Skip to main content
. 2017 Aug 17;101(3):428–440. doi: 10.1016/j.ajhg.2017.07.010

Figure 3.

Figure 3

Effects of TBC1D23 Downregulation on Neuronal Migration and Positioning

Short hairpin RNA and cDNA constructions were co-electroporated with a RFP-encoding reporter construction (Tomato) in progenitor cells adjacent to the ventricle in E14.5 embryonic mouse cortices. Analyses were done at E18.5 or P3.

(A) Coronal sections of E18.5 brains electroporated at E14.5 with either a control shRNA or one of the three different anti-TBC1D23 shRNAs. Sections were stained with DAPI. CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone. Scale bar, 50 μm.

(B) Quantification of fluorescent neurons positioning in four different regions: up CP, down CP, IZ, and VZ/SVZ. Data are represented as mean ± SEM. shRNA-1 versus control, p = 0.0165 (IZ), p = 0.041 (up CP); shRNA-2 versus control, p < 0.0001 (IZ), p < 0.0001 (up CP); shRNA-3 versus control, p < 0.0001 (IZ), p < 0.0001 (up CP).

(C) Coronal sections of E18.5 brains electroporated at E14.5 with empty vector, mouse Tbc1d23-WT cDNA, control shRNA, shRNA-2, or shRNA-2 together with Tbc1d23-WT (Rescue). For rescue experiments, two different concentration ratios were tested: 1 μg/μL shRNA-2 + 1 μg/μL Tbc1d23-WT and 1 μg/μL shRNA-2 + 3 μg/μL Tbc1d23-WT. Sections were stained with DAPI. Scale bar, 50 μm.

(D) Quantification of fluorescent neurons positioning in four different regions: up CP, down CP, IZ, and VZ/SVZ. Data are represented as mean ± SEM. shRNA-2 versus shRNA-2 + Tbc1d23-WT p = 0.0078 (IZ); shRNA-2 versus shRNA-2+3∗Tbc1d23-WT p < 0.0001 (IZ), p = 0.0113 (down CP).

(E) Coronal sections of P3 brains electroporated at E14.5 with either a control shRNA or shRNA-2. Sections were stained for the upper layer marker Cux1 (cyan) and with DAPI in order to define upper and deep cortical layers and the white matter (WM). Scale bar, 100 μm.

(F) Fluorescent neurons were counted in four different regions: Cux1 layers (II–IV), layer V, layer VI, white matter. Data are represented as mean ± SEM. Control versus shRNA-2: p < 0.0001 (Cux1 layers), p = 0.025 (WM).