Integrative multi-omics increase resolution of the sea urchin posterior gut gene regulatory network at single cell level

Gene regulatory networks (GRNs) can be used to describe gene interactions of various complex biological processes such as embryonic development, regeneration and disease. The GRN orchestrating endomesoderm development in the purple sea urchin Strongylocentrotus purpuratus pre-gastrula embryo has been the subject of multiple studies. Drafting GRNs requires embryological knowledge pertaining to the cell type families, information on the regulatory genes that control the regulatory state of the cell type family, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis, which helps identify the cis-regulatory modules (CRMs) of the regulatory genes. We use multi-omics involving next-generation sequencing (-seq) to obtain the necessary information for GRN drafting. Here we present an update to the sea urchin posterior gut GRN using i) a single cell RNA-seq derived cell atlas highlighting the 2 day post fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at single cell level, ii) a set of putative CRMs and transcription factor (TF) binding sites obtained from chromatin accessibility ATAC-seq data, and iii) interactions directionality obtained from differential bulk RNA-seq following knockdown of the TF Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1 positive cells in the 2 dpf gastrula embryo. Overall, our data resolves the complex connectivity of the posterior gut GRN and increases the resolution of gene regulatory cascades operating within it.