Figure 6. Cortical basal progenitors are increased and display columnar distribution in the cortex of TBC1D3-transgenic mice.

(A) Staining for Pax6, Sox2, and Tbr2 in E12.5 WT and TG mice. Dash lines indicate pial surfaces. Note the increase in Pax6⁺Sox2⁺Tbr2^- cells (white arrowheads) in the basal region of TG mice. Scale bar, 50 μm. (B) Quantification for the density of Pax6⁺Sox2⁺Tbr2^- cells in extra-VZ regions (WT: n = 9 slices from 3 mice, mean = 0.26, SEM = 0.09; TG: n = 11 slices from 4 mice, mean = 4.01, SEM = 0.57). p<0.0001. (C) E13.5 TG mice were subjected to IUE with EGFP-expressing plasmids to label cell morphology, and brain sections were stained for Pax6 at E16.5 (left panel). Scale bar, 5 μm. Three types of oRG-like cells constitute more than half of Pax6⁺ BPs (right panel, 82 Pax6⁺EGFP⁺ cells from 5 brains were analyzed). (D and E) DiI labeling (D) and quantification (E) of RG cells in E14.5 WT and TG mice (WT: n = 9 slices, mean = 0.72, SEM = 0.37; TG: n = 13 slices, mean = 3.92, SEM = 0.88). p = 0.009. White arrows indicate typical oRG-like cells with soma located in the SVZ/IZ and a basal process attached to the pial surface. Scale bar, 50 μm. (F) E12.5 TG mice were stained with Sox2, and Tbr2. Note the columnar distribution of Sox2⁺Tbr2^- cells in basal regions, as illustrated for the boxed area. Scale bar, 50 μm. (G) Distribution profile of Sox2⁺Tbr2^- cells in the basal region of TG mice cortex ranging from dorsal to lateral cortical regions. (H) Immunostaining for Sox2, Pax6, Tbr2 in E14.5 WT and TG mice cortex. Yellow dash lines indicate the brain surface. Note the apparent columnar distribution of Sox2⁺Pax6⁺Tbr2^- cells (yellow dotted circles) in the boxed area below a cortical gyrus-like structure (white arrow). Scale bars, 50 μm. (I and J) Immunostaining (I) and quantification (J) of HOPX cells in the extra-VZ (white arrowheads) of E14.5 WT and TG mice cortices (WT: n = 3 brains, mean = 0.03, SEM = 0.03; TG: n = 3 brains, mean = 2.66, SEM = 0.08). p<0.0001. Scale bar, 20 μm.

DOI: http://dx.doi.org/10.7554/eLife.18197.022

Figure 6—figure supplement 1. Cortical basal progenitors are increased in the cortex of TBC1D3-transgenic mice.

(A and B) Immunostaining for PH3 in WT and TBC1D3 TG mice at E12.5. White dash lines in enlarged areas (B) indicate cortical surfaces, and yellow dash lines indicate the boundary between apical and basal regions. Scale bars, 50 μm. (C and D) Quantification for the density of PH3⁺ cells distributed radially (C, from ventricular to pial surface) or in apical/basal regions (D), respectively. Apical: n = 3 mice, mean = 453.37, SEM = 24.89 for WT, n = 4 mice, mean = 432.36, SEM = 24.99 for TG; Basal: mean = 251.14, SEM = 13.09 for WT; mean = 378.62, SEM = 42.49 for TG. p-values are 0.570 (apical) and 0.017 (basal). (E) Staining for Pax6, Sox2, and Tbr2 in E12.5 WT and TG mice. Scale bar, 50 μm. (F) The densitys of Pax6⁺ cells in the cortex of WT and TG mice were quantified (WT: n = 3 mice, mean = 55.5, SEM = 1.67; TG: n = 4 mice, mean = 61.59, SEM = 1.43). p = 0.014.

DOI: http://dx.doi.org/10.7554/eLife.18197.023

Figure 6—figure supplement 2. Increased proliferation potency of BPs in TBC1D3-transgenic Mice.

(A) BrdU and EdU were sequentially administered into TG or WT mice, at E13.5 and E16.5, respectively. Double positive cells from cerebral cortex of mice 2 hr after EdU injection were analyzed. Note the cells indicated by white arrows in magnified areas (A1 and A2). Dash lines indicate the boundary of apical and basal regions in cortex. Scale bars, 50 μm. (B) Quantification for the density of BrdU/EdU double positive (BrdU⁺EdU⁺) cells in the dorsal cerebral cortex of WT (apical: mean = 319.94, SEM = 52.36; basal: mean = 20.34, SEM = 5.09) and TG (apical: mean = 276.19, SEM = 43.31; basal: mean = 237.93, SEM = 81.52) mice. p-values WT vs TG are 0.544 (apical) and 0.029 (basal) (n = 4 mice in each group).

DOI: http://dx.doi.org/10.7554/eLife.18197.024

Figure 6—figure supplement 3. Increased neurons in the superficial layer of the cortex of TBC1D3-transgenic mice.

(A) Schematic for the motor cortex (M1/M2) and sensory (S) cortex, and the brain regions (blue or red rectangle) for the staining analysis. (B) Staining for the superfical layer marker Cux1 and the deep layer marker Ctip2 in the motor cortex of P3.5 mice. Scale bar = 50 μm. (C) Staining for NeuN and GFAP signals in the motor cortex of P28 mice. Scale bar = 50 μm. (D) Quantification for the density of Cux1⁺ (WT: mean = 44.02, SEM = 5.91; TG: mean = 60.83, SEM = 0.92; p = 0.031) or Ctip2⁺ (WT: mean = 30.17, SEM = 2.43; TG: mean = 31.19, SEM = 2.64; p = 0.786) neurons in the motor cortex of P3.5 mice (n = 4 mice). (E and F) Quantification for the density of NeuN⁺ neurons (WT: n = 4 mice, mean = 14.70, SEM = 0.43; TG: n = 3 mice, mean = 16.79, SEM = 2.85; p = 0.014) and GFAP⁺ astrocytes (WT: n = 4 mice, mean = 1.19, SEM = 0.02; TG: n = 3 mice, mean = 1.25, SEM = 0.16; p = 0.645) in the motor cortex of P28 mice. (G and H) Immunostaining and quantification for the superficial layer marker Cux1 in the sensory cortex (red rectangle in A) of P3.5 mice. Scale bar, 50 μm. n = 4 mice, mean = 69.33, SEM = 3.71 for WT; n = 5 mice, mean = 81.07, SEM = 1.59 for TG. p = 0.016.

DOI: http://dx.doi.org/10.7554/eLife.18197.025