Intracellular pH dynamics regulates intestinal stem cell lineage specification

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. Overall design: Both wild-type (WT) and inducible-CRISPR NHE1 knockout (KO) small intestinal organoids were derived from adult mice. Upon NHE1 inhibition, WT organoids were treated with 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) and KO organoids were treated with doxycycline (Dox) post-plating. All WT, WT+EIPA, KO, KO+Dox organoids were dissociated into single cell suspension and sorted by Fluorescence-activated cell sorting (FACS) prior to scRNAseq.