Unravelling the Mesp1/2 Shared Regulatory Landscape - Insights into Developmental Gene Regulation by Using Gastruloids

Embryogenesis is a complex process that starts from a single-cell fertilised egg and ends with a completely formed embryo. During embryo development, tissues are formed, patterned, and matured to create a fully functional organism. Central to these processes is the regulation of gene expression, orchestrated by various regulatory elements, including enhancers. Enhancers play a pivotal role in fine-tuning gene expression patterns during development, ensuring that genes are activated or repressed at the appropriate times and locations. In this study, we focused on a dynamic genomic locus that is active during embryonic development: the Mesp1/2 locus, constituted of two genes: Mesp1 and Mesp2. These genes are essential for proper mesoderm specification, cardiac differentiation and somitogenesis. Mesp1 and Mesp2 display a complex spatio-temporal expression pattern, reflecting their intricate roles in diverse tissues and at diverse stages of embryonic development and suggesting a tight control of their expression. Although several regulatory elements involved in the regulation of Mesp1 and Mesp2 have been identified, the complete regulatory landscape orchestrating Mesp gene expression and their rule of engagement has yet to be elucidated. In this work, we used gastruloids to study and understand the mechanisms underlying spatio-temporal regulation of Mesp genes. Gastruloids are an embryonic stem cell-based model that recapitulate key processes of early mammalian development - symmetry breaking, diversification of cellular identities and establishment of body axes. They provide a simplified yet powerful system for studying post-gastrulation processes, enabling us to do high-throughput experiments, real-time imaging of developmental dynamics and facilitated genetic manipulations. In gastruloids, similarly to the embryos, Mesp1 and Mesp2 expression occurs in two waves: a first wave ofMesp1 at early stages and a second wave of Mesp1 and Mesp2, partially co-localising, at later time points. We therefore took advantage of gastruloids to clarify the dynamic of Mesp1 and Mesp2 expression in early embryogenesis and investigate how these two genes share their regulatory landscape. To achieve this, we employed a combination of in vitro gene editing techniques, live imaging of reporter lines, chromatin accessibility assays (ATAC), transcriptomics, and reporter gene analysis. Our work provided new insights in the regulatory mechanisms orchestrating this shared genomic landscape. First, we documented with high resolution the patterning of the Mesp1 and Mesp2 lineages in gastruloids. Then, we scanned the Mesp1/2 locus for enhancers and discovered two new enhancers critical for the precise temporal and spatial regulation of Mesp genes. We analysed the dynamic and function of Mesp enhancers and found that enhancers were cooperating in a synergistic fashion. These insights not only enhance our understanding of mesodermal development but also underscore the different fl