Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1 (2021)
Figure 1

Figure 1 - Characterization of AT+GCPs and ND+GCPs

A. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs.

B. Schematic illustration of AT+GCPs and ND+GCPs. KI67-positive GCPs in the EGL were distinguished by the expression of two bHLH transcriptional factors, ATOH1 (AT+GCPs) and NEUROD1 (ND+GCPs).

C. Pax6-positive and Pcna-positive cells were extracted from published scRNA-seq data. UMAP-based dimensional reduction and cluster analysis grouped extracted cells into 5 clusters.

D. Normalized expression of selected genes (Atoh1, Neurod1) were mapped onto the UMAP dimension.

E. Featured molecules in each cluster were determined and visualized by heatmap. The values of each molecule were normalized by Z-score. Molecular features of each cluster suggested that clusters 0 and 1 were GCP lineage cells, cluster 3 was GCs and clusters 2 and 4 were likely to be interneuron precursors as Pax3 and Id3 expression were strong. In addition, the strong and featured expression of Atoh1 suggested that cluster 0 and cluster 1 might be AT+GCPs and ND+GCPs.

F. Cumulative labeling of AT+GCPs and ND+GCPs in P6 mice. Total cell cycle length (Tc) and S phase length (Ts) were calculated as previously described. Calculated time of Tc, Ts, TG1, TG2 and TM were indicated in the table (F'). N=3 for each time point. Error bars are representing as Mean ± SEM. Two-way ANOVA analysis was performed against Cumulating rate from 0hrs to 9hrs in each cell, followed by Bonferroni test. *p < 0.05, **p < 0.01. Number of cells: 113,101 and 159 AT+GCPs from N=3 mice(0hrs), 116,115 and 95 AT+GCPs from N=3 mice(3hrs), 106,105 and 100 AT+GCPs from N=3 mice(6hrs), 122,168 and 170 AT+GCPs from N=3 mice(9hrs). 196,167 and 158 ND+GCPs from N=3 mice(0hrs), 139,110 and 101 ND+GCPs from N=3 mice(3hrs), 98,163 and 81 ND+GCPs from N=3 mice(6hrs), 136,124 and 115 ND+GCPs from N=3 mice(9hrs).

G-J. Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67. (I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).

Data information: Scale bars: 100µm(A),20µm (A'-A'''', G, H).