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. We recently showed that the ECM expansion unique to the right side precedes the cellular asymmetries taking place on the left, making expansion the first symmetry-breaking event and pointing to a novel pathway during gut rotation initiated by the right side of the embryo. This pathway is driven by Hyaluronan (HA), a unique and highly conserved glycosaminoglycan. HA within the right DM is covalently modified by the enzyme Tsg6 forming stable heavy chain (HC-HA) matrices. HC-HA then triggers a dramatic ECM expansion that simultaneously initiates gut rotation and asymmetric gut arterial growth, which also depends on Pitx2. Whereas Tsg6 is responsible for the timely activation of HC-HA, what triggers Tsg6 expression on the right remains unknown. Moreover, although Pitx2 has been thoroughly studied, how asymmetric Pitx2 expression within the DM is established remains undefined. Pitx2 directs oriented mesenchymal cell compaction on the left, suggesting key roles in morphogenesis, but the essential role for Pitx2 in left fate specification hampers our ability to test this hypothesis. We recently made several important discoveries that frame the questions in our proposed studies. We uncovered BMP4 as a driver of the right-sided program, which Pitx2 suppresses on the left. We identified the latent TGFβ complex as a mechanosensor in the DM that activates a “second wave” of Pitx2 expression in response to forces from expansion. Moreover, we discovered that exogenous Noggin on the left inhibits TGFβ signaling and Pitx2 expression, and simultaneously prevents de-repression of the default BMP4 right-side program. Thus, for the first time, we are able to study Pitx2-dependent mechanisms on the left without the confounding emergence of a “double-right” phenotype. Using a panel of Pitx2 hypomorph mouse lines, in Aim 1 we titrate the dosage of Pitx2 expression in the DM to uncouple the confounding BMP4-driven right side identity gain-of-function from the loss of Pitx2 cell on the left side. In Aim 3, we elucidate the mechanism controlling TGFβ regulation of Pitx2 dosage on the left side and measure stiffness and cortical cell tension of the live DM. Together, these studies will significantly advance our understanding of the transcriptional and mechanical control of asymmetric gut and vascular morphogenesis.