ABSTRACT The mechanisms discovered through the study of embryogenesis have been fundamental to understanding disease. We use classic chicken embryology and sophisticated mouse genetics to elucidate how basic cellular processes define the shape and function of organs. We are most fascinated by left-right (LR) organ asymmetry, as errors of organ laterality are linked to life-threatening birth defects. The counterclockwise rotation of the gut is an excellent model to study organ laterality. A critical aspect of this rotation is initiation of a leftward tilt directed by the master regulator of LR asymmetry, Pitx2. Failure to do so leads to gut malrotation and catastrophic volvulus in pediatric patients. Whereas rotation forces had long been assumed intrinsic to the gut tube, we instead discovered that gut rotation is driven by asymmetric deformation of the adjacent dorsal mesentery (DM) that suspends the gut, and whose cellular architecture is downstream of Pitx2. A key property of the DM is its exquisite binary organization, with distinct LR compartments that are readily accessible to genetic manipulation. Cellular and extracellular matrix (ECM) changes in each compartment cause the DM to deform and tilt the attached gut tube leftward. This critical bias determines gut chirality and frames a model to explain how LR gene expression is ultimately responsible for changes in cell behavior that initiate asymmetric organogenesis. The DM is also the sole conduit for blood and lymphatic vessels that serve the gut. We discovered that Pitx2-dependent mechanisms directing gut tilting are also crucial for patterning the gut vasculature and provide a mechanism to coordinate these two processes. Whereas most situs-specific organogenesis depends on Pitx2, mechanistic studies downstream have been hampered by a confounding “double-right” isomerism in Pitx2 mutants. Whereas Pitx2 expression in all vertebrates is activated by Nodal, Nodal disappears before asymmetric morphogenesis, leaving unresolved the question of how Pitx2 directs organogenesis. We discovered that Pitx2 expression in the gut is not an extension of previous induction by Nodal. Instead, we demonstrate that gut rotation requires a “second wave” of Pitx2 that is subject to mechanoregulation by the latent TGFb, linking LR gene expression to force translation. In aim 1, we determine the mechanism of Pitx2 dose response during gut, vascular, and lymphatic development and identify two distinct roles for Pitx2 dependent on its repressive threshold on BMP4 signaling. In aim 2, we define the molecular mechanism by which the formin Daam2, a Pitx2 target, directs mesenchymal cell polarity, actomyosin contractile asymmetry, and thereby steers the forces to polarize tilting. In aim 3, we combine cutting-edge physical measurements of tissue properties with quantitative imaging and computational modeling, to elucidate how stiffness and force anisotropies drive gut rotation through mechanical feedback. Together, the...