Figure 1.

hCOs Reveal an Early Change from Weak to Strong Nuclear UBE3A in Neurons

(A) Immunostaining of neurotypical hCO neurodevelopment. Strong nuclear UBE3A in neurons (arrows) and weak nuclear UBE3A in SOX2⁺ cells (arrowheads) are seen. Cytoplasmic UBE3A decreases over time (double arrows). Dotted white lines delineate boundaries between TUJ1⁺ and SOX2⁺ cells.

(B) Percentage of strong nuclear UBE3A increases during hCO development. Immunostaining quantification. ^∗p < 0.05 between all groups, one-way ANOVA with Tukey-Kramer post hoc analysis, n = 3 independent experiments with two organoids in each. Error bars, 95% confidence intervals.

(C) Strong (arrows) and weak (double arrows) nuclear UBE3A in PAX6⁺ cells. Strong nuclear UBE3A in PAX6⁻/weak cells (arrowheads) is seen.

(D) UBE3A localization by cell type identified by immunostaining. Error bars, 95% confidence intervals. n = 3 independent experiments with two organoids in each.

(E) H1- and hiPSC-derived hCOs. Strong nuclear UBE3A in neurons (arrows) is seen.

(F) Immunoblot analysis of UBEA, GAPDH, and H3 using nuclear (NE) and cytoplasmic (CE) extracts isolated from H9 hCOs. n = 2 independent experiments with 15–25 organoids in each. Error bars, 95% confidence intervals.

(G) 2D immunostaining of dissociated H9 hCOs. Strong nuclear UBE3A in neurons (arrows) and weaker diffuse staining in progenitors (double arrows) are seen.

(See also Figures S1 and S2)