A critical aspect of gut rotation is initiation of a leftward tilt directed by the conserved left-­‐‑right (LR) Pitx2 transcription factor. Failure to establish proper gut chirality leads to gut malrotation and catastrophic volvulus in pediatric patients. Whereas the direction of rotation has long been assumed to be intrinsic to the tube itself we demonstrated that rotation is instead driven by asymmetric cellular behavior within the dorsal mesentery (DM) that suspends the gut tube and whose cellular architecture is downstream of Pitx2 expressed strictly on the DM left side. In contrast, the mechanisms governing the right side of the DM remain entirely unknown and are the major goal of this proposal. We recently showed that the ECM expansion unique to the right side precedes the cellular asymmetries taking place on the left, making ECM expansion the first symmetry-­‐‑breaking event and pointing to a novel pathway during gut rotation initiated by the right side of the embryo. Hyaluronan (HA), a unique and highly conserved glycosaminoglycan, predominates in the ECM of the right DM and is a strong candidate for driving ECM expansion. Inhibition of HA synthesis, or, loss of the inflammatory enzyme tumor necrosis factor stimulated gene 6 (Tsg6), the only enzyme that covalently modifies HA, prevents DM expansion and results in randomized gut looping. Tsg6 transfers heavy chains (HCs) to HA and transforms the normally inert HA matrix into a unique HC-­‐‑HA complex implicated in a variety of inflammatory diseases. The only known developmental function of HC-­‐‑HA is to ensure female fertility. Blood vessels in the gut mesentery are the cause of tissue death during midgut volvulus, but how these vessels become positioned inside the DM isn’t known. We now show that gut arteries develop only on the left side because they are progressively excluded from the right side, dependent on HA and Tsg6. Thus, HA may be a common link in the global phenotype observed on the right side of the DM. In this proposal, we hypothesize that ECM changes on the right initiate gut rotation. Using chicken and mouse DM, our goal is to identify the mechanisms regulating HA function in the DM, the relationship between Tsg6 and HA production, and the changes in tissue architecture that establish gut and vascular chirality. In Aim 1, we propose to define the role of Tsg6 during ECM expansion and vascular exclusion. In Aim 2, we characterize the mechanisms by which HA inhibits vascular development on the right. We hypothesize that HA regulates endothelial migration by inhibiting the Cxcl12/Cxcr4 axis in the right DM. In Aim 3, we propose a mechanism responsible for the opposing pro-­‐‑angiogenic and anti-­‐‑angiogenic phenotypes within the LR DM. Our studies will identify mechanisms that govern LR organ and vascular morphogenesis downstream of HA-­‐‑matrices and may shed light on the origin of gut and vascular anomalies. Vasculogenesis -­‐‑ induction of new vessels and destruction of exiting ones -­‐‑ is among the most important processes in human biology. Our DM system is perfectly suited to study these dynamic and clinically relevant events in the intestine.