Figure 7.
Figure 7. Overactivation of β-catenin in PGCs leads to an increase in proliferation and misregulation of genes in common with Ror2Y324C. (A) Schematic of breeding and tamoxifen exposure to generate control (Cnt, β-cateninGOF/+; Pou5f1+/+) and βcatGOF (β-cateninGOF/+; Pou5f1Cre-ER/+) embryos in vivo and cells ex vivo. (B) Mean number of PGCs counted in histological sections of Cnt and βcatGOF embryos at different ages. n = 13 Cnt embryos and 15 βcatGOF embryos. P-values by Student’s t test for section counts. Estimates for total numbers of Cnt and βcatGOF PGCs per embryo were calculated using the multiplier of 100× for E9.5, 125× for E10.5, and 150× for E11.5 based on cell counts reported in the literature. (C) Oct4-ΔPE-GFP+ PGCs (gray) in E9.5 littermates. Bar, 100 µm. (D) Rate of in vitro EdU incorporation in Cnt and βcatGOF PGCs cultured for 10–22 h with 4-OHT. n = 4 litters; 794–818 cells; *, P < 0.05 by Student’s t test; error bars indicate standard deviation. (E) Microarray data from βcatGOF PGCs compared with WT shown as log fold change versus log p-values for all annotated microarray probes (Gladstone Bioinformatics Core [GBC]). Each dot represents a single probe. Blue dots show genes with P < 0.05 and a fold-change >1.2 (not log scale). Orange dots show significant genes that overlap with Ror2Y324C misregulated genes. (F) Functional analysis of >1.2-fold microarray hits by Ingenuity Pathway Analysis find cell cycle–associated genes to be significantly misregulated. Number of genes associated with each Gene Ontology category is shown on the right. Example genes of interest for cell cycle and cell growth and proliferation categories are listed. (G) Number of genes that overlap between Ror2Y324C microarray hits (dChip) and βcatGOF microarray hits (GBC). (H) GSEA comparison of the Ror2Y324C microarray dataset with βcatGOF misregulated genes suggests an enrichment for genes associated with both mutations. P-value, enrichment score (ES), and normalized enrichment score (NES) are specified. Heatmap shows normalized microarray intensity values for the 20 most and 20 least enriched genes in Ror2Y324C PGCs that are misregulated in the β-cateninGOF microarray dataset. For each gene, dark blue color indicates the lowest probe intensity value (zero percentile), dark red indicates the highest probe intensity value (100th percentile), and white indicates the middle 50th percentile of intensity values.

Overactivation of β-catenin in PGCs leads to an increase in proliferation and misregulation of genes in common with Ror2Y324C. (A) Schematic of breeding and tamoxifen exposure to generate control (Cnt, β-cateninGOF/+; Pou5f1+/+) and βcatGOF (β-cateninGOF/+; Pou5f1Cre-ER/+) embryos in vivo and cells ex vivo. (B) Mean number of PGCs counted in histological sections of Cnt and βcatGOF embryos at different ages. n = 13 Cnt embryos and 15 βcatGOF embryos. P-values by Student’s t test for section counts. Estimates for total numbers of Cnt and βcatGOF PGCs per embryo were calculated using the multiplier of 100× for E9.5, 125× for E10.5, and 150× for E11.5 based on cell counts reported in the literature. (C) Oct4-ΔPE-GFP+ PGCs (gray) in E9.5 littermates. Bar, 100 µm. (D) Rate of in vitro EdU incorporation in Cnt and βcatGOF PGCs cultured for 10–22 h with 4-OHT. n = 4 litters; 794–818 cells; *, P < 0.05 by Student’s t test; error bars indicate standard deviation. (E) Microarray data from βcatGOF PGCs compared with WT shown as log fold change versus log p-values for all annotated microarray probes (Gladstone Bioinformatics Core [GBC]). Each dot represents a single probe. Blue dots show genes with P < 0.05 and a fold-change >1.2 (not log scale). Orange dots show significant genes that overlap with Ror2Y324C misregulated genes. (F) Functional analysis of >1.2-fold microarray hits by Ingenuity Pathway Analysis find cell cycle–associated genes to be significantly misregulated. Number of genes associated with each Gene Ontology category is shown on the right. Example genes of interest for cell cycle and cell growth and proliferation categories are listed. (G) Number of genes that overlap between Ror2Y324C microarray hits (dChip) and βcatGOF microarray hits (GBC). (H) GSEA comparison of the Ror2Y324C microarray dataset with βcatGOF misregulated genes suggests an enrichment for genes associated with both mutations. P-value, enrichment score (ES), and normalized enrichment score (NES) are specified. Heatmap shows normalized microarray intensity values for the 20 most and 20 least enriched genes in Ror2Y324C PGCs that are misregulated in the β-cateninGOF microarray dataset. For each gene, dark blue color indicates the lowest probe intensity value (zero percentile), dark red indicates the highest probe intensity value (100th percentile), and white indicates the middle 50th percentile of intensity values.

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