ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin

Effects of the knockdown of Arf6 on cell proliferation and neuronal differentiation. A, Representative micrographs showing the distribution of BrdU-positive cells in the VZ. Embryos were electroporated with the indicated shRNAs and EGFP at E14.5 and killed at E15.5 1 h after the intraperitoneal administration of BrdU to pregnant mice. Arrowheads show BrdU-incorporated mCherry-positive cells in the VZ. Note no apparent differences in the cell number or position of BrdU-positive transfected cells between Control and Arf6 knockdown (Arf6 KD). B, Representative micrographs showing the expression of PSA-NCAM in control and Arf6-KD neurons in the IZ at E17.5. C, Representative micrographs showing that the expression of Cux1 in control and Arf6-KD neurons in the superficial cortical layers at P0. Scale bars: A, 25 μm; B, 50 μm; C, 100 μm.

Arf6 is important for neuronal migration in the IZ. A, C, Representative time-lapse images of transfected migrating neurons in the CP and IZ. Embryos were electroporated with the indicated shRNA in combination with pCAGGS-Cre recombinase and pCAGGS-Floxp carrying EGFP, EGFP-NLS, and EGFP-Fyn at E14.5, and brains were subjected to slice culture and time-lapse observations at E17.5. Arrowheads indicate transfected neurons. B, D, Quantification of the migration speed of transfected neurons in the CP (B, n = 115 cells from 3 embryos) and IZ [D, Control, n = 41 cells from 3 embryos; Arf6 knockdown (Arf6 KD), n = 43 cells from 3 embryos]. E, Tracking analysis of migration of transfected neurons in the IZ. The graphs show the trajectory of individual migrating neurons (Control, 41 cells; Arf6 KD, 43 cells from 3 embryos) for 10–13 h. F, The orientation of the Golgi apparatus in migrating neurons in the IZ. Arrowheads indicate the Golgi apparatus labeled by GM130 in transfected neurons in the IZ. G, Quantification of the proportion of cells in the IZ with the Golgi apparatus facing the CP. Note the disorientation of Golgi apparatus in Arf6-KD neurons (n = 3 embryos). H, Representative micrographs showing the morphology of transfected neurons in the IZ at E16.5. Brains were electroporated with the indicated shRNA plasmids in combination with pCAGGS-EGFP at E14.5 and immunostained with an anti-EGFP antibody. I, Quantification of the number of primary processes. Note the increase in primary processes in migrating neurons transfected with Arf6-KD plasmid (Control, n = 195 cells from 3 embryos; Arf6 KD, n = 208 cells from 3 embryos). Data were presented as mean ± SD, and statistically analyzed by unpaired t test (*P < 0.05, ***P < 0.0005). Scale bars: A, C, 50 μm; F, H, 20 μm.

Arf6 localizes at endosomes and regulates the distribution of syntaxin12-positive endosomes in migrating neurons. A, Immunoblot analysis showing the specificity of rabbit anti-Arf6 antibody. The lysates of the mouse cerebral cortex (Cx) at E14.5 (10 μg) and HeLa cells transfected with the indicated FLAG-tagged Arf plasmids were immunoblotted with antibodies against Arf6 or FLAG. The positions and sizes of the molecular weight markers are indicated on the left. B, Representative micrographs showing endosomal localization of Arf6 in migrating neurons in the IZ at E17.5. Coronal sections of the E17.5 cerebral cortex, which had been electroporated with pCAGGS-mCherry at E14.5, were subjected to immunofluorescent staining with antibodies against Arf6 (magenta), the indicated markers (green), and mCherry (red). Nuclei were counterstained with DAPI (blue). Insets show the high-magnification views of boxed areas. C, D, Representative micrographs showing the subcellular localization of EEA1-positive (C) and syntaxin12 (STX12)-positive (D) endosomes in migrating neurons transfected with Control or Arf6 knockdown (Arf6 KD) plasmid in the IZ at E17.5. Arrowheads in D indicate STX12-positive puncta in transfected neurons. E, Quantification of the relative immunofluorescence intensity for STX12 in the perinuclear region. The relative immunofluorescence intensity was calculated by normalizing the immunofluorescence intensity for perinuclear STX12 in transfected neurons to that in the surrounding untransfected neurons. Note the significant increase in perinuclear STX12 in Arf6-KD neurons (Control, n = 155 cells; Arf6 KD, n = 239 cells). Data collected from three embryos in each condition were presented as mean ± SD and statistically analyzed with unpaired t test (*** P < 0.0001). Scale bars: B–D, 10 μm.

Arf6 regulates endosomal trafficking of N-cadherin in cortical neurons. A, Immunoblot analysis showing the specificity of guinea pig anti-N-cadherin antibody. The lysates of the adult mouse brain (10 µg) and HeLa cells transfected with or without C-terminally FLAG-tagged N-cadherin were immunoblotted with antibodies against N-cadherin or FLAG. B, Representative micrographs showing the subcellular localization of N-cadherin in migrating neurons transfected with Control or Arf6 knockdown (Arf6 KD)in the IZ at E17.5. Arrowheads indicate the localization of N-cadherin in transfected neurons. C, Quantification of the relative immunofluorescence intensity for cytoplasmic N-cadherin. The relative intensity was calculated by normalizing the immunofluorescence intensity for cytoplasmic N-cadherin in transfected neurons to that in the surrounding untransfected neurons (Control, n = 137 cells from 3 embryos; Arf6 KD, 174 cells from 3 embryos). Note the significant increase in cytoplasmic N-cadherin in Arf6-KD neurons. D, Internalization assay for N-cadherin in cultured cortical neurons. A bottom graph shows the quantification of biotinylated N-cadherin internalized in neurons transfected with Control (open bars) or Arf6 KD(black bars; Control, n = 5 independent experiments; Arf6 KD, n = 5). E, Recycling assay for N-cadherin in cultured cortical neurons. A bottom graph shows the quantification of biotinylated N-cadherin retained inside neurons transfected with Control (open bars) or Arf6 KD (black bars; Control, n = 5 independent experiments; Arf6 KD, n = 6). Data were presented as mean ± SD in C and mean ± SEM in D and E, and statistically analyzed by unpaired t test in C (***P < 0.0001), and Mann-Whitney U test in D and E (*P < 0.05). GSH, Glutathione. Scale bar: B, 20 μm.

Arf6 regulates neuronal migration through the interaction with class II FIPs. A, Representative micrographs showing rescue experiments of impaired neuronal migration caused by the knockdown of Arf6 with separation-of-function Arf6 mutants. Brains were electroporated with the indicated pCAGGS plasmids carrying Mock, Arf6^iSW, Arf6^N48I, Arf6^W168L/L169V in combination with Arf6 shRNA and pCAGGS-EGFP at E14.5, and fixed at P0. Bottom graphs show the quantification of the distribution of EGFP-positive cells in cortical layers. B, Comparison of the percentage of EGFP-positive cells transfected with the indicated plasmids in the superficial layers II–IV at P0. Data were presented as mean ± SEM and statistically analyzed using one-way ANOVA followed by post hoc Tukey-Kramer’s test (*P < 0.05). n in the graph indicates the number of embryos examined. Scale bar, 200 μm.

Expression of FIP3 in the developing cerebral cortex. A, Representative micrographs showing the gene expression of Arf6, FIP3, and FIP4 in the developing cerebral cortex. Coronal sections of mouse cerebral cortices at E14.5 and E16.5 were subjected to nonradioactive in situ hybridization analysis for Arf6, FIP3, and FIP4. Right, Magnified views of the boxed region in the middle panel. Note the widespread gene expression of FIP3 and Arf6 in the VZ, IZ, and CP in contrast to the preferential gene expression of FIP4 in the CP. B, Immunoblot analysis of developing cerebral cortices with anti-FIP3 and anti-α-tubulin antibodies. Note the gradual increase in FIP3 during cortical development. The positions and sizes of the molecular weight markers are indicated on the right. C, Representative micrographs showing the subcellular localization of FIP3 in migrating neurons in the IZ at E17.5. Coronal sections of the cerebral cortex, which had been electroporated with pCAGGS-mCherry at E14.5, were subjected to immunofluorescent staining with antibodies against FIP3 (magenta), the indicated markers (green), and mCherry (red). Nuclei were counterstained with DAPI (blue). Insets show the high-magnification views of boxed areas. Scale bars: A, left, middle, 500 μm; A, right, 10 μm; C, 20 μm.

FIP3 regulates neuronal migration in the IZ through the interaction with Arf6 and Rab11. A, Immunoblot analysis showing the efficiency of FIP3 shRNAs (FIP3 KD#1 and #2) in primary cortical neurons. The numbers indicate the percentage of endogenous FIP3 expression level relative to α-tubulin. Note that the expression of FIP3 KD#1 and #2 decreased endogenous FIP3 by 37.0 and 65.6%, respectively. B, C, E, Representative images of coronal sections of E17.5 (B and E) and P0 (C) cerebral cortices electroporated with Control, FIP3 knockdown (B and C), or FIP3 KD#2 and FIP3^WT, FIP3^ΔABD, or FIP3^ΔRBD (E) in combination with pCAGGS-EGFP at E14.5. The bottom graphs show the quantification of the distribution of EGFP-positive neurons in each layer. n in the graph indicates the number of embryos examined. D, Immunoprecipitation. HeLa cells were transfected with the indicated combinations of plasmids and subjected to immunoprecipitation with anti-FLAG affinity gel. Immunoprecipitates and total lysates were subjected to an immunoblot analysis with anti-HA or anti-FLAG antibodies. Note the lack of the ability of FIP3^ΔABD and FIP3^ΔRBD to interact with Arf6 and Rab11, respectively. WB, Western blot. F, Comparison of the percentage of EGFP-positive cells transfected with the indicated plasmids in the CP at E17.5. Note the ability of FIP3^WT but not FIP3^ΔABD or FIP3^ΔRBD to partially rescue the disturbed cortical layer formation caused by the knockdown of FIP3. G, Representative time-lapse images of transfected neurons in the IZ. Embryos were electroporated with the indicated shRNAs, pCAGGS-Cre recombinase, and pCAGGS-Floxp carrying EGFP, EGFP-NLS, and EGFP-Fyn at E14.5, and subjected to time-lapse observations at E17.5. Arrowheads indicate transfected neurons. H, Quantification of the migration speed of transfected neurons in the IZ (n = 40 cells from 3 embryos). Note the decrease in the migration speed of FIP3 KD#2-transfected neurons compared with that of control neurons. I, Tracking analysis of migration of transfected neurons in the IZ. The graphs show the trajectory of migrating neurons for 10–13 h (n = 40 cells from 3 embryos). Data were presented as mean ± SEM (B, C, E, F) and mean ± SD (H), and statistically analyzed using one-way ANOVA followed by post hoc Tukey-Kramer’s test in B (vs control) and E (vs FIP3 KD#2 in B; *P < 0.05, ** P < 0.01), and unpaired t test in C and H (vs control; *P < 0.05, ** P < 0.005, ***P < 0.0005). The total numbers of examined animals were indicated in the graphs in B, C, and E. Scale bars: B, C, E, 200 μm; F, 50 μm.