ventricle (LV) lumen), while 60% presenting a spherical morphology were distant from the LV and were defined as non-RGCs. Morphologically-defined RGCs express the NSC marker Vimentin (88.4 ± 0.7) and were EGFP positive in Hes5-EGFP reporter mice (94.2 ± 2.4), but negative in Ascl1-EGFP reporter mice, in which type-B or type-C cells are labelled, respectively [33, 34]. In contrast, non-RGCs consisted of a heterogeneous population of type-C (i.e., Ascl1⁺, 53.6 ± 4.3) and type-A cells (i.e., Dcx⁺, 45.9 ± 0.2). Non-RGCs were frequently proliferative (i.e., Ki67⁺, 58.2 ± 5.7). (C) A subsequent characterization of cycling non-RGCs revealed that most Ki67⁺ cells were type-C cells (Ascl1⁺, 60.1 ± 4.4), whereas fewer were type-A cells (Dcx⁺, 22.9 ± 4.0). Notably, a minor population of cycling cells were double-positive for Ascl1 and Dcx (10.1 ± 1.4), probably representing a transition phase in between type-C and type-A phenotypes. (D–G) Antigenic assessment of RGC differentiation blockade following dnE47 overexpression. Electroporation of dnE47 in the postnatal SVZ reduced RGC differentiation, as revealed by the increased number of Vimentin⁺/RFP⁺ RGCs (D) and concomitant decrease of Ascl1⁺/RFP⁺(E) and Dcx⁺/RFP⁺(F) non-RGCs, respectively, when compared to an empty RFP control plasmid 2 dpe (Vimentin: 100 ± 3.2 vs. 150.5 ± 1.6; Ascl1: 100 ± 8.4 vs. 48.2 ± 3.1; Dcx: 100 ± 3.3 vs. 39.5 ± 6.3). This blockade of differentiation was further supported by an increase in Dcx⁺/RFP⁺ type-A cells retaining expression of the type-C cell marker Ascl1 (Dcx⁺Ascl1⁺, 100 ± 25.7 vs. 308.5 ± 50.3) ( G ). P values: *P <0.05; **P <0.01; ***P <0.001. Quantifications were normalized to control conditions (D–G). Scale bars: A, 10 μm.