Deciphering when the pivotal transcription factor Dorsal exerts patterning effects using optogenetics

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The Drosophila transcription factor and morphogen, Dorsal, assumes a graded nuclear distribution along the dorsal-ventral (DV) axis of the early embryo with high levels in ventrally-positioned nuclei and progressively lower concentrations in lateral and dorsal nuclei. Activating some genes and repressing others, Dorsal determines the pattern of expression of zygotic target genes along the DV axis in a nuclear concentration- dependent manner. This nuclear gradient is maintained over several nuclear mitotic divisions occurring during the syncytial stage of embryogenesis, but it is unclear whether Dorsal function is required continuously during this period or only transiently, or whether the temporal requirements for Dorsal function differ among its target genes. Moreover, only recently has it come to light that the concentration of Dorsal exhibits dynamic changes in concentration in individual nuclei within single nuclear cycles and that the concentration of Dorsal present within nuclei at different positions along the DV axis changes over the course of syncytial development. These dynamic behaviors of Dorsal likely contribute to robust and reproducible spatial and temporal control of Dorsal's many target genes through regulatory mechanisms that remain uncharacterized. To provide insight into how levels as well as time of exposure to concentration-dependent morphogens may influence patterning activity, the following two experimental aims will be pursued: Specific Aim 1: To develop optogenetic approaches to control activity and/or levels of Dorsal. Several approaches will be used to either activate or inactivate Dorsal with fine-scale temporal and spatial precision by illumination with blue light. These approaches will use different strategies to modulate either the stability or integrity of Dorsal protein, or its nuclear versus cytoplasmic localization. Specific Aim 2: To elucidate the relationship between Dorsal protein dynamic behavior and the expression of its zygotic target genes along the embryonic dorsal- ventral axis. The expression of ~70 target genes will be examined using NanoString technology applied to fixed embryos that have been subjected to optogenetic modulation for varying durations, and MS2-MCP RNA stem-loop based imaging methods will be used to further characterize dynamic behaviors associated with a subset of these genes in live embryos. The ways in which target genes respond when Dorsal activity is either activated or perturbed at specific times during early embryogenesis will provide important insight into the role of Dorsal dynamics on development. In addition to providing new insight into Dorsal's role in controlling this paradigmatic gene regulatory network, these investigations will optimize and validate powerful new and widely applicable optogenetic approaches for the study of other dynamic transcriptional/regulatory factors operating in the embryo as well as a wide variety of other contexts, particularly those for which dynamic behavior is an important component of function.