Fig. 7: PTEN rescues cortical development defects caused by Foxj3 knockdown.

A Electroporation of shCtrl or shFoxj3 or shFoxj3 + Pten constructs along with GFP (green) was performed at E12.5, with brains fixed at E14.5, and Immunostaining for the neural stem cell marker SOX2 (red) in E14.5 cortices. Scale bar: 50 μm. B EdU was injected at E13.5, and brain tissues were fixed at E14.5. Immunostaining for Ki67 (red) and EdU (blue) to assess cell cycle exit in E14.5 cortices. EdU + /Ki67- cells were considered exited from the cell cycle. Scale bar: 50 μm. C Bar graph showing the percentage of SOX2 + /GFP+ cells among GFP+ cells, indicating enhanced NSC maintenance with Foxj3 knockdown, and restoration with Pten rescue. Error bars represent S.E.M. *: p < 0.05, **: p < 0.01 (shCtrl: n = 4, shFoxj3: n = 3, shFoxj3+Pten: n = 3 independent experiments; one-way ANOVA with post-hoc Tukey test; p = 0.0179, 0.0032). D The bar graph shows that Foxj3 knockdown reduced the cell cycle exit (EdU + /Ki67-) percentage, which is restored by PTEN expression. Error bars represent S.E.M. **: p < 0.01, ***: p < 0.001 (shCtrl: n = 3, shFoxj3: n = 3, shFoxj3+Pten: n = 4 independent experiments; one-way ANOVA with post-hoc Tukey test; p = 0.002, 0.0004) (E) Foxj3 knockdown disrupted neuronal migration (GFP+ cells in the IZ), while co-expression with PTEN restored cortical layer distribution, confirmed by TBR1+ immunostaining (red). Scale bar: 100 μm. F Bar graph showing GFP+ cells distributed across the CP, IZ, and SVZ/VZ regions. PTEN expression increased the proportion of cells in the CP compared to the shFoxj3 group. Error bars represent S.E.M. **: p < 0.01 (n = 3 independent experiments; one-way ANOVA with post-hoc Tukey test; p = 0.0023). G At P7, Foxj3 knockdown resulted in a shift toward SATB2+ (red) cell fate. PTEN expression effectively rescued this phenotype, restoring TBR1 (magenta) and CTIP2 (red) marker expression. Scale bar: 200 μm. H Bar graph showing the percentage of SATB2, CTIP2, and TBR1 markers colocalized with GFP+ cells. Error bars represent S.E.M., **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 (TBR1: shCtrl: n = 3, shFoxj3: n = 3, shFoxj3+Pten: n = 3 independent experiments; one-way ANOVA with post-hoc Tukey test; p = 0.0001, p < 0.0001; CTIP2: shCtrl: n = 4, shFoxj3: n = 3, shFoxj3+Pten: n = 3 independent experiments; one-way ANOVA with post-hoc Tukey test; p = 0.0023, 0.0032; SATB2: shCtrl: n = 4, shFoxj3: n = 3, shFoxj3+Pten: n = 3 independent experiments; one-way ANOVA with post-hoc Tukey test; p = 0.0004, 0.0004).