PROJECT SUMMARY In the developing embryos, the formation of organs and tissues arises from the collective actions of many individual cells which may change their shape, size, adhesion, and motility. Our long-term goal is to understand how these cellular behaviors are regulated in both space and time to correctly build each tissue and organ. In this project, we will leverage two closely related yet phenotypically distinct cell types, the endodermal and dorsal forerunner cells in zebrafish embryos, to better understand the regulation between migratory mesenchymal states and coherent epithelia. During early gastrulation, endodermal cells exhibit mesenchymal characteristics; they are highly migratory, spatially dispersed, and avoid contact with each other. In contrast the dorsal forerunner cells are tightly associated with each other and display epithelial characteristics, even as they migrate collectively. Remarkably, both cell types are specified by the same developmental signals and express many of the same marker genes, despite their divergent migration behaviors. In Aim 1 of this project, we will use single- cell RNA sequencing and bioinformatic analysis to compare the transcriptional profiles of endodermal and dorsal forerunner cells and identify differentially expressed candidate genes likely involved in the regulation of cell migration modes. In Aim 2, we will generate split fluorescent protein knock-in zebrafish lines to characterize the expression and localization of key endodermal and dorsal forerunner genes. This approach is based on technology developed in the parent grant to this diversity supplement application. If successful, this study will identify key genes responsible for different modes of migration in two closely related, yet distinct cell types. Our findings will shed light on the broader question of how small differences in transcriptional states translate into major differences in cellular behavior.