Fig 2: POGZ regulates the mouse cortical neuronal development.a Temporal expression patterns of Pogz mRNA in the brain (each n = 3). E embryonic day; wk week-old. b Regional expression pattern of Pogz mRNA in the E16.5 brain (each n = 3). VZ ventricular zone; SVZ subventricular zone. c Fluorescence in situ hybridization of coronal sections of the E16.5 brain for Pogz and immunostaining with antibodies against PAX6, SOX2, and TBR2. Scale bars, 100 µm. LV lateral ventricle; VZ ventricular zone; SVZ subventricular zone; IZ intermediate zone. d Impairment of neuronal migration by shRNA-mediated knockdown of Pogz in E18.5 mouse cortices electroporated at E14.5. CP cortical plate; IZ intermediate zone; SVZ subventricular zone. Scale bars, 50 µm. e Quantification of GFP+ cells in each layer (each n = 4). f GFP+ neurons were co-labeled for SATB2 (a layer II/III marker). g Quantification of GFP+ SATB2+ neurons (n = 4). h GFP+ neurons were co-labeled for CTIP2 (a layer V marker). i Quantification of GFP+ CTIP2+ neurons (n = 4). j GFP+ neurons were co-labeled for TBR1 (a layer VI marker). k Quantification of GFP+ TBR1+ neurons (n = 4). Note that Pogz knockdown had little effect on the proportion of SATB2+ GFP+, CTIP2+ GFP+ or TBR1+ GFP+ neurons and that the GFP+ cells with delayed migration were mostly SATB2+ neurons. h Same slice as in f. f, h, j Right panels, magnifications of the areas outlined with orange boxes. White scale bars, 50 µm; orange scale bars, 10 µm. a, b, g, i, k One-way ANOVA with Bonferroni–Dunn post hoc tests; a F10, 22 = 48.52; b F7, 16 = 73.23; g F2, 9 = 0.393; i F2, 9 = 1.079; k F2, 9 = 0.239. e Two-way repeated-measures ANOVA with Bonferroni–Dunn post hoc tests, F4, 27 = 39.28. ***P < 0.001. Data are presented as the mean ± s.e.m.

Fig 3: Analysis of neural marker expression in the 3D bioprinted construct. (A) Phase contrast images of cells within the 3D bioprinted construct (“3D printed” panels), at the indicated days post printing, and cells in conventional monolayer conditions (“2D” panels) or resuspended in the bioink (“3D bulk” panels) and maintained for the same time of differentiation. (B) RT-PCR analysis of neuronal progenitor markers (PAX6, FOXG1, TBR2), a cortical neuron marker (TBR1) and an astrocyte marker (GFAP). GAPDH was used as a housekeeping control. (C) Immunostaining analysis of bioprinted cells at DPP7. MAP2 (green), TBR1 (red) and DAPI (blue) signals are shown. Scalebar: 150 µm. (D) Mosaic reconstruction of confocal images of bioprinted neural cells at DPP70, showing the entire sample, stained with MAP2 (green), TBR1 (white) and GFAP (red) antibodies. Scalebar: 2 mm. (D’) Mosaic reconstruction of confocal images of the region inside the white box in panel D, acquired at higher resolution. Scalebar: 300 µm.

Fig 4: Evaluation of cellular identity and morphology in 3D brain organoid.A Representative immunostaining showing the presence of a N-CADHERIN-positive (green) germinal layer in correspondence of the apical membrane of the ventricular zone layer in a FMRP-WT brain organoid at day 50. B, C Representative images revealing forebrain progenitor markers in developing cortical plates within FMRP-WT brain organoid at day 50: B SOX2 (green), DAPI (blue); C PAX6 (red), FOXG1 (green) and DAPI (blue). D Representative staining of early-born deep-layer cortical neurons positive for TBR1 (red), CTIP2 (green) markers, and pan-neuronal MAP2 marker (white) in a FMRP-WT brain organoid at day 50. E Representative images of a cortical plate from a FMRP-WT brain organoid at day 100 revealing heterogeneous cellular population, labeled for specific cortical neuronal marker (CTIP2, green), astrocytic marker (GFAP, red), and neuronal progenitor marker (PAX6, white). Nuclei were stained with DAPI (blue). Scale bar: 100 µm.