Fig. 3. Regulation of endoderm genes by Nodal proteins, Mixer and Sox17. (A) The expression of the 301 endoderm-enriched sequences was determined by microarray analysis at stage 11 in three experimental conditions. (1) Nodal-, embryos where nodal signaling was inhibited by injection of Cerberus-S RNA (1 ng/embryo). (2) Mixer-, embryos injected with Mixer antisense morpholino oligos (40 ng/embryo). (3) Sox17-, embryos injected with antisense morpholino oligos to Sox171 + Sox172 + Sox17ß (20 ng each/embryo). The average intensity, normalized to stage 11 whole embryo (We) is shown on a log scale. An, animal cap ectoderm; Veg, endoderm enriched vegetal tissue; Eq, mesendoderm enriched equatorial tissue. The yellow lines indicate the 1.4-fold change threshold from the control. (B) Hierarchical clustering indicates which conditions have the most similar expression profiles. Low expression is indicated in blue and high expression in red. A transcript was considered `regulated' in a given condition if its expression was more than 1.4-fold changed from whole embryo control. (C) The Venn diagram shows that based on this 1.4-fold criteria, 112/301 endoderm transcripts are Nodal regulated, 168/301 are Mixer regulated and 100/301 are Sox17 regulated, with a total of 223/301 endoderm transcripts that are regulated by either Nodal proteins Mixer or Sox17. Only 36/301 of transcripts (white) are regulated in a manner consistent with the simple linear model of endoderm development.