Gut malrotation is a birth defect of abnormal intestinal rotation that occurs once in approximately 500 live births. While malrotation predisposes affected babies to volvulus, a catastrophic strangulation of the intestine and its blood supply, the origin of this anomaly remains entirely unknown. It has long been assumed that rotation is intrinsic to the tube itself; however, our research has shown that gut tilting in mice and birds is driven by cell and extracellular matrix (ECM) asymmetries in the dorsal mesentery (DM), a mesodermal structure that suspends the gut. Specifically, the left DM condenses while the right expands causing the DM to tilt the gut tube leftward. This critical leftward bias determines the conserved gut chirality. Whereas the transcription factor Pitx2, a master regulator of left-right (L-R) organogenesis, drives all left-sided cellular changes in the DM, the mechanisms governing the right sided expansion remain entirely unknown. Importantly, this right expansion precedes all other DM asymmetries, suggesting that the key initiator for leftward gut tilting derives from the right side. Hence, the major objective of our proposal is to identify the mechanisms regulating the right-sided expansion of the DM. 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. We have shown that HA synthesis is inhibited by Pitx2, and that pharmacological inhibition of HA synthesis ablates both DM expansion and the leftward tilt. Moreover, the product of tumor necrosis factor a-stimulated gene 6 (Tsg6), an enzyme that can increase synthesis and stabilize HA matrices, is restricted to the right (expanded) DM, and that forced Tsg6 expression in the left (condensed) DM causes expansion akin to the right side. Based on these findings, we hypothesize that changes on the right initiate gut rotation and that synthesis of HA and Tsg6 provide the symmetry-breaking event responsible for the right-sided DM expansion. We propose three specific aims to address our hypothesis. We leverage the strengths of multiple model systems to address all aims, and our specialized, technical mastery of targeting different compartments of the chicken DM offers us a singular advantage by introducing gain- or reduction- of function gene constructs into each DM compartment (Aims 1 and 3). We combine the use of egg with mouse genetics to allow for direct analyses of our candidates in a mammalian system. To attack Aim 2 we have established a productive collaboration with the group of Dr. Vince Hascall, a world-renowned expert in HA biochemistry, which enables us to combine classical developmental biology techniques with rigorous biochemical analyses that will provide the mechanistic insights into HA regulation of gut rotation. Collectively, this work will increase our understanding of the mechanistic basis of gut malrotation and may ultimately improve diagnosis in neonates.