Wnt signaling pathway interactions in early anterior-posterior specification and patterning In most animal embryos the establishment of the anterior-posterior (AP) axis provides the necessary initial coordinates for building an embryo, and as such, it is the most critical step during embryonic development. A large body of work has determined that the AP axis is initially established by the localized activation of Wnt/β- catenin signaling at the future posterior end of the embryo in many animals. In general, posterior restriction of Wnt/β-catenin signaling creates a posterior-to-anterior morphogen gradient that activates and positions early gene regulatory networks (GRNs) along the AP axis. These include the endomesodermal GRN at the posterior pole, an equatorial mostly non-neural ectoderm GRN, and the anterior neuroectoderm (ANE) GRN around the anterior pole. For the first time in any animal, we have discovered that this fundamental developmental process depends on 3 different, but interconnected, Wnt signaling pathways (Wnt/β-catenin, Wnt/JNK and Wnt/PKC) in the sea urchin embryo. Importantly, comparison of functional and expression studies among multiple deuterostome species, including vertebrates, strongly suggests that aspects of this AP Wnt signaling network are conserved. The long-term goal of the studies in our lab is to use systems biology approaches along with functional analyses to characterize the extracellular, intracellular and transcriptional components of this network. These discoveries will likely provide insight into how dysregulation of Wnt-mediated AP patterning can lead to developmental disruptions, including human birth defects. The objective of this proposal is to establish the transcriptional GRNs activated downstream of the non-canonical Wnt/JNK and Wnt/PKC pathways in the network and to uncover how extracellular and intracellular Wnt modulators influence the activity of the these GRNs. The central hypothesis is that key interactions among the Wnt signaling pathways occur at the extracellular, intracellular, and transcriptional level. The rationale is that by generating the transcriptional GRNs activated by the non-canonical Wnt pathways in the network, we can uncover interactions at that level to be used to determine how the extracellular and intracellular Wnt modulators are integrated in the overall network. Aim1 will generate a model of the transcriptional GRNs activated by the Wnt/JNK and Wnt/PKC signaling pathways, combining information from temporal ATAC-seq data with existing temporal differential screen data that compared wild type embryos with Wnt/JNK and Wnt/PKC knockdown embryos. In Aim2 we will perform functional gene perturbation studies on key nodes in our network model from Aim 1 to further establish the initial GRN scaffold downstream of Wnt/JNK and Wnt/PKC signaling and to define any key interactions among the pathways at the transcriptional level. Aim 3 will use gene perturbation analyses on putative extr...