(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 analyzed for mRNA levels of indicated genes relative to GAPDH with values from control groups normalized as 1.0 (n = 3 experiments for each group). Trnp1: mean = 1.00, SEM = 0.03 for control; mean = 0.57, SEM = 0.07 for TBC1D3 (p = 0.0003). Hes1: mean = 1.00, SEM = 0.02 for control; mean = 0.96, SEM = 0.05 for TBC1D3 (p = 0.561). Hes5: mean = 1.00, SEM = 0.20 for control; mean = 0.85, SEM = 0.34 for TBC1D3 (p = 0.762). Numb: mean = 1.00, SEM = 0.17 for control; mean = 1.12, SEM = 0.14 for TBC1D3 (p = 0.612). (E) ReNeuron cells were transfected with constructs encoding siRNA targeting TBC1D3 or scrambled sequence. After 3 days, transfected cells were treated with actinomycin D for 4 hr and the mRNA level of Trnp1 relative to Hprt was quantified (n = 4 experiments; mean = 94.40, SEM =0.89 for control; mean = 94.67, SEM = 1.05 for siTBC1D3; p = 0.851). Data are presented as mean ± SEM of percentage of Trnp1 mRNA compared to the value prior to actinomycin D treatment. (F) Cortices of E14.5 WT and TG mice were stained for pERK1/2 and TBC1D3. Scale bars, 20 μm (F), 10 μm (F1). (G) Quantification for the ratio of pERK1/2 intensity in VZ/SVZ regions to that in CP (WT: n = 3 mice, mean = 1.17, SEM = 0.07; TG: n = 5 mice, mean = 1.50, SEM = 0.05). p = 0.006. (H) GW15.5 human brain slice was stained with pERK1/2, Pax6, and Tbr2 antibodies. Scale bars, 50 μm (H) and 10 μm (H1). (I) Quantification of pERK1/2 levels in different types of OSVZ progenitors (n = 2 slices). Note that almost all pERK1/2 signals are detected in Pax6+Tbr2- cells. (J) Proposed model for the role of TBC1D3 in cortical folding. TBC1D3 expression causes delamination of vRG cells, through down-regulating the level of N-cadherin and Trnp1, and promotes proliferation of oRG-like cells by regulating cell stemness pathways, including Ras/ERK signaling. The increased generation of the oRG-like cells, the IP cells, and subsequently regional increase in the density of new born neurons, induces cortical folding in mice.