Investigating the potential non-cell autonomous Robo2 function during lumen formation of the Drosophila melanogaster dorsal vessel. Judith J Canabal Alvear^1,2, Sunita G Kramer^1,2. 1) Pathology Department , UMDNJ/RWJMS, Piscataway , NJ; 2) Cell and Developmental Biology, Rutgers University, Piscataway, NJ.

   Biological tubes are required for the development of complex organisms given that they distribute important molecules to different parts of the organism. In this study, we investigate a previously unknown function for the transmembrane receptor Roundabout2 (Robo2) during lumen formation of the Drosophila dorsal vessel, a simple linear tube required to pump hemolymph throughout the embryo. Two major steps are required for dorsal vessel formation. First, specified cardioblasts (CBs) migrate in rows toward the dorsal midline of the embryo and second, the CBs undergo a series of cell shape changes to form a linear tube with a central lumen. The two rows of CBs are flanked on either side by two rows of non-muscle pericardial cells (PCs). While the PCs have been shown to be important for dorsal CB migration, a potential role for the PCs in mediating lumen formation in the adjacent CBs is unclear. CBs express a single Roundabout receptor (Robo1), while PCs express both Robo1 and Robo2. Our lab has shown that loss of Robo1 results in defects in lumen formation. However the role for Robo2 in this process has not been explored. The present work investigates the role for Robo2 in lumen formation through loss-of-function (LOF) and gain-of-function (GOF) studies. In robo2 LOF embryos, we observe defects in CB lumen formation. Because Robo2 is expressed by the PCs, these findings suggest a non-cell autonomous role for Robo2 in this process. Furthermore, ectopic expression of robo2 at low levels in the CBs results in a robo LOF phenotype, while expression of Robo2 at high levels in the CBs produces a strong robo GOF phenotype. These results suggest that Robo2 has the ability to both antagonize as well as mimic Robo function in the dorsal vessel. We are currently investigating the significance of this biphasic nature of Robo2, as well as determining its intrinsic role during lumen formation using a combination of genetic, structure function and live imaging analysis.