During embryogenesis, tissues must be correctly patterned in order to properly develop. The fruit fly embryo is patterned along the dorsal-ventral (DV) axis by the morphogen Dorsal, a transcription factor that forms a nuclear concentration gradient with high levels present ventrally and little to none present dorsally. Dorsal protein is also known to be phosphorylated; however, it is unknown where the phosphorylation occurs, and whether it plays an important role in development. Both Dorsal, and the protein that binds and inhibits it, Cactus, are downstream of the Toll receptor. The textbook view is that Toll signaling acts only on Cactus, whereas we hypothesize that Toll signaling may also act to phosphorylate Dorsal. Study of the regulatory relationships of these three factors may elucidate general signaling mechanisms in which an activated signaling complex phosphorylates both inhibitor (Cactus protein which retains Dorsal in the cytoplasm) and activator (Dorsal transcription factor that functions in the nucleus to turn on gene expression upon being freed from Cactus). We propose that this regulatory circuit acts to shape the developmentally appropriate cellular response required for patterning the DV axis: fast increase in Dorsal nuclear levels to support zygotic transcription across the DV axis followed by a precipitous drop in Dorsal nuclear levels at the onset of gastrulation once patterning is complete. To provide support for this hypothesized alternate role of Toll signaling in the regulation of Dorsal activity through phosphorylation, our proposal has two experimental aims. The goal of Aim 1 is to determine if phosphorylation of C-terminal serines alters Dorsal protein function or key, Dorsal-dependent developmental outcomes; whereas the goal of Aim 2 is to discern the relationship between Toll signaling and Dorsal C-terminal phosphorylation. The overarching goal of the proposed research is to probe the mechanisms by which signaling pathways regulate transcription factor dynamics, and how this leads to the proper morphogenesis of tissues. Phosphorylation as a result of Toll signaling may serve to both increase nuclear residence of Dorsal and degrade Cactus, which frees Dorsal to enter the nucleus. This dual role would represent a signaling mechanism that functions to amplify a specific downstream outcome and may allow the cell or embryo to perform a nuanced regulatory computation. The insights gained here will set the groundwork for additional studies of regulation of signaling and transcription factor dynamics by phosphorylation. Specifically, the mammalian homolog of Dorsal, NF-kB, is also phosphorylated, is important for diverse developmental processes such as inflammation during the immune response, and has been implicated in cancer. Therefore, understanding where Dorsal is phosphorylated and how this post-translational modification affects its function has the potential for far-reaching insights.
The regulation of transcription factors that pattern developing organisms is not well understood and this proposal focuses on understanding the post-translational regulation of the transcription factor Dorsal, which patterns the dorsal-ventral (DV) axis of fruit fly embryos. Understanding how Dorsal is regulated post-translationally may lead to a better understanding of how transcription factors are regulated, in general, with wide-ranging implications from understanding embryonic development to the inflammation response. The mammalian homolog of Dorsal, NF-kB, plays an important role in many developmental processes in vertebrates, is involved in the immune response, and has been implicated in cancer and autoimmune disease.
