Intestinal absorption of dietary nutrients is an important process contributing to the etiology of multiple metabolic diseases that represent ~30% of worldwide mortality. An organism’s response to a high-fat meal (HFM) requires coordination between multiple cell types in digestive tissues as well as the gut microbiota. Enterocytes (EC) are the absorptive cells of the intestine that coordinate this response by temporarily storing lipids in cytosolic lipid droplets (LD) and ultimately preparing them for circulation to distal tissues in ApoB- containing lipoproteins (BLP). In parallel, enteroendocrine cells (EEC) sense and communicate nutrient information to other cells and tissues via calcium-dependent hormone and neurotransmitter release. While the roles of these cells in postprandial physiology are broadly acknowledged, the specific mechanisms mediating EC and EEC postprandial responses to high-fat meal (HFM) and how microbiota influence those responses remain unresolved. Our research team pioneered the zebrafish system for studies of lipid metabolism and host-microbiota interaction. Over the past 10 years of this award, we have developed and applied highly innovative tools in the zebrafish system to reveal new mechanisms of lipid metabolism, EEC physiology, and host-microbiota interaction. The central hypothesis of this renewal is that a coordinated dynamic response to HFM between EC and EEC is mediated by a suite of microbe-influenced transcription factors that ultimately alter BLP and LD levels and turnover. Our research team has developed a set of novel tools to measure digestive organ lipid uptake, transport, storage, and signaling. These include the first family of in vivo reporter lines to quantify BLP size, numbers and turnover, as well as LD protein dynamics. In Aim 1, we will apply these new tools along with genetic analyses to define the dynamics underlying LD and BLP responses to HFM and microbes. We have previously used genetic approaches in zebrafish to identify host transcription factors mediating responses to HFM and microbiota, and recently performed the first single cell nuclei RNAseq analysis of an entire animal at a range of timepoints following a HFM. This approach allows us to interrogate the transcriptional networks driving the postprandial response of digestive organs at unprecedented levels of cellular and temporal granularity. In Aim 2, we will use these resources to define the transcriptional regulatory pathways mediating intestinal epithelial response to HFM. We have also established tools for measuring EEC responses to nutrients and microbes in zebrafish, and used them to reveal a novel microbiota-mediated EEC postprandial adaptation after a HFM. In Aim 3, we will define the mechanisms underlying this microbiota- induced EEC adaptation. This competitive renewal leverages long-standing partnerships between three field- leading labs with a powerful set of mutant and novel transgenic reporter lines to elucidate the relat...