Integrin mutant FSCs are lost over time. Quantitation of stem cell maintenance over time. Data are presented as the percentage of germaria containing marked stem cells for each time point. (top) GSC time course. (middle and bottom) FSC time course. The day 2 ACI time point is the initial rate of clone induction and includes both FSCs and prefollicle cells.

Model for integrin function in FSCs. (1) FSCs (yellow triangles) secrete LanA (green) into the ECM to generate a critical niche component. (2) Engagement of αPS1βPS (mew/mys, white/pink) by LanA and of αPS2βPS (if/mys, white/blue) by an RGD peptide–containing extracellular ligand (ECM, red) anchors the FSC to the basal lamina, maintaining its position within the niche. (3) LanA binding to αPS1βPS (mew/mys, white/pink) receptors also controls FSC proliferation rates. Together, these three mechanisms contribute to FSC maintenance regulation.

mys(βPS) mutant FSCs produce defective progeny. (A) Germaria 4 d ACI bearing WT or mys mutant cells (no GFP) labeled with Fas3 antibodies (purple). (B) Germaria 10 d ACI bearing WT or mys mutant cells labeled as in A. White arrowheads indicate WT cells at the germarial surface. Fas3 localizes to lateral membranes but is excluded from apical (a) or basal (b) membranes. Round mys mutant cells (green arrows) pile up, and Fas3 localizes to all nonapical membranes. Invasive mys mutant cells separate three germ cells from the cyst (outlined in blue). (C) Germaria 10 d ACI bearing WT or mys mutant cells (no GFP) are labeled with anti-Bazooka antibodies (purple). Apical domains form normally (green arrows). Bars, 10 μm.

mew and lanA mutant FSCs exhibit normal positioning, shape, and actin organization. (A–C) Labeled mew^P13(αPS1) (A), if^B2(αPS2) (B), or lanA^6-36 (C) mutant FSCs (no GFP; outlined in white). In B, GFP⁻ germline clones also are visible. (D–G) GFP⁻ cells labeled for actin (D′–G′) or both GFP (green) and actin (purple; D″–G″). (D and F) Single confocal section at the center of the germarium. FSCs are magnified at the right. Basal actin (red arrowheads) and internal cortical membranes (green arrowheads) are indicated. (D) mew^P13 mutant cells (no GFP). (F) lanA^6-36 mutant cells (no GFP). (E and G) Basal surface of the germaria in D and F, respectively. FSCs are magnified at the right. Yellow arrows indicated GFP⁻ clones. Bars, 10 μm.

Integrin mutant FSCs are lost over time. (A) 3-wk time course of FSC maintenance. Examples of WT (red asterisk) and mys mutant (green asterisk) FSCs that lack GFP (white arrowheads; outlined in white) and a GFP-expressing FSC (yellow arrowheads) are indicated. GFP⁻ progeny (outlined in white) are further to the posterior (red bracket). Days 2, 6, 10, 14, and 21 ACI are shown for germaria bearing clones of the following genotypes: WT (GFP FRT^19A), mys^M2(βPS), mew^M6 if^k27e(αPS1αPS2), mew^P13(αPS1), and if^B2(αPS2). Nuclei are stained with propidium iodide (blue). Prefollicle cells are stained with anti-Fas3 antibody (red). (B) WT and temperature-sensitive mys^nj42(βPS) mutants stained with anti-Fas3 antibody (purple) and anti-Vasa antibody (green) are shown. A single confocal section from the exact center of each germarium is shown. Bars, 10 μm.

Aberrant positioning and morphology of mys^XG43 mutant FSCs. (A and B) Wild type (WT; A) or mys^XG43(βPS) mutant (B) FSCs (no GFP; outlined in white). (C) mys^XG43 mutant FSCs lack GFP (white arrows). The P element PZ1444 leads to expression of β-gal in escort cells (yellow arrows; red). (D and E) WT (D) or mys^XG43 mutant (E) FSCs (no GFP; white boxes) stained with anti–E-cadherin antibodies (purple). Insets are magnified views of boxed areas. (F–I) WT or mys^XG43 mutant cells (no GFP) labeled for actin (F′–I′) or both GFP and actin (purple; F″–I″). (F and H) Single confocal section at the center of the germarium. Boxed FSCs are magnified at the right. (G and I) Basal surface of the germaria in F and H. FSCs are magnified at the right. (F and F″) WT cells (no GFP) at the germarial center. Basal actin (red arrowheads) and internal cortical membranes (green arrowheads) are indicated. (G–G″) Basal surface of the germarium in F. (H–H″) mys^XG43 mutant cells (no GFP) at the germarial center. Actin on all cortical membranes (green arrowheads) is equal. (I–I″) Basal surface of the germarium in H. Areas of nonmutant cells are magnified at the right. Bars, 10 μm.

FSCs lacking lanA are lost from the niche. (A) High levels of LanA (purple) localize to the basal lamina starting near the region 2A/2B border (arrows). Nuclei are labeled with GFP. (B) LanA (red) and βPS(mys) (blue) localization is highest beginning near FSCs (no GFP; arrowheads). (C and D) LanA (purple) localizes normally in mys(βPS) cells (C; no GFP) or lanA^6-36 cells (D; no GFP). (E) Quantitation of lanA^6-36, lanA^9-32, and lanA¹²⁶⁴¹ mutant FSC loss over time. The percentage of germaria containing marked FSCs for each time point is shown. The day 2 ACI time point is the initial rate of clone induction and includes both FSCs and prefollicle cells. (F) 3-wk time course of FSC maintenance. Days 2, 6, 10, 14, and 21 ACI are shown for germaria bearing GFP FRT^2A clones or lanA^6-36 mutant clones (no GFP; outlined in white). Nuclei are stained with propidium iodide (blue), and prefollicle cells are stained with anti-Fas3 antibody (red). A single confocal section from the exact center of each germarium is shown. Bars, 10 μm.

Integrins localize to the basal surface of the germarium. (A) Oogenesis schematic. Germline stem cells (GSCs; green) and escort stem cells (ESCs; light pink) reside in a niche consisting of terminal filament (black) and cap cells (purple) in germarium region 1. GSCs generate nonstem cell daughters that produce 16-cell cysts (teal) containing one oocyte (blue). ESCs generate escort cells (dark pink) that accompany cysts through region 1. Follicle stem cells (FSCs; yellow) associate with escort cells in region 2A. FSC daughters, prefollicle cells (pFC; red) that contact germ cells, form an epithelium around the cyst. Prefollicle cells that fail to contact germ cells form stalk cells (SS). Germ cell–follicle cell units bud from the germarium as egg chambers that subsequently develop through 14 stages to form mature eggs. (B) Germaria stained with anti-integrin antibodies (top) or both anti-Vasa, a germ cell marker (bottom, green), and anti-integrin (purple). Three integrin subunits, βPS, αPS1, and αPS2, colocalize to the germarial basal surface with high levels starting at the region 2A/2B border (arrowheads). (C) Prefollicle cell clones (no GFP, green; outlined in white) in germaria 6 d ACI stained with anti-integrin antibodies (purple) as indicated. Localization of βPS, αPS1, and αPS2 was analyzed in prefollicle cell cells bearing mys^XG43, mew^P13, and if^B2 or mew^M6 if^k27e mutations, respectively. Bar, 10 μm.

Reduced proliferation rates in integrin mutant FSCs. (A–C) Box and whisker plots represent the distribution of marked control or integrin cells in individual germaria containing a GFP⁻ FSC. The top of the box is the 75th percentile, and the bottom of the box is the 25th percentile. Whiskers extend above the box to the maximum cell number and below the box to the minimum cell number. Red lines represent the mean, and blue diamonds represent the median. At least 10 germaria were scored per genotype. Statistically significant differences calculated using a t test in the numbers of WT versus mutant cell numbers are indicated with asterisks (*, P < 0.002). The appropriate GFP FRT clones are WT controls for integrin mutants on the same FRT chromosome. (D and E) Rates of FSC proliferation. Phosphohistone H3 (PH3)–positive WT or mutant FSCs (no GFP) were divided by the total number of germaria. Error = twice the SEM (± 2[SEM]). Fold reduction = WT proliferation divided by the mutant rate. N = total number of germaria scored/genotype. (E) FSC proliferation rates calculated by BrdU labeling. The percentage of BrdU WT FSCs from mosaic germaria containing mutant FSCs is the control and is based on the presence of two FSCs/germarium (Nystul and Spradling, 2007). (F) Rates of prefollicle cell proliferation. The rate equals the number of GFP⁻ phosphohistone H3⁺ cells in regions 2B and 3 of germaria containing a marked FSC divided by the total number of germaria scored. Error = ±2(SEM). Arrows indicate dividing prefollicle cells. (G) Mosaic germaria containing phosphohistone H3⁺ (red) WT (GFP positive) or mys(βPS) mutant (GFP⁻) FSCs. FSCs (arrowheads) express low levels of Fas3 (blue). Bars, 10 μm.