Cdx is crucial for the timing mechanism driving colinear Hox activation and defines a trunk segment in the Hox cluster topology

•Cdx is essential for temporally colinear induction of clustered Hox genes in epiblast stem cells.•Cdx is required for chromatin opening and H3K27ac positions around central Hox genes.•HoxA exhibits distinct domains according to both its topology and inducing signals.•An early Wnt-dependent initiating transcription phase is followed by a Cdx-dependent phase.

Cdx and Hox transcription factors are important regulators of axial patterning and are required for tissue generation along the vertebrate body axis. Cdx genes have been demonstrated to act upstream of Hox genes in midgestation embryos. Here, we investigate the role of Cdx transcription factors in the gradual colinear activation of the Hox clusters. We found that Hox temporally colinear expression is severely affected in epiblast stem cells derived from Cdx null embryos. We demonstrate that after initiation of 3′ Hox gene transcription, Cdx activity is crucial for H3K27ac deposition and for accessibility of cis-regulatory elements around the central - or 'trunk' - Hox genes. We thereby identify a Cdx-responsive segment of HoxA, immediately 5′ to the recently defined regulatory domain orchestrating initial transcription of the first Hox gene. We propose that this partition of HoxA into a Wnt-driven 3′ part and the newly found Cdx-dependent middle segment of the cluster, forms a structural fundament of Hox colinearity of expression. Subsequently to initial Wnt-induced activation of 3′ Hox genes, Cdx transcription factors would act as crucial effectors for activating central Hox genes, until the last gene of the cluster arrests the process.