In all vertebrates, including humans, the determination of the left-right (L-R) body axis is critical for the morphogenesis and physiological function of the cardiovascular and digestive systems, as well as many other visceral tissues. Molecular genetic studies have recently shown that the L-R laterality of specific tissues is specified by a pathway of regulatory genes that acts during early stages of embryogenesis. In our preliminary studies, we have found that L-R axis specification requires the activity of Cryptic, which encodes a member of the EGF-CFC family of extracellular proteins. Targeted disruption of mouse Cryptic results in L-R laterality defects including randomization of abdominal situs, hyposplenia, and pulmonary right isomerism, as well as randomized embryo turning and cardiac looping. Since mutations in the human Cryptic ortholog occur in patients with similar phenotypes, these Cryptic mutant mice represent a unique model for studying human L-R laterality defects. Furthermore, in Cryptic mutant embryos, we find that asymmetric expression of genes in the L-R pathway does not occur in the lateral plate mesoderm. This suggests that Cryptic is essential for activation of L-R asymmetric gene expression, perhaps by acting in concert with Nodal, a member of the TGF-beta superfamily that requires EGF-CFC function for its activity. Finally, we have found that proteolytic cleavage of EGF-CFC proteins is essential for their activity in cell culture, which may represent an important mechanism for regulating their function and localization in vivo. Consequently, our proposal is focused around three specific aims that address the molecular mechanisms for establishment of the L-R axis in the vertebrate embryo: I) Investigation of Cryptic activity in L-R axis formation by determining its position in a genetic pathway; II) Analysis of Cryptic mutant mice as a model system to understand the molecular basis for tissue-specific laterality during organogenesis, and to identify novel asymmetrically expressed genes in the L-R pathway; III) Analysis of processing and activity of EGF-CFC proteins to examine the regulation of EGF-CFC protein activity and localization, and their potential interactions with NODAL and its signaling pathway. By elucidating the molecular mechanisms for EGF-CFC function in embryonic axis formation, our proposed experiments should provide fundamental insights into signaling pathways involved in human development.
