(703c) Constructing a Synthetic Gene Network to Model and Understand Signaling Interactions

In multicellular organisms, cellular signaling events are crucial for patterning tissues, as well as for maintaining healthy adult tissues, while improper signaling can lead to disease states, such as cancer.  Therefore, cellular signaling process must be tightly regulated.  A complex system of gene regulatory circuits controls this signaling process and acts to buffer this system against noise, thereby minimizing mistakes in gene expression and preventing patterning defects or disease states.  Despite their importance to patterning and development, hypotheses regarding these gene regulatory circuits have been difficult to test experimentally due to their complexity and high connectivity.  Therefore to better understand the fundamental processes involved, we created a synthetic gene network in the fruit fly Drosophila melanogaster.  This network utilizes genes from yeast and E. coli, namely, gal4, gal80, and lacZ.  We expressed gal4 in a graded fashion along the anterior-posterior axis of the embryo, mimicking the intracellular diffusion of an endogenous transcription factor, Bicoid.  The gal4 activates expression of UAS-linked gal80 and lacZ.  gal80 inhibits gal4 activation, creating a negative feedback loop in our system.  These genes were chosen since they are not endogenous to Drosophila, so all interactions in this network will be fully understood.  Our goal is to measure the variability in location of the lacZ domain both with and without the negative feedback loop.  This system provides a direct experimental test of whether negative feedback loops in cellular signaling events can provide robustness to noisy, diffusive systems.  It is this robustness that is important for combating diseases and defects in development and maintenance of expression in the organism.