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Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

ABSTRACT: We performed 7 single-cell RNAseq experiments to identify progenitor cell populations in the small intestine. To also capture rare intestinal cell populations such as stem cells and secretory cells we not only analyzed crypt cells from wildtype mice but also used two reporter mouse lines: i) the FltpZV/+ mouse line of which we mixed live crypt cells with Fltp Venus reporter (FVR) positive cells at different ratios and ii) Foxa2-Venus fusion (FVF) reporter mice. The FVF-enriched samples are part of another manuscript (link to GEO# will be provided). Using this FACS-based enrichment strategy, we could identify a Paneth cell-primed ISC population and potential progenitor populations for all intestinal lineages. To assess the role of Wnt/PCP signaling for enteroendocrine and Paneth cell differentiation, we performed 4 single-cell RNAseq experiments from crypt cells of Celsr1crsh/+; FltpZV/ZV compound mutant mice. When comparing control and mutant cells we found specific transcriptional alterations in the Paneth cell lineage.

ORGANISM(S): Mus musculus

PROVIDER: GSE152325 | GEO | 2020/09/11

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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Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

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