Developmental enhancers are marked independently of zygotic Nodal signals in Xenopus

• Enhancers marked by H3K4me1 and H3K27ac are established widely by blastula stages.
• Smad2/3 does not associate strongly with developmental genes before gastrulation.
• Nodal-regulated genes establish enhancer marks first, and Smad2 is bound later.
• Enhancer marks at most genes are preserved when Nodal signaling is ablated.

To determine the hierarchy of transcriptional regulation within the in vivo vertebrate embryo, we examined whether developmental enhancers were influenced by Nodal signaling during early embryogenesis in Xenopus tropicalis. We find that developmental enhancers, defined by the active enhancer chromatin marks H3K4me1 and H3K27ac, are established as early as blastula stage and that Smad2/3 only strongly associates with these regions at gastrula stages. Significantly, when we perturb Nodal signaling using the drug SB431542, most enhancers remain marked, including at genes known to be sensitive to Nodal signaling. Overall, as enhancers are in an active conformation prior to Nodal signaling and are established independently of Nodal signaling, we suggest that many developmental enhancers are marked maternally, prior to exposure to extrinsic signals.