Fig 2: TBC1D3 expression in developing mice cortex disrupts adherens junctions and induces formation of column-like structures.(A) RT-PCR analysis for the expression of TBC1D3 mRNA in developing (GW26 - 40) and adult (21–29 years) human whole brain with GAPDH as control. (B) The expression pattern of TBC1D3 in fetal human cortex at GW18. VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars, 50 µm. (C–E) In utero electroporation (IUE) of the mouse cerebral cortex was performed at E13.5 and analyzed at E15.5 (C and D) or E16.5 (E). TBC1D3 or control plasmid was mixed with YFP. (C) In situ hybridization analysis for the expression of TBC1D3 in mouse brain sections. Scale bar, 500 µm. (D) Adherens junctions in the ventricular surface were marked by N-cadherin staining. Scale bars, 50 µm. (E) Distribution of GFP+ cells in E16.5 mice cortex. The right panels show magnified regions indicated by asterisks in left panels, with histograms outlining relative fluorescence intensity of tangentially distributed GFP+ cells. Scale bars, 50 µm.DOI: http://dx.doi.org/10.7554/eLife.18197.003

Fig 3: Expression of TBC1D3 induces an increase in cell proliferation in basal regions of developing mouse cortex.(A) Schematic of BrdU pulse labeling. (B) E15.5 whole brain section incorporated with BrdU after IUE at E13.5. Dashed rectangle indicates cortical area for quantification. Scale bar, 200 µm. (C) BrdU incorporation in mice subjected to IUE with indicated plasmids. Scale bar, 50 µm. (D) Staining for phosphorylated Histone H3 (PH3) in E15.5 mice subjected to IUE at E13.5. Scale bar, 50 µm. (E) Quantification for the percentage of BrdU+ cells among electroporated YFP-labeled cells (control: n = 5 mice, mean = 19.18, SEM = 0.69; TBC1D3: n = 10 mice, mean = 31.91, SEM = 2.02; ?286–353: n = 9 mice, mean = 21.62, SEM = 1.36). Respective p-values are 0.0003 (control vs TBC1D3) and 0.0004 (TBC1D3 vs ?286–353). (F) Mean cortical distribution of BrdU+ cells among electroporated YFP-labeled cells (n = 5 mice). (G) Quantification for the percentage of PH3+ cells among electroporated YFP-labeled cells attached to ventricular surface (VZ surface) or out of ventricular zone (extra-VZ). Total number of PH3+ cells in VZ surface and extra-VZ were normalized to 100 for each group (n = 8 mice, mean = 28.10, SEM = 3.43 for extra-VZ of control; n = 10 mice, mean = 57.22, SEM = 2.24 for extra-VZ of TBC1D3; n = 8 mice, mean = 35.99, SEM = 3.26 for extra-VZ of TBC1D3 plus N-cadherin). ***p<0.001.DOI: http://dx.doi.org/10.7554/eLife.18197.006

Fig 4: Blockade of TBC1D3 interaction with G9a down-regulates expression of genes associated with NP proliferation.(A) Top six enriched KEGG pathways by clustering analysis of down-regulated genes in T-T groups compared with T-S groups. (B) Heatmap showing representative down-regulated genes in T-T–treated organoids. (C) Relative mRNA levels of indicated genes to GAPDH measured by quantitative PCR analysis for D16 organoids treated with T-T or T-S peptides. Data are presented as means ± SD of at least five experiments in each group with values from T-S group normalized as 1. Unpaired Student’s t test. **P < 0.01; ***P < 0.001. (D) ChIP-seq showing the count and distribution of peaks around TSSs in T-S– or T-T–treated organoids at D16. (E) H3K9me2-binding peaks increase in T-T–treated organoids. (F) Genomic tracks showing differential H3K9me2 enrichment regions near TSS of FZD10 and WNT4. (G and H) Venn diagrams (G) and GO biological enrichment analysis (H) of genes corresponding to ChIP-seq peaks and DEGs identified by RNA-seq. (I) A proposed model showing a role of TBC1D3 in promoting NP proliferation and cortex expansion through down-regulating the level of H3K9me2. PI3K, phosphatidylinositol 3-kinase.

Fig 5: 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