Regulatory factor identification for genes in zebrafish by causal inference.

Activation of genes is critical for mesoderm and endoderm induction. Our previous study reported that zebrafish genes / and / are coordinately regulated by maternal Eomesa, Hwa-activated β-catenin (Hwa/β-catenin) signaling, and Nodal autoregulation (Nodal/Smad2) signaling. However, the exact contribution and underlying mechanisms are still elusive. Here, we applied "causal inference" to evaluate the causal between the independent and dependent variables, and we found that Hwa/β-catenin and Smad2 are the cause of activation, while Eomesa is the cause of activation. Mechanistically, the different -regulatory regions of and bound by Eomesa, β-catenin, and Smad2 were screened out ChIP-qPCR and verified by the transgene constructs. The marginal GFP expression driven by could be diminished without both maternal Eomesa and Hwa/β-catenin, while Eomesa, not β-catenin, could bind and activate demonstrated by transgenesis. Thus, the distinct regulation of / relies on different -regulatory regions.