Wnt signaling promotes hindgut fate commitment through regulating multi-lineage genes during hESC differentiation.

Wnt signaling plays essential roles in both embryonic pattern formation and postembryonic tissue homoestasis. High levels of Wnt activity repress foregut identity and facilitate hindgut fate through forming a gradient of Wnt signaling activity along the anterior-posterior axis. Here, we examined the mechanisms of Wnt signaling in hindgut development by differentiating human embryonic stem cells (hESCs) into the hindgut progenitors. We observed severe morphological changes when Wnt signaling was blocked by using Wnt antagonist Dkk1. We performed deep-transcriptome sequencing (RNA-seq) and identified 240 Wnt-activated genes and 2023 Wnt-repressed genes, respectively. Clusters of Wnt targets showed enrichment in specific biological functions, such as "gastrointestinal or skeletal development" in the Wnt-activated targets and "neural or immune system development" in the Wnt-repressed targets. Moreover, we adopted a high-throughput chromatin immunoprecipitation and deep sequencing (ChIP-seq) approach to identify the genomic regions through which Wnt-activated transcription factor TCF7L2 regulated transcription. We identified 83 Wnt direct target candidates, including the hindgut marker CDX2 and the genes relevant to morphogenesis (MSX1, MSX2, LEF1, T, PDGFRB etc.) through combinatorial analysis of the RNA-seq and ChIP-seq data. Together, our study identified a series of direct and indirect Wnt targets in hindgut differentiation, and uncovered the diverse mechanisms of Wnt signaling in regulating multi-lineage differentiation.