The role of intracellular pH in the specification of cell fate

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Non-Technical Paragraph Stem cells in embryos and adults have the capacity to change to specific cell types, such as pancreatic cells that make insulin, intestinal cells that absorb nutrients, and master blood cells that can become an immune cell or a cell that carries oxygen. How stem cells change their identity is important for understanding human development, developmental defects, and approaches for regenerative medicine. For development, research is aimed at understanding how an embryonic stem cell becomes all the diverse cell types that make organs, like the heart and liver. Development “gone wrong” is often the cause of defects such as cleft-lip palate and heart malformations, as well as diseases such as cancer and diabetes. And regenerative medicine holds promise to repair damaged organs or treat diseases like Alzheimer’s. Hence, how stem cells change their identity or “fate” is a highly significant question to resolve. One new answer to this question is that when stem cells change their identity they change their internal acid-base balance. The current proposal tests predictions on how these changes in acid-base balance ensure a correct cell fate. Predictions will be tested by using new tools to accurately measure acid-base balance in live isolated stem cells and in stem cells in whole animals, and by using new computational and experimental approaches to understand how changes in acid-base balance change the shape and function of proteins as well as the expression of genes previously recognized for controlling how a cell fate is specified. Technical Paragraph Studies on how cell fate is specified from naïve stem cells mostly focus on regulation by signaling circuits, transcriptional programs, and epigenetic changes. New findings reveal that intracellular pH (pHi) dynamics is a previously unrecognized critical regulator of stem cell fates in three stem cell models: mouse embryonic stem cells (mESCs), adult Drosophila follicle stem cells (FSCs), and adult mouse intestinal stem cells (ISCs). Building on these findings will generate a mechanistic understanding of how pHi dynamics specifies cell fate. Aim 1 addresses how pHi dynamics regulates mESC pluripotency and FSC differentiation by testing the hypothesis that pHi regulates distinct stem cell states through pHi-dependent effects on the activity or ligand-binding affinity of selective endogenous proteins with established roles in regulating cell fate decisions. Roles for known (-catenin, DIDO3, phosphofructokinase-1) and predicted (BCL9 and FOXM1) pH sensitive proteins will be resolved at molecular, cellular and animal scales. Aim 2 determines the conservation of pHi dynamics in cell fate decisions through studies using ISC organoids. Preliminary data support testing the hypothesis that pHi-dependent differentiation of ISCs occurs at two steps; crypt budding and lineage specification. Crypt budding will be determined by focusing on pHi-regulated actinomyosin contractility and Wnt signaling, and lineage specification to secretory cells will be resolved by using lineage-specific fluorescent reporters and reporters for Wnt and Notch pathway activity. Outcomes will generate mechanistic insights on how pHi dynamics regulates cell fate decisions that will be significant to understand human development, developmental defects, and approaches for regenerative medicine.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.