Fig. 2. Validation of endoderm-enriched transcripts. (A) At stage 11, RNA was isolated from animal cap ectoderm (An), endoderm-enriched vegetal tissue (Veg) and mesendoderm enriched equatorial tissue (Eq), and assayed by RT-PCR to validate the expression profile of endoderm-enriched transcripts. The black histogram shows relative normalized expression from the array and the grey histograms show the relative expression levels in RT-PCR normalized to the loading control, ODC. Sox17, Xbra and Epidermal keratin (Epi-K) are positive controls for the dissections. Fifty-one out of 54 genes were confirmed to have vegetal expression that was three times greater than animal cap expression (see Table S4 in the supplementary material) and 12 representative genes are shown [Xl.10408, Xl.13381, Xl.14891, Xl.15171, Xl.16410, Xl.16875 (Mixer-b), Xl.2410, Xl.2554, Xl.4709, Xl.4935, Xl.5999, Xl.8924 (gap junction subunit 6)]. (B) In situ hybridization to bisected gastrula with probes to the indicated genes validates their endoderm-enriched expression. Xl.1191 and Xl.15054 are not detected until neurula stage (st15).