Fig 1.
Foxp1 is expressed at CP in mouse embryonic brain and repressed by shRNA constructs.
Brain sections (14 μm) obtained from E16.5 (A) and E18.5 (B) mice were immunostained with antibodies against FOXP1 (green) and Satb2 (red). a, High-magnification images of the boxed regions in the left panels. Scale bar, 20μm in A, 50μm in B. (C) and (D), Relative mRNA expression of Foxp1 in N2a cells (C) and in cultured cortical cells (D) transfected with shRNA-Scr or two Foxp1 shRNAs (shRNA-a and shRNA-b). ***p<0.001, one-way ANOVA. (E), GFP-pCAGGS was cotransfected into N2a cells with shRNA-Scr, Foxp1 shRNA-a or shRNA-b. After 48h, cells were collected and analyzed for the expression of FOXP1 and GAPDH by western blot. The expressions of FOXP1 were quantitated as rations of the background-subtracted intensities for FOXP1 to GAPDH. (F), GFP-pCAGGS was electroporated with shRNA-Scr, Foxp1 shRNA-a or shRNA-b into cerebral cortices at E14.5. Coronal sections were obtained from the cerebral cortices at E17.5 and immunostained with anti-GFP (green) and anti-FOXP1 (red). a, b, c, High-magnification images of the boxed regions in the left panels. Arrows indicate GFP-positive neurons. Scale bar, 50μm in F, 20μm in c.
Fig 2.
Suppression of Foxp1 inhibits neuronal migration.
(A), E14.5 mouse embryos electroporated with a GFP expression plasmid along with shRNA-Scr, Foxp1 shRNA-a or shRNA-b together with pCMV-mFoxp1 were allowed to develop until E17.5. Coronal sections (14 μm) of E17.5 brains were immunostained with an antibody against GFP (green). Nuclei were stained with DAPI (blue). Short black lines indicate the borders between CP, IZ, SVZ/VZ. Scale bar, 50μm. (B), Statistical analysis of the percentages of GFP-positive cells in the indicated regions of the cerebral cortex as showed in (A). *p<0.05, ***p<0.001, one-way ANOVA. (C), E14.5 mice receiving mir30-ScrRNA or mir30-shRNA-b were examined at E17.5. Scale bar, 50μm. (C)-a and (C)-b, Representative images of migrating neurons (Green) stained with FOXP1 antibody (Red) showed FOXP1 expression in control neurons but not in mir30-shRNA-b expressing neurons. (D), Statistical analysis of the percentages of electroporated cells in the indicated regions of the cerebral cortex as showed in (C). ***p<0.001, Student’s t-test. (E), GFP-pCAGGS was coelectroporated with pX330 or pX330-gFoxp1 into E14.5 mouse brains. Sections were examined at E17.5. Scale bar, 50μm. Immunostaining for FOXP1 (Red) indicated a lack of FOXP1 expression in pX330-gFoxp1 transfected neurons (E-b). Vector pX330 transfected neurons expressed FOXP1 in the CP normally (E-a). (F), Quantitative analysis of the distribution of electroporated cells in cortical layers as showed in (E). **p<0.01 and ***p<0.001, Student’s t-test.
Fig 3.
Effects of Foxp1 knockdown on the placement of cortical neurons.
(A), (B) E14.5 embryos were electroporated with the control or Foxp1 shRNA-b, together with GFP-pCAGGS, and analyzed at P2, P4, P7, and P14. Scale bar, 50μm. (C), (D), (E), (F) Histograms showed the percentage of transfected cells in different regions of the cerebral cortex at P2, P4, P7, and P14. *p<0.05 and ***p<0.001 for comparisons between shRNA-b and the corresponding control, Student’s t-test.
Fig 4.
Foxp1 insufficiency does not affect cell division and specification of neuronal progenitors.
Foxp1 shRNA-b or shRNA-Scr was transfected into progenitor cells of the mouse cortex at E14.5 by IUE, and brains were fixed at E17.5. Coronal sections were immunostained for neuronal marker β-III-tubulin (A), M-phase marker pH3 (B), intermediate/basal progenitor marker Tbr2 (D). (C), Quantification of the percentage of pH3 positive, GFP-positive neurons. (E), Quantification of the percentage of Tbr2 positive, GFP-positive neurons in the SVZ/VZ of the cerebral cortex. Scale bar, 50μm in A, B and D. Student’s t-test.
Fig 5.
Foxp1 knockdown does not affect neuronal differentiation.
E14.5 mouse embryos were electroporated with Foxp1 shRNA-b or shRNA-Scr, and brains were fixed at P2. Coronal sections were immunostained for Satb2 (A) and Cux1 (B). a, b, c, High-magnification images of the boxed regions in the left panels. a, b, c in (A) and (B) represent layer II-IV, layer V and white matter, respectively. (C), (D), Immunofluorescence staining of Ctip2 and Tbr1 in brain sections from control and Foxp1 shRNA-b. (C)-a, (D)-a, High-magnification images of the boxed regions in the left panels. Scale bar, 50μm in (A), (B), (C) and (D).
Fig 6.
Foxp1 regulates neuronal morphogenesis in vitro.
(A), Cortical neurons were transfected in utero at E14.5 and isolated at E15.5 for primary cultures in vitro. At day four in vitro, the neurons transfected with GFP-pCAGGS together with shRNA-Scr (n = 39) or Foxp1 shRNA-b (n = 53) were analyzed by Neurolucida. Scale bar, 50μm. (B), Quantitative analysis of total lengths of the axon and dendrites from images in (A). ***p<0.001, Student’s t-test.
Fig 7.
Multipolar to bipolar transition of migrating neurons is compromised by Foxp1 knockdown.
(A), Brains were electroporated in utero with control shRNA or shRNA-b at E14.5 and examined at E17.5. (B), Representative GFP-labeled neurons in the IZ region in each group. White and red lines show cells with uni/bipolar and multipolar morphologies, respectively. (C), Quantitative and statistical analysis of the percentages of uni/bipolar and multipolar neurons in the IZ. Scale bar, 50μm in (A) and 20μm in (B). ***p<0.001, Student’s t-test.
Fig 8.
Knockdown of Foxp1 in pyramidal neurons leads to abnormal dendritic development at P30.
(A), Coronal sections (80 μm) from Foxp1 knockdown and the control mice. Scale bar: 50μm. (B), Representative images and Neurolucida tracings of GFP-positive, layer II-III neurons from the control (n = 18) and Foxp1 knockdown (n = 17) mice at E15.5. Scale bar: 50μm. (C), Representative images and Neurolucida tracing of GFP-positive neurons stalled in layer V (n = 15) from shRNA-b transfected mice at E15.5 or from the control (n = 11) transfected mice at E13.5. (D), (E), (F), Quantitative and statistical analysis of dendritic parameters in layer II-III. (G), (H), (I), Quantitative and statistical analysis of dendritic parameters in layer V at P30. *p<0.05, **p<0.01, Student’s t-test.