An analysis of the WntD signaling pathway in inhibition of dorsal activity and in embryonic primordial germ cell guidance [electronic resource]

Only a few signaling pathways control the majority of biological processes throughout animal development and homeostasis, indicating that each pathway regulates multiple processes. The Wnt family of secreted ligands is one interesting and important example of this paradigm. Evidence abounds that Wnt signaling impacts numerous biological events including embryonic patterning, stem cell self- renewal, cell proliferation, and tissue regeneration. While only a few Wnt proteins have been extensively characterized, it is likely that each family member signals to control a variety of critical biological processes; therefore, it is clear that by understanding the signaling mechanism used by a single Wnt protein, we can understand how many aspects of biology are regulated. WntD is one of 7 Wnt homologs encoded by the Drosophila genome. While most of these Wnt proteins likely signal through a pathway dependent on the [Beta]-catenin homolog Armadillo (Arm), WntD very likely utilizes an Arm-independent mechanism to control embryonic dorsal/ventral (D/V) patterning, migration of the primordial germ cells (PGC) to the embryonic gonad, and the innate immune response. It is therefore of great interest to identify genes involved in this signal transduction pathway and better characterize the effect WntD has on these important biological processes. To this end, a suppressor/enhancer deficiency screen based upon the role of WntD in D/V patterning was previously performed in the laboratory. From this screen, 30 strong candidates for suppressors and 13 strong candidates for enhancers were identified. Here, I present further characterization of two candidate WntD pathway members identified in the screen: Fz4, a likely WntD receptor and Dcerk, a lipid kinase. I show by both in vitro and in vivo assays that both Fz4 and Dcerk function in the WntD signal transduction pathway, and present evidence suggesting that this WntD-Fz4-Dcerk signaling cassette is utilized during both D/V patterning and PGC migration, yet another instance of a single signaling cascade mediating multiple developmental events. Interestingly, I show that both of these novel WntD signal transducers appear to act redundantly with a homologous protein: in both D/V patterning and PGC migration, WntD likely signals through Fz3 and Fz4 receptors to activate the lipid kinases Dcerk and Dmulk. I further show, in collaboration with the Saba Laboratory at Children's Hospital Oakland Research Institute, that these kinases are necessary for wild-type ceramide-1-phosphate (C1P) levels in the adult, and either is sufficient to increase embryonic C1P levels, suggesting that both Dcerk and Dmulk are bona fide ceramide kinases. My data linking WntD-ceramide kinase signaling to PGC migration is consistent with a model in which WntD signaling is responsible for generation of optimal levels of embryonic C1P, which is likely to be the long-undiscovered substrate that is formed into a gradient by the lipid phosphate phosphatases encoded by Wunen and Wunen2. Future experiments will be required to further test this model. Importantly, these results may suggest a mechanism for the regulation of a broad range of migratory cell types, including other cells that migrate to generate a specific tissue or organ during embryonic development, and immune cells that must travel long distances to fight off infections.

Keywords: Developmental Biology.
Advisor: Nusse Roel
Kingsley David M. (David Mark) advisor. Thesis advisor
Schneider David (David Samuel)
Talbot William