Formation of vascular structures involves a series of complex changes in cellular architecture. Blood vessels are essentially small tubes formed by a monolayer of endothelial cells enclosing a central lumen. How do groups of unorganized endothelial cells migrate to their proper location and then arrange themselves into a linear tube with a central lumen? While experiments in cultured cells have led to a speculative model for lumen formation, surprisingly the cellular and molecular mechanisms that occur in the developing organism remain relatively unknown. The heart tube of the fruit fly, Drosophila melanogaster, provides a simple and elegant genetic model to discover the molecules and mechanisms of lumen formation. During Drosophila heart tube development, the alignment of cardiac cells at the dorsal midline of the embryo is the first of two important steps in heart tube assembly. Following alignment, these cells meet at the midline to form a tube with a central lumen. Dr. Kramer has established that the guidance molecule Slit is a good candidate for playing an important role in heart tube lumen formation. Slit is a secreted extracellular molecule that is common to both insects and humans and has been shown to guide migrating cells in many different tissues in the developing organism. Dr. Kramer has found that Slit and its receptor protein Roundabout are expressed in the developing Drosophila heart at the time of lumen formation, and that defects in lumen formation occur in embryos missing these genes. These results indicate that Slit and Roundabout play an important role in this process. However, the precise mechanism by which they affect heart tube formation has yet to be determined. To address this problem, Dr. Kramer will carefully analyze heart tube formation in embryos that are mutant for these genes as well as use the power of Drosophila genetics to identify other genes that are functioning in the Slit and Roundabout pathway. It is highly likely that these mechanisms are conserved and will shed light on lumen formation in vertebrate vascular structures. In terms of Broader Impacts, the fruit fly provides a powerful and tractable genetic model system not only for the study of vascular development, but also as a teaching tool for training PhD students and for introducing undergraduate students to basic science. Dr. Kramer has a strong record of involving undergraduate and graduate students in her research and is committed to training students from diverse backgrounds.
Vasculogenesis refers to the formation of new blood vessels from endothelial precursor cells. In the fruit fly, Drosophila melanogaster, Dorsal Vessel morphogenesis shares many similarities with the early events of vertebrate heart and blood vessel development and has proven to be an important system for the study of vascular assembly due to its relatively simple structure and the availability of a variety of genetic approaches. Blood vessels are essentially small tubes formed by a monolayer of endothelial cells enclosing a central space or lumen. How do groups of unorganized endothelial cells migrate to their proper location and then arrange themselves into a linear tube with a central lumen? In this study, we have built upon our previous findings that the Slit and Roundabout (Robo) ligand and receptor proteins are required for vascular lumen formation. Both Slit and Robo are expressed in the lumen of the Dorsal Vessel and mutations in either gene results in a lumen-less phenotype. We have shown that Slit and Robo function in lumen formation by negatively regulating the adhesion of opposing cells in the vessel, allowing for a space to form in between. Specifically we have found that Slit/Robo signaling negatively regulates the cell adhesion molecule E-cadherin, which is normally required to hold together the vessel at specific sites of attachment. In the absence of Slit/Robo, E-cadherin is up regulated, resulting in inappropriate adhesion between Dorsal Vessel cells. We have strong evidence that the regulation of E-cadherin by Slit/Robo signaling occurs at the level of membrane trafficking. Furthermore, we found that the Enabled protein, which regulates the actin cytoskeleton, is required for the positive regulation of E-cadherin-mediated contacts and co-localizes with E-Cadherin in the Dorsal Vessel. In enabled mutants, the Dorsal Vessel cells often fail to make appropriate contacts. These results, together with Enabled's known function downstream of Robo during repulsive axon guidance suggest that Enabled is normally required to maintain stable E-Cad-mediated adhesions at sites of CB cell contact through interaction with the actin cytoskeleton and that in the presence of Robo, this link is destabilized. Finally, we have discovered a new role for Slit and Robo in regulating microvillus growth during morphogenesis of the Drosophila embryonic hindgut. Specifically, we have shown that Robo is required in the mesoderm for proper microvillus length in the endoderm, suggesting a non-autonomous role for Robo signaling in directing apical membrane growth. The fruit fly is an excellent and tractable model system not only for the study of heart development, but also as a teaching tool for training PhD students and for introducing undergraduate students to research. This project relied heavily on the participation of graduate students and undergraduate students from diverse backgrounds. In addition to providing funding for the training of several graduate and undergraduate students, another activity that resulted was the creation of a first year undergraduate seminar course specifically designed to introduce college freshman to basic research at an early point in their training. The seminar is called: "Of Tin Man and Scarecrow: Understanding the Heart and Brain." In the class, which is limited to 20 students, we explored the use of model systems to study human development and disease and introduced the students to research by taking them to our labs and giving them some hands on experience.
