Figure 4: Loss of the Dvls Disrupts Mesoderm Differentiation but Endoderm and Ectoderm Induction Remain Intact.

A-C) Heatmaps of unsupervised hierarchical clustering of genes involved in germ lineage differentiation. A) In Dvl TKO EBs, significant down-regulation is observed in genes involved in mesoderm formation. Genes involved in B) endoderm and C) ectoderm formation are not exclusively down-regulated in Dvl TKO EBs, but display differential expression compared to WT EBs. All heatmap scales represent the Z-score of gene expression. D) Immunostaining of Brachyury (green), Otx2 (red), and DAPI (blue) in WT and Dvl TKO EBs at day 6. Mesodermal Brachyury-positive cells are undetectable in the Dvl TKO EBs. The outer ectodermal ring of cells that is present in Dvl TKO EBs, but not WT EBs, is positive for Otx2. Scale bar = 100 μm. E-F) Quantification of cells expressing Brachyury and Otx2 protein in WT and Dvl TKO EBs that were differentiated for 5–7 days (n=20/group). G-I) Expression levels of germ lineage markers Brachyury (mesoderm), GATA4 (endoderm), and Otx2 (ectoderm) were measured by qRT-PCR analysis (n=3). G) WT EBs display significant increases in Brachyury expression over time, whereas Brachyury levels in Dvl TKO EBs are at baseline throughout the differentiation process, indicating a lack of mesoderm differentiation in the absence of the Dvls that is consistent with defects observed in vivo. H) GATA4 (endoderm) expression is slightly higher in Dvl TKO EBs compared to WT throughout the course of differentiation, indicating that endoderm differentiation is not disrupted in the absence of the Dvls. However, these differences are not statistically significantly. I) The levels of Otx2 mRNA decline over time in WT EBs, but Dvl TKO EBs display a delay in decreased Otx2 expression and remain higher over the course of differentiation until day 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.