Optogenetic control of spatio-temporal patterning in early embryogenesis

Princeton University, Princeton NJ

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Abstract

? DESCRIPTION (provided by applicant): Development is a complex process that requires spatial and temporal coordination of signals to specify local cell fates and properly map out body and organ formation. In the last decade, much work has been done to quantitatively characterize these patterns - measuring shape of gradients and the kinetics of their establishment. Yet the genetic tools available to study these patterns are very poorly suited to perturb them. Genetic perturbations typically destroy patterns completely, or alter multiple features of their spatiotemporal distribution simultaneously. In contrast, the emerging tools of optogenetics make it possible for the first time to directly control the spatial patterns and dynamics of protein activity. Unlike chemical stimuli which undergo diffusion, light patterns can be sharply defined and removed at will. Here we will aim to apply light based control over a developmental signal, MAPK activation, in Drosophila melanogaster. With this fine control we will measure the consequences of varying the amplitude, duration and spatial range of the signal, and quantitatively compare how the signal is interpreted at different developmental stages. This proposal aims to engineer the first optogenetic inputs to MAPK in a developmental context. After generating and validating transgenic flies which express our optogenetic system, we will cross these flies with ones which are depleted of the MAPK activating ligand which normally specifies the head and tail structures. This will enable us to test if local light-induced MAPK activity can rescue head/tail formation in these embryos. We can then systematically perturb the spatial range and dynamics of light to determine which of these parameters is sensed by the cell to specify its fate. To better understand how the MAPK signal is repurposed in a very different developmental event, that of the neurogenic ectoderm specification, we will once again introduce our optogenetic system into flies depleted of the ligand which controls this patterning. This will enable us to compare not only the way the signal is interpreted but also how it processed between the two embryogenesis events. Successful completion of these aims will mark the first use of optogenetics in an in vivo developmental system and shed light on two fundamental questions: understanding what parameters of a signal actually specify cell fate as well as how the same molecular signal specifies fates in different contexts.