Identification of intestinal enteroendocrine cell subtypes and their associated hormones in zebrafish

Enteroendocrine cells (EECs) are rare sensory cells in the intestinal epithelium that coordinate digestive physiology by secreting a diverse repertoire of peptide hormones. These hormones are the main effectors of EEC function, and their characterization requires direct observation by mass spectrometry due to the specialized protein cleavage and posttranslational modifications that yield their mature forms. Based on the distinct subset of hormones they predominantly secrete, EECs can be categorized into subtypes. How each EEC subtype is specified, however, remains poorly understood. Here, we describe EEC subtype differentiation and hormone production in the zebrafish. Using single-cell RNA sequencing data, we identified EEC progenitors and six EEC subtypes in zebrafish and revealed that their expression profiles are consistent across larval and adult stages. Mass spectrometry analysis of isolated zebrafish EECs identified highly processed peptides derived from 19 of 23 hormone-coding genes expressed by EECs, including a previously undescribed zebrafish secretin ortholog. We assembled reporters for zebrafish EEC subtypes to test the lineage relationships between EEC subtypes and the EEC progenitor population, which expresses neurogenin 3 (neurog3). Despite its essential role in mammalian EEC differentiation, we found that selective cytotoxic ablation of neurog3+ cells in zebrafish only reduced a subset of EEC subtypes and loss of the neurog3 gene had no impact on EEC numbers. Finally, we discovered that selective ablation of ghrelin+ EECs reduced a different subset of EEC subtypes, together suggesting that neurog3+ and ghrelin+ cells serve as distinct precursors for separate EEC subtypes. We anticipate these observations and resources will facilitate future studies in the zebrafish to discern the developmental biology, physiology, and endocrinology of EEC subtypes.