Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1 (2021)
Figure 7

Figure 7 Canonical WNT signaling controls the AT-ND transition

A, B. Immunostaining of P5 cerebellum with indicated antibodies. Dashed lines indicate the upper and bottom of EGL. Low magnification images (A) and high magnification images showing the upper, the middle and the inner part of the EGL(B-B'').

C. Quantification of the relative intensity of NP-β-catenin in the outer and the inner parts of EGL. Bars and error bars are representing as Mean± SEM. Student T-test between the outer and the inner part of EGL was performed. ***p < 0.001. 9 sections from N=3 mice were measured for the quantification.

D-F. Over-expression analysis of the activated form of β-catenin (βcat S33Y). (D, E) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of H2B-GFP (D, Control) and βcat S33Y (E, S33Y). Arrows indicate the cells of interest where GFP signals are co-localized with PROX1. (F) Proportions of PROX1-positive cells in electroporated cells. Bars and error bars are representing as Mean± SEM. Student T-test between Control and S33Y was performed. ***p < 0.001. Number of cells: 89, 90 and 55 cells from N=3 mice (Control), 32, 111 and 56 cells from N=3 mice (S33Y).

G, H. Immunostaining of P5 or P10 cerebellum with indicated antibodies.

I. Schematic illustrations of dynamic activity of WNT signaling estimated by the intensity of NP-β-catenin. Solid lines indicate the upper and the bottom of EGL and dashed line indicates the bottom of ATOH1 positive region.

J-L. Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids (4D) to P5 mice EGL and immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Images of βcat S33Y (J, S33Y) and βcat S33Y with Ccnd1 and Cdk4 (K). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1.Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. Control value is the same as Fig 2G-K. Bars and error bars are representing as Mean± SEM. Tukey-Kramer test was performed between Control, S33Y and S33Y+4D (L). ***p < 0.001, N.S: Not Significant. Number of cells: 123, 138 and 113 cells from N=3 mice (S33Y), 144, 223 and 180 cells from N=3 mice (S33Y+4D).

M. Schematic illustrations summarizing this study.

Data information: Scale bars: 20µm (A-E, G, H, J, K)

Expanded View Figure legends

Expanded View Figure legend 1 Cell cycle status and molecular profile of AT+GCPs and ND+GCPs

A. Immunostaining with indicated antibodies to P6 mice. 50mg/kg EdU was administered intraperitoneally 30 min before fixation. Solid white arrowheads indicate NEUROD1-positive and EdU-positive GCPs (ND+GCPs in S-phase). White hollow arrowheads indicate NEUROD1-negative and EdU-positive GCPs (AT+GCPs in S-phase).

B. Immunostaining with indicated antibodies to P6 mice. Solid white arrowheads indicate NEUROD1-positive and PH3-positive GCPs (ND+GCPs in M-phase). White hollow arrowheads indicate ATOH1-positive and EdU-positive GCPs (AT+GCPs in M-phase).

C. Normalized expression of selected genes (Pax6, Pcna, Plxnb2, Barhl1, Mki67, Mcm6, Ccnd1 and Cntn2) were mapped onto the UMAP dimension. Pax6 (Shiraishi et al., 2019) and Pcna (Migheli et al., 1995) were used as threshold genes to extract the population that included GCPs. Plxnb2 (Friedel et al., 2007) and Barhl1 (Li et al., 2004) were used as markers for granule cell lineage cells. Mki67 and Mcm6 were used as markers for mitotic cells (Giotti et al., 2018). Ccnd1 (Pogoriler et al., 2006) and Cntn2 (Xenaki et al., 2011) were used as markers for undifferentiated and differentiated cells of granule cell lineage, respectively.
D. Mitotic labelling experiments were performed with P6 mice. Rates of PH3+ cells among the EdU+ cells were determined 1 or 2hrs after EdU incorporation in AT+GCPs and ND+GCPs (D). Error bars are representing as Mean± SEM from three mice. Percentages of PH3+ cells among the AT+GCPs and ND+GCPs were determined with P6 mice (D').

E,F. Efficiency of shRNAs was confirmed by immunoblotting. Lysates of N2a cells transfected with either Atoh1 expression plasmids with scramble (Scr) shRNA plasmids or Atoh1 expression plasmids with Atoh1 shRNA (sh#1, sh#2 or sh#3) were subjected to immunoblotting with anti Atoh1 or β-actin (E). Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-tests between Scr and sh#1 or Scr and sh#2 were performed. Lysates of N2a cells transfected with either GFP expression plasmids fused to Atoh1 3' untranslated region (UTR) (GFP-3'UTR) with scramble (Scr) shRNA plasmids or GFP-3'UTR with Atoh1 sh#3 targeting 3'UTR region of Atoh1 were subjected to immunoblotting with anti GFP or β-actin (F). Quantification of relative levels of GFP to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between Scr and sh#3 was performed.

Data information: Scale bars: 20µm(A,B)

Expanded View Figure legend 2 Expression and the function of Ccnd1 in the EGL

A. Cell cycle-related expression of Ccnd1 in GCPs categorized to cluster 0 (AT+GCPs) or cluster 1 (ND+GCPs, Fig1C).

B,C. Immunostaining with indicated antibodies in P6 mice. Strong CCND1 expression was observed in dividing AT+GCPs (B, NEUROD1-negative and PH3-positive), while CCND1 expression was faint in ND+GCPs (C, NEUROD1-positive and PH3-positive).

D. Efficiency of shRNAs was confirmed by immunoblotting. Lysates of N2a cells transfected with either sh-scramble plasmid (scr) or Ccnd1 shRNA (sh#1 or sh#2) were subjected to immunoblotting with anti-Ccnd1 or β-actin to examine endogenous expression of CCND1. Quantification of relative levels of CCND1 to the β-ACTIN (right panel). Mean± s.e.m. The Student T-tests between Scr and sh#1 or Scr and sh#2 were performed.

E. Rates of KI67+ cells 2days after electroporation. Ccnd1 shRNAs were introduced to P5 EGL, followed by immunostaining of KI67 in P7 mice. Ccnd1 KD significantly reduced the rate of KI67+, GFP+ / GFP+ cells. The Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Error bar sem.

F-H. Ccnd1 KD analysis related to Fig. 2C-F. (F, G) Ccnd1 shRNAs were introduced to P5 EGL with H2B-EGFP expressing plasmid and immunostaining for Neurod1 (magenta) and Ki67 (cyan) were performed the following day. Nuclei of electroporated cells were detected with GFP (yellow). Images of Control (F) and Ccnd1 KD (G). (H) Rates of ND+GCPs (NEUROD1-positive and KI67-positive cells) among the electroporated GCPs (GFP+, KI67+ cells). The Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Error bar sem.

I, J. CCND1 expression in GCPs electroporated either Ccnd1 (CCND1 OE) or Ccnd1 and Cdk4 (4D OE), related to Fig. 2G-I.

K. Rates of NEUROD1+ cells related to Fig. 2G-I. Immunostaining of Neurod1 was performed in the following day of electroporation. Rates of NEUROD1 positive cells among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly reduced the rates of NEUROD1+ cells compared to Control. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Mean ± s.e.m.

Data information: Scale bars: 10µm(B, C), 20µm (F, G, I, J).

Expanded View Figure legend 3 Generation and validation of specific antibody against S309-phosphorylated ATOH1

A. GST-tagged ATOH1 or ATOH1S309A were purified from N2a cells with glutathione sepharose beads. Purified ATOH1 was incubated with λ-phosphatase (λPP) for 30 min. Purified proteins were resuspended and subjected to immunoblotting with indicated antibodies. As expected, p-S309-Ab recognized WT-ATOH1 but not S309A-ATOH1. However, when the purified WT-ATOH1 was de-phosphorylated by λ-phosphatase, no bands were detected.

B. Immunoblotting with the indicated antibodies against lysates of N2a cells transfected with either Atoh1-GST or Atoh1 S309A expression plasmids. No signals were detected with p-S309-Ab antibody in N2a cell lysates transfected with S309A-ATOH1, while WT-ATOH1 gave strong signals. Quantification of relative levels of phosphorylated ATOH1 at S309 to the total ATOH1 (right panel). Mean± s.e.m. Student T-tests between WT and S309A was performed. These findings confirmed that our p-S309-Ab antibody specifically recognized phosphorylation at S309 of ATOH1.

Expanded View Figure legend 4 Generation and validation of specific antibody against S328-phosphorylated ATOH1

A. GST-tagged ATOH1 was purified from N2a cells. Purified ATOH1 was incubated with λ-phosphatase (λPP). Purified proteins were resuspended and subjected to immunoblotting with indicated antibodies. As expected, p-S328-Ab robustly detected purified GST-WT-ATOH1, but signals were lost upon λ-phosphatase coincubation.

B. Immunoblotting with the indicated antibodies against lysates of N2a cells transfected with either Atoh1-GST or Atoh1 S328A expression plasmids. p-S328-Ab detected strong bands in WT-ATOH1-transfected N2a cells but not in those transfected with S328A-ATOH1, a S328 phosphorylation-resistant form of ATOH1. Quantification of relative levels of phosphorylated ATOH1 at S328 to the total ATOH1 (right panel). Mean± s.e.m. Student T-tests between WT and S309A was performed. These observations suggest that our p-S328-Ab specifically recognizes phosphorylated ATOH1 at S328.

Expanded View Figure legend 5 Expression of PROX1 and the function of WNT-PROX1 signaling axis

A-D. Immunostaining of P6 mice with the indicated antibodies.

E Schematic illustration of PROX1 expression in the EGL.

F. The rates of KI67-positive cells 1 day after electroporation of Prox1 expression plasmid related to Fig. 5B-D.

G. The rates of ND+GCPs (NEUROD1-positive and KI67-positive cells) 1 day after electroporation of Prox1 expression plasmid related to Fig. 5B-D.

H. Prox1 expression in GCPs electroporated with Prox1 expression vector related to Fig.5B-D.

I. Prox1 expression in Prox1fl/fl mice electroporated with Cre expression vector to related to Fig.5E-G.

J. ChIP assay in P7 mouse cerebellum with anti-Prox1 or goat IgG as a negative control. Primer set used in this experiment was designed to amplify Ccnd1 gene-body (outside of the promoter region), related to Fig.5L.

K-M. Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids to P5 mice EGL and immunostaining for NEUROD1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Images of βcat S33Y (K, S33Y) and βcat S33Y with Ccnd1 and Cdk4 (L, S33Y+4D). Rates of NEUROD1+ cells among the electroporated cells. Tukey-Kramer test was performed between Control, S33Y and S33Y+4D (M).

Data information: Scale bars: 20µm (A-D).