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. 2020 Sep 2;9:e58737. doi: 10.7554/eLife.58737

Figure 3. Manipulation of mTOR signaling results in migration defects.

(A) shRNAs were delivered along and electroporated onto the ventricular surface of primary cortical tissue which was then acutely sectioned and cultured for six days prior to collection. (B) After electroporation of RPTOR or TSC2 shRNAs, GFP+ HOPX+ oRGs migrate less from the ventricular edge (n = 37 control, n = 33 Raptor and n = 27 TSC2-shRNA electroporated GFP+Hopx+ cells from three independent experiments; D’Agostino Pearson Normality Test: normally distributed; one-way ANOVA with multiple comparisons: Raptor shRNA: ****p<0.0001, TSC2 shRNA: ****p<0.0001, error bars represent SD). The distance of each HOPX+ GFP+ cell away from the VZ edge was measured as indicated by white brackets. (C) For dynamic imaging studies, primary cortical tissue was collected, dissociated, infected with a CMV::GFP adenovirus, and plated on glass-bottom 12 well plates. Small molecules were added one day later, two hours before the start of dynamic imaging. (D) GFP+ oRG cells undergo division via MST. Yellow arrowheads indicate cell body, white dot indicates initial position of cell body and pink arrowheads indicate two cell bodies after division. After inhibition of mTOR signaling oRG cells have shorter MSTs (n = 10 control and n = 8 rapamycin cells across two independent experiments; D’Agostino Pearson Normality Test: normally distributed; unpaired two-tailed student’s t-tests: **p<0.0082, error bars represent SD). (E) oRGs migrate less from their original position after mTOR inhibition (n = 9 vehicle treated and n = 12 rapamycin treated cells from two independent experiments; D’Agostino Pearson Normality Test: normally distributed; unpaired two-tailed student’s t-tests: **p<0.0058 error bars represent SD). White, yellow, and pink arrowheads indicate cell bodies at starting time-point. Multiple arrowheads of the same color over time indicate daughter cells from the same parent cell.

Figure 3—source data 1.

Figure 3.

Figure 3—figure supplement 1. shRNAs against mTOR pathway components cell autonomously regulate oRG process length.

Figure 3—figure supplement 1.

(A) shRNAs were delivered along the ventricle of primary cortical tissue which was acutely sectioned and cultured for six days prior to collection. (B) Organoids were bathed in medium containing shRNAs, electroporated inside a cuvette and then cultured for 4–10 days prior to collection. (C) Electroporation of either RPTOR or TSC2 shRNAs resulted in a decrease in the length of the GFP+ basal process in HOPX positive cells (n = 19 control, 20 Raptor and 12 TSC2-shRNA electroporated GFP+Hopx+ cells from three independent experiments, one-way ANOVA: ****p<0.0001 Raptor, ****p<0.0001 TSC2, error bars represent SD). RPTOR electroporation resulted in a decrease in mTOR signaling indicated by fewer pS6+GFP+ cells, while TSC2 electroporation resulted in an increase in pS6+GFP+ cells (n = 2 tissue slices from two independent experiments; error bars represent SD). Cyan arrowheads indicate GFP+HOPX+ or GFP+pS6+ cells and white arrowheads indicate cells that express either GFP or pS6. Magenta arrowheads indicate the basal processes of GFP+ cells. (D) Electroporation of RPTOR and TSC2 shRNAs in week 8–10 cortical organoids. RPTOR and TSC2 shRNA electroporation decreased the GFP+ primary process length in oRG cells (n = 49 control, n = 39 Raptor, n = 27 TSC2 electroporated GFP+HOPX+ cells from nine organoids per condition across four independent experiments, one-way ANOVA: ***p<0.0009 Raptor, ***p<0.0003 TSC2, error bars represent SD). There were fewer pS6+ GFP+ cells after RPTOR shRNA electroporation, while TSC2 electroporation did not significantly affect the number of pS6+GFP+ cells in the organoid (n = 21 control, n = 22 Raptor, n = 23 TSC2 sections from nine organoids per condition across four independent experiments; one-way ANOVA: **p<0.0065 Raptor, p=0.8 TSC2, error bars represent SD). Cyan arrowheads indicate GFP+HOPX+ or GFP+pS6+ cells and white arrowheads indicate cells that express either GFP or pS6. Magenta arrowheads indicate the basal processes of GFP+ cells. (E) Constructs are dropped along the ventricular edge of intact primary tissue and pulsed with electricity before sectioning and culturing. GFP+ electroporated cells (white arrowheads) are initially restricted to the ventricular edge demonstrated by their position one and two days post- electroporation. By five days post-electroporation GFP+ cells from the ventricular edge have migrated to the oSVZ (n > 2 slices/group across two independent experiments).
Figure 3—figure supplement 1—source data 1.