Fig 2: Mest promoter activity is required for miR-335 expression in brain organoids (A) Immunofluorescence staining on brain organoids derived from mESCs using antibodies for brain primordium markers NESTIN/PAX6 (middle panels) and TUBB3/TBR1 (right panels) after eight and 15 days of differentiation. The top panels show entire organoids and the bottom panels are zoom-in insets. Scale bars: 200 µm for organoids (top panels) and 50 µm for insets (bottom panels). (B) Time course of expression of Mest and miR-335 mature products during the development of brain organoids from CRISPRi ESCs stably expressing either control sgRNA or Mest sgRNA. The heatmap shows the mean of four independent experiments performed on two CRISPRa sgRNA control and two CRISPRa sgRNA ESC clones. Heatmap was built using Morpheus. https://software.broadinstitute.org/morpheus/.

Fig 3: Gigyf1 disruption in the developing brain disturbs neurogenesis.(A) Quantitative comparison of brain weight, cortical anterior-dorsal (A-D) length, anterior-posterior (A-P) length, and area in Gigyf1fl/fl (n = 11), cHET (n = 10), and cKO (n = 6) mice at P2. (B) Littermate cortices stained with Satb2 from Gigyf1fl/fl (n = 4), cHET (n = 4), and cKO (n = 3) mice at E18.5. Satb2+ cells per 100 μm of apical surface in L2–L4 were compared. (C) Littermate cortices stained with Brn2 from Gigyf1fl/fl (n = 4), cHET (n = 4), and cKO (n = 3) mice at E18.5. Brn2+ cells per 100 μm of apical surface in L2–L4 were compared. (D) Littermate cortices stained with Tbr1 and Ctip2 from Gigyf1fl/fl (n = 4), cHET (n = 4), and cKO (n = 3) mice at E18.5. Ctip2+ cells and Tbr1+ cells per 100 μm in L5 and L6 were compared. (E) Comparison of Pax6+ RGC (VZ) and Tbr2+ IPC (SVZ) populations per 100 μm of apical surface in Gigyf1fl/fl (n = 3), cHET (n = 3), and cKO (n = 3) mice at E14.5. (F) Comparison of Edu+ (SVZ) population, Ph3+ and Pax6+Ph3+ (VZ) population, Pax6+Edu+/Pax6+ proportion of apical surface from Gigyf1fl/fl (n = 3), cHET (n = 3), and cKO (n = 3) mice at E14.5. (G) Ki67 and Edu antibodies after 24 hour Edu pulse at E13.5. All cells that exited the cell cycles (Edu+/Ki 67–) were counted. The percentage of total Edu+ cells evaluated 24 hours after injection was analyzed. (H) S phase sequential labeling analysis of NPCs. EdU-Brdu double-stained cortical sections at E14.5 are shown. S phase durations were calculated (Ts=Ti/(Lcells/Scells)) and compared. All statistics were performed by 1-way ANOVA. Scale bars represent 50 μm. All data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: Neural Progenitor marker vimentin is decreased, and neural rosette lumens are enlarged, in TSC2 mutant day 9 organoids.(a) Cartoon depicting the genotypes of the cell lines used in this study.(b) Representative immunofluorescence maximum intensity images of day 9 organoids showing expression of nestin (green), TBR1 (orange), vimentin (red) and Hoechst (blue) to identify neural progenitor cells. Scale bars = 50 µm.(c) Quantification of mean intensity (A.U.) of vimentin. N = 10 organoids across multiple differentiations. +/+ vs. +/LOF * p = 0.0203, +/+ vs. LOF/LOF * p = 0.0229, genotype ** p = 0.0015 [ANOVA, mixed effects]. Error bars = mean with SD.(d) Quantification of mean intensity (A.U.) of nestin. N = 10 organoids across multiple differentiations. No significance [ANOVA, mixed effects]. Error bars = mean with SD.(e) Representative phase DIC microscopy images of day 1 and day 9 organoids treated with vehicle or rapamycin. Scale bar = 200 µm.(f) Representative immunofluorescence maximum intensity images of day 9 organoids treated with vehicle or rapamycin showing expression of ZO-1 (green), CDK5RAP2 (red) and Hoechst (blue) to identify neural rosette lumens. Scale bars = 50 µm.(g) Quantification of area (µm2) of day 9 organoids treated with vehicle or rapamycin. N = 25 organoids across multiple differentiations. Vehicle +/+ vs. LOF/LOF ** p = 0.0057, Vehicle +/LOF vs. LOF/LOF * p = 0.0156, Rapamycin +/+ vs LOF/LOF **** p = <0.0001, Rapamycin +/LOF vs LOF/LOF *** p = 0.0002, genotype **** p = <0.0001 [two way ANOVA]. Log 10 scale. Error bars = mean with SD.

Fig 5: Spp1 is enriched in adult human RGCs with large somata, and SPP1 expression correlates with the glaucoma severity in human patients(A) Sample images of SPP1 (green) and RBPMS (red) in the retina from an adult donor sample eye with no notable ocular history, including expression in RGC and horizontal cell subsets.(B) Sample images of SPP1 (green), TBR1 (red, a previously characterized transcription factor found in T-RGCs/midget OFF-RGCs),28 and Nissl (gray) in the retina from a donor in (A).(C) Percentage of SPP1-positive and TBR1-positive RGCs in the adult human retina from the donor in (A) and (B). n = 2 retinas.(D) Quantification of RGC soma size of SPP1-positive, TBR1-positive, and the average Nissl (NeuroTrace)-positive RGCs in the adult human retina. n = 2 retinas.(E) SPP1 concentration in aqueous humor was quantified using ELISA from patients with mild (n = 11) and severe (n = 13) forms of primary open-angle glaucoma (POAG) and age-matched controls (n = 16). Significance was assessed by one-way ANOVA followed byTukey’s Honest Significant Difference (HSD) test for pairwise comparisons, demonstrating a significant difference between patients with severe glaucoma and control patients (p = 0.02). AH, aqueous humor.(F) Model illustrating the differential actions of Spp1 and survival of different RGC subclasses between SOHU and ONC treatments. In the SOHU model, Spp1 expression is upregulated, and ectopic Spp1 expression provides protection for RGCs. In the ONC model, Spp1 expression is downregulated, and restricted Spp1 expression in αRGCs only provides limited protection for αRGCs.Scale bars (A and B), 50 μm.