Formation of vascular structures such as blood vessels involves a series of complex cell shape changes. 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 in the developing embryo 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 responsible for lumen formation in the developing organism remain relatively unknown. The heart of the fruit fly, Drosophila melanogaster, provides a simple and elegant genetic model to discover the molecules and mechanisms of vascular assembly and 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 migrate to the dorsal midline to form a linear tube with a central lumen. Dr. Kramer has established that the guidance molecule Slit and its receptors Robo and Robo2 are required during both steps of heart tube assembly. During the first step, Slit, Robo and Robo2 are required to maintain adhesion between cardiac cells during their dorsal migration. During the second step, Dr. Kramer has found that Slit and Robo are also required to negatively regulate E-Cadherin-mediated cell adhesion between neighboring cardiac cells, allowing for the formation of a lumen. However, the precise mechanism by which Slit and Robo are regulating cell adhesion in the heart 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 proteins that are functioning with Slit and Robo receptors during cell adhesion. It is highly likely that these mechanisms are conserved and that the outcomes of this work will shed light on lumen formation in vertebrate vascular structures. The broader impacts of this work rest on the fact that the fruit fly is a powerful and tractable 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 teaching and training students from diverse backgrounds.
The focus of this project was to elucidate the cellular mechanisms underlying the development of the Drosophila embryonic heart tube, which forms by the merging of two rows of cardiac cells at the embryonic midline. Our preliminary data showed strong evidence that the secreted guidance molecule Slit and its transmembrane receptor Roundabout direct lumen formation in the heart by negatively regulating E-cadherin- mediated cell adhesion between opposing pairs of cardiac cells. In this study, we extended these findings by examining potential downstream cytoskeletal regulatory molecules for their roles in this process. Specifically, we found that the Rho GTPase protein Cdc42 is required in a genetically distinct subset of cardiac cells for their forward migration and adhesion to the opposing row. This subset of cells makes initial contact across the midline with their contralateral partners in a Cdc42-dependant manner. In embryos in which Cdc42 activity is depleted, these specific cardiac cells lack cell protrusions and fail to make cell adhesions with the opposing row. Furthermore, we demonstrated that the cytoskeletal regulatory protein Ena/Vasp also plays a role in the process, specifically in sealing the transient holes that remain during the final stages of heart tube closure. In embryos lacking functional Ena protein, these holes fail to seal, resulting in holes in the final structure of the heart tube. Interestingly, Ena is required in a subset of cardiac cells that is distinct from the cells that require Cdc42. Thus, cardiac tube formation requires the activity of multiple cytoskeletal regulatory proteins in distinct cardiac cell subtypes. An integral part of the activities relating to this award has been the mentoring of underrepresented graduate and undergraduate students as well as in the introduction of undergraduate students to basic science. This was achieved both through direct participation in laboratory research activities as well as through the teaching of a freshman undergraduate seminar course designed to introduce students to basic science and laboratory research. Several undergraduates from this seminar course have subsequently joined the lab to participate in the research described above.
