Project Summary: All cell types in an organism share the same basic genetic information, yet they execute remarkably diverse gene expression programs and behaviors. Much of this diversity is derived from cell type-specific and condition-specific usage of gene-distal regulatory regions known as cis-regulatory modules (CRMs) (also known as enhancers). Typical mammalian genomes contain hundreds of thousands of CRMs distributed across large genomic distances that act collectively and collaboratively to produce differential gene expression patterns of extreme complexity. At present, our understanding of the biological roles of most of these CRMs remains limited. For instance, how are specific CRMs selected to regulate a given gene's expression? How do different CRMs coordinate and activate various target genes in a spatially and temporally regulated manner? Our lack of understanding is in large part due to current limitations in delivering simultaneous experimental measurements of the activities of large numbers of CRMs during animal development. The major goal of this proposal is the successful use of Xenopus gastrula-stage embryos as a model system for quantifying CRM activities involved in delivering precise spatiotemporal expression patterns of target genes. While CRM activities are to be assayed at a genome-wide level, their associated gene regulatory mechanisms would be determined with single-cell resolution. Applying advanced computational biology algorithms to that data will deliver mapping of CRM activities sufficient to infer the TFs and associated signaling processes involved in regulation of differential cell states in gastrulae at single-cell resolution. We propose to address two specific aims. First, we will apply a modified STARR-seq approach to identify CRMs that regulate spatiotemporal gene expression patterns in gastrula embryos. We plan to mutate potential TF binding sites within CRMs to aid in identifying their biological functions. Second, we will combine scRNA-seq with STARR-seq as a means of uncovering CRM-centric gene regulatory structures critical for specifying cell states for every cell in gastrula-stage embryos. In sum, we will use Xenopus tropicalis gastrula-stage embryos as the model system for generating a system-level understanding of CRM activities in single cells because of the key phylogenetic position amphibians occupy in the vertebrate evolutionary lineage. The approach proposed here would be much more difficult to perform in mammals, because their embryos are not easily accessed for certain necessary experimental manipulations, and because mammalian embryo availability is rate limiting for some genomic work.