Mechanisms of Hand2-mediated cell migration in mesothelia - PROJECT SUMMARY Mesothelia are epithelial sheets that envelop internal organs and line body cavities, providing essential structural support, immune response, and friction-reducing serous fluids which enable proper organ physiology. Despite their fundamental roles in body plan organization and disease, mesothelial biology remains poorly characterized due to limited understanding of their developmental origins, structure, and migration dynamics. Discovering migratory mechanisms of this under-appreciated tissue promises to inform our collective understanding of organogenesis, body plan organization, and congenital mesothelial defects including gastroschisis, a congenital defect in which the developing organs herniate through the mesothelial body wall. My project seeks to define how mesothelial progenitors migrate and participate in organ development and body plan organization. Mesothelial cells arise from the lateral plate mesoderm (LPM) and express the transcription factor Hand2, which plays a critical role in early mesodermal patterning. We recently demonstrated that pericardial mesothelium formation is not dependent on heart formation, indicating decoupled development. Preliminary single-cell RNA-seq data show that mesothelial progenitors are compartmentalized along the anterior-posterior axis, further indicating early regionalization of mesothelial progenitors before organ formation. Live imaging further indicates that these progenitors migrate along defined, non-mixing trajectories, yet the regulatory basis of this segregation is unknown. Our Hand2 ChIP-seq data reveal that Hand2 binds regulatory regions of key epithelial–mesenchymal transition (EMT) genes, including Twist1 and Zeb1, suggesting direct transcriptional control of migratory programs. Because Zeb1 drives lateral plate mesoderm migration around the developing gut, Hand2 regulation of EMT may underlie mesothelial movement across organs. Together, these data support the hypothesis that Hand2 directs compartmentalized mesothelial migration and formation. Aim 1 will define the compartmentalization and migration of mesothelial progenitors. I will perform 4D live imaging of i) mesothelium alongside endoderm and ii) mesothelium-specific Cre/lox lineage tracing to determine clonality. Additionally, I will define the interplay between mesothelium and organs such as the gut tube, pancreas, and liver during organogenesis. Aim 2 will determine the molecular mechanisms driving mesothelial cell migration, namely epithelial-mesenchymal transition (EMT) and the reverse process, MET. I will use my lab’s novel dual- transgenic EMT reporter to map EMT dynamics in both time and space with 4D microscopy. I hypothesize Hand2 controls the gene regulatory program responsible for driving these cell property changes and subsequent migration, and I will test this with putative enhancer reporters and mutants. Collectively, these studies will reveal how Hand2 orchestrates mesothelial patterning, organ association, and drives EMT/MET activation.
