The long-term goal of this work is to understand molecular mechanisms underlying endoderm development during vertebrate embryogenesis. Most of our current understanding about vertebrate endoderm formation comes from studies in Xenopus laevis. These studies have led to the identification fo several proteins critical for endoderm formation including the transcription factors VegT, Milk, Milk and Xsos17 as well ast he signaling molecules Veg1, Xnr1, Xnr2 and Derriere. Genetic analyses in zebrafish have also revealed the requirement of the nodal-related signals Cyclops and Squint as of One-eved pinhead, a transmembrane protein essential for nodal signaling. Work in out laboratory indicates that several additional zebrafish loci play important roles in endoderm formation. These loci include casanova (cas), bonnie and clyde (bon), faust (fau), natter (nat) and miles apart (mil). We have recently shown that bon encodes a zebrafish homologue of Xenopus Mixer and that fau encodes Gata5 and have assembled several of the above regulators of endoderm formation into a molecular pathway. We aim to continue out investigation of endoderm development in zebrafish by using bon/mixer, fau/gata5, cas, nat and mil as additional entry points into the process and propose the following specific aims: 1) analyze the role of bon/mixer and fau/gata5 in endoderm formation. We hypothesize that Mixer and Gata5 act in parallel pathways to regulate sox17 expression and will conduct loss-as well as gain-of-function studies to test this hypothesis; 2) analyze the role of cas in endoderm formation. We will isolate cas by positional cloning and expand our analysis of the cas phenotype. Once we have isolated cas we will perform gain-of-function studies to gain a full understanding of the role it plays in endoderm formation; 3) screen for and analyze additional regulators of endoderm formation through a combination of genetic and molecular approaches. a) we will identify additional loc by analyzing in detail a novel collection of endoderm mutants we have recently generated in the laboratory. b) we will use a variety of molecular techniques to identify genes regulated by bon/mixer, fau/gata5 or cas and analyze their role through overexpression studies. These molecular genetic studies of endoderm formation will help provide the necessary context for further investigating the pathophysiology of malformations and malfunctions of the human gut and its associated organs, and may also facilitate differentiation-based strategies for treatment of a variety of diseased states.