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Intracellular pH dynamics regulates intestinal stem cell lineage specification

ABSTRACT: Emerging evidence is revealing critical roles of intracellular pH (pHi) in development (), but it remains unclear whether pHi regulates adult stem cell lineage specification. We find that pHi dynamics is a key regulator of cell fate in the mouse intestinal stem cell (ISC) lineage. We identify a pHi gradient along the crypt axis in intestinal organoids and find that dissipating this gradient by inhibiting Na-H exchanger 1 (NHE1) activity genetically or pharmacologically abolishes crypt budding. Using lineage tracing and single cell RNA sequencing we demonstrate that pHi dynamics acts downstream of Atoh1, with increased pHi promoting differentiation toward the secretory lineage, while reduced pHi biases differentiation into absorptive lineage. Consistent with this, disrupting the pHi gradient blocks new Paneth cell differentiation. Paneth cells provide an essential Wnt signal to ISCs in organoids, and we find that the loss of crypt budding upon NHE1 inhibition can be rescued with exogenous WNTs. Altogether, our findings indicate that pHi is tightly regulated in the ISC lineage and that an increase in pHi is required for Paneth cell specification and thus tissue maintenance. These observations reveal a previously unrecognized role for pHi dynamics in the cell fate specification within an adult stem cell lineage.

ORGANISM(S): Mus musculus

PROVIDER: GSE211097 | GEO | 2023/05/10

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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Intracellular pH dynamics regulates intestinal stem cell lineage specification

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