Generation of neural organoids with different anterior-posterior identities (see also Figures S1-2).

(A) Schematic representation of an embryo; the forming brain is shown in purple, the spinal cord in yellow and the somites are shown in green. Gradients of morphogenetic signals are shown. (B, C) Schematic representation of organoid culture, guiding drugs are indicated at the appropriate days in culture. Selected images of anterior (B) and posterior (C) organoids stained for SOX2 (red), BRA (green), aPKC (grey). Scale bars = 30 µm. (D) Quantification of organoids expressing the indicated markerş SOX2 (red) and BRA (green), after 3, 5 and 7 days (D) in culture. Guiding drugs are indicated in each column, as well as days (D) of treatments.

Anterior and Posterior neural organoids show different transcriptome identity.

(A) Schematic representation of organoid culture and RNA extraction for bulk RNA Sequencing. (B) Heatmap of the pairwise Pearson correlation coefficients of gene expression in the three replicates for both Anterior and Posterior organoids. (C) Volcano plot representation of differential expression analysis of genes in Posterior and Anterior NT organoids. (D) Bubble plots the expression levels of selected neural, mesodermal and endodermal genes in the anterior (blue) and posterior (yellow) organoid samples. (E) Principal Component Analysis (PCA) of RNAseq-based expression data of selected genes for forebrain (purple), midbrain (lilac), hindbrain (purple) and spinal cord (mustard) samples isolated from human embryos (week 5 of development) and anterior (blue) and posterior (yellow) neural organoid samples. (F) Schematic representation of an embryo; the Anterior forming brain is shown in purple, Posterior forming spinal cord in yellow, and the somites are shown in green. (G) Heatmap of the expression of the AP domain-associated selected genes in human forebrain, midbrain, hindbrain and spinal cord samples isolated from human embryos (week 5 of development) and compared to the Anterior and Posterior neural organoid samples. In the x-axis it is represented the hierarchical clustering of the AP domain-associated genes. In the y-axis it is represented the hierarchical clustering of the samples.

Posterior neural organoids are composed by epithelial polarized cells.

(A) Selected images of Posterior (day 7) organoids stained for the ciliary membrane marker ARL13B (green) and the junctional protein βCatenin (βCAT, magenta), DAPI (white). Selected images of the βCAT staining alone are shown in A’. Scale bars = 10 µm. (B) Plots major axis/ minor axis of the cell (aspect ratio) in organoids with mesenchymal-like identity, Anterior, and Posterior organoids (horizontal bold lines show the median; n=100, 100 cells from 5 organoids; ****p<0.0001 Kruskal-Wallis test and Dunn’s multiple comparison test). (C) Selected images of Posterior organoids stained for the centrosome marker pericentrin (PCNT, red) and the junctional complexes protein N-Cadherin (NCAD, blue). Higher magnifications of transversal views are shown in B’. Scale bars = 10 µm. En face views is shown in B’’. Scale bars = 5 µm. (D) Plots nuclei to centrosome distance in cells from organoids with mesenchymal identity, Anterior, and Posterior organoids (horizontal bold lines show the median; n=187 cells from 5 organoids; ****p<0.0001 Kruskal-Wallis test and Dunn’s multiple comparison test). (E) Selected images of transversal views of Posterior organoids stained for ARL13B (green) and βCAT (magenta), DAPI (white). Scale bars = 10 µm. Higher magnifications of mitosis in transversal views are shown in E’. Scale bars = 5 µm. 3D reconstructions of the apical end foot of the cells are shown in E’’. (F) Plots cilia length in cells from organoids with mesenchymal identity, Anterior, and Posterior organoids (horizontal bold lines show the median; n=100, 100 cells from 5 organoids; ****p<0.0001 Kruskal-Wallis test and Dunn’s multiple comparison test). (G) Selected images of transversal views of Posterior organoids stained for the fate-determining factor aPKC (yellow) and the zonular protein ZO1 (cyan). Higher magnifications of transversal views are shown in G’. Scale bars = 10 µm. 3D reconstructions of the apical surface of the epithelium are shown in G’’. (G’’’) Plots show the fluorescence intensity to the distance from the organoid lumen (zero). Yellow line represents aPKC, cyan line represents ZO1 and grey line represents DAPI (white). (H) Scheme of polarized neuroepithelial cell in transversal and en face views. The centrosome (red) is apically localized. Cilia (green) are nucleated from the centrosomes pointing to the lumen of the tissue (red dots). Apical polarity components are organized in the apical end foot, being aPKC (yellow) apical to ZO1 (cyan) and NCAD (blue). βCAT (magenta) is localized in the cell membrane.

The cell and tissue dynamics of neural progenitor cells within the human organoids mimic the early embryonic NT

(A) Selected images of GFP-expressing posterior organoids, at the beginning and the end of the time-lapse video microscopy, where GFP+ cells are tracked undergoing INM. In vivo, organoids are stained for ACTIN (magenta) and GFP (green to blue). Scale bars = 10 µm. (N) Plots the cell cycle length estimation from the time-lapses (n=5). (B) Zoom in of the sequential time point following a NPC (marked with the red asterisk) undergoing INM. NT organoids are labelled for GFP (green to blue). L= Lumen; M= Matrigel. Scale bars = 10 µm. (C) Plot shows the changes in circularity that the NPC undergoes in the time-lapse showed in B. Orange bars highlight the mitosis time points. (D) Plots the cell cycle length estimation from the time-lapses (n=5). (E) Plots the position of the cell nuclei within the organoid, as the distance to the lumen surface in the time-lapse showed in B. Orange bars highlight the mitosis time points. (F) Selected images of posterior NT organoids, 10 days in culture showing laterally positioned Tuj1 (magenta) expressing differentiated neurons, DAPI (white). Scale bar= 10 µm.

Modelling de novo lumen formation in posterior organoids and tracking cell intercalation for lumen resolution

(A) Scheme representing the pre-aggregation step protocol for modelling secondary neurulation in posterior NT organoids. The violet square represents the live imaging time window showed in C and D. (B) Selected images of the general view of posterior organoids maintained 48h in agitation and then transferred to a Matrigel droplet. B’’ shows a selected image of posterior organoids undergoing multiple lumen formation that spontaneously will later resolve into a single lumen (B’’’). Organoids are stained for ACTIN (grey), GFP (green) and DAPI (blue). Scale bars = 50 µm. (C) Selected images of the general view of posterior organoids at the beginning and the end of the time-lapse. Organoids are stained for ACTIN (magenta) and GFP (green to blue). Scale bars = 30 µm. (D) Zoom in of the sequential time point following a NPCs (marked with the red asterisk) undergoing cell intercalation in the posterior organoids showed in C. Taking the ACTIN staining as reference, a long discontinuous line was drawn to mark the BM while the short discontinuous line represents the apical limit of the epithelia. L= Lumen; M= Matrigel. Scale bars = 10 µm. (E) Plot shows the minimum distance of the intercalating cell to the BM showed in D as time goes on. Pink areas highlight the intercalation time points. (F) Plots the changes in cell shape assessed by the ratio between major and minor axis of the intercalating cell showed in D as time goes on. Pink areas highlight the intercalation time points.

Lumen resolution in posterior neural organoids requires YAP activity.

(A) Selected images of peripheral and central cells from Posterior organoids maintained 48h in agitation and then grown in a Matrigel droplet for 24h. Organoids are stained for ACTIN (grey), YAP (red) and DAPI (blue). Dot line represents the limits of the cells based on the ACTIN staining. Scale bars = 5 µm (B) Plots the ratio of the integrated density of the nuclear YAP over the integrated density of YAP in the whole cell (horizontal bold lines show the median; n=23 (peripheral cells) n=18 (central cells), from 7 organoids; *p<0.05 Mann-Whitney test). (C) Plots the mean area occupied by peripheral and central cells (horizontal bold lines show the median; n=10, 10 images from 5 organoids; ***p<0.001 Mann-Whitney test). (D) 3D reconstructions of organoid lumen (ventricle) in control and Dasatinib (DASA) treated cultures. (E) Plots the ratio of lumen perimeter mean over the organoid perimeter at day 1, day 2, day 3 and day 4 of culture in control (black) and DASA (orange) (horizontal bold lines show the median; * p<0.05, ** p<0.005, **** p<0.0001, two-way ANOVA). (F) Schematic representation of the culture conditions Pie charts represent the proportion of organoids with single (grey) and multiple (purple) lumen. (G,H) Selected images of the general view of posterior organoids maintained 48h in agitation and then transferred to a Matrigel droplet in control conditions or with a YAP inhibitor (DASA). Organoids are stained for ACTIN (grey), GFP (green) and DAPI (blue). Scale bars = 20 µm. White dot line delimitates the lumen perimeter while, the white discontinuous line delimitates the organoid perimeter. (I) Schematic representation of the culture conditions. Pie charts represent the proportion of organoids with single (grey) and multiple (purple) lumen.

List of runs for each sample

Screening for the guidance of hESCs towards NMPs lineage and the subsequent restriction to NPCs in 3D cultures (related to Figure 1).

(A) Schematic representation of organoid culture, guiding drugs are indicated at the appropriate days in culture. Selected images of Anterior organoids stained for CDX2 (Yellow) and DAPI (blue) after 3, 5 and 7 days (D). (B, C) Guiding drugs are indicated at the appropriate days in culture. Selected images of organoids stained for SOX2 (red), BRA (green). (D) Guiding drugs are indicated at the appropriate days in culture. Selected images of Posterior organoids stained for CDX2 (Yellow) and DAPI (blue) after 3, 5 and 7 days (D). (E,F) Guiding drugs are indicated at the appropriate days in culture. Selected images of organoids stained for SOX2 (red), BRA (green), after 3, 5 and 7 days (D). Scale bars = 30 µm.

iPSC generated neural organoids with different anterior-posterior identities (related to Figure 1).

(A) Schematic representation of anterior neural organoid culture, guiding drugs are indicated at the appropriate days (D) in culture (0-2). (B) Quantification of organoids expressing the indicated markerş SOX2 (red), BRA (green), and CDX2 (yellow) after 5 and 7 days (D) in culture. iPSC cell lines (Kolf2C1 and CRTD1) are indicated. Guiding drugs are indicated in each column, as well as days (D) of treatments. (C) Schematic representation of posterior neural organoid culture, guiding drugs are indicated at the appropriate days (D) in culture (0-2). (D) Selected images of anterior organoids stained for SOX2 (red) and BRA (green) and DAPI (blue), after 5 and 7 days (D) in culture. (E) Selected images of anterior organoids stained for CDX2 (yellow) and DAPI (blue). (F) Selected images of posterior organoids stained for SOX2 (red) and BRA (green) and DAPI (blue), after 5 and 7 days (D) in culture. (G) Selected images of posterior organoids stained for CDX2 (yellow) and DAPI (blue). Scale bars = 30 µm.

RNAseq of neural organoids revealed dorsal neural progenitor identities (related to Figure 2).

(A) Bubble plots the expression levels in the Anterior (blue) and Posterior (yellow) neural organoid samples, selected genes identified in the Forebrain, Midbrain, Hindbrain, and Spinal Cord of the early human embryos (week 3 of development). (B) Diagram of the DV domains in the developing NT highlighting the selected genes responsible for the identity of the ventral (p3, pMN, p2-0) and the dorsal (dP6-dP1). (C) Heatmap of the expression of the DV domain-associated genes highlighted in A in spinal cord samples isolated from CS14 (week 5 of development) human embryos and Posterior neural organoids samples.

Aosterior neural organoids are composed by epithelial polarized cells (related to Figure 3).

(A, B) Selected images of mesenchyme_like (A) and Anterior neural (B) organoids stained for the ciliary membrane marker ARL13B (green), βCAT (magenta), and DAPI (white). Scale bars = 10 µm. Higher magnifications crops are shown. (C, D) Selected images of mesenchyme-like (C) and Anterior neural (D) organoids stained for the centrosome marker pericentrin (PCNT, red), the junctional complexes protein N-Cadherin (NCAD, blue), and DAPI (white). Higher magnifications are shown. Scale bars = 10 µm. (E, F) Selected images of mesenchyme-like (E) and Anterior neural (F) organoids stained for the fate-determining factor aPKC (yellow) and ZO1 (cyan). Higher magnifications are shown. Scale bars = 10 µm.

Multilumen formation is associated to Posterior organoids, and single lumen resolution is independent on apoptosis (related to Figure 5).

(A) Schematic representation of the culture conditions Pie charts represent the proportion of organoids with single (grey) and multiple (purple) lumen. (B) Selected images of the general view of Anterior organoids maintained 48h in agitation and then transferred to a Matrigel droplet, that spontaneously organize a single lumen. Organoids are stained for ACTIN (grey) and DAPI (blue). Scale bars = 50 µm. (C) Selected images of the general view of Posterior organoids maintained 48h in agitation and then transferred to a Matrigel droplet, that open multiple lumen foci (day 2) that spontaneously organize a single lumen (day 7). Organoids are stained for ACTIN (grey) and DAPI (blue). (D) Schematic representation of the culture conditions Pie charts represent the proportion of organoids with single (grey) and multiple (purple) lumen. (E) Plots Circularity of the cells (4π area/perimeter) in anterior (A) compared to posterior (P) organoids. As the value approaches 0,0 it indicates increasingly elongated shape. (horizontal bold lines show the graphicn; n=100, 100 cells from 5 organoids; ****p<0.0001 Kruskal-Wallis test and Dunn’s multiple comparison test). (F) Plots major axis/ minor axis of the cell (aspect ratio) in anterior (A) compared to posterior (P) organoids (horizontal bold lines show the graphicn; n=100, 100 cells from 5 organoids; ****p<0.0001 Kruskal-Wallis test and Dunn’s multiple comparison test). (G) Schematic representation of the culture conditions. GFP-expressing Posterior organoids were maintained 48h in agitation and then transferred to a Matrigel droplet and fixed after 24h for staining. (H) Selected image of Posterior organoids stained with Phalloidin (grey), Caspase3 (red), GFP (green) and DAPI (blue). Scale bars = 10 µm. (I) Plot shows the percentage of Caspase positive cells in central vs peripheral cells. Horizontal bold lines show the graphicn; n=8 organoids; son significant differences. Mann-Whitney test.

Lumen resolution in posterior neural organoids is associated with an increase in cell density and decrease in nuclear YAP (related to Figure 6).

(A) Scheme representing central and peripheral cells in relation to cell density. (B) Selected image of the general view of posterior organoid maintained 48h in agitation and then maintained in a Matrigel droplet for 24h. Scale bars = 20 µm. Zoom in of peripheral and central cells. Scale bars = 5 µm. Organoids are stained for ACTIN (grey), GFP (green) and DAPI (blue). (C‘, C”) Plots the fluorescence intensity of YAP (red line) to the distance from the organoid lumen (zero) for the central cell and the peripheral cell. Grey boxes represents the limits of the cell based on the ACTIN staining, blue box represents the nuclei localization based on DAPI staining.