Fig 2: Enhanced Notch activation and efficient radial organization co-localize with cortical markers in Triple-i derived monolayer cultures.a. Phase contrast (top) and HES5::eGFP (bottom) images of representative day 12 neural monolayer progenitors derived from small EBs (sEBs) subjected to neural induction under Inhibitor-free conditions (Inhibitor-free), WNT inhibition using XAV-939 (WNT-i), Dual SMAD inhibition using SB-431542 and NOG (Dual SMAD-i) and combined Dual SMAD and WNT inhibition (Triple-i) (representative images from N=3 biological replicates for each protocol). Scale bar: 50 µm. b. Merged bright field images and matched H9 HES5::eGFP confocal image taken from a representative day 17 organoid derived under Triple-i protocol (representative image from N=9 biological replicates). Shown is an individual image from the z-stack of the representative organoid shown in Fig. 2a. A radially organized Notch active region is outlined in the dashed line. Scale bar: 100 µm. c. Immunostaining of FOXG1 (top) and PAX6 and EMX2 (bottom) with respect to Notch activation (HES5::eGFP) and radial organization in representative day 12 monolayer sEB protocol neural progenitors derived by indicated treatments (representative images from N=3 biological replicates for each protocol). The right image represents a magnified rosette from Triple-i derived progenitors. Scale bar: 50 µm. Cell counts and co-localization analysis of markers are presented in the right panel. See Source Data Extended Data Fig. 2 for immunostaining counts. d. Immunostaining for cortical markers PAX6 and EMX1 in Triple-i derived organoids generated using ZIP8K8 hiPSC line (representative images from N=2 biological replicates). Bars represent mean. Cell counts were generated from 26 rosettes across 2 organoids. Scale bar: 200 µm. See Source Data Extended Data Fig. 2 for immunostaining counts. Source data

Fig 3: mRNA expression of the Plaur-miR1-3p and Plaur-miR1-5p target genes Mef2d, Snrnp200, Emx2, and Nrip3 in mouse striatum. qPCR analysis of (A) Mef2d, (B) Snrnp200, (C) Emx2 and (D) Nrip3 expression in the striatum of control mice (treated with saline, 0 h) and mice 0.5, 1, 3, 6, 24, and 72 h after endogenous Plaur induction. The data are presented as mean ± SEM (n = 4), normalized to Actb (encodes β-actin) expression as a reference gene. The data were analyzed by using analysis of variance followed by Dunnett’s multiple comparisons test with GraphPad Prism software. Statistical significance is indicated by bars and asterisks as follows: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: Enhanced NOTCH activation and efficient radial organization co-localize with cortical markers in organoids derived by Triple-i.a, Merged bright-field images and their matched H9 HES5::eGFP confocal images taken of representative day 17 organoids (n = 9). Maximum intensity projections for H9 HES5::eGFP confocal images taken at intervals of 15 µm are shown. Green arrows, enhanced NOTCH-active and radially organized regions; white arrows, radially organized regions lacking enhanced NOTCH activation; red arrows, non-neuro-epithelial extensions. b, Mean HES5::eGFP intensity levels measured for the images from a (n = 9). c, Number of rosettes expressing HES5::eGFP in the organoid images from a. b,c, Statistical test, two-sided t-test with Benjamini–Hochberg correction; *P < 0.05; ***P < 0.001; ****P < 0.0001; and NS, not significant. The boxplots display the median and interquartile range (box boundaries) with whiskers extending to 1.5× the interquartile range; n = 9. d, Immunostaining of representative H9-derived day 30 organoids (left) and counts within rosettes of SOX1, FOXG1 and HES5::eGFP (top right; Triple-i, n = 13 from two replicates; Dual SMAD-i, n = 4 from one replicate; and Inhibitor-free, n = 5 from one replicate) as well as PAX6, EMX2 and HES5::eGFP (bottom right; Triple-i and Dual SMAD-i, n = 10 from one replicate; and Inhibitor-free, n = 18 from two replicates). e, Immunostaining of representative ZIP8K8-derived day 27 organoids (left) as well as counts within rosettes of EMX2 (Triple-i, n = 8 from one replicate; Dual SMAD-i, n = 38 from three replicates; and Inhibitor-free, n = 14 from one replicate), SP8 (Triple-i, n = 35 from three replicates; Dual SMAD-i, n = 23 from three replicates; and Inhibitor-free, n = 12 rosettes from one replicate) and NR2F1 (Triple-i, n = 36 from three replicates; Dual SMAD-i, n = 44 from three replicates; and Inhibitor-free, n = 12 from one replicate) with PAX6, FOXG1 and SOX1 (Triple-i, n = 40 from two replicates; Dual SMAD-i, n = 29 from two replicates; and Inhibitor-free, n = 14 from one replicate; right). f, Immunostaining of representative ZIP8K8-derived day 27 organoids (left) and counts within rosettes of the cortical marker MEF2C with SOX2 and DCX (top right; Triple-i, n = 19 from two replicates; Dual SMAD-i, n = 22 from two replicates; and Inhibitor-free, n = 9 from one replicate), OLIG3 and TCF7L2 (middle right; Triple-i and Dual SMAD-i, n = 7 from one replicate; and Inhibitor-free, n = 11 from one replicate), and TTR and LMX1A (bottom right; Triple-i, n = 10; Dual SMAD-i, n = 13; and Inhibitor-free, n = 9; all from one replicate). a,d–f, Scale bars, 100 µm. d–f, The bars represent the mean. Immunostaining counts for b–f are provided.Source data

Fig 5: Alignment of Plaur-miR1-5p (A) and Plaur-miR1-3p (B) with promoter regions and introns of the predicted target genes Mef2d, Emx2, Snrnp200, and Nrip3 with the M-coffee web service T-COFFEE Multiple Sequence Alignment Server.