Birth defects are a leading cause of infant mortality, yet in most cases, their etiology is unknown. Some of the most common and complex malformations are found in families with abnormal left-right (LR) asymmetry, suggesting that many congenital defects may result from perturbed laterality. The initial embryonic events that determine the LR body axis, including the early breaking of bilateral symmetry, and subsequent left-sided expression of determinants such as nodal and Pitx2, are now well understood. However, the genetic and morphogenetic events involved in the final phases of LR development, at the organ level, remain largely unknown. The long term goal is to ascertain the mechanisms of LR asymmetric organogenesis. The objective in this application is to identify the molecular and cellular processes that generate LR asymmetry (curvature) within an individual organ, the stomach. Preliminary analyses identified LR asymmetries in radial cell rearrangements in the developing stomach as the driving force for its curvature. To identify the proximate effectors of this novel asymmetric morphogenetic program, a new model organism (Lepidobatrachus laevis) was employed. The extra-large embryos of this species facilitated a gene-discovery approach that would be intractable in most models: transcriptome profiling (RNASeq) of tissues dissected from left vs. right halves of the embryonic stomach. Pilot datasets include genes with LR asymmetric expression patterns and functions during stomach curvature. The central hypothesis is that stomach curvature is determined by distinct left and right regulatory networks which differentially modulate the cellular events controlling radial cell rearrangement. The unique experimental amenability of frog embryos will be used to test this hypothesis via three specific aims: 1) Generate molecular signatures of normal and abnormal stomach curvature. Comprehensive spatiotemporal profiles of LR stomach genes will be generated and compared in the context of both normal LR asymmetry and experimentally-induced LR axis defects. 2) Determine the cellular function of stomach- specific LR genes. Select genes will be tested in loss- and gain-of-function assays to determine their influence on radial cell rearrangement in the developing stomach. 3) Determine the regulatory hierarchy that controls stomach curvature. Experimental perturbations combined with spatiotemporal profiling will reveal core gene regulatory interactions governing asymmetric morphogenesis. The overall approach is innovative because it takes advantage of distinctive attributes of a unique species to address one of the key unanswered questions in LR development: what are the proximate mechanisms by which developing organs become LR asymmetric entities? The proposed research is significant because it will immediately advance our understanding of normal laterality and laterality-related birth defects by defining the genes, morphogenetic processes and regulatory logic that govern the emergence of LR asymmetry at the organ level.
As the leading cause of infant mortality in the United States, birth defects incur significant economic burden, yet the origin of most malformations is still unknown. Defining the etiology of some of the most common and severe structural birth defects, such as those associated with abnormal left-right (LR) asymmetry, is therefore of significant clinical importance. A major contribution of the proposed project will be the revelation of new classes of molecules and morphogenetic mechanisms that control the development of LR asymmetry at the organ level. This contribution will be complemented by the construction of an organ-specific LR gene regulatory network with potential to explain the complex and variable phenotypic manifestations of laterality- related birth defects.
