Morphogenetic mechanisms of intestinal lymphatic smooth muscle-lacteal complex function and dysfunction

ABSTRACT The lymphatic vasculature is essential for organogenesis and dietary fat absorption and the intestinal villus plays the leading role in this process. However, the developmental programs governing the formation and organ-level role of villus lacteals, specialized lymphatic vessels responsible for lipid absorption, remain poorly understood.

We recently discovered that the master left-right transcription factor Pitx2 governs lacteal function through a non-cell autonomous pathway involving the smooth muscle (SM). Pitx2-derived SM cells secrete growth factors to guide lymphatic development, forming the muscular-lacteal complex that is essential for lipid transport and

villus maintenance. Pitx2 mutant mice develop abnormal SM and lacteals, and surprisingly shunt dietary lipids into villus blood capillaries of the portal circulation, the major blood supply to the liver. This causes fatty liver disease in Pitx2 mutants, the most common human liver disorder. Compared to the villus epithelium, research

on the mesenchyme is scarce, and we lack a sufficient understanding of villus SM origin, assembly alongside lacteals, self-repair, and how villus SM dysfunction is linked to abnormal fat trafficking. Our research aims to address these critical gaps in understanding. In Aim 1, we will elucidate how Pitx2 patterns the villus SM

program by studying a fibroblast-to-myofibroblast transition and its effectors as a potential mechanism. We will test the hypothesis that intestinal myofibroblasts are the major source of renewal of villus SM crucial to villus maintenance and repair. In Aim 2, we will define the role of Notch receptor-ligand signaling in SM development,

assembly, and physiology. The focus is on understanding the expression and function of Notch3 and Jag1 in the villus vasculature and how they mediate cellular interactions between the endothelial and mesenchymal cells. In Aim 3, we will investigate the mechanism of gut-derived fatty liver disease in Pitx2 deficient mice. We aim to

understand how lipids gain access to the portal vein in Pitx2 mutants. The potential role of Pitx2 in governing blood endothelial cell permeability and its contribution to fatty liver disease in mutant mice will also be explored. This research combines our demonstrated strengths in single-cell analysis, quantitative lineage tracing,

functional assays, imaging, genetic manipulation, and targeted interventions. At the completion of these aims, our research will uncover how the muscular-lacteal complex is built and repaired through the complex intercellular interactions within the intestinal villus, opening new avenues for therapeutic interventions

targeting lymphatic-related metabolic disorders.