The Roles of Definitive Endoderm in Establishment of Left-Right Asymmetry

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DESCRIPTION (provided by applicant): Defects in human left right (LR) axis formation cause birth defects of the heart, vasculature, lungs, and gastrointestinal tract that are life-threatening and costly to the healthcare system. To understand how laterality defects develop, it is essential to understand the molecular mechanisms by which LR asymmetry is established. My long-term goal is to understand the mechanism of how LR asymmetry is established during early embryogenesis. My previous work including collaboration with my colleagues in this field has revealed important steps in this process in the mouse model. However, there are still gaps in our understanding of the entire process, such as the mechanisms by which the node structure and rotating node cilia develop, and how LR polarity in the node is transferred to the left LPM. Here I will address these questions by investigating roles of definitive endoderm in LR determination. To date, no report has been focused on roles of endoderm in LR asymmetry. But several series of evidence suggest importance of endoderm in this process. Our hypothesis is that endoderm cells regulate LR determination in two ways: regulating node formation and/or function, and transferring LR signal from the node to the LPM. This hypothesis will be addressed by analyzing Sox17 mutants because Sox17 is the only gene that specifically expressed in the endoderm and primary defects of the mutants are specific to the definitive endoderm. Aim 1 will study the nature of the initial LR determination defects occurring in Sox17 mutants by characterizing the node and cilia formation, in addition to the development of nodal flow. Aim 2 will examine the possible defects occurring in the process of transferring LR signals from the node to the LPM regarding the gap junctional transport and intracellular Ca2+ signaling. In addition to chemical blockers, the roles of gap junctions will be tested by a sophisticated transgenic approach using a dominant negative form of connexin that can be temporally and/or spatially controlled. Conditional mutants of Sox17 will be studied to identify the importance of endoderm cells in the signal transfer from the node to the LPM, being separated from the initial abnormalities in the node. We have found that the Nodal gene continues to be expressed in migrating endoderm in the Sox17 mutants, which could be the cause of endoderm defects in Sox17 mutants that affect LR determination. In Aim 3, I will test this possibility by transgenic techniques. I will rescue Sox17 mutants by expressing Nodal antagonists in endoderm and the Sox17 mutant phenotype will be reproduced by ectopically expressing the Nodal gene in the endoderm of wild type embryos. The 20 yrs of experience that the PI possesses in this model system has enabled us to design and optimize novel tools and techniques that are ideal for addressing the questions I have put forth in this proposal. The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality. PUBLIC HEALTH RELEVANCE: The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality.