Fig 2: Bidirectional Molecular Communication between IPs and cINs Shapes Neocortex Histogenesis(A and B) Number of proliferating cells and cINs in the pallium (A), 1 hr post EdU injection at several distances from the CSB in CCP1 WT and cKO E13.5 brain sections (B) (n = 3 embryos from 3 females donors; two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001).(C) EdU immunodetection 48 hr post-injection in E13.5 brain sections from CCP1 WT and cKO embryos. cINs express Cre-GFP and the CSB is highlighted by dashed yellow lines. Scale bar, 500 µm.(D) EdU, Sox2, and Tbr2 immunodetection on brain sections from E13.5 CCP1 WT and cKO embryos. cINs express Cre-GFP.(E) Number of Tbr2+ and Sox2+ normalized on total number of progenitor cells of the pallium in E13.5 brain sections from CCP1 WT and cKO embryos (n = 5 embryos from at least 3 females; non-parametric t test, p > 0.05).(F and G) Number of proliferating EdU+/Sox2+ (F) and EdU+/ Tbr2+ (G) in E13.5 brain sections from CCP1 WT and CCP1 cKO embryos, normalized on total number of progenitor cells of the pallium (n = 5 embryos from at least 3 females; non-parametric t test, p < 0.01).(H) EdU, Sox2, and Tbr2 immunodetection on brain sections from E13.5 Nkx2.1 WT and Nkx2.1 KO embryos.(I) Number of Tbr2+ and Sox2+ normalized on total number of pallial progenitor in E13.5 brain sections from Nkx2.1 WT and Nkx2.1 KO embryos (n = 4 embryos from at least 3 females; non parametric t test, p > 0.05).(J and K) Number of proliferating EdU+/Sox2+ (J) and EdU+/Tbr2+ (K) in E13.5 brain sections from Nkx2.1 WT and KO embryos, normalized on total number of progenitors (n = 4 embryos from at least 3 females; non-parametric t test, p < 0.01). Scale bar, 10 µm.(L) Immunodetection of Ctip2+ and Cux1+ at E18.5 and P21 on cortical sections from CCP1 WT and cKO brains. Nuclei are DAPI+. Scale bar, 100 µm.(M and N) Quantification in E18.5 (M) and P21 (N) CCP1 WT and cKO cortex, of the number of Ctip2+ and Cux1+ neurons in the cortical layers normalized on DAPI cell in the cortical plate (n = 4–8 embryos/adolescent mice; non-parametric t test, p < 0.01).All graphs contain bars representing SEM.

Fig 3: Development of DG: Notch activation and sequential IP-NSC migrations.(A) Schematics of DG development and cell migrations (see also Figure 1—figure supplement 1). (B) E12.5 coronal sections immunolabeled for Dll1 and IP and NSC markers: the DNe is marked by intense Dll1+ immunoreactivity (red arrowheads and brackets) with increased IPs (top: Dll1+,Tbr2+, Dll3+, Tbr2GFP+ TG) and Notch active NSCs (bottom: Sox9+ Hes1+). (B1) Boxed region from Hes1 rotated 90o shows Dll1+/Hes1-/GFP+ IPs dividing in basal VZ and extending long-range branched processes from SVZ into VZ (arrows), and mitotic-Hes1low NSCs at the VZ surface encased by neighboring Dll1 puncta (arrowheads). (C) On E14.5, Tbr2+ IPs pioneered the DMS (inset arrow) in advance of Sox9+ NSCs (inset arrowhead). (D) Most cells in E14.5 DMS were IPs (Tbr2+); NSCs (Pax6+/Tbr2-) were sparse (inset arrowhead). (E) By E16.5, the DMS contained Sox9+ NSCs, as well as Tbr2+ IPs: the FDJ and SPNZ had developed and likewise contained both progenitor types. Bottom panel: mitotic Tbr2+ IPs (arrows) and Sox9+ NSC (arrowhead) in the DMS. (F) By E18.5, transient neurogenic niches (DMS?FDJ?SPNZ,Hi) were well defined and the SPB was visible. Note Sox9+ mitosis in DMS (inset). (G) The E18.5 SPB (top panel) was delimited by rows of NSCs (Sox9+) in the SPNZ and nascent SGZ. Some NSCs were proliferative (PCNA+), others quiescent (PCNA-). PCNA+/Sox9- proliferative IPs (bottom panel, double arrow) were also seen in E18.5 SPNZ. (H) On P2, Tbr2+ IPs and Sox+ NSCs were abundant in the DMS, SPNZ, Hi, and FDJ; and IPB formation was apparent. Boxed area shown at higher magnification in right panel. Note mitotic Tbr2+ IP in the nascent SGZ (yellow arrowhead). (I) Rare triple-labeled Tbr2+/Sox9+/Pax6+ mitotic figure in the FDJ, in transition from NSC to IP. (J) By P10, the SGZ contained abundant Tbr2+ IPs and Sox9+ NSCs (arrows). Transient niches (SPNZ, FDJ, Hi) were declining. Sox9+ astrocytes were scattered in the Hi and molecular layer (ML). (K) In P10 SGZ, Tbr2+ IPs clustered near Sox9+ NSCs with GFAP+ radial processes (arrows). (L) GFAP+/Sox9+ astrocytes, including mitotic forms (arrows), were also present (shown in ML).

Fig 4: BAF53a regulates cell cycle progression in the developing cortex. (A) Representative coronal sections through the developing mouse brain at E14.5 depicting expression of BAF53a (top left) and BAF53b (bottom left). Dashed line delineates the wall of the lateral ventricle (LV). (Middle) Immunofluorescence staining shows BAF53A expression in SOX2+ (blue) and TBR2+ (green) neural stem and intermediate progenitor cells, respectively. (Right) Immunolabeling for BAF53b and MAP2 (green) reveals strong BAF53b expression in postmitotic neurons in the cortical plate at E14.5. Scale bars, 100 µm. Note: The BAF53a antibody cross-reacts with BAF53b in immunostainings of brain sections. (B) BAF53a mutant brains at P0. Photomicrographs of control, heterozygous, and BAF53a mutant brains at P0 (left) and Nissl stains of sagittal sections (right). Closed arrow points to cerebellum and open arrow indicates the neocortex. (C, top panel) Immunoprecipitation and western blot analysis of BAF proteins in control and Baf53a mutant nuclear extracts using an anti-BRG1 antibody. (Bottom panel) ?-32P labeled-ATP was used as substrate to assess BAF ATPase activity, separated by chromatography and visualized by autoradiography. (D) Western blot on BAF53a-deficient and control nuclear glycerol gradient (0%–30%) fractions analyzed for BAF, PRC2, and TIP60 complex components. (E, top panel) Growth curve of Actin-CreER; Baf53afl/- neurospheres for 6 d after tamoxifen (Tam) or ethanol (EtOH) control treatment. (Bottom panel) Quantification of cell cycle FACS analysis of Actin-CreER; Baf53afl/- neurospheres. Cells were treated with BrdU 2 h before collection and harvested 72 h after tamoxifen administration for FACS. n = 3. Error bars represent SEM. (*) P < 0.05.

Fig 5: TBR2 removal impairs preferential chemical synaptic connectivity. a Schematic diagram of in utero intraventricular injection of retrovirus expressing EGFP (green, Ctrl) or Cre/tdTomato (red, Tbr2 Mut) into the Tbr2fl/fl mice at E12-13. Acute brain slices were prepared at P14–25 for quadruple whole-cell patch-clamp recording. b, e Representative confocal images of two radially situated EGFP-expressing sister excitatory neurons (1 and 3, green, Ctrl, b) or Cre/tdTomato-expressing sister excitatory neurons (1 and 3, red, Tbr2 Mut, e) and two adjacent unlabeled non-sister excitatory neurons (2 and 4). Scale bars: 50 µm. c, f Example traces of four excitatory neurons recorded in (b) and (e), respectively. Brief and long duration depolarizing currents were injected into one of the four neurons to elicit action potentials (presynaptic, red) and postsynaptic responses were monitored in the other three neurons. Individual traces are shown in gray and average traces are shown in black. Arrows indicate the reliable postsynaptic responses in control sister excitatory neuron 1 elicited by the presynaptic action potentials in control sister excitatory neuron 3. Scale bars: 100 mV (red), 20 pA (black), and 200 ms. d Zoom-in traces of the presynaptic action potentials and postsynaptic responses between EGFP-expressing control sister excitatory neurons 1 and 3 in (c). Scale bars: 25 mV (red), 10 pA (black), and 25 ms. g Summary of the frequency of chemical synaptic connections between the control (green) or Tbr2 mutant (red) sister excitatory neurons and nearby non-sister excitatory neurons. The numbers of recorded pairs are shown in the bar graph. A similar display is used in subsequent figures (**P < 0.01; ****P < 0.0001; NS not significant; chi-square test)