Failure to properly pattern the left-right axis of the human embryonic body plan results in a variety of birth defects, including the most common category of human birth defects, congenital heart defects. Despite the importance of left-right patterning of the body plan for human health, the mechanisms that generate it are only partially understood. Left-right patterning in mammals begins in the node where symmetry breaking occurs by the rotation of motile cilia, which causes a leftward fluid flow. Cells on the left side of the node sense this asymmetry and respond, through a process that remains largely unclear, by signaling to the lateral plate mesoderm (LPM) cells on the left side of the body. The signals used are the TGF-? family members Nodal and GDF-1. In the LPM, Nodal induces its own expression, which propagates through the left LPM, as well as the expression of its inhibitors, Lefty1 and Lefty2. Activation of the Nodal pathway in the left LPM drives morphological asymmetry with respect to the placement of visceral organs as well as structural asymmetries with an organ (e.g., the heart and lungs). Our data indicate the vesicle trafficking protein Rab23 is critical for the production of functional TGF-? signals that relay information to the left LPM in both mouse and zebrafish. Our data indicate that Rab23 functions in both the node and the LPM and that cells of the left node may respond to asymmetric fluid flow by regulating the level of Rab23 activity. Under this model, active Rab23 traffics Nodal and GDF-1 proteins through the secretory pathway so they may be processed and efficiently released to signal cells in the left LPM. This second part of the model is the subject of the research proposed here. Based on human and mouse phenotypic data, we hypothesize that this process also involves the multiple EGF-repeat protein Megf8. Under the current proposal, a set of three aims will address fundamental aspects of this model. Under the first aim, the mechanism by which Rab23 regulates Nodal/GDF-1 trafficking and secretion will be investigated using microscopy and biochemical approaches. Under the second aim, we will determin the localization of Rab23 and Nodal in the embryo proper. Under the final aim, we will investigate the hypothesis that Megf8 functions together with Rab23 to control trafficking and secretion of the TGF-beta signals Nodal and GDF-1. Collectively, these experiments will provide the basis for addressing the long-standing question in the left-right patterning field regarding how biophysical information in the form of cilium-driven fluid flow is translated into asymmetric gene expression and, subsequently, asymmetric morphogenesis of the internal organs.
Althoughourbodiesappearsymmetricalfromtheoutside,weknowthatthe placementandstructureourinternalorgansareoftendifferentontheleftandright sides.Failureinlateralityspecificationleadstoavarietyofbirthdefectssuchas congenitalheartdefects(CHD).Ourstudiesareaimedatunderstandingthe molecularmechanismthatdistinguishesbetweentheleftandrightsidesduring embryonicdevelopment.
