This project continues long term studies into mechanisms of morphgenesis. Here the focus is on an investigation into an Epithelial to mesenchymal transition (EMT). The goal is to understand how this complex morphogenetic movement works at a molecular level. The EMT is an essential morphogenetic movement used by almost all animals. Its failure contributes to a number of birth defects, and it is also co-opted, at least in part, by metastatic processes of carcinomas leading to the spread of tumors. The model system used here is the sea urchin skeletogenic cells which undergo an EMT synchronously at 9 hr after fertilization. Knowledge of the cells, the known timing of the EMT, and many experimental tools for this system provide an excellent model for discovery of how the process works. A number of assays and approaches enable the EMT process to be studied in detail at a molecular level as it occurs in this system. Prior research identified essential transcription factors that regulate the component processes of the EMT. Distinct transcription factors regulate activation of motility, basement membrane remodeling, cell polarity change, and de-adhesion. To build on this foundation, the proposal is for studies to identify molecular effectors downstream of those transcription factors. The effectors already obtained, and those sought, control the on- time delivery of signals to activate the motility, conduct the basement membrane remodeling, regulate the de-adhesion, coordinate the several component processes, and convert the epithelial membrane into a functioning mesenchymal membrane. The project is enabled by a number of advances in the past funding cycle. RNA-seq and scRNA-seq databases recording temporal transects through the EMT interval were obtained. A pipeline was established to identify candidate molecules, authenticate their participation, and identify the role they play in the EMT process. Additional candidates will be obtained using these systems level databases. New molecular tools were advanced so that multiple approaches are available for testing and authenticating candidate participation and function. In addition, two other tissues that undergo later synchronous EMTs have been added to the project. Pigment cells and blastocoelar cells each undergo EMT 3 hrs and 7 hrs after the skeletogenic cells. These additions enable us to determine which effectors are used for one, two, or all three of the EMTs. Although preliminary evidence indicates that even though the three cell types are derived from mesodermal precursors in the embryo, their EMTs are conducted by largely independent molecular components. Nevertheless, there is some overlap so it is important to learn which components are unique to a particular EMT and which may be more universal. Before translational advances can be applied it is necessary to understand the EMT sequence in detail and to know which molecular pathways are universal. This project therefore is of great value in gaining...