Intestinal Enteroendocrine Cell Differentiation and Hormone Production in Zebrafish

Abstract

Enteroendocrine cells (EECs) are rare sensory cells in the intestinal epithelium thatcoordinate digestive physiology by secreting a diverse repertoire of peptide hormones. These hormones are the main effectors of EEC function. EEC hormones are highly conserved across evolution, and studies in diverse model systems have been pivotal to understanding their fundamental biology and leveraging it for therapeutic design. EEC hormone function is dependent on highly specific and regulated processing of the initial precursor pro-protein into functional peptide hormones. As such, sequencing data is not sufficient to predict peptide hormone forms, and instead their characterization requires direct observation by mass spectrometry. 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 22 hormone coding genes expressed by EECs, including peptides from a previously undescribed putative zebrafish secretin. We assembled reporters for zebrafish EEC subtypes to test the lineage relationships between EEC subtypes and the EEC progenitor population, which expresses neurogenin3. Despite its essential role in mammalian EEC differentiation, we found that selective cytotoxic ablation of neurogenin3+ cells in zebrafish only reduced a subset of EEC subtypes. Finally, we discovered that selective ablation of ghrelin+ EECs reduced a different subset of EEC subtypes, together suggesting that neurogenin3+ 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.