The homeobox gene hhex is one of the earliest markers of the anterior endoderm, which gives rise to foregut organs such as the liver, ventral pancreas, thyroid, and lungs. The regulatory networks controlling hhex transcription are poorly understood. In an extensive cis-regulatory analysis of the Xenopus hhex promoter, we determined how the Nodal, Wnt, and BMP pathways and their downstream transcription factors regulate hhex expression in the gastrula organizer. We show that Nodal signaling, present throughout the endoderm, directly activates hhex transcription via FoxH1/Smad2 binding sites in the proximal -0.44 Kb promoter. This positive action of Nodal is suppressed in the ventral-posterior endoderm by Vent 1 and Vent2, homeodomain repressors that are induced by BMP signaling. Maternal Wnt/β-catenin on the dorsal side of the embryo cooperates with Nodal and indirectly activates hhex expression via the homeodomain activators Siamois and Twin. Siamois/Twin stimulate hhex transcription through two mechanisms: (1) they induce the expression of Otx2 and Lim1 and together Siamois, Twin, Otx2, and Lim1 appear to promote hhex transcription through homeobox sites in a Wnt-responsive element located between -0.65 to -0.55 Kb of the hhex promoter. (2) Siamois/Twin also induce the expression of the BMP-antagonists Chordin and Noggin, which are required to exclude Vents from the organizer allowing hhex transcription. This study reveals a complex network regulating anterior endoderm transcription in the early embryo.
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