The intestinal villi are key functional units enabling dietary nutrient absorption, defects in which lead to a host of congenital and lifestyle disorders. It consists of two critical components - capillary lymphatic projections called lacteals that absorb packaged lipids and contractile axial smooth muscles (ASM), which wrap around and squeeze the lacteals to facilitate lipid transport. Importantly, genetic compromise of the ASM perturbs the structure of their adjacent lacteals and leads to abnormal lipid transport and non-alcoholic fatty liver disease (NAFLD) due to shunting of lipids into the hepatic portal (venous) circulation. Moreover, given their regenerative potential, mapping the developmental origin of the lacteal-ASM system would make them excellent therapeutic targets for metabolic disorders such as Inflammatory Bowel Disease (IBD). Lack of unique markers and the nonspecificity of existing Cre recombinase mouse strains (Cre-drivers) that restrict expression to specific cell types hinder our understanding of intestinal cell-fates. Here I propose to use newly identified markers from my time-resolved single cell transcriptomics of the developing intestine to build cell lineage trajectories of intestinal lymphatic vasculature and their adjacent smooth muscle. Aim 1: To map the molecular, cellular, and spatial organization of distinct gut lymphatic subsets in mouse embryos. Gut-specific lymphatics have been controversially proposed to have non-venous sources against a century-old dogma that all lymphatics are of venous-origin. I will perform lineage tracing with newly validated marker genes of venous (AplnR-CreER), arterial (Gja4-CreERT2) and hemogenic (cKit-CreER) endothelium from single-cell trajectories, overcoming the nonspecificity of previously used drivers. I will co-label these trajectories with immunofluorescence for newly identified and previously established markers of all endothelial cell subsets to construct the first accurate spatiotemporal map of intestinal lymphatic endothelial cell lineage, delineating sources and contributions. Aim 2: To define the intercellular interactions of the villus muscular-lymphatic milieu. Despite their crucial interplay with lacteal structure and function, the origins and patterning mechanisms of the ASM remain unexplored. Based on their gene expression and in vivo location, I hypothesize that myofibroblasts of the intestinal mucosa act a source of ASM precursors. I will perform sharply timed, inducible lineage tracing of mucosal myofibroblasts with Credrivers informed by single-cell transcriptomics (Pdgfra- and Postn- CreERT2) to delineate the contribution of myofibroblasts to the intestinal fat absorption apparatus. Importantly, Notch3 is specifically expressed in myofibroblast derived ASM precursors whereas its receptor Dll4 is expressed by villus capillary vasculature. I will perform spatiotemporally restricted deletions of Notch3 and Dll4 to test if juxtracrine (contact-dependent) interactions between villus capillary vasculature and myofibroblasts from the mucosal bed enable the structural development and physiological function of the lacteal-ASM fat absorption system.