Dynamics of Inductive ERK Signaling in the Drosophila Embryo

Abstract

Transient activation of the highly conserved Extracellular Signal Regulated Kinase (ERK) establishes precise patterns of cell fates and morphologies in developing tissues. Quantitative parameters of these transients are essentially unknown, but a growing number of studies suggest that changes in these parameters can lead to a broad spectrum of developmental abnormalities. In the thesis, we provide a detailed quantitative picture of an ERK-dependent inductive signaling event in the early Drosophila embryo, an experimental system that offers unique opportunities for high-throughput studies of developmental signaling. We analyzed ERK signaling during the 3rd and 4th hour of development, when ERK activation induces the gene intermediate neuroblasts defective (ind), which is responsible for the formation of nervous system in flies, via relief of repression from transcriptional repressor Capicua (Cic).

Since none of the existing techniques for real-time monitoring of ERK activity are working at this time in the embryo, we developed an alternative approach for reconstructing the in vivo dynamics of ERK signaling from snapshots of embryo that are arrested at different time points (Chapter 2). Our reconstruction showed that ERK is activated as a pulse that consists of power-law growth and exponential decay. This pulse induces a stable transcriptional response of ind. Going beyond reconstructing the dynamics of ERK signaling, we investigated the regulatory origins of the observed kinetics and how it controls transcriptional responses (Chapter 3). With a simple mathematical model and experimental validations, we suggest that in the early Drosophila embryo, ERK activation kinetics can be explained solely by the dynamics of a single short ranged ligand, despite the highly complicated gene regulatory network in the system. The pulse of ERK signaling acts as a switch in controlling the expression of the ERK-target gene. Then we established that ERK relieves gene repression by Cic through a two-tiered mechanism (Chapter 4): First, gene repression by Cic is relieved within minutes of ERK activation, while Cic is still in the nucleus. On a longer time-scale, Cic is exported from the nucleus and degraded. We also found that Cic-binding sites within the regulatory DNA of its target genes provide quantitative control of the timing and the spatial extent of the gene induced by ERK activation.

In conclusion, with a combination of high-throughput genetic experiments and data mining, we present a compact and quantitative portrait of ERK signaling and subsequent gene regulation within 1.5 hours of embryonic development. Based on this study, an extended mathematical model that describes the signaling behavior in the entire tissue can be built and be used to make predictions (Chapter 5). This system can serve as a platform to study other space and time-dependent signaling events in quantitative manner.