Ulmschneider et al. demonstrate that intracellular pH increases during differentiation of Drosophila ovarian epithelial stem cells and mouse embryonic stem cells, that blocking this increase impairs differentiation, and that intracellular pH may regulate the strength of Hedgehog signaling in epithelial stem cells.

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Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. We found previously that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, we named the gene ae2. Here, we generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, we find that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, we find that the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and our studies provide new genetic tools to investigate the mechanisms by which this occurs.

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SHORT ABSTRACT We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values. LONG ABSTRACT Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating of pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin.1 Here, we describe a protocol we routinely use for culturing and imaging Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage from mCherry::pHluorin transgenic wild type and mutant lines; however the methods we describe can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, culturing ovarian tissue, and acquiring and analyzing images to obtain pHi values.

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