In utero knockdown of Abba expression is linked to radial glial disruption and alters neuronal migration.

A. Representative coronal sections of E16-E18 mice brains electroporated at E14 with either scramble or Abba-shRNA3 and immunostained for vimentin (in blue). In Abba-shRNA3 brain sections, vimentin staining revealed a marked disruption of radial glial apical fibers. B. Representative neocortical coronal sections showing migration of transfected cells 3 days after electroporation at E14 with either scramble or Abba-shRNA. C. Quantification of RFP-positive cell distribution in the cortex at E17 (VZ/SVZ: scramble 24.33±2.13%, Abba-shRNA3 63.71±1.03%; IZ: scramble 39.80±2.22%, Abba-shRNA3 29.71±0.85%; CP: scramble 35.86±1.73%; Abba-shRNA3 6.58±0.53%; n=9 scramble and 8 Abba-shRNA3; (VZ/SVZ: p=<0.0001: IZ: p=0.0002; CP: p=<0.0001) showing a significant increase of Abba knockdown cells in SVZ/lower IZ. D. Higher magnifications of regions indicated by a square in C. E. Total number of RFP positive cells in mouse neocortex at E17 (scramble 969.2±109.0%; Abba-shRNA3 311.3±28.61%; n=9 scramble and 8 Abba-shRNA3) showing a striking reduction of RFP positive cells in Abba knockdown electroporated brains. Error bars represent mean ± s.d. **P < 0.002, ***P < 0.001, Scale bars 100μm (A). F. Quantification of radial glial progenitor cells at the VZ (scramble 126.50±13.36%, Abba-shRNA3 65.14±9.91%). mean ±SD. VZ: ventricular Zone, IZ: Intermediate Zone, CP: Cortical Plate. Error bars represent mean ± s.d. **P < 0.002. Scale bars represent100μm (A), 50μm (B).

Abba down-regulation induced inhibition of basal nuclear migration in RGP cells blocks cell cycle progression.

A. Quantification of the nuclear distance from the VS for RGP cells after electroporation of scrambled or Abba-shRNA3. Nuclear distribution of RFP RGP cells was significantly altered after electroporation of Abba-shRNA3 (0–10: scramble, 30.20 ± 1.21%; Abba-shRNA3, 38.34 ± 0.89%; 10–20: scramble, 34.35 ± 0,69%; Abba-shRNA3, 22.80 ± 1.03%; 20–30: scramble, 23.42 ± 1.00%; Abba-shRNA3, 17.00 ± 1.10%; >30: scramble, 12.04 ± 1.27%; Abba-shRNA3, 21.85 ± 0.74%; (0–10: P = 0,000156; 10-20: P=<0.000001; 20-30:P= 0,001030; >30:P= 0,000023). B-C. E14 mice brains were subjected to in utero electroporation with either scramble or Abba-shRNA3. Brains were then fixed at E17 and stained with the RGP cell marker Pax6. We observe an important decrease of Pax6 RGP cells expressing Abba-shRNA3(scramble, 31.87 ± 0.57%; Abba-shRNA3, 21.08 ± 0.63%; n = 8 scramble; 5 Abba-shRNA3). D-G. E14 mice brains were subjected to in utero electroporation with either scramble or Abba-shRNA3. Brains were then fixed at E17 and stained with the cell cycle marker Ki67 (D)and PH3 (F). We observe a striking decrease in the percent of cycling (E: Ki67; scramble, 46.54 ± 0.79%, n=7; Abba-shRNA3, 23.71 ± 1.37%, n=5 Abba-shRNA3)) and but not in mitotic (G:PH3; scramble, 4.50 ± 0.17%; Abba-shRNA3, 4.25 ± 0.65%) cells with low expression of Abba. G. Flow cytometry analysis of cells electroporated with Abba-shRNA3 show accumulation in S-phase and recovery after additional expression of Abba-FL (G1: scramble 45,2 ± 1,4 %, Abba-shRNA3 37,4 ± 1,0 %, Abba-shRNA3+Abba-FL 42,8 ± 1.3 %; S: scramble 34,3 ± 2,8 %, Abba-shRNA3 48,2 ± 2.7 %, Abba-shRNA3+Abba-FL 38,9 ± 3.4; G2: scramble 17,1 ± 2,1 %, Abba-shRNA3 14,4 ± 2,7%, Abba-shRNA3+Abba-FL 15,9 ± 1,9 %; n = 10). Error bars represent mean ± s.d. **P < 0.002, ***P < 0.001. Scale bar represents 20μm.

Abba down-regulation blocks cytokinesis

A. Cellular distribution of Abba in C6 cells during cell division. Upper panel shows two example immunofluorescent microscopy images of Abba (red). Left most figure shows the projection images at the cross line of the image. Mid and lower panels show the distribution of Abba at different cell division stages in relation to β-tubulin (green) aggregation B. Immunofluorescence microscopy images from C6 cells 72 hours after transfection with Abba-shRNA3 shows an absence of Abba expression at cytokinesis. C. Brain slices prepared from in utero electroporated animals with either Abba-shRNA or scramble were cultured at E17 and subjected to live imaging for a duration of 15-20 hours. The length of the time lapse was adjusted as necessary to capture significant events during interkinetic nuclear migration (INM). In the case of the scramble group, an RGP cell underwent a single mitotic event at the ventricular surface of the brain slice, observed at timepoints 1:50 and 2:20. At timepoint 3:00, two cells can be observed (indicated by arrows), along with the presence of basal fibers (indicated by arrowheads). Conversely, in the Abba knockdown group, the nucleus of the RGP (indicated by an arrow) initially underwent apical INM and subsequently divided at 8:30. Remarkably, the resulting daughter cells remained at the ventricular surface for at least 7 hours, suggesting that the absence of Abba hinders cells from exiting the mitotic state. D. Abba-shRNA3-expressing cells show increase in cytokinesis (scramble 0,8285 ± 0,1161%, n=12; Abba-shRNA3 2,078 ± 0,2682%, n=12) E. Quantification of the total number of RFP positive cells in the neocortex illustrating the impact of cytokinesis block on cell survival. We observed a lower total number of cells 4 days after transfection with Abba-shRNA3 compared with control scramble (scramble 380,3± 48,16%; n=12, Abba-shRNA3 155,8± 22,32%; p= <0.0001; n=12). Error bars represent mean ± s.d. *P < 0.03, **P < 0.002, ***P < 0.001. Scale bars 50μm (A), 20μm (D).

Role of Abba-Nedd9 complex in RGP cells division

A. Good and high confidence interactions detected in yeast two-hybrid screen performed in mouse brain embryo library using full length mouse Abba as a bait. B. Schematic representation of the Abba and Nedd9 proteins. C. E18 cortical homogenates were subjected to a direct pulldown assay with endogenous Abba and Nedd9. Nedd9 co-immunoprecipitated with Abba, but not in control pulldowns showing the specificity of the interaction. D. immunocytochemistry on C6 cells 72 hours after transfection with Abba-shRNA3 shows that lack of Abba did not decrease Nedd9 expression (green) but rather its localization. Scale bars 20μm. E. Representative coronal sections of E17 mice brains transfected at E14 with Nedd9-shRNA and immunostained for vimentin (in blue). Vimentin staining revealed a marked disruption of radial glial apical fibers. F. Quantification of RFP-positive cell distribution in the cortex at E17 (VZ/SVZ: scramble 25 ± 2.30%, Nedd9-shRNA 46.92±2.78%; IZ: scramble 38.94±2.32%, Nedd9-shRNA 37.37±2.96%; CP: scramble 36.06±1.95%; Nedd9-shRNA 15.71±2.25%; n=8 scramble and 7 Nedd9-shRNA; (VZ/SVZ: p=<0.0001: IZ: p=0.0002; CP: p=<0.0001) showing a significant increase of Nedd9 knockdown cells in SVZ/lower IZ. Scale bars 100μm (E), 50μm (D).

Abba downregulation decreases RhoA activity.

A. Confocal images were captured of C6 cells 72 hours post-transfection with the Raichu-RhoA vector for monitoring RhoA activity. Six experimental groups were examined: transfected with scramble control, Abba-shRNA3, Abba-fl, rescue of Abba-shRNA3 with Abba-fl and expression of the mutant Abba-R671W. In addition to testing the sensitivity of the assay to RhoA activation by calpeptin. B. Quantification of FRET density (Raichu-RhoA activity) showed a decreased of Raichu-RhoA activity in C6 cells transfected with Abba-shRNA3 as compared to transfected with the scramble (scramble: 103,1.03 ± 5.3; n=102, Abba-shRNA3: 68,5 ± 2,3; n=46; p<0.0001, Abba-shRNA + ABBA-FL: 139,8 ± 19,6 n=26, Abba-FL: 105,2 ± 16,0; n=37, Abba-R671W: 142 ± 17,9 n=15). Error bars represent mean ± s.d. **P < 0.002, ***P < 0.001. Scale bar 25 μm (A).

Effect of ABBA R671W human mutation on neuronal migration and mitosis

A. ABBA is composed of an N-terminal BAR domain, a serine-rich region, three proline-rich motifs and a C-terminal WH2 domain. B. Evolutionary conservation analysis revealed that the Arg671 site is conserved from zebrafish to humans. C. Representative brain imaging features of one patient carrying ABBA R671W variant. D. 3D structure of ABBA denoting the position of Arginine 571. Lower panel show the high probability of disruption of the α-helix conformation by an Arginine to Tryptophane mutation. E. Coronal sections of E17 mice brains electroporated at E14 with cDNAs encoding the human wild-type, ABBA-FL, or the human mutant form of ABBA, ABBA-R671W. Expression of ABBA-R671W results in a defect in neuronal migration as indicated by the accumulation of neurons in the SVZ/lower IZ as well as a disorganization of the radial glial cell fibers using vimentin staining. F. Quantification of cell distribution in the cortex at E17 (VZ/SVZ: ABBA-FL 18.67±1.44%, ABBA-R671W 39.77±2.73%; IZ: ABBA-FL 53.07±2.37%, ABBA-R671W 43.40±1.40%; CP: ABBA-FL 28.25±1.25%, ABBA-R671W 16.8±2.03%; n=6) showing a significant increase of mutant cells in SVZ/lower IZ. G. Quantification of the distance of RGP nuclei from the ventricular surface (VS) at E17 (0–10: ABBA-FL 32.56±2.58%, ABBA-R671W 50,04±2.04%; 10–20: ABBA-FL 34.37±0,59%, ABBA-R671W 25.76±1.97%; 20–30: ABBA-FL 22.08±1.59%, ABBA-R671W 16.12±2.12%; >30: ABBA-FL 10.99±1.52%, ABBA-R671W 7.71±1.38%; n=8 ABBA-FL and n=6 ABBA-R671W), revealing accumulation at this site for the mutant, but not wild-type ABBA. (VZ/SVZ: p=0.000001: IZ: p=0.0014; CP: p=0.0003; 0–10: p=0.0002; 10–20: p=0.0005; 20–30: p=0.040; >30: p=0.150); mean ± SD. H. Brains were fixed at E17 and stained with the mitotic marker Phopho-Histone3 (PH3). I. The percentage of PH3-positive nuclei located at the ventricular surface (VS) did not differ in RGP cells expressing ABBA-R671W (ABBA-FL, 4.50±0.17%, n=9; ABBA-R671W, 4.71±0.88%, n=5, P=0.761;). Error bars represent mean ± s.d. **P < 0.01, ***P < 0.001. VZ: ventricular Zone, IZ: Intermediate Zone, CP: Cortical Plate. Scale bars 100μm (E), 20μm (H).