Optogenetic Approaches for Determining the Temporal Role of Morphogen Inputs on Target Gene Expression

Citation

McGehee, James Mitchell (2023) Optogenetic Approaches for Determining the Temporal Role of Morphogen Inputs on Target Gene Expression. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/c610-za20. https://resolver.caltech.edu/CaltechTHESIS:05302023-162053583

Abstract

The Dorsal transcription factor and morphogen is important for patterning the Dorsal- Ventral axis of Drosophila melanogaster and while it has been extensively studied, the temporal dynamics of Dorsal are not well understood. There are many processes that contribute to Dorsal nuclear concentration levels, including Toll signaling and Cactus degradation, interactions with other proteins, shuttling of Dorsal to the ventral side, DNA binding, and nuclear spacing. Dorsal nuclear levels are known to activate or repress target gene expression in a concentration or threshold dependent manner. To test how Dorsal dynamics and changes to the Dorsal gradient over time affect target gene expression, we added two optogenetic tags to Dorsal at the endogenous locus to control Dorsal nuclear levels: Blue Light Inducible Degradation (BLID) and Light Inducible Nuclear Export System (LEXY). We found that upon degradation of Dorsal using blue light and BLID that a downstream ratchet was able to maintain the expression of high threshold target genes. Using blue light and LEXY to export Dorsal, we identified an important window where Dorsal activity is required to allow activation of high threshold target genes at later stages. In comparing BLID and LEXY in conjunction with mutations to a nuclear export sequence, we also identified how rapid nuclear import and export of Dorsal is sufficient for low threshold target gene expression but actively disrupts high threshold target gene expression. We conclude that not only are final concentration levels, but also the dynamics leading to those levels are important for proper gene expression.

Thesis Files