FOXO/Fringe is necessary for maintenance of the germline stem cell niche in response to insulin insufficiency

AbstractThe stem cell niche houses and regulates stem cells by providing both physical contact and local factors that regulate stem cell identity. The stem cell niche also plays a role in integrating niche-local and systemic signals, thereby ensuring that the balance of stem cells meets the needs of the organism. However, it is not clear how these signals are merged within the niche. Nutrient-sensing insulin/FOXO signaling has been previously shown to directly control Notch activation in the Drosophila female germline stem cell (GSC) niche, which maintains the niche and GSC identity. Here, we demonstrate that FOXO directly activates transcription of fringe, a gene encoding a glycosyltransferase that modulates Notch glycosylation. Fringe facilitates Notch inactivation in the GSC niche when insulin signaling is low. We also show that the Notch ligand predominantly involved is GSC niche-derived Delta. These results reveal that FOXO-mediated regulation of fringe links the insulin and Notch signaling pathways in the GSC niche in response to nutrition, and emphasize that stem cells are regulated by complex interactions between niche-local and systemic signals

Generation of Inducible Gene-Switched GAL4 Expressed in the Drosophila Female Germline Stem Cell Niche

The stem cell niche, a regulatory microenvironment, houses and regulates stem cells for maintenance of tissues throughout an organism’s lifespan. While it is known that stem cell function declines with age, the role of niche cells in this decline is not completely understood. Drosophila exhibits a short lifespan with well-characterized ovarian germline stem cells (GSCs) and niche compartments, providing a good model with which to study stem cell biology. However, no inducible tools for temporal and spatial control of gene expression in the GSC-niche unit have been previously developed for aging studies. The current UAS-GAL4 systems are not ideal for aging studies because fly physiological aging may be affected by the temperature shifts used to manipulate GAL4 activity. Additionally, the actual needs of the aged niche may be masked by continuously driven gene expression. Since GeneSwitch GAL4 is conveniently activated by the steroid RU486 (mifepristone), we conducted an enhancer-trap screen to isolate GeneSwitch GAL4 lines with expression in the GSC-niche unit. We identified six lines with expression in germarial somatic cells, and two lines (#2305 and #2261) with expression in niche cap cells, the major constituent of the GSC niche. The use of lines #2305 or #2261 to overexpress Drosophila insulin-like peptide 2, which maintains GSC lifespan, in aged niche cap cells significantly delayed age-dependent GSC loss. These results support the notion that insulin signaling is beneficial for maintaining aged stem cells and also validate the utility of our GeneSwitch GAL4 lines for studying stem cell aging

Decreased expression of <i>lethal giant larvae</i> causes ovarian follicle cell outgrowth in the <i>Drosophila Scutoid</i> mutant

<div><p>Snail, a zinc-finger transcription factor, controls the process of epithelial-mesenchymal transition, and ectopic expression of this protein may produce cells with stem cell properties. Because the effect of Snail expression in ovarian epithelial cells remains unclear, we generated <i>Drosophila</i> ovarian follicle stem cells (FSCs) with homozygous <i>Scutoid</i> (<i>Sco</i>) mutation. The <i>Sco</i> mutation is a reciprocal transposition that is known to induce ectopic Snail activity. We found that <i>Sco</i> mutant FSCs showed excess proliferation and high competitiveness for niche occupancy, and the descendants of this lineage formed outgrowths that failed to enter the endocycle. Surprisingly, such phenotypes were not rescued by suppressing Snail expression, but were completely restored by supplying Lethal giant larvae (Lgl). The <i>lgl</i> allele is a cell polarity gene that is often mutated in the genome. Importantly, <i>Sco</i> mutants survived in a complementation test with <i>lgl</i>. This result was probably obtained because the <i>Sco</i>-associated <i>lgl</i> allele appears to diminish, but not ablate <i>lgl</i> expression. While our data do not rule out the possibility that the <i>Sco</i> mutation disrupts a regulator of <i>lgl</i> transcription, our results strongly suggest that the phenotypes we found in <i>Sco</i> mutants are more closely associated with the <i>lgl</i> allele than ectopic Snail activity.</p></div

<i>Sco</i> follicle cells lose cell polarity and are delaminated.

<p>(A-F) One-week (w)-old control (ctrl) (A, C and E) and <i>Sco</i> mosaic (B, D and F) stage (S) 6 and 7 egg chambers: GFP (green, wild-type cells) and E-cadherin (gray, E-cad) in A and B; Disc large (gray, Dlg) in C and D; aPKC (E and F); DAPI (blue, DNA). A’ and B’ show E-cad channel only; C’ and D’ show Dlg channel only; E’ and F’ show aPKC only. Dashed lines indicate follicle cell clones. Inserts are enlarged images from the area indicated by asterisks. Scale bar, 20 μm. The genotype of A, C and E is <i>hs-flp</i>/+; <i>ubigfp FRT40A</i>/ <i>FRT40A</i>, of B, D and F is <i>hs-flp/+; ubi-gfpFRT40A/ScoFRT40A</i>.</p

Snail and Noc do not account for the multiple-layered phenotype of <i>Sco</i> follicle cells.

<p>(A-D) One-week-old control (ctrl) mosaic (A), <i>Sco</i> mosaic (B) and <i>Sco</i> mosaic ovarioles with <i>snail</i> knockdown (C and D). (E-J) One-week (W)-old control (ctrl) (E and F), <i>10930>noc</i>^<i>RNAi</i> (G), <i>GR1> noc</i>^<i>RNAi</i> (H), <i>10930>snail & gfp</i> (I), and <i>10930>snail & noc</i>^<i>RNAi</i> ovarioles (J): FasIII (green, follicle cell lineages), Traffic Jam (Tj) (gray, follicle cells) in I and J, and DAPI (blue, DNA). Arrows in I and J indicated overexpression of Snail increase cell number in the stalk that connects two egg chambers. The scale bar is 20 μm. The genotype of A is <i>c587-GAL4/UAS-flp; ubi-gfpFRT40A/FRT40A</i>, of B, C, and D is <i>c587-GAL4/UAS-flp</i>: <i>ScoFRT40A/ubi-gfpFRT40A</i>.</p

Exogenous supplement of <i>lgl</i> prevents <i>Sco</i> follicle cells forming multiple layers.

<p>(A) Mosaic Analysis with Repressible Cell Marker (MARCM) was used to generate <i>Sco</i> homozygous mutant cells expressing <i>lgl-gfp</i>. Females carried wild-type alleles linked to <i>tubulin promoter-GAL80</i> (a GAL4 suppressor) <i>in trans</i> with the <i>Sco</i> mutant allele on the second chromosome, <i>actin promoter-GAl4</i>, <i>UAS-nuclear (n) LacZ</i> and <i>UAS-lgl-gfp</i> on the third chromosome. FLP-mediated recombination between two FRT sites during mitotic division generated <i>Sco</i> homozygous mutant cells lacking GAL80, allowing <i>UAS</i>-trangenes to be expressed by GAL4 driven by an <i>actin</i> promoter. (B-C) Mosaic egg chambers in one-week (W)-old <i>Sco</i> mutant (B), and <i>Sco</i> mutant with <i>lgl</i> overexpression: LacZ (green, mutant cells) and DAPI (blue, DNA). Dashed lines mark follicle cell clones. The scale bar is 20 μm. The genotype of B is <i>hs-flp</i>/+; <i>tub-Gal80FRT40A</i>/ <i>ScoFRT40A</i>; <i>act-GAL4UAS-nlacZ/+</i>, and of C is <i>hs-flp</i>/+; <i>tub-Gal80FRT40A</i>/ <i>ScoFRT40A</i>; <i>act-GAL4UAS-nlacZ/UAS-lgl-gfp</i>.</p

<i>Sco</i> follicle cells are hyperproliferative and do not enter the endocycle.

<p>Control (Ctrl) (A, C, F and H) and <i>Sco</i> mosaic ovarioles (B, D, G and I) at one week (1W) after clone induction (ACI) are labeled with GFP (green, wild-type cells) and FasIII (gray, membranes of follicle cell lineages) in A and B, phospho-Histone 3 (PH3, gray, mitotic marker) and DAPI (blue, DNA) in C, D, F and G, and Cyclin B (CycB, gray, G2/M phase marker) in H and I. Wild-type cells are outlined by yellow dashed lines. The scale bar in A is 10 μm, and scale bars in C, F and H are 20 μm. (A and B) <i>Sco</i> mosaic ovarioles contain stalk cell overgrowths that are completely composed of excessive numbers of irregularly-shaped <i>Sco</i> cells, as compared to the control. (C and D) <i>Sco</i> follicle cells formed multiple layers in stage (S) 4 and 6 egg chambers. (E) Percentage (%) of mosaic ovarioles exhibiting PH3 signal in GFP-negative follicle cell clones. The number of ovarioles analyzed is shown above each bar. (F and G) <i>Sco</i> follicle cells of the stage 8 egg chamber formed multiple layers, and continued to undergo mitosis. F’ and G’ show the outermost layers of egg chambers. Asterisks indicate nurse cells. (H and I) <i>Sco</i> follicle cells of the stage 7 egg chamber retained greater CycB signals than the controls. Wild-type cells are outlined by yellow dashed lines. Asterisks indicate anterior or posterior poles of egg chambers. The genotype of the controls in A, C, E, F and H is <i>hs-flp</i>/+; <i>ubi-gfpFRT40A</i>/ <i>FRT40A</i>, and of the <i>Sco</i> mosaic mutant in B, D, E, G and I is <i>hs-flp/+; ubi-gfpFRT40A/ScoFRT40A</i>.</p

Notch signaling is disturbed in the outer layers of <i>Sco</i> follicle cells.

<p>(A) Notch signaling is required for the transition of follicle cells from the mitotic phase to endocycle phase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188917#pone.0188917.ref010" target="_blank">10</a>]. In mitotic follicle cells (up to stage 6), Notch signaling activity is low because germ cells produce low amounts of Delta (a Notch ligand, shown as yellow triangles). In addition, Cut is expressed in follicle cells to suppress the mitosis-endocytosis transition. After stage 7, Notch signaling is activated to promote Hindsight expression, which suppresses Cut expression and thereby permits the mitosis-endocytosis transition. Red squares indicate Notch receptors. (B-G) One-week (w)-old control (ctrl) (B, D and F) and <i>Sco</i> mutant mosaic egg chambers (C, E and G) at stages (S) 6 and 7: GFP (green, wild-type cells), <i>Esplm7</i>-<i>lacZ</i> (gray, a Notch signaling reporter) in B and C, Cut (gray) in D and E, Hindsight (gray) in F and G. Expression of <i>E(spl)m7-lacZ</i> is decreased in ectopic layers of <i>Sco</i> mutant follicle cells located far from the germline at the anterior and posterior poles. In the control, Cut is mainly expressed in stage 6 follicle cells and is downregulated in stage 7 follicle cells. However, Cut expression is weaker in the inner layer as compared to the outer layer of <i>Sco</i> mutant follicle cells. In contrast, Hindsight expression is stronger in the inner layer as compared to the outer layer of <i>Sco</i> follicle cells at stage 7, indicating a non-cell autonomous effect of <i>Sco</i> on the mitosis-endocycle transition. Arrows show the boundary between germ cells and follicle cells. Asterisks indicate outer layers of ectopic follicle cells. Scale bar, 20 μm. The genotype of B, D and F is <i>hs-flp</i>/+; <i>ubi-gfpFRT40A</i>/<i>FRT40A</i>, of C, E and G is <i>hs-flp/+; ubi-gfpFRT40A/ScoFRT40A</i>.</p

<i>Sco</i> FSCs exhibit extended lifespan, enhanced proliferation, and increased competitiveness for niche occupancy.

<p>(A) Schematic of the <i>Drosophila</i> ovariole. The anterior-most structure of the ovariole, the germarium, contains germ cells that are enveloped by prefollicle cells (light green), to form egg chambers. The prefollicle cells are derived from two FSCs (yellow), which are located at the 2a/2b boundary of the germarium. Follicle cells of egg chambers up to stage (S) 6 (light blue) undergo mitotic cycles, while follicle cells of egg chambers after stage 7 (orange) enter the endocycle. (B) Mitotic recombination was used to generate <i>Sco</i> FSCs. Females were generated, carrying a wild-type allele linked to a marker gene (GFP) <i>in trans</i> with the <i>Sco</i> allele. FLP-mediated recombination between <i>FRT</i> sites during mitotic division generated a homozygous <i>Sco</i> FSC that could be identified by the absence of GFP. (C-F) Control (Ctrl) (C and E) and <i>Sco</i> mosaic germaria (D and F) shown at one week (W) after clone induction (ACI): GFP (green, wild-type cells), FasIII (red, follicle cell lineages), DAPI (blue, DNA), and Edu (white in E and F, indicating proliferating cells). Solid and empty triangles indicate GFP-positive and GFP-negative FSCs, respectively. Scale bar, 10 μm. (G) Relative percentage (%) of germaria carrying GFP-negative follicle cell clones at 1, 2 and 3 weeks ACI. (H) Percentage of Edu-positive FSC clones in total FSC clones at one week ACI. The number of FSC clones analyzed is shown above the bar. (I) Percentage of germaria carrying two GFP-negative FSCs in control (black line) and <i>Sco</i> (red line) at 1, 2 and 3 weeks ACI. The blue squares in G and I indicate significant differences as compared to the initial time point. * <i>P</i><0.05. *** <i>P</i><0.001. Statistical analysis was carried out with student <i>t</i>-test. Data are shown as mean ± SEM. The genotype of C and E is <i>hs-flp</i>/+; <i>ubi-gfpFRT40A</i>/<i>FRT40A</i>, of D and F is <i>hs-flp/+; ubi-gfpFRT40A/ScoFRT40A</i>.</p