RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex (2021)
Figure 3

Figure 3. Deletion of RGCC diminishes the VZ-like area and exhibits premature differentiation in hCOs

A, Representative pictures of H9 and RGCC-KO hCOs at 5, 11, 14, 24, 30, 40, 50 and 60 days. Scale bar: 500 μm.

B, Area changes of hCOs of different groups at different time points. The area of single hCO from all different batches were analyzed. Each plot represented one hCOs (Day 5, n=12; day 11, n=12; day 14, n=44; day 24, n=26; day 30, n=46; day 40, n=98; day 50, n=63; day 60, n=24 for each group from 4 independent experiments).

C, Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with SOX2. Scale bar: 200 μm

D, Quantification of the VZ-like SOX2-positive rosette area. Each plot represented an individual rosette. H9, n=19; #6, n=24; #23, n=19 from 2 independent experiments.

E, Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with PAX6 and Phospho-Histone 3 (PH3). Scale bar: 200 μm

F, Quantification of the ratio of PH3 and PAX6 double positive cells versus the total number of PAX6 positive cells of each rosette. Each plot represented an individual organoid (H9, n=6; #6, n=8; #23, n=8 from 2 independent experiments). Three to five rosettes were analyzed for each organoid.

G, Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with Ki67. Scale bar: 200 μm.

H, Quantification of the VZ-like Ki67 positive rosette area. Each plot represented an individual rosette. H9, n=60; #6, n=54; #23, n=96 from 2 independent experiments.

I, Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TUJ1 and PAX6. Scale bar: 200 μm.

J, Quantification of the ratio of TUJ1 area versus PAX6 area. (H9, n=15; #6, n=6; #23, n=9 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.

K, Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TBR1 and PAX6. Scale bar: 200 μm.

L, Quantification of the ratio of TBR1-positive area around PAX6-positive area versus PAX6-positive area in each rosette. (H9, n=10; #6, n=10; #23, n=7 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.

M, Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with CTIP2 and PAX6. Scale bar: 200 μm.

N, Quantification of the ratio of CTIP2 area versus PAX6 area. (H9, n=8; #6, n=8; #23, n=8 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.

Data information: The proliferating rosettes and apical surface adjust to ventricle-like regions were highlight by white dash lines. VZ like rosette area were indicated by white dash lines, and the CP like area around VZ were illustrated by notes. Data are presented as mean ± SEM. Statistical significance was tested by one-way ANOVA. *, p<0.1; **, p<0.01; ***, p<0.001; ****, p<0.0001. Abbreviation: VZ, ventricular zone; CP, cortical plate.