PMC

Dominant negative STAT3 rescues KLF4-induced migration defect. Brains were electroporated at E14.5 and examined at E18.5. Coexpression of a dominant negative form of STAT3 (dnSTAT3) with KLF4 significantly improved cellular migration (red signal) to the cortical plate (CP). IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone. n = 3; **, P < 0.01. Scale bar, 200 μm.

Knocking down KLF4 has no long-term effect on differentiated neurons. Representative images of coronal brain sections, which were electroporated at E14.5 and examined at P3, are shown. A pCAG-EGFP plasmid was coelectroporated to label transfected cells. Sections were counterstained with Hoechst (blue signal). Arrowhead marks axonal bundles that crossed the midline. Images in panels B and D are higher-magnification views of boxed regions in panels A and C, respectively. Scale bar, 50 μm.

Downregulation of KLF4 promotes multipolar-to-bipolar transitions of migrating neurons. (A) Representative confocal images showing morphological changes induced by knocking down endogenous KLF4 by shRNA. Arrows and arrowheads mark cells with bipolar and multipolar morphologies, respectively. Scale bar, 40 μm. (B) Quantification of neurons with bipolar, round, or multipolar morphology in the intermediate zone. n = 3; **, P < 0.01. (C and D) Knockdown of KLF4 has no effect on cell proliferation, which was quantified by staining for PCNA. Brains were electroporated at E14.5 and examined at E18.5 (n = 3). Scale bar, 50 μm.

Radial neuronal migration requires downregulation of KLF4. (A and B) Constitutive KLF4 expression inhibits proliferation of neural progenitors in vivo. Brains were electroporated at E14.5 and examined at E17.5. Proliferating cells were labeled by PCNA staining (red signal in panel A) and quantified by counting PCNA⁺ GFP⁺ cells in ventricular zone/subventricular zone. n = 5; **, P < 0.01. Scale bar, 50 μm. (C and D) Radial neuronal migration is impaired by constitutive KLF4 expression. E14.5 mouse embryos were electroporated in utero with GFP or KLF4-GFP plasmids and examined at E17.5. Coronal sections were counterstained with Hoechst (blue signal). Very few KLF4-expressing cells migrated to the cortical plate (CP). n = 5; **, P < 0.01; ***, P < 0.001. Scale bar, 100 μm. (E and F) Multipolar-to-bipolar transition of migrating neurons is compromised by KLF4. Images were taken from the intermediate zone. Brains were electroporated at E14.5 and examined at E17.5. Arrows and arrowheads show cells with uni- or bipolar and multipolar morphologies, respectively. n = 5; *, P < 0.05; **, P < 0.01. Scale bar, 20 μm. IZ, intermediate zone; VZ, ventricular zone; SVZ, subventricular zone.

Neurogenesis is sensitive to constitutive expression of KLF4. (A and B) Representative coronal sections taken from P7 brains, which were electroporated at E14.5 with plasmids constitutively expressing GFP or KLF4-GFP, are shown. Most KLF4-expressing cells were identified along the corpus callosum, whereas control GFP-expressing cells migrated and settled in layers II/III. Nuclei were counterstained with Hoechst (blue). Scale bar, 100 μm. (C) Quantification of cellular distribution, including layer I, layers II/III, layers IV to VI, and white matter (WM) of cerebral cortices at P7. n = 3; ***, P < 0.001. (D to I) Immunohistochemistry analysis of KLF4-expressing cells at P7. They are neither neurons (NeuN⁺) (D) nor stem cells (Sox2⁺) (I). In contrast, they express markers for glial cells, such as GS (E and F), NG2 (G), and GFAP (H). Magnified views of cells in boxed regions are also shown. Arrows indicate a KLF4-expressing cell colabeled with the glial marker GS in layer I (E). Scale bars, 100 μm (D), 20 μm (E), and 50 μm (F to I).

Regulation of the JAK-STAT3 pathway by KLF4. (A) qPCR analysis of endogenous KLF4 expression in NSCs or under differentiation conditions. KLF4 is induced by LIF but downregulated by FSK and RA treatment. n = 3; **, P < 0.01. (B) qPCR analysis of gene expression in NSCs 36 h posttransduction with lentiviruses expressing either GFP or KLF4-GFP. n = 3; *, P < 0.05; **, P < 0.01. Lifr, leukemia inhibitory factor receptor; Metrn, meteorin; Rest, RE1-silencing transcription factor; Ncam1, neural cell adhesion molecule 1; Nfia, nuclear factor I/A; Nfib, nuclear factor I/B. (C) Western blot analysis. The expression of GFAP and pSTAT3 was induced by KLF4 in NSCs 3 days after lentiviral transduction. β-Actin was used as a loading control. (D) Confocal images taken from coronal brain sections after staining for GFP (green) and pSTAT3 (red). Brains were electroporated at E14.5 and examined at E17.5. Strong pSTAT3 staining was detected in KLF4-expressing but not control GFP-expressing cells. Scale bar, 20 μm.

Knockdown of KLF4 enhances radial migration of cortical neurons. (A) Two shRNAs against KLF4 (shRNA-Klf4) efficiently silenced the expression of transfected KLF4 in HEK293 cells by Western blot analysis. GFP and β-tubulin were used as loading controls. A modified form of KLF4 with a higher molecular mass is also indicated. (B) shRNA-Klf4 partially rescued the neuronal migration defect induced by constitutive KLF4. Brains were coelectroporated with KLF4-GFP and shRNA-Klf4 or a control at E14.5 and analyzed at E17.5. Scale bar, 100 μm. (C to E) Enhanced radial neuronal migration by knocking down endogenous KLF4. Brains were electroporated at E14.5 and analyzed at E18.5. A GFP marker driven by the constitutive CAG promoter was coelectroporated to label transfected cells. Representative traces of migrating neurons with leading and trailing processes are shown at the far right in panel C. (D and E) Quantification of regional distribution and processes of migrating neurons. CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone. n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bar, 200 μm.