DOCK7 interacts with TACC3 to regulate interkinetic nuclear migration and cortical neurogenesis

Neurogenesis in the developing neocortex relies on the ability of radial glial progenitor cells (RGCs) to switch from proliferative to differentiative neuron-generating divisions, but the molecular mechanisms that control this switch in a correct temporal manner are not well understood. Here, we show that DOCK7, a member of the DOCK180 family of proteins, plays an important role in the regulation of RGC proliferation versus differentiation. Silencing of DOCK7 in RGCs of developing mouse embryos impedes neuronal differentiation and maintains cells as cycling progenitors. In contrast, DOCK7 overexpression promotes RGC differentiation to basal progenitors and neurons. We further present evidence that DOCK7 influences neurogenesis by controlling apically directed interkinetic nuclear migration (INM) of RGCs. Importantly, DOCK7 exerts its effects by antagonizing the microtubule growth-promoting function of the centrosome-associated protein TACC3. Thus, DOCK7 interaction with TACC3 controls INM and the genesis of neurons from RGCs during cortical development.


Supplementary Videos
Supplementary Video 1: Example of a control vector expressing RGC (top left cell) undergoing bl-to-ap INM in VZ of neocortex. Time-lapse imaging was carried out on acute cortical slices 2 d after in utero electroporation of plasmids expressing empty control vector, and EGFP (green) and mKO2-F (red) fluorescent markers (at E15.5). Images acquired over an 8 h time period are shown; time (in min) is indicated on the top. Note: the cell body of the RGC migrates steadily toward the ventricle. After about 5 h, it reaches the ventricular (apical) surface, and the cell subsequently divides at the ventricular surface.
Supplementary Video 2: Example 1 of a DOCK7 overexpressing RGC (cell in the middle) undergoing bl-to-ap INM in VZ of neocortex. Time-lapse imaging was carried out on acute cortical slices 2 d after in utero electroporation of plasmids expressing FLAG-DOCK7, and EGFP (green) and mKO2-F (red) fluorescent markers (at E15.5). Images acquired over an 8 h time period are shown; time (in min) is indicated on the top. Note: the cell body of the RGC remains at its original basal position for about 5 h, which is followed by cell division away from the ventricular surface.

Supplementary Video 3:
Example 2 of a DOCK7 overexpressing RGC (cell in upper middle) undergoing bl-to-ap INM in VZ of neocortex. Time-lapse imaging was carried out on acute cortical slices 2 d after in utero electroporation of plasmids expressing FLAG-DOCK7, and EGFP (green) and mKO2-F (red) fluorescent markers (at E15.5). Images acquired over an 8 h time period are shown; time (in min) is indicated on the top. Note: the cell body of the RGC remains at its basal position for about 4.5 h and then moves over a very short distance toward the ventricle, which is followed by cell division away from the ventricular surface.

Supplementary Video 4:
Example of a control scr#1 shRNA expressing RGC (cell in the middle) undergoing bl-to-ap INM in VZ of neocortex. Time-lapse imaging was carried out on acute cortical slices 2 d after in utero electroporation of plasmids expressing scr#1 shRNA, and EGFP (green) and mKO2-F (red) fluorescent markers (at E15.5). Images acquired over an 8 h time period are shown; time (in min) is indicated on the top. Note: the cell body of the RGC migrates steadily toward the ventricle. After about 5 h, it reaches the ventricular (apical) surface and the cell subsequently divides at the ventricular surface.

Supplementary Video 5:
Example 1 of a Dock7#2 shRNA expressing RGC (cell in the middle) undergoing bl-to-ap INM in VZ of neocortex. Time-lapse imaging was carried out on acute cortical slices 2 d after in utero electroporation of plasmids expressing Dock7#2 shRNA, and EGFP (green) and mKO2-F (red) fluorescent markers (at E15.5). Images acquired over an 8 h time period are shown; time (in min) is indicated on the top. Note: the cell body of the RGC migrates considerably faster to the ventricle surface than that of the control scr#1 shRNA expressing RGC, where it then remains for about 3 h before the cell undergoes apical mitosis.