Fig. 5
From: Olfactory bulb anomalies in KBG syndrome mouse model and patients
Loss of Ankrd11 results in reduced SVZ NPC migration in vitro. A Schematic: P7 SVZ primary neurospheres were cultured from naive uninjected Ankrd11fl/fl and Ankrd11fl/fl;NestinCreERT2 pups for 6 DIV. Neurospheres were treated with 4OH-TMX for 24 h prior to dissociation on 6 DIV resulting in Ankrd11Control and Ankrd11nscKO NPCs. Treated primary neurospheres were directly plated onto adherent wells for primary neurosphere migration assay (H–M). Treated primary neurospheres were also dissociated and cultured as adherent NPC monolayer for scratch assay (C–G) or secondary neurospheres (B). B Quantitative RT-PCR for Ankrd11 mRNA in P7 SVZ secondary neurosphere cultures from Ankrd11control and Ankrd11nscKO NPCs. Data were normalized to Gapdh mRNA and calibrated against Ankrd11Control samples. C Schematic: 4OH-TMX treated P7 SVZ primary neurosphere cells were cultured as adherent NPCs and subjected to scratch assay. D, E Representative images of Ankrd11control and Ankrd11nscKO NPC scratch assay at 0, 1, and 2 DIV. 0 DIV indicates images taken immediately after scratch, and 1 and 2 DIV indicate images taken 1–2 days after scratch. Scratch region at 0 DIV is outlined in black. F, G Quantification of cell migration into scratch region at 1 DIV (F) and 2 DIV (G) normalized to control. H Schematic: 4 OH-TMX treated selected P7 SVZ primary neurosphere cells were allowed to adhere and neurosphere cell migration was assessed at 1 DIV. I, J Representative images of Ankrd11control and Ankrd11nscKO primary neurospheres assay at 0 and 1 DIV. Black outline shows the area of the neurosphere boundary (K). L Quantification of primary neurosphere area at 0 DIV. M Quantification of percent area change on 1 DIV relative to 0 DIV and normalized to control. Error bars represent SEM. Data was analyzed the non-parametric Mann–Whitney test. ns not significant, *p < 0.05, **p < 0.01 n = 4–6 mice per genotype from at least two independent litters. Scale bars represent 100 μm (D, E), 200 μm (I, J). DIV day in vitro, NPC neural precursor cells, NS neurospheres, 4OH-TMX 4-hydroxytamoxifen, P postnatal day, SVZ subventricular zone