PROJECT SUMMARY Properly patterned lymphatic vessels are critical regulators of fluid and cellular traffic to support an effective inflammation and wound healing response. However, the mechanisms that govern lymphatic vessel patterning are not fully understood, and new therapeutics are needed to treat dysregulations of lymphangiogenesis, for example in neovascularized corneal transplant rejection, lymphedema, and tumor-associated lymphangiogenesis. The long-term goal of this research program is to develop lymphatic-targeted strategies to promote wound healing and prevent or reverse refractory inflammation. In the course of work examining lymphatic vessel pathology, the current investigators recently identified primary cilia on lymphatic endothelial cells in vitro and in vivo. In other cell types, this organelle is a hub of cell signaling and regulates cellular processes such as proliferation, cell polarity, and migration. Preliminary data in knockout mouse models of IFT20, an intraflagellar transport protein important for primary cilia function, show that loss of primary cilia exacerbates lymphangiogenesis. This suggests that previously unappreciated primary cilia-dependent mechanisms may complement and modulate canonical mechanisms of lymphangiogenesis regulation. The central hypothesis of this proposal is that unknown primary cilia signaling pathways negatively regulate excessive lymphangiogenesis by suppressing sprouting, proliferation, and migration to form vasculature appropriately tuned to local demand. This hypothesis will be tested with two specific aims: (1) Identify the cellular mechanisms by which loss of primary cilia dysregulates lymphangiogenesis; and (2) Identify lymphatic endothelial cell signaling pathways that are disrupted by loss of primary cilia. In aim 1, intravital imaging in mice with IFT20 knockout and tdTomato reporter fluorescence along with in vitro lymphangiogenesis assays will be used to identify the timing and extent of dysregulation of specific lymphatic endothelial cell activities that comprise a functional lymphangiogenesis response, such as sprouting, cell-cell junction organization, and chemotaxis. In aim 2, two next generation sequencing technologies, RNA-Seq and spatial transcriptomics, will be used in combination with pathway analysis and gene ontology strategies and rational hypothesis-driven prioritization of hits to identify candidate primary cilia-dependent regulators of inflammation-associated corneal lymphangiogenesis. Candidates will be validated in vitro in CRISPR knockout lymphatic endothelial cell lines. The combination of intravital imaging, transcriptomics, gene editing, and a panel of in vitro lymphangiogenesis assays is an innovative strategy to comprehensively interrogate the cellular mechanistic basis of primary cilia regulation of lymphangiogenesis. The proposed project is significant because it will provide the first mechanistic insight into primary cilia function in lymphatic endothelial c...