Using Optogenetics to Manipulate ERK Signaling in the Zebrafish Embryo

Abstract

The Ras/MAPK pathway plays a crucial role in cell differentiation, cell patterning, and morphogenesis movements. Recent work done on Drosophila in the Shvartsman lab has focused on extrapolating the spatiotemporal limits of ERK signaling – a key component of the MAPK pathway – using optogenetics. This provided motive to uncover these limits in a different animal model: zebrafish. The objective of this thesis is to analyze the spatiotemporal limits of ERK signaling in zebrafish embryos in early morphogenesis, while also seeking to characterize the optogenetic tool used to perform these experiments. The aspect ratio assay published in PNAS to quantitatively measure mutant MEK effects on a common scale was the assay primarily used in determining the limits of ERK signaling [6]. Released in Science in early 2017, there was a design for a photoswitchable MEK tool that allowed MEK1 to be constitutively turned on or off depending on the wavelength of light the tool was exposed to: 500 nm turned it on, 400 nm turned it off [20]. We were able to modify this tool by incorporating various other mutants to finally arrive at psMEK tool containing MEK mutations that could then be manipulated with light, just as it was originally designed in Science. This thesis shows that this psMEK tool was successful in turning on and off MEK mutants but depended on various other mutations and conditions that the psMEK mutant was exposed to, including different wavelengths of light, whether or not the construct included phosphomimetic mutations, and where the inserted mutation was on the MEK enzyme in relation to the photoswitchable Dronpa domains – the part of psMEK that controls whether or not the mutant would be able to be turned off or not. Through preliminary experimentation, we found that rapid elongation causing an abnormal aspect ratio due to mutant MEK activity occurs later on in morphogenesis but that the time window of when the embryos must be exposed to mutant MEK activity, which is controlled via light, actually occurs much earlier on. The results here provide the basis for fully characterizing an optogenetic tool that could be used in various other experiments to fully explore the spatiotemporal limits of ERK signaling in the zebrafish and possibly other animal models. These limits could then be used to shed more light on the limits of ERK signaling in humans and the implications of these limits in cancers and RASopathies.