Lymphatic vessel function is critical for organ development and disease, yet the mechanisms governing organ-level lymphatic function remain poorly understood. Within the gut villus - the body’s densest lymphatic network - specialized lacteal capillaries absorb and transport dietary fats, vitamins, and drugs. Disruption of lacteal function contributes to metabolic and cardiovascular diseases, highlighting the urgent need to understand these critical pathways. Our lab focuses on gut morphogenesis directed by the left-right symmetry-breaking transcription factor Pitx2. We recently discovered that Pitx2 governs lacteal function non-cell autonomously via the muscular-lacteal complex (MLC), a lymphatic structure critical for nutrient and fluid transport and metabolic homeostasis. Pitx2 deficiency disrupts the lacteal-associated villus smooth muscle (SM) and formation and lacteal function leading to lipid malabsorption and postnatal lethality. Despite advances in epithelial biology, gut mesenchyme remains poorly defined, limiting therapeutic targeting of lymphatic muscle cells. Using single-cell and quantitative lineage-tracing approaches, we discovered that PDGFRa+ fibroblast progenitors undergo a fibroblast-to-myofibroblast transition to form the villus lymphatic muscle. This continuum persists as the major mechanism for MLC renewal throughout adult life. Our overarching goal is to define the mechanisms of MLC specification, differentiation, and function in gut health and metabolic disease. In Aim 1, we will define how Pitx2 specifies villus lymphatic muscle during villification in coordination with lacteal development. In Aim 2, we will elucidate the distinct and complementary roles of Notch3 in PDGFRa and PDGFRb lineage-dependent MLC formation, lipid absorptive function, and Notch3 relationship to gut laterality. The goal of Aim 3 is to establish MLC as a hallmark of gut lymphatic disorders. Using genetically tractable mice, we will assess systemic metabolic consequences of MLC disruption, including fatty liver progression, and test reversibility. Findings will be integrated with human intestinal organoid data to inform translational relevance. By combining developmental analyses, single-nucleus genomics, lineage tracing, and multi-organ metabolic assessments, our studies will provide novel insights into the formation and function of the villus lymphatic muscle and its role in metabolic malabsorption disorders of the lymphatic system.