Temporal coordination of two enhancers relies on the modulation of a common inductive signal. Lily S. Cheung¹, Alisa Fuchs², David S. A. Simakov³, Len M. Pismen³, Giorgos Pyrowolakis², Stanislav Y. Shvartsman¹. 1) Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ; 2) Institute for Biology I, Albert-Ludwigs University of Freiburg, Germany; 3) Department of Chemical Engineering, Technion-Israel Institute of Technology, Israel.

   Development is directed by temporal and spatial changes in gene expression. Although it is recognized that changes in gene expression patterns can result from stage-specific use of different enhancers, the mechanisms of coordination between enhancers remain less understood. We explore this question in Drosophila oogenesis, at stages when the expression of the transcription factor broad (br) evolves from an spatially uniform to a more complex, two-domain pattern that determines the formation of the dorsal respiratory structures in the eggshell. We identified two enhancers of br activated sequentially during mid-oogenesis that together recapitulate the dynamics of the gene. In addition to their temporal coordination, these enhancers also exhibit complementary spatial patterns due to differential regulation by the EGFR pathway. Here, we use RNAi-mediated downregulation of br to show that the activity of the early enhancer is required for the activation of the late one. Using a shorter version of the late enhancer, we also show that this effect does not depend on a direct positive autoregulation. Instead, we propose that modulation of the EGFR pathway by the protein produced by the early enhancer acts as a permissive signal for expression of the late one. We have formulated a computational model combining transient inductive signals with multiple enhancers that supports the feasibility of our mechanism. We use this model to guide our experimental effort, and to investigate the more general question of how a single signaling pathway is used recurrently during development to establish complex patterns of gene expression.