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. 2015 Nov 25;35(47):15666–15681. doi: 10.1523/JNEUROSCI.2172-15.2015

Figure 3.

Figure 3.

Smaug2 overexpression in vitro and in vivo is sufficient to enhance cortical precursor self-renewal. A–C, Cultured E12.5 precursors were cotransfected with a nuclear EGFP construct and control (con) or murine Smaug2 (Smg2-OE) expression vectors, and analyzed by immunostaining 3 d later. A, Images of transfected precursors immunostained for EGFP (green) and Ki67 or βIII-tubulin (both red). Arrows and arrowheads indicate EGFP-positive, marker-positive cells and EGFP-positive, marker-negative cells, respectively. Scale bar, 10 μm. B, C, Quantification of cultures as in A for the percentage of EGFP-positive cells expressing Ki67 (B) or βIII-tubulin (C). **p < 0.01. n = 3 experiments. D, Cortical precursor cultures were cotransfected with the piggybac EGFP labeling system plus control (con) or murine Smaug2 (Smg-OE) expression vectors. Cultures were immunostained for EGFP 3 d later and clone size was scored. *p < 0.05. n = 3 experiments. E–J, E13/E14 murine cortices were coelectroporated with a nuclear EGFP construct and control (con) or mouse Smaug2 (Smg2-OE) expression vectors and coronal cortical sections were analyzed 3 d later at E16/E17. E, Confocal images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 30 μm. F, Quantification of sections similar to those in E for the percentage of EGFP-positive cells located in the different cortical regions. *p < 0.05. ns, Nonsignificant. n = 3 embryos each, at least 3 sections per embryo. G, Confocal images of the VZ/SVZ (two top rows) or CP (bottom row) of electroporated sections immunostained for EGFP (green) and Pax6, Tbr2, or Satb2 (all red). Arrows indicate double-labeled cells. Arrowheads indicate EGFP-positive, marker-negative cells. v, Ventricle. Scale bar, 10 μm. H–J, Quantification of sections as in G for the percentage of EGFP-positive cells that were positive for Pax6 (H), Tbr2 (I), or Satb2 (J). *p < 0.05. **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. Statistics were performed with Student's t test. Error bars indicate SEM.