Role of Force-directed Lipid Metabolism in the Endothelial-to-Hematopoietic Transition - Project Summary/Abstract In vertebrates, self-renewing hematopoietic stem cells (HSCs) are produced from a developmental event called endothelial-to-hematopoietic transition (EHT). EHT consists of a cellular and transcriptional reprogramming that allows hemogenic endothelial cells (HECs) from a subset of embryonic arteries to leave the vessel and become blood stem cells. HSCs have the capacity to replace and restore the complete blood system upon transplant, making HSC transplant the only curative therapy available for blood diseases like leukemia and lymphoma. Given this therapeutic need, great effort has focused on the development of in vitro protocols that attempt to recapitulate the conditions of EHT for clinical expansion or de novo production of stem cells in the dish. To date none efficiently produce long-lived multipotent HSCs, suggesting that one or more developmental signals for this process remain to be defined. Mechanical forces from blood flow are an essential cue for HSC production via EHT, and the zebrafish Danio rerio provides an excellent animal model in which to study this contribution to hematopoiesis due to conserved molecular genetics of EHT in this species and the ability to observe live embryos with active circulation. Flow-driven EHT is mediated in part by the Yes-associated protein (YAP) transcription factor (TF), a transcriptional coregulator that has roles in organ growth, nutrient regulation and cell fate specification. YAP can be directed to the nucleus as a direct result of physical forces acting on the cell, but the molecular mechanisms by which this promotes EHT and HSC production are unclear. In preliminary data generated under K01 support, single-cell transcriptional analysis of wildtype, yap -/- and YAP-overexpressing HECs from zebrafish point to a role for YAP in regulating a battery of self-renewal hematopoietic TFs, cell cycling and metabolic processes. In examining these YAP gain- and loss-of-function (GOF/LOF) transcriptomes, gene module scores suggest an impaired glycolysis-to-oxidative phosphorylation rewiring in HECs. Genes related to lipid metabolism are also dysregulated by YAP perturbation and can be identified in ‘no flow’ datasets from mouse models. This R03 application will investigate the role of force-directed lipid metabolism in developmental EHT using zebrafish as a model. We hypothesize that hemodynamic forces alter lipid usage in HE to drive the metabolically intensive process of EHT. In the first aim, an unbiased approach of mass spectrometry-based lipidomic profiling will be used to quantify the abundance of lipid species in wildtype and YAP gain or loss of function (GOF/LOF) whole-embryo and sorted endothelial cell populations to determine those metabolites that are YAP-regulated (as a proxy for a major cellular transducer of mechanical force). In the second aim a candidate pathway, the secreted sphingosine-1-phosphate lipid mediator, will be studied for its role in EHT by live-imaging, chemical perturbation and state-of-the-art genome editing technologies to create tissue-specific LOF zebrafish lines. Findings from this proposal will uncover force-driven metabolic responses that might enhance production of HSCs via EHT and generate critical preliminary data to support R01 applications.
