Illuminating developmental gene regulation with optimized photoswitchable MEK

Abstract

We are becoming ever closer to describing the complete list of parts required to build a living organism. Many of these components have been identified from genetic studies in embryos, where mutations in DNA can be mapped to biochemical, cellular, and tissue-level functions. Studies of protein-coding genes in embryos have revealed complicated networks among regulatory enzymes, most of which are involved in signal transduction in the cytoplasm, or control transcription in the nucleus. There are many quantitative questions about these networks that require a more engineered approach - to perturb signals and measure dynamic responses in vivo. Here, we embark upon these questions by focusing on one ubiquitous signaling system, the Ras/ERK pathway found in flatworms to humans. The first part of the thesis describes a new methodology for engineering signal inputs into living embryos using light-based manipulation of enzymes. This work focuses on a single signaling enzyme, MEK, that transduces signals from a cascade of interactions starting at the cell surface to a generalist enzyme, ERK. Since this signaling system is found in almost every eukaryotic cell, the approaches presented are powerful and widely applicable. We take advantage of molecular, kinetic, and embryological understanding of disease-causing mutations, and use it in invertebrate and vertebrate model systems. The second half of the thesis uses engineered Ras/ERK signaling inputs to characterize transcriptional regulation at multiple levels. In zebrafish, genomic studies in large populations of cells from young embryos revealed sets of gene responses constituting cell fate identities. Since the signals can be manipulated with light, we also studied the return to a normal signaling state with live readouts of gene activity in single cells of the early fruit fly embryo. As a result, the time scale of gene control by a transcriptional repressor that interprets Ras/ERK signals was accessed with unprecedented resolution. This thesis combines optogenetics, imaging at multiple scales, and genomics. The approaches described here will be important references for the future cutting edge of quantitative biology.