Gut lymphatics are essential for dietary fat uptake and the intestinal villus plays the leading role in this process. However, the organ-level role of villus lacteals, the specialized lymphatic capillaries responsible for lipid absorption, remains poorly understood. We recently uncovered an intestinal lymphatic program that is linked to the left-right asymmetric transcription factor Pitx2. Pitx2 governs lacteal development and function through a non-cell autonomous pathway involving the villus smooth muscle (SM). Pitx2-derived SM cells guide lymphangiogenesis, forming the muscular-lacteal complex that is essential for lipid transport throughout life. Pitx2-deficient mice exhibit abnormal SM morphogenesis and lacteal growth, leading to lipid malabsorption. These discoveries highlight a novel role for Pitx2 in lymphatic transport of lipids and emphasize the critical interplay between gut lymphatics and their mesenchymal neighbors. While studies have transformed our knowledge of the intestinal epithelium, research on the underlying mesenchyme remains scarce. We lack details of the mechanisms underlying the formation, differentiation, renewal, and interactions between villus musculature and adjacent endothelium. To address this gap, we constructed a single-cell atlas of the mouse intestine, uncovering a hierarchical organization of fibroblasts that differentiate into lacteal-associated villus SM fibers dependent on Notch3. In Aim 1, we will elucidate how Pitx2 patterns the lymphatic SM program by studying a fibroblast-to-myofibroblast transition and its effectors as a potential mechanism. We will test the
hypothesis that myofibroblasts are the major source of renewal of lymphatic SM crucial to villus maintenance and repair. In Aim 2, we will define the role of Notch signaling in lymphatic SM development, assembly, and physiology. In Aim 3, we will investigate how Pitx2 regulates lacteal button junctions that are crucial for lipid uptake. VEGFA directs these junctions by reducing lacteal permeability while increasing permeability in BECs. Pitx2 loss not only reduces lacteal transport but also shifts lipid uptake to BECs, causing steatohepatitis through the portal vein, the major blood supply to the liver. We will investigate if Pitx2 deficiency leads to lipid malabsorption by affecting VEGFA/VEGFR2. We will also investigate if malabsorption in Pitx2 and Notch3 mutants results from flaws in muscular-lacteal contractile function. These aims will involve a range of experimental approaches, including quantitative lineage tracing, state-of-the-art contractility imaging, functional assays, and mouse models. Our goal is to delineate the mechanisms behind intestinal fibroblast and myofibroblast contributions to lymphatic SM formation and renewal, the assembly and maintenance of the muscular-lacteal complex, and its role in regulating lacteal junctions and contractile function during lymphatic fat transport.