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Fig. 5. The Cerberus gene signature identifies early dorsal β-catenin genes that require endomesodermal induction. (A) MA-plot comparing gene expression of 16,729 mRNAs between control and Cerberus mRNA-injected embryos. The average log2 fold change in expression of transcripts in Cerberus mRNA-injected embryos over control embryos is plotted on the y axis; the average gene expression in RPKM of uninjected control embryos is on the x axis. This MA-plot confirms the proper normalization of the RNA-seq data because the horizontal nature of the line across the zero baseline shows that there is no systematic bias. (B) Cerberus overexpression greatly inhibits Xbra, xSox17β, and xWnt8 mRNA expression when examined by in situ hybridization. (Magnification: 10×.) (C) Heat map showing how gene signatures repressed or induced by Cerberus mRNA were differentially regulated by various dorsalizing or ventralizing conditions. Note that a large block of Spemann organizer genes strongly activated in D/V regenerating half-embryos (in red, top of the heat map) are repressed by Cerberus-mediated endomesodermal inhibition. (D–G) GSEA analysis of the Cerberus-repressed gene set. Approximately 16,000 transcripts were ranked according to β-CatMO, D/V expression, and Wnt8 and Siamois mRNA induction and were examined for the ranking of Cerberus-repressed genes (vertical lines). Cer, Cerberus; con, control; Ctrl, control; D/V, dorsal/ventral ratio; Sia, Siamois. |
