Fig 2: Transcriptional signature of the transient transcriptional state (TTS) which enriches after DOT1L inhibition in electroporated data set Left: Heatmap showing expression levels of selected cell type/state markers clustered for progenitor cells only. Right: Heatmap showing expression levels of selected cell type/state markers clustered for TTS, BPs and neurons. Scales represent z-scores.UMAP plots showing differentiation trajectories of control and EPZ-treated cells inferred from RNA velocity, with proportions of spliced and unspliced reads per cluster displayed below. 0 = APs, 1 = BPs, 2,4 = neurons and 3 = TTS.Slingshot pseudo-time trajectory plots showing cell lineage structure from APs to neurons for control and EPZ-treated cells.Monocle3 trajectories showing differentiation trajectories from APs to neurons.UMAP representation showing the co-expression of Eomes and Tubb3 in the electroporation data set.

Fig 3: Increased ASNS expression mediates differentiative effects in response to DOT1L inhibition Overview of mCherry-positive cells in IF staining for TUBB3 in control (Con), EPZ and co-inhibition (EPZ + L-Albizziine (L-Alb)) conditions. Scale bars in main panels: 200 µm, in insets: 50 µm.Fraction of TUBB3-positive and -negative cells expressed as percentage of total cells per condition. Quantifications were made from independent experiments (n = 3), 183 cells in total (Con: 118 cells, EPZ: 182 cells, L-Alb/EPZ: 174 cells). Fisher's exact test, ****P < 0.0001, error bars represent SEM. MIP—maximum-intensity projections. Ex utero electroporated cells expressing GFP (green) or TUBB3 (magenta) in control (Con; pCMV-GFP) or in ASNS overexpression (ASNS OE; pCMV-ASNS-GFP) condition 24 h after slice culture. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily a GFP and TUBB3 double-positive cell upon ASNS overexpression. Dotted shapes indicate the border of cell soma based on GFP expression.Fraction of TUBB3-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from independent experiments (n = 3). Student's t-test, **P < 0.01, error bars represent SEM.Representative images of mCherry (red) or TBR1-expressing (green) cells in control (Con; pCAG-mCherry) or in ASNS overexpression (ASNS OE; pCAG-ASNS) condition 48 h after in utero electroporation. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily mCherry and TBR1 double- or single-positive cells upon ASNS overexpression.Fraction of TBR1-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from 1,141 cells in control group and 606 cells in ASNS OE group. Fisher's exact test, ****P < 0.0001. Source data are available online for this figure.

Fig 4: DOT1L inhibition leads to neurogenic commitment of VZ progenitors Microinjected cells in the VZ (magenta) of control (Con) and EPZ-treated slices 24 h after microinjection and slice culture. Left: daughter cell pair in control section with asymmetric GFP expression, with cell 1 (basal location) being mild GFP-positive, and cell 2 (apical location) GFP-negative. Right: EPZ-treated section showing a daughter cell pair with symmetric mild GFP-positive expression. Scale bars: 5 µm. Dotted shapes indicate the contour of Dx-A555 cell soma.Graph showing proportion of labelled cells expressing GFP positive, mild positive or negative, expressed as percentage of total cells per condition.Symmetry or asymmetry in daughter cells' apical contact (yes/yes: both cells have apical contact; yes/no: one cell has apical contact and the other not; no/no: both cells lack apical contact).Symmetry (pos/pos, neg/neg) or asymmetry (pos/neg) in GFP expression in daughter cells expressed as percentage of total cells per condition.Symmetry or asymmetry in TUBB3 expression in daughter cells plotted (pos/pos: both cells express TUBB3; pos/neg: one cell expresses TUBB3 and the other not; and neg/neg: none of the cells expresses TUBB3).Phalloidin staining of control and EPZ-treated sections showing cells during mitosis. Scale bar: 5 µm.Pie chart showing the proportion of cells with different angles of the cleavage plane in respect to the ventricular surface, divided into groups with increments of 30°.Percentage of cells undergoing symmetric or asymmetric cell division, defined by the cleavage plane bypassing or bisecting the apical plasma membrane, in control and EPZ-treated samples. Data information: Quantifications shown in Fig 4B–E are based on three independent experiments (n = 3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***P < 0.001, ****P < 0.0001, alpha = 0.05. VZ—ventricular zone. The following number of cells were scored: (B) TIS21-GFP—267 total cells were quantified (Con: 86 cells, EPZ: 181 cells). (C) Apical contact—85 total daughter cell pairs (Con: 31 pairs, EPZ: 54 pairs). (D) TIS21-GFP—58 total daughter cell pairs (Con: 21 pairs, EPZ: 37 pairs). (E) TUBB3—50 total daughter cell pairs (Con: 17 pairs, EPZ: 33 pairs). (G) For control condition, 41 cells from four independent samples, five sections and 15 different images were assessed. EPZ-treated quantifications were performed on a total of 26 cells from three independent samples, five sections and 16 images. (H) For control condition, a total of 37 cells from four independent samples, five sections and 15 different images were assessed. EPZ-treated quantifications were performed on a total of 23 cells from three independent samples, five sections and 16 images. Source data are available online for this figure.

Fig 5: Lineage tracing of microinjected cortical APs shows DOT1L inhibition increases neurogenesis and apical delamination Microinjected cells in the VZ (magenta) in sections treated with DMSO (Con) (upper panel) or EPZ (lower panel) showing Dx-A555-labelled cells after 24 h in slice culture. White arrow indicates the apical process contacting the ventricular surface. White arrowheads indicate the apical-directed process of delaminated cells. Scale bars: 10 µm.Immunofluorescence staining of microinjected sections showing the progeny of microinjected cells (a daughter cell pair) and expression of EOMES and TUBB3. White arrows indicate the apical-directed processes. Note that in the top panel, the apical-directed process contacts the ventricular surface (dotted white line) (EOMES and TUBB3 negative), while in the bottom panel, it does not contact the ventricular surface, indicating that the cell has delaminated (EOMES and TUBB3 positive). Scale bars MIP: 10 µm; insets: 5 µm.Quantification of microinjected cells found as single-cell or two-cell clusters, expressed as percentage of total Dx-A555-positive cells per condition.Quantification of microinjected cells with or without contact to the apical surface of the organotypic slice expressed as percentage of total Dx-A555-positive cells per condition. Total number of independent microinjection experiments: 6 (n = 3 wild-type embryos, n = 3 in Tis21-GFP embryos). Total number of cells scored: Con, 156 cells; EPZ, 287 cells.EOMES-positive and EOMES-negative cells plotted as a percentage of total cells scored per condition.TUBB3-positive and TUBB3-negative cells in control and EPZ conditions plotted as a percentage of total cells scored per condition. Data information: Cell scoring based on TUBB3 or EOMES expression is from three independent experiments (n = 3; Con: 70 cells, EPZ: 113 cells). Fisher's exact test was performed for all quantifications, ***P < 0.001, error bars represent SEM. MIP—maximum intensity projection. VZ—Ventricular Zone, SVZ—Subventricular Zone, CP—Cortical Plate. Scale bars in all main panels: 20 µm, and all insets: 5 µm. Source data are available online for this figure.