RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex (2021)
Figure 5

Figure 5. Knockdown of Rgcc affects cell cycle progress and deletion of Rgcc leads to mitotic spindle misorientation of NSCs in both the mouse brains and the hCOs

A, Schematic of the IUE and cumulative EdU incorporation.

B, Liner fit of EdU and Ki67 double positive NSC ratio for 6 hours cumulative EdU incorporation. ShRNA-Scrb: y =9.21x + 23.23; shRNA-Rgcc: y =7.98x + 16.46. At least 3 brains were taken for each time points/group.

C, Representitive immunofluorescent staining for KI67, EdU, GFP of coronal sections of the E14.5 mouse cortex after IUE and 6 h EdU corporation. Scale bar: 20 μm.

D, Immunofluorescent staining of coronal sections of the E14.5 mouse cortex one-day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. PH3 were co-stained with GFP. The punctuated PH3 cells were at G2-phase, while PH3 cells with continuous pattern were at M-phase. To quantify the ratio between PH3 and GFP double positive cells and GFP cells, only the cells in VZ and SVZ are taken into account. Scale bar: 20 μm. n≥3 mouse brains for each group. Data are presented as mean ± SEM; n≥3 mouse brains for each group; Statistical significance was tested by Student's t-test. **, p<0.01; ***, p<0.001;

E, Cell cycle length of E14.5 NSCs in mouse cortex 24h post IUE of ShRNA-Scrb (upper column) and shRNA-Rgcc (lower colum). This were calculated by cumulative EdU labeling and PH3 analysis. Data are presented as mean ± SEM; n≥3 mouse brains for each group; Statistical significance was tested by Student's t-test.

F, Metascape analysis of differentially expressed Spindle-related genes between wildtype H9 hCOs and RGCC-KO hCOs. (adjusted absolute log2(FC)>0.5, padj <0.05). List of DEGs under the GO term Spindle from 3D RNAseq results were clustered into sub-terms to make enrichment network. The top 20 significantly enriched sub-terms were showed in the right panel. Each color of the node represents a sub-term. The size of the node corresponds to the number of genes within this sub-term were shown in the middle panel. Sub-terms with with smaller p-value was shown in the right panel. Nodes with kappa similarity over 0.3 are linked by edges.

G, Representative pictures of mitotic NSC marked by P-Vimentin and PH3 revealed by DAPI at the apical surface of the E14.5 mouse cortex after IUE. A representative vertically dividing cell was shown in upper panel, and a representative horizontally dividing cell was shown in lower panel. White dash line indicates the apical surface and splitting plane. Scale bar: 5 μm.

H, Distribution of mitotic spindle orientation of dividing cells from shRNA-Scrb group and shRNA-Rgcc group. Each plot represents a dividing cell (shRNA-Scrb, n=29; shRNA-Rgcc, n=32).

I, Statistic analysis of splitting plane angle after Rgcc KD. Data were presented as mean ± SEM. Each plot represents a dividing cell (shRNA-Scrb, n=29; shRNA-Rgcc, n=39). Statistical significance was tested by Student's t-test t. **p<0.01.

J, Representative pictures of mitotic NSC marked by P-Vimentin and PH3 revealed by DAPI at the apical surface of the VZ-like area of hCOs. A representative vertically dividing cell was shown in upper panel, and a representative horizontally dividing cell was shown in lower panel. White dash line indicates the apical surface and splitting plane. Immunofluorescent staining was performed on sections of day 45 wildtype H9 hCOs and RGCC-KO hCOs. Scale bar: 5 μm.

K, Distribution of mitotic spindle orientation of dividing cells from wildtype H9 hCOs, #6 and #23 RGCC-KO hCOs. Each plot represents a dividing cell (H9, n=22; #6, n=27; #23, n=39 from 2 independent experiments).

L, Statistic analysis of splitting plane angle in RGCC-KO hCOs. Data were presented as mean ± SEM. Each plot represents a dividing cell (H9, n=22; #6, n=27; #23, n=39 from 2 independent experiments).Statistical significance was tested by one-way ANOVA. **, p<0.01; ***, p<0.001; ****, p<0.0001.

Data information: Abbreviation: n.s., not significant.