Fig. 6

Loss of Ankrd11 decreases SVZ NPC proliferation and causes aberrant neurogenesis in vitro. A Schematic: P7 SVZ primary neurospheres were cultured from Ankrd11^fl/fl and Ankrd11^fl/fl; NestinCre^ERT2 pups for 6 DIV. Neurospheres were treated with 4OH-TMX for 24 h prior to dissociation on 6 DIV resulting in Control and nscKO NPCs. Primary neurospheres were dissociated and further cultured as secondary neurospheres for 7 DIV or adherent NPC monolayer for 4 DIV. B Representative images of Ankrd11^control and Ankrd11^nscKO secondary neurospheres at 7 DIV. Inset is shown at the bottom left corner in higher magnification. C, D Quantification of B for secondary neurosphere number (C) and diameter (D). E–L Representative images and quantification of Ankrd11^control and Ankrd11^nscKO 4 DIV NPC monolayers immunostained for βIII (yellow) (E, F), GFAP (green) (G, H), PDGFRα (white) (I, J), CC3 (red) (K, L). Arrowheads indicate marker + cells. Yellow insets in K are shown at higher magnification on the right side. Cells were counterstained with Hoechst (blue). Data are presented as % marker + cells from healthy (non-condensed nuclei) except % CC3 + cells is presented as % from all nuclei (healthy and condensed). Examples of condensed nuclei positive for CC3 are shown in K and insets (yellow boxes) on right side. Error bars represent SEM. Data was analyzed using unpaired t-test. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–4 mice per genotype from at least two independent litters. Scale bars represent 500 μm (B), 100 μm (inset of B), 50 μm (E, G, I, K). DIV day in vitro, NPC neural precursor cells, NS neurospheres, 4OH-TMX 4-hydroxytamoxifen, P postnatal day, SVZ subventricular zone