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Multi-omic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals microRNA markers of almost all major cell types

ABSTRACT: MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal epithelium. However, it is not yet known which microRNAs are expressed in which cell types of the small intestinal epithelium. To bridge this important knowledge gap, in this study we performed a comprehensive profiling analysis of microRNAs in all major epithelial cell types of the mouse small intestine. We first used flow cytometry and fluorescence activated cell sorting with multiple different reporter mouse models to isolate intestinal stem cells (ISCs), enterocytes, goblet cells, Paneth cells, enteroendocrine cells (EECs), tuft cells and secretory progenitor cells. We then subjected these cell populations to small RNA-seq. We identified highly enriched microRNA markers for every major small intestinal epithelial cell type except goblet cells. Interestingly, one of the secretory progenitor cell enriched microRNAs, miR-672, is deleted in hominin species. Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin run-on sequencing (ChRO-seq) to determine which of these LEMs are likely co-transcribed with their host genes. We then employed single cell RNA-sequencing (scRNA-seq) in order to define the cell type specificity of the host genes, and by proxy, the LEMs as well. Finally, using four additional in vivo models, we validated that miR-375 is highly enriched in secretory progenitor cells that give rise to enteroendocrine and tuft cells and that miR-152 is a Paneth cell-specific microRNA.

ORGANISM(S): Mus musculus

PROVIDER: GSE178826 | GEO | 2022/03/29

REPOSITORIES: GEO

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Project description:A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required for better treatment options for a variety of chronic intestinal diseases, however, current models of ISC lineage hierarchy and segregation are still under debate. Here we report the identification of Lgr5+ ISCs that express Flattop (Fltp), a Wnt/planar cell polarity (PCP) reporter and effector gene. Functional analysis and lineage tracing revealed that Wnt/PCP-activated Fltp+ ISCs are primed towards either the enteroendocrine or Paneth cell lineage in vivo, while retaining self-renewal and multi-lineage capacity in vitro. Surprisingly, canonical Wnt/beta-catenin- and non-canonical Wnt/PCP-activated Lgr5+ ISCs are indistinguishable by the expression of stem-cell signature or secretory lineage-specifying genes, suggesting that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch of canonical to non-canonical Wnt signalling. Pseudotemporal ordering of targeted single-cell gene expression data allowed us to delineate the ISC differentiation path into enteroendocrine and Paneth cells. Strikingly, both lineages are directly recruited from ISCs via unipotent transition states, excluding the existence of formerly predicted bi- or multipotent secretory progenitors. Transitory cells that mature into Paneth cells are defined by label-retention and co-expression of stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ label-retaining cells (LRCs). Taken together, we identified the Wnt/PCP pathway as a new niche signal and polarity cue regulating stem cell fate. Active Wnt/PCP signalling represents one of the earliest events in ISC lineage priming towards the Paneth and enteroendocrine cell fate, preceding lateral inhibition and expression of secretory lineage-specifying genes. Thus, our findings provide a better understanding of the niche signals and redefine the mechanisms underlying ISC lineage hierarchy and segregation. Here we establish the Wnt/Planar cell polarity (PCP) gene Flattop (Fltp) as a unique marker for intestinal LRCs and their distinct secretory fate. We show that Fltp+ cells are characterized by a combined stem cell and secretory gene signature. A subset of Fltp+ cells is classified by label-retention and predominantly locates at position +4, indicative of quiescent stem cells. Strikingly, Fltp+ LRCs are specified by Wnt/PCP signaling in contrast to actively cycling stem cells that rely on the canonical Wnt/β-catenin pathway. In mice with disturbed Wnt/PCP signaling, the differentiation of enteroendocrine cells from LRCs is impaired. These findings establish Fltp as a novel marker for intestinal LRCs and implicate Wnt/PCP signaling in cell-cycle exit and commitment of ISCs to the secretory lineage. Taken together, we not only provide a marker to study LRCs in homeostatic and diseased conditions, but also identify the Wnt/PCP signaling pathway as a therapeutic target for colorectal cancer and metabolic disease.

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Multi-omic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals microRNA markers of almost all major cell types

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