Drosophila anion exchanger 2 is required for proper ovary development and oogenesis

Abstract

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.

Intracellular pH dynamics regulates intestinal stem cell lineage specification

“…Increased pHi is necessary for the differentiation of clonal mouse embryonic stem cells from naïve to primed states (Ulmschneider et al, 2016), mesenchymal stems cells to cardiomyocytes (Li et al, 2009), and CD4 + T helper 9 cells (Singh et al, 2016). Additionally, in vivo studies confirm that increased pHi is necessary for differentiation of Drosophila follicle stem cells (Benitez et al, 2019; Ulmschneider et al, 2016), melanocytes during zebrafish neural crest development (Raja et al, 2020), and mesoderm progenitors in the chicken embryo (Oginuma et al, 2020). Similar to our findings, an increasing pHi gradient is seen in murine colonic crypts (Amiri et al, 2021; Nikolovska et al, 2022), and reducing pHi is associated with decreased expression of absorptive fate genes (Nikolovska et al, 2022).…”

“…To quantify the pHi of cells in the ISC lineage, we generated organoids stably expressing a genetically encoded pHi biosensor, mCherry-SEpHluorin (Benitez et al, 2019; Grillo-Hill et al, 2014, 2015; Ulmschneider et al, 2016). We acquired ratiometric (SEpHluorin/mCherry) images at days 1 and 3 of organoid growth (Figures 1A and 1B; Figure S1A) and calibrated fluorescent ratios to pHi by perfusing with nigericin-containing buffers of known pH values at the end of each imaging set (Grillo-Hill et al, 2014).…”

“…Intracellular pH (pHi) dynamics is increasingly recognized as a key regulator of diverse cell behaviors, including epithelial to mesenchymal transition (Amith et al, 2016; Raja et al, 2020), transformation and dysplasia (Amith et al, 2016; Grillo-Hill et al, 2015; Liu et al, 2020; White et al, 2017), and differentiation (Benitez et al, 2019; Liu et al, 2020; Oginuma et al, 2020; Raja et al, 2020; Ulmschneider et al, 2016). We previously reported that increased pHi is required for differentiation of clonal mouse embryonic stem cells and Drosophila adult ovary follicle stem cells (Ulmschneider et al 2016; Benitez et al 2019). However, it remains unknown whether pHi dynamics regulates lineage specification of mammalian adult stem cells.…”