Tools for studying enteroendocrine cell subtype development and physiology in zebrafish

Abstract

Enteroendocrine cells (EECs) are sensory cells in the intestinal epithelium that respond to ingested nutrients and microbial products in the gut lumen by releasing over 30 different hormones and neurotransmitters. These signaling molecules travel throughout the body to influence a wide variety of metabolic functions, including glucose metabolism, satiety, and gut motility. EECs are typically divided into subtypes based on their predominant hormone. As enteroendocrine hormones have different and sometimes antagonistic metabolic effects, this subdivision enables fine-tuned control of metabolism in response to a variety of stimuli. While many aspects of EEC development and physiology have been defined, the mechanisms underlying EEC subtype-specific development and physiologic adaptations to diet and microbiota remain largely unknown. To investigate these questions, we recently established zebrafish as a model system for studying EEC physiology. The optical transparency of larval zebrafish enables live imaging to observe EEC adaptations in vivo and in real time. We used single-cell RNA sequencing to profile sorted intestinal epithelial cells from zebrafish larvae and identified secretory lineages including 7 EEC subtypes that we identified by expression of EEC hormones and transcription factors. Many zebrafish EEC subtypes we identified correlate with subtypes described in the mammalian small intestine, suggesting an evolutionary preserved role for EEC subtypes in regulating animal physiology. We also conducted LC-MS/MS peptidomics on EECs sorted from adult zebrafish intestine to reveal extensive homology in EEC hormone peptide sequence and post-translational processing. Using this information, we have begun to create transgenic lines to label and manipulate each of the EEC subtypes. For example, we identify a single EEC subtype that expresses genes encoding ghrelin, somatostatin, and glucose dependent insulinotropic peptides. Peptidomic analysis reveals striking conservation in their posttranslational processing including octanoylated and decanoylated isoforms of the ghrelin peptide. We have generated a transgenic line using the ghrelin gene and are using it to test how the number, distribution, and function of ghrelin positive cells vary as a function of diet and microbial colonization status. Establishment of a toolkit for zebrafish EEC subtype biology will provide an important alternative to the prevailing mammalian models and help unravel their unique developmental programs, physiologic functions, and adaptations to environmental variables. NIH R01-DK093399 and the Gordon & Betty Moore Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.