Fig 2: NFIA and NFIB display overlapping regulation.mRNA sequencing was performed on neocortical tissue dissected from E16 Nfia and Nfib knockout mice. The 91 shared mis-regulated genes in Nfia and Nfib knockout mice were enriched for the presence of an NFI binding motif in their promoter, as compared to all genes in the genome, as well as all differentially regulated genes in either Nfia or Nfib knockout mice (a) Furthermore, the promoter regions of shared mis-regulated genes also displayed an enrichment for putative NFI binding sites, based on an increase in NFIB binding peaks in their promoter observed in ChIP-sequence data from mouse hair follicle stem cells (Chang et al., 2013) and E16 lung tissue (Lajoie et al., 2014) (b and c). This enrichment of putative NFI binding sites was predominantly observed in shared down-regulated genes observed between Nfia and Nfib knockout mice (striped pattern). Furthermore, NFIA and NFIB can form heterodimers in vivo, as both NFIA and NFIB proteins co-immunoprecipitate in nuclear lysate derived from E13 neocortical tissue (d).*<0.05, **<0.005,***<0.001 in a Yates’ corrected chi-squared test.

Fig 3: Roles of Nuclear factor I proteins in YAP1-MAMLD1-driven tumorigenesis. a, b Immunoprecipitation assay of Flag-tagged a Nfia and b Nfib and HA-tagged YAP1 fusion-related proteins in HEK293T cells. Asterisks indicate overexpressed proteins according to their predicted molecular weights. c An enriched heatmap of YAP1 and NFIA ChIP-seq signals within the regions of ST-EPN-YAP1 group-specific YAP1 peaks. d–g IHC of P0 brains with anti-GFP (green) and anti-YAP1 (red) antibodies after electroporation of YAP1-MAMLD1-IRES-Luc and either d, f EGFP alone or e, g Nfia-En-IRES-EGFP. Arrowheads in g indicate the cells expressing exogenous YAP1-MAMLD1 but Nfia-En-EGFP. f and g are high magnification views of the area outlined by a rectangle in d and e, respectively. PB Piggybac transposase. h–k IHC of P0 brains with anti-GFP (green) and anti-Ki67 (red) antibodies after electroporation of YAP1-MAMLD1-IRES-Luc and either h, j EGFP alone or I, k Nfia-En-IRES-EGFP. Arrowheads in k indicate abnormally proliferating cells lacking Nfia-En expression. j and k are high magnification views of the area outlined by a rectangle in h and i, respectively. Scale bar in i is 100 µm (for d, e, h, and i) and the scale bar in k is 25 µm (for f, g, j, and k). l The graph represents the percentages of YAP1- and Ki67-positive cells within EGFP-positive cells. Data obtained from three independent brain samples. **p < 0.0001, n.s., not significant. The error bars indicate mean ± S.D. (n = 6). m The graph indicates the percentage of YAP1- and cleaved caspase 3-positive cells within EGFP-positive cells. p = 0.554. The error bars indicate mean ± S.D. (n = 4). n The graph indicates the percentage of YAP1- and Ki67-positive cells lacking EGFP expression. **p < 0.0001. The error bars indicate mean ± S.D. (n = 6). o, p Dorsal views of the brains bearing tumors from the mice electroporated with YAP1-MAMLD1-Luc with EGFP (o) and Nfia-Rn-IRES-EGFP (p). EGFP signals are detected only in (o). Scale bar, 1 mm. q The graph shows the percentage of EGFP-positive cells in the tumor tissues. **p < 0.0001. The error bars indicate mean ± S.D. (n = 6). Significant differences for l–n, q were assessed by t-test

Fig 4: Double knockout of Nfia and Nfib results in a reduction of neurons and axonal projections within the cerebral cortex.Coronal sections of wildtype (a, d), Nfia+/-;Nfib+/- (b, e) and Nfia-/-;Nfib-/- (c, f) mice at E16 stained for TBR1 (a–c) and GAP43 (d–f). Within the neocortex, the subplate was visible within wildtype and Nfia+/-;Nfib+/- mice (arrows in a' and b'). However, no subplate was evident within Nfia-/-;Nfib-/- mice at this age (c'). There were also fewer TBR1-expressing cortical neurons present within Nfia-/-;Nfib-/- mice at this age compared to that in wildtype and Nfia+/-;Nfib+/- mice. GAP43 staining revealed extensive axonal projections within the neocortex of wildtype and Nfia+/-;Nfib+/- mice (arrows in d' and e'), some of which extended into the cingulate cortex (arrows in d? and e?). In Nfia-/-;Nfib-/-mice, few GAP43-expressing axons were seen within the neocortex (double arrowhead in f'), and none were observed within the cingulate cortex (f?). Moreover, some ectopic axonal projections were observed in the marginal zone of Nfia-/-;Nfib-/- mice.Scale bar (in f?) a–f = 300 µm; a'–c', d'–f? = 80 µm. Nct: neocortex; S: septum; CPu: striatum; MZ: marginal zone; V, VI: cortical layers V and VI; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone; CP: cortical plate; Cg: cingulate cortex; SP: subplate; Pir: piriform cortex.

Fig 5: The reduction of cortical astroglia in Nfia+/-;Nfib+/- mice is comparable to that in single homozygous knockout animals.Immunohistochemical staining of E18 coronal sections showing the expression of the astroglial marker GFAP at the midline (a-d) and hippocampus (e-h). In wildtype embryos (A, A’), GFAP expression was detected in the indusium griseum, the glial wedge and the midline zipper glia at the midline, as well as within cortical radial glia. In Nfia+/-;Nfib+/-mice (b, B’), GFAP expression was absent in the cortex and reduced at the midline (B’; asterisks). The GFAP-positive glial wedge cell population was reduced (arrow), while only sparse GFAP labelling was detected at the unfused midline (arrow head in B’). This phenotype was comparable to that observed in Nfia-/- (c) or Nfib-/- (d) mice, in which GFAP immunoreactivity within the indusium griseum glia and midline was absent, and was markedly reduced in the region of the glial wedge (open-headed arrows in C and D). Similarly, GFAP staining was reduced throughout the hippocampus of Nfia+/-;Nfib+/-mice (F, F’) compared to that in wildtype controls (e, E’). The remaining staining was localized in the ammonic neuroepithelium and fimbrial glioepithelium (F’). This expression pattern was reminiscent of that in Nfia-/- mice (g), whereas Nfib-/- embryos only retained GFAP immunoreactivity within the fimbrial glioepithelium (arrowhead in h). Scale bar (in H): A, B, E, F = 500 µm; A’, B’, C, D, E’, F’, G, H = 125 µm. CC = corpus callosum; Cx = cortex; S = septum; CPu = striatum, Pir = piriform cortex; IG = indusium griseum; GW = glial wedge; MZG = midline zipper glia; Nct = neocortex, Hp = hippocampus, Th = thalamus, DG = dentate gyrus; CA1-3 = hippocampal subfields, AN = ammonic neuroepithelium; FG = fimbrial glioepithelium.