Whereas the role of the homeodomain transcription factor Pitx2 has been traditionally studied in the context of embryogenesis, the main purpose of my proposal is to study its role beyond embryonic development. As the master regulator of left-right asymmetry, Pitx2 works early in embryonic development to direct the asymmetric morphogenesis of all visceral organs, including the intestine. Here, Pitx2 is also necessary for the formation and organization of the gut vasculature including intestinal lacteals, the highly specialized lymphatic capillaries crucial to dietary fat uptake and gut immune surveillance. Mutant pups lacking the asymmetric enhancer of Pitx2 fail to gain weight and have aberrant lipid transport, shunting dietary lipids from the gut directly to the liver via the portal vein, resulting in diet-induced Fatty Liver Disease (FLD). Thus, in addition to the established role of Pitx2 in early embryogenesis, Pitx2 is necessary for nutrient absorption postnatally. However, whether the role of Pitx2 in nutrient absorption is solely related to its function in embryonic development, or, whether Pitx2 is continuously required in the postnatal intestine remains unknown. Recently, I discovered that inducible loss of Pitx2 in postnatal mice (Pitx2 iKO) results in failure to gain weight, significant changes in cell type distribution of the intestinal epithelium, and early postnatal lethality. To our knowledge, this is the first evidence that Pitx2 plays a role in the postnatal intestine. Moreover, Pitx2 expression is not limited to the mesoderm during embryonic development – I have recently demonstrated a robust expression of Pitx2 in the early gut endoderm and identified Pitx2 daughter cells in the endodermally-derived absorptive enterocytes that are paramount to nutrient absorption, suggesting that Pitx2 is a hidden player in this germ layer. Pitx2 has also been implicated in the regenerative process following injury in mouse neonatal hearts and may play a similar role during regeneration of intestinal tissues following injury. In intestinal disorders like Inflammatory Bowel Disease (IBD), regeneration of the epithelium is necessary to repair damage. Importantly, in organoids generated from tissue samples of patients with Crohn’s disease, Pitx2 is one of the most differentially expressed genes, further implicating Pitx2 in intestinal diseases. Based on these exciting preliminary findings, I will test the hypothesis that Pitx2 is necessary for the absorptive function, homeostasis, and regeneration of the postnatal intestinal epithelium. To accomplish this, Aim 1 will dissect the role of Pitx2 in the intestinal epithelium using the Intestinal Stem Cell (ISC)-specific Lgr5-CreERT2 mice along with single-cell transcriptomics, and chromatin binding analyses. In Aim 2, I will define the intestinal regenerative function of Pitx2 following injury by administering Dextran Sulfate Sodium (DSS), Doxorubicin, and irradiation to Pitx2 mutant mice. In summary, this is the first time Pitx2 function is dissected in the intestinal epithelium and in the context of postnatal development. Moreover, my research will develop tools to address Pitx2 function in the context of debilitating intestinal metabolic diseases.