Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans

Oxygen deprivation is a major cause of cellular damage and death. Here we demonstrate that Caenorhabditis elegans embryos, which can survive both in anoxia (<0.001 kPa O(2)) by entering into suspended animation and in mild hypoxia (0.25-1 kPa O(2)) through a hypoxia-inducible factor 1-mediated response, cannot survive in intermediate concentrations of oxygen, between 0.01 and 0.1 kPa O(2). Moreover, we show that carbon monoxide can protect C. elegans embryos against hypoxic damage in this sensitive range. Carbon monoxide can also rescue the hypoxia-sensitive mutant hif-1(ia04) from lethality in hypoxia. This work defines the oxygen tensions over which hypoxic damage occurs in C. elegans embryos and demonstrates that carbon monoxide can prevent this damage by inducing suspended animation

Data from: Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche

In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. We have found that the phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period

RNA-Seq of early follicle cells – EGFRact Rep1 Read2

Paired-end RNA-Sequencing data from early follicle cells Genotype 109-30-Gal4, UAS-mCD8::GFP, UAS-EGFR[lambda]top Replicate #1, Read

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

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 anion exchanger 2 (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

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

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 anion exchanger 2 (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

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation.

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms

Quantification of marked FSC clone frequency at 7, 14 and 21 days post heat shock.

<p>Quantification of marked FSC clone frequency at 7, 14 and 21 days post heat shock.</p

Follicle stem cells undergo neutral drift.

<p><b>A</b>. A Drosophila ovariole stained for Hu li tai shao (green) to highlight cell membranes, Traffic jam (red) to highlight somatic cell nuclei and DAPI (blue) to highlight DNA. In each ovariole, new follicles are produced by the germarium (boxed region, diagramed below) at the anterior tip. Follicles move toward the posterior as they mature. The germarium is divided into four sections as indicated, and the FSCs reside at the Region 2a/2b border. Anterior is to the left. <b>B</b>. Mosaic, uniformly marked (GFP^-), and uniformly unmarked (GFP⁺) wildtype ovarioles stained for FasIII (red) to label follicle cells, GFP (green) to mark FSC clones, and DAPI (blue). <b>C</b>. Mosaic ovarioles become uniformly marked when the GFP⁺ FSC is replaced by a daughter of the GFP^- FSC, or uniformly unmarked when the GFP^- replaced by a daughter of the GFP⁺ FSC. These events are referred to as clone expansion (r₊) or clone loss (r_-), respectively. <b>D</b>. An alignment of the observed fraction of ovarioles with 0, 1 or 2 GFP^- FSCs at 7, 14 and 21 dphs with the values predicted by the neutral competition model. The points indicate the actual data, and the error bars indicate the S.E. The solid lines indicate the values predicted by the model and the grey shaded areas indicate the 95% confidence interval ranges.</p