Regulatory factor identification for nodal genes in zebrafish by causal inference

Activation of nodal genes is critical for mesoderm and endoderm induction. Our previous study reported that zebrafish nodal genes ndr1/squint and ndr2/cyclops 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 ndr1 activation, while Eomesa is the cause of ndr2 activation. Mechanistically, the different cis-regulatory regions of ndr1 and ndr2 bound by Eomesa, β-catenin, and Smad2 were screened out via ChIP-qPCR and verified by the transgene constructs. The marginal GFP expression driven by ndr1 transgenesis could be diminished without both maternal Eomesa and Hwa/β-catenin, while Eomesa, not β-catenin, could bind and activate ndr2 demonstrated by ndr2 transgenesis. Thus, the distinct regulation of ndr1/ndr2 relies on different cis-regulatory regions.