During early embryogenesis the vertebrate embryo extends from anterior to posterior due to the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for the NMps to retain mesodermal potential, and hence normal axis development. We recently showed that the Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor binding sites, we identified over 500 potential Hox13 targets. Importantly, we found two highly conserved Hox13 binding elements far from the tbxta transcription start site, which also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis stage expression to a tbxta promoter that alone only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.

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