Figure 5. Transient delay in neuronal differentiation expands the NP pool.

A) Immuno-staining for DCX and Nestin at 4 weeks in control and PTEN mutant WIBR3 cerebral organoids. Scale bar, 200um.

B-C) Representative images of EdU-Ki67 co-staining in 4-week-old control and mutant WIBR3 organoids (B), and quantification of cell cycle exit ratio (EdU+Ki67-/all EdU+, C) in 4 and 6 weeks old organoids. Scale bars, 20um.

D) Immuno-staining for DCX and EdU in 4-week-old control and mutant WIBR3 organoids. Scale bars, 20um.

E-F) Quantitative cell fate analysis of EdU+ cells in 4-week-old organoids, showing PTEN mutants have increased retention as Sox2+ NP (E) and decreased propensity for differentiation into DCX+ immature neurons (F).

G-H) Immuno-staining for DCX and Nestin at 8 weeks. Scale bar, 200um.

I-K) Differential gene expression analyses by quantitative RT-PCR on control and mutant WIBR3 organoids at 4, 10 and 16 weeks. Genes analyzed are representative of NPs (PAX6, TBR2, HOPX), pan-neuronal markers (TUBB3, DCX, MAP2, RBFOX3), early-born neurons (CTIP2, TBR1) and late-born neurons (BRN2, SATB2, CUX1, CUX2). Majority of neuronal markers were transiently down-regulated in PTEN mutants at 4 weeks, but greatly normalized at 16 weeks. Gene expressions are normalized to control-1 at each time point. P value reflects controls vs. mutants.

L-N) Immuno-staining for markers of outer radial glia (Hopx), early-born neurons (Tbr1 and Ctip2) and late-born neurons (Satb2 and Brn2) in control and mutant WIBR3 organoids at 12 weeks. Scale bars, 50um.

O) PTEN mutant WIBR3 organoids had folded cortical plate at 16 weeks. Images show immuno-staining for markers of neurons (Ctip2) and astrocytes (GFAP). White dash lines outline the cortical surface. Scale bars, 50um.

Results are mean +/− SEM. *p<0.05, **p<0.01, ***p<0.001. See also Figure S6.