Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: Expression of DOCK7 in the postnatal mouse forebrain. (A) A cartoon representation of the V-SVZ–RMS–OB pathway. (B) Coronal sections of forebrains of P10 mice at positions indicated in the cartoon in A immunostained with antibodies to DOCK7 and DCX and counterstained with DAPI. Boxed regions in III are enlarged and shown at the bottom. Bars: (top) 50 µm; (bottom) 10 µm. (C) Coronal sections of the V-SVZ region of P10 mice immunostained with antibodies to DOCK7 and GFAP, MASH1, or nestin and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. The yellow dashed line indicates the boundary region between the V-SVZ and the lateral ventricle. Bars: (top) 25 µm; (bottom) 10 µm. (D) Localization of DOCK7 in cultured V-SVZ–derived neuroblasts. Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstaining with DAPI. Bar, 10 µm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Expressing, Cell Culture, Derivative Assay, In Vitro, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 function is required for the migration of neuroblasts along the RMS. (A) Schematic drawing of postnatal in vivo electroporation. A virtual line (red) connecting the right eye to the craniometrical landmark λ serves as positional marker for plasmid injection into the lateral ventricle (LV). Lateral bars indicate position of electrodes. (B) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and nontargeting shRNA (scr#1), Dock7-targeting shRNAs (Dock7#1 and Dock7#2), or Dock7#2 shRNA together with DOCK7 cDNA (Dock7#2 + DOCK7) and sacrificed at P10 showing distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (C) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway in the forebrains of mice electroporated at P3 and sacrificed at P10. n = 1,316–2,511 cells from at least three animals for each condition. (D) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway in the forebrains of mice electroporated at P3 and sacrificed at P17. n = 2,379–2,462 cells from at least three animals for each condition. (E) Enlarged images of neuroblasts in RMSp regions of mice electroporated with indicated constructs at P3 and sacrificed at P10. Arrowheads indicate branching of LP. Bar, 20 µm. (F) Quantification of the length of the LP of neuroblasts in the RMS of mice electroporated with indicated constructs at P3 and sacrificed at P10. n = 91–680 cells from at least three animals for each condition. Data are shown as means ± SEM. **, P < 0.01; ***, P < 0.001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test (C and F) or Student’s t test (D).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Migration, In Vivo, Electroporation, Marker, Plasmid Preparation, Injection, Expressing, shRNA, Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: Knockdown of DOCK7 affects the migration distance, displacement, speed, and LP branching frequency of migrating neuroblasts. (A–F) P2–3 mouse pups were electroporated with plasmids expressing tdTomato and scr#1, Dock7#2 shRNA, or the rescue construct (Dock7#2 + DOCK7). After 5 d, acute sagittal brain slices were prepared, and tdTomato + transfected neuroblasts were imaged by spinning-disk confocal microscopy over a 4-h time period. (A) Time-lapse sequence of scr#1 or Dock7#2 shRNA-expressing cells migrating in the lower vertical arm of the RMSp. Five cells in each slice are labeled in the 0-min panel, and their tracks over time are indicated by lines of the same color. Bars, 70 µm. (B–D) Quantifications of the mean migrated distance (B), displacement (C), and velocity (D) of neuroblasts expressing indicated constructs. n = 50–61 cells from five to seven animals for each condition. (E) Examples of time-lapse series of scr#1 or Dock7#2 shRNA-expressing cells in the lower vertical arm of the RMSp (higher magnification). Arrowheads indicate branching of LP. Bars, 50 µm. (F) Quantification of the number of branching events per h for neuroblasts expressing indicated constructs. n = 50–61 cells from five to seven animals for each condition. Data are shown as means ± SEM. **, P < 0.01; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Migration, Expressing, shRNA, Construct, Transfection, Confocal Microscopy, Sequencing, Labeling

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 controls different cellular aspects of neuroblast migration via DHR2/Rac-dependent and R2-dependent pathways. (A) DOCK7 domain structure and deletion and point mutant constructs. ΔR2 comprises the DHR1 domain (amino acids 561–727) and TACC3-binding domain (amino acids 933–1,164). The asterisk indicates the DOCK7(V2022A) mutation. (B) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and Dock7#2 shRNA + DOCK7, Dock7#2 shRNA + DOCK7ΔDHR2, Dock7#2 shRNA + DOCK7V2022A, or Dock7#2 shRNA + DOCK7ΔR2 and sacrificed at P10, showing distribution of EGFP + transfected neuroblasts along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (C) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. n = 1,316–1,870 cells from at least three animals for each condition. (D) Quantification of the length of the LP of neuroblasts expressing indicated constructs. n = 91–680 cells from at least three animals for each condition. (E–H) P2–3 mouse pups were electroporated with a tdTomato-expressing plasmid together with one of the indicated constructs. Acute sagittal brain slices were prepared 5 d later and subjected to confocal live-cell imaging. Quantifications of the mean migrated distance (E), displacement (F), velocity (G), and number of branching events per h (H) for the tdTomato + transfected neuroblasts. n = 42–61 cells from four to seven animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Migration, Mutagenesis, Construct, Binding Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 interacts with p116 Rip . (A) DOCK7 and p116 Rip domain structure. (Top) DOCK7 fragments used in YTH screen/testing. CC, coiled-coil domain; PH, pleckstrin homology domain. (B) YTH interaction between DOCK7-R2 and p116 Rip . Yeast transformed with plasmids expressing DOCK7-R2 or DOCK7-R3 fused to GAL4 DNA–binding domain (GBD) and p116 Rip -C (amino acids 505–933) fused to the GAL4-activation domain (GAD) were grown on medium lacking histidine. Lamin served as negative and H-Ras and phosphatidylinositol 3-OH–kinase-δ (PI 3 KD) as positive controls. (C) Expression of p116 Rip in DCX-positive neuroblasts along the V-SVZ–RMS–OB pathway. Coronal sections of P10 mouse forebrain at positions indicated in the cartoon in immunostained with antibodies to p116 Rip and DCX and counterstained with DAPI. (D) DOCK7–p116 Rip interaction in mammalian cells. Lysates from HEK293 cells transiently expressing Flag-DOCK7 or empty control vector were immunoprecipitated (IP) with an antibody to Flag and analyzed by immunoblotting with antibodies to Flag and p116 Rip . TL, total lysate. (E) GST-p116 Rip -C (amino acids 505–1,024) fusion protein or GST alone (bottom, Coomassie brilliant blue [CBB] staining), immobilized on beads, was incubated with lysates from P5 mouse brains. Bound DOCK7 was detected by immunoblotting with an antibody to DOCK7. (F) Interaction of DOCK7 and p116 Rip in the brain. P10 mouse whole-brain extracts were immunoprecipitated with normal rabbit IgG, an anti-DOCK7 (DOCK7-Ab), or an anti-p116 Rip (p116 Rip -Ab) antibody and then were analyzed by immunoblotting with antibodies to DOCK7 and p116 Rip . Molecular masses are shown in kilodaltons. (G and H) Colocalization of DOCK7 and p116 Rip in V-SVZ–derived neuroblasts. (G) Coronal sections of P10 mouse forebrain at position III in the RMS as indicated in the cartoon in immunostained with antibodies to DOCK7 and p116 Rip and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. (H) Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstained with DAPI. Bars: (C and G, top) 50 µm; (G, bottom, and H) 10 µm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Transformation Assay, Expressing, Binding Assay, Activation Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Derivative Assay, In Vitro, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 interaction with p116 Rip is essential for neuroblast migration along the RMS. (A) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and Dock7#2, Dock7#2 + DOCK7, or Dock7#2 + DOCK7Δp116 Rip and sacrificed at P10, showing distribution of EGFP + transfected neuroblasts along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (B) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. n = 1,316–1,791 cells from at least three animals for each condition. (C) Quantification of the length of the LP of neuroblasts expressing indicated constructs. n = 91–680 cells from at least three animals for each condition. (D–G) P2–3 mouse pups were electroporated with a tdTomato-expressing plasmid together with one of the indicated constructs. Acute sagittal brain slices were prepared 5 d later and subjected to confocal live-cell imaging. Graphs show quantifications of the mean migrated distance (D), displacement (E), velocity (F), and number of branching events per h (G) for the tdTomato + transfected neuroblasts. n = 50–61 cells from five to seven animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Migration, Expressing, Transfection, Construct, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7/p116 Rip signaling controls somal translocation. (A–F) Neuroblasts dissociated from the V-SVZ of P1–3 mice were coelectroporated with one of the indicated constructs and a plasmid coexpressing EGFP and PACT-mKO1, a marker for the centrosome. Cells were reaggregated, embedded in Matrigel, and subjected to confocal live-cell imaging. (A) Examples of time-lapse series showing centrosome and cell soma movements during migration in neuroblasts transfected with indicated constructs. Images are taken from Videos 8–12. Signals of EGFP and PACT-mKO1 are shown in green and red, respectively. White dashed lines indicate advancement of the rear edge of each cell. Arrowheads indicate centrosome position. Bars, 10 µm. (B and C) Temporal changes in the distance between the nucleus and the centrosome (N–C distance; B) and velocity of the cell body (green lines) and the centrosome (red lines; C) in migrating neuroblasts expressing indicated constructs. (D–F) Quantification of velocity (D), somal translocation events (E), and maximum nucleus–centrosome distance (F) for neuroblasts expressing indicated constructs. n = 6 cells (scr#2); n = 5 cells (p116 Rip #1); n = 7 cells (Dock7#2); n = 12 cells (Dock7#2 + DOCK7); and n = 8 cells (Dock7#2 + DOCK7Δp116 Rip ); from 5–12 animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7/p116 Rip signaling controls somal translocation through F-actin remodeling at the cell rear. (A) Neuroblasts dissociated from the V-SVZ of P1–3 mice were coelectroporated with one of the indicated constructs and a plasmid coexpressing Lifeact-EGFP, a marker for F-actin, and PACT-mKO1. Cells were reaggregated, embedded in Matrigel, and subjected to confocal live-cell imaging. Examples of time-lapse series showing F-actin remodeling during migration in neuroblasts transfected with indicated constructs. Signals of Lifeact-EGFP and PACT-mKO1 are shown in green and red, respectively. White dashed lines indicate advancement of the rear edge (RE) of each cell. White and yellow arrowheads indicate position of centrosome ahead of the nucleus and formation of F-actin condensation at the cell rear, respectively. Linescan profiles to the left of each image show the signal intensities of Lifeact-EGFP (green lines) and PACT-mKO1 (red lines) from the rear edge of the cell soma to the tip of the LP in the corresponding images. Representative images of a total of 5–12 independent experiments/condition are shown. (B) Enrichment of DOCK7 and p116 Rip signals at the cell rear during somal translocation. Reaggregated neuroblasts isolated from V-SVZ tissue of P1–3 mice were embedded in Matrigel and allowed to migrate for 30 h before immunostaining for DOCK7 and p116 Rip . Representative images of neuroblasts captured before (top two rows) and during somal translocation (bottom row) are shown. Enrichment of DOCK7 and p116 Rip signals at the cell rear just before and during somal translocation is indicated by arrowheads. Bars, 10 µm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Isolation, Immunostaining

Journal: EMBO Reports

Article Title: Autocrine Wingless constricts the Drosophila embryonic gut by Ca +2 -mediated repolarisation of mesoderm cells

doi: 10.1038/s44319-025-00411-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse anti-KDEL , Stressmarq Biosciences , SMC-539.

Techniques: