The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice
- Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
- Chinese Academy of Sciences, China
- Chinese Academy of Sciences University, China
- Shanghai University, China
- State Key Laboratory of Medical Neurobiology, Fudan University, China
- CAS Center for Excellence in Brain Science and Intelligence Technology, China
- ShanghaiTech University, China
Figures
Figure 1 with 2 supplements
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 …
Figure 1—figure supplement 1
The mRNA level of N-cadherin gene is reduced by TBC1D3 expression in mouse cortex.
(A) In utero electroporation (IUE) of TBC1D3 or control plasmid, together with YFP, in mouse at E13.5 and examined at E15.5. Adjacent sections were subjected to in situ hybridization (ISH) to …
Figure 1—figure supplement 2
Dislocalization of adherens junctions proteins upon TBC1D3 expression in the VZ.
E13.5 mouse brain was subject to IUE with pCAGGS-TBC1D3-IRES-EGFP or vehicle control plasmids, following by staining at E15.5 with antibodies of Numb or integrin beta 1 (ITGB1), or …
Figure 2 with 4 supplements
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) …
Figure 2—figure supplement 1
Colocalization between BrdU and Ki67 in TBC1D3-expressing BPs.
E13.5 mouse brain was subjected to IUE with TBC1D3 and YFP, followed by BrdU injection for 2 hr at E15.5, and immunostaining with Ki67 or NeuN antibody. Yellow arrows show BrdU+YFP+ cells co-labeled …
Figure 2—figure supplement 2
Cell autonomous and non-cell autonomous effects of TBC1D3 on neural progenitors.
E13.5 mice were subjected to IUE with pCAGGS-TBC1D3-IRES-EGFP plasmid or pCAGGS-IRES-EGFP as the control, followed by 2 hr BrdU incorporation (A) or staining with PH3 antibody (C) at E15.5. Number …
Figure 2—figure supplement 3
Detachment of vRGs induced by blocking N-cadherin-mediated adhesion does not promote generation of BPs.
(A) E13.5 mice were subjected to IUE with Myc-EC1 or vehicle plasmid, together with YFP plasmid to mark cell morphology. Dash lines indicate endfeet of vRG cells attached to the ventricular surface …
Figure 2—figure supplement 4
Effect of dominant-negative form of Ras on proliferation of TBC1D3-induced BPs.
(A) E13.5 mice were subjected to IUE with TBC1D3, Ras-DN (RasS17N), TBC1D3 plus Ras-DN, or control plasmids, mixed with EGFP, followed by BrdU incorporation for 2 hr at E15.5 and staining. Shown are …
Figure 3 with 2 supplements
TBC1D3 expression in the mouse cortex elevates proliferative oRG cells.
(A) Staining for Pax6 in E15.5 mice after IUE at E13.5 with pE/nestin-TBC1D3 or pE/nestin (control), together with YFP to mark cell morphology. Scale bars, 50 μm. (B) Quantification for the …
Figure 3—figure supplement 1
oRG-like cells and IPs increase in the basal region of TBC1D3-expressing mouse cortex.
(A and C) Staining for phospho-Vimention (p-Vim) and Sox2 (A) or p-Vim and Pax6 (C) in E15.5 mice subjected to IUE at E13.5. Dash lines indicate the boundary between basal (outer VZ) and apical (VZ) …
Figure 3—figure supplement 2
Birth dating analysis for division patterns of neuroprogenitors after IUE.
(A) Schematic of BrdU/EdU double labeling. (B) E13.5 mice were subjected to IUE with TBC1D3 or vehicle plasmid, together with YFP plasmid, and then injected with BrdU at E14.5 and EdU at E15.5, to …
Figure 4
Knockdown of TBC1D3 in human vRGs inhibits the generation of oRGs.
(A) Detection of TBC1D3 protein levels in Hela cells transfected with pSuper-siTBC1D3 plasmids, with a scramble sequence as the control. (B) Paradigm of culture and electroporation of human brain …
Figure 5 with 4 supplements
Cortical folding and gyrification induced by TBC1D3 expression in mice.
(A) E13.5 mice were subjected to IUE with pE/nestin-TBC1D3 or vehicle control plasmids, together with RFP to mark electroporated cells, and analyzed at the indicated time. White arrows indicate …
Figure 5—figure supplement 1
TBC1D3 electroporation has no effect on the pial basement membrane integrity.
Shown are representative images of E15.5 brain slices (IUE with pCAGGS-TBC1D3-IRES-EGFP or control vehicle at E13.5) stained with laminin antibody. PS, pial surface; CP, cortical plate. Scale bar, …
Figure 5—figure supplement 2
Generation of TBC1D3 transgenic mouse.
(A) Schematic structure of the construct used for generating TBC1D3 transgenic mice. The expression of TBC1D3 was under the control of promoter composed of the second intron enhancer of rat nestin …
Figure 5—figure supplement 3
Normal neuronal migration in the cortical plate of TG mice.
(A) E13.5 WT or TG mice were subjected to IUE with EGFP expression plasmids, and the cortical plates of mice at E16.5 were examined for the distribution of EGFP+ cells. (B) Percentage of EGFP+ cells …
Figure 5—figure supplement 4
Cortical folding mainly occurs in the motor cortex of TBC1D3-transgenic mice.
(A) Top view of a P3.5 TG mouse brain hemisphere shows the position of a cortical fold (left panel) and a summary distribution of cortical folds (right panel) on the brain surfaces from 3 TG mice at …
Figure 6 with 3 supplements
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 …
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 …
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 …
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 …
Figure 7
Effect of TBC1D3 on intrinsic stemness signaling pathways and model for the TBC1D3 function in cortical folding.
(A–D) E13.5 mice were subjected to IUE with TBC1D3 or vehicle control plasmid, together with YFP plasmid, and at E15.5, FACS-sorted transfected cells (see Figure 1—figure supplement 1B,C) were …
Videos
Video 1
oRGs in cultured organotypic slices from control mice.
This movie illustrates the behavior of the sparse oRGs in organotypic slice cultures from control mice electroporated with vehicle plasmid. IUE was done at E13.5 and organotypic slice culture was …
Video 2
oRGs in cultured organotypic slices from TBC1D3-electroporated mice.
This movie illustrates the behavior of TBC1D3-expressing oRGs. IUE was done at E13.5 and organotypic slice culture prepared at E14.5 and observed 2–3 hr after culture preparation. White arrows …
