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

Figure 5.

Figure 5.

Nanos1 is necessary and sufficient to promote neurogenesis in vivo. A, Western blots of HEK-293T cell lysates cotransfected with murine Nanos1 or Flag-tagged murine Nanos2 or Nanos3 expression constructs and a control shRNA (Con) or a Nanos1 shRNA (shNos1) and probed with anti-Nanos1 or anti-Flag, as indicated. The blots were reprobed with ERK1/2 as a loading control. B–H, E13/E14 murine cortices were coelectroporated with a nuclear EGFP construct, and either a control (con) or Nanos1 shRNA (shNos1) and coronal sections were analyzed 3 d later at E16/E17. B, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 μm. C, Quantification of sections similar to those in B for the percentage of EGFP-positive cells located in the different cortical regions. **p < 0.01. n = 3 embryos each, at least 3 sections per embryo. D, Confocal micrographs of the VZ/SVZ (three top rows) or CP (bottom row) of electroporated sections immunostained for EGFP (green) and Pax6, Ki67, Tbr2, or Satb2 (all red). Arrows indicate double-labeled cells. v, Ventricle. Scale bar, 10 μm. E–H, Quantification of sections similar to those in D for the percentage of EGFP-positive cells that expressed Pax6 (E), Ki67 (F), Tbr2 (G), or Satb2 (H). **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. I–K, E13/E14 cortices were coelectroporated with a nuclear EGFP construct and a control (con) or Nanos1 shRNA (shNos1) ± an shRNA-resistant human Nanos1 expression vector (resc) and coronal sections were analyzed 3 d later at E16/E17. I, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 μm. J, K, Sections similar to those in I were immunostained for EGFP and Pax6 or Satb2 and the proportion of EGFP-positive cells that were also positive for the marker was quantified. **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. L–R, E13/E14 cortices were coelectroporated with a nuclear EGFP construct and either a control (con) or murine Nanos1 (Nos1-OE) expression vector, and coronal sections were analyzed 3 d later at E16/E17. L, Images of electroporated sections immunostained for EGFP (green). v, Ventricle. Scale bar, 10 μm. M, Quantification of sections as in L 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. N, Confocal images of the VZ/SVZ (top three rows) or CP (bottom row) of electroporated sections similar to those in L immunostained for EGFP (green) and Pax6, Ki67, Tbr2, or Satb2 (all red). Arrows indicate double-labeled cells. Arrowheads indicate EGFP-positive, marker-negative cells. v, Ventricle. Scale bar, 10 μm. O–R, Quantification of sections as in N for the percentage of EGFP-positive cells that were also positive for Pax6 (O), Ki67 (P), Tbr2 (Q), or Satb2 (R). *p < 0.05. **p < 0.01. ***p < 0.001. n = 3 embryos each, at least 3 sections per embryo. J, K, Statistics were performed with ANOVA and Tukey's post hoc multiple comparisons test. All other panels, Statistics were performed with Student's t test. Error bars indicate SEM.