Summary: Regulation of gene expression along the dorsal-ventral (DV) axis of Drosophila embryos serves as a paradigm of developmental patterning. Comparative studies of cis-regulatory elements that support expression along the DV axis from many research groups have made it clear that combinatorial input into enhancers by multiple transcription factors drives distinct spatial-outputs of gene expression. A pivotal regulator of this patterning process is the maternally provided transcription factor Dorsal (Dl), homolog of NFκB. Dl functions as a morphogen to activate target gene expression in a concentration-dependent manner along the DV axis, contributing to the initiation of zygotic gene expression at the maternal-to-zygotic transition (MZT). Using live imaging, we quantified the Dl gradient in embryos and found, surprisingly, that levels change not only in space but also build in time. Our focus during the previous funding period was to study the impact of these Dl dynamics on target gene expression using quantitative approaches involving analysis of live imaging or fixed embryo time- series data to provide insight. In the current proposal, we follow three new and exciting directions, which relate to the timing of cell actions in early embryos and arose as a result of the previous work. Project 1 involves studying how broadly expressed activators and repressors cooperate to control the onset of zygotic gene expression during the MZT. We hypothesize that broadly expressed repressors are equally important to pioneer activators in the control of chromatin accessibility and thereby also regulate initiation of zygotic gene expression. Project 2 focuses on dissecting the function of short transcripts for long genes that are expressed specifically in the early syncytial embryo. We hypothesize that these short transcripts act to regulate timing of cell signaling pathway activation by functioning as dominant-negative variants of signaling molecules. Project 3 focuses on identifying the mechanism by which FGF signaling regulates adherens junctions (AJs) and their interaction with the actin cytoskeleton to contribute to the first epithelial-to-mesenchymal transition (EMT) in embryos; in particular, to understand how a degron associated with one FGF ligand, Pyramus, limits signaling time. The overarching goal of the proposed research program is to understand how the timing of these cell activities - patterning, signaling, and movement - are controlled in developing Drosophila embryos and to provide general insights applicable to higher animals. While many studies have focused on spatial outputs of gene expression, less is known about the temporal dynamics of patterning. Drosophila embryos are a tractable system to study MZT as it occurs in 3-4 hours, in contrast to taking days in preimplantation mammalian embryos. The Drosophila embryo is also amenable to live in vivo imaging and tracking analyses making it well-suited to the study of nascent transcription a...