Figure 1.
Further characterization of the Lsd1 mutant phenotype.
(A) Schematic of a Drosophila germarium. Two germline stem cells (GSCs) reside next to terminal filament and cap cells labeled in blue. GSCs carry a round endoplasmic reticulum-like organelle called a fusome or spectrosome. These fusomes become branched in appearance as GSC daughters form multicellular cysts. Lining the anterior end of the germarium next to the cap cells are the escort cells labeled in green. Germaria from (B) c587-gal4>mCD8::GFP and (C,D) c587-gal4>mCD8::GFP+Lsd1RNAi three day old females stained for Hts (red), GFP (green) and DNA (blue). RNAi knockdown of Lsd1 results in the accumulation single cells with round fusomes and the gradual retraction of escort cell extensions. (E) c587-gal4>mCD8::GFP and (F) c587-gal4>mCD8::GFP+Lsd1RNAi germaria from females shifted to 29°C for 3 days stained for activated Caspase 3 (Casp3), GFP (green) and DNA (blue). (G–L) MARCM Lsd1ΔN clones stained for Hts (red), GFP (green) and DNA (blue). Lsd1 mutant escort cell clones are associated with undifferentiated germ cells with round fusomes. Scale bars = 10 µM.
Figure 2.
Defining the targets of Lsd1 in the cap cell niche and the surrounding escort cells within Drosophila germaria.
(A,B) Germaria stained for HA (green), to label the transgene, Hts (red), to label fusomes, and DNA (blue) (Scale bars, 10 µM). (A) hh-gal4>UASt-HA::Lsd1 germarium showing expression in cap cells and (B) c587-gal4>UASt-HA::Lsd1 germarium displaying expression in the escort cells and early follicle cells, but not in the cap cells. (C) Venn diagram showing the number of Lsd1 binding sites in escort cells and cap cells. (D) Lsd1 binding peaks on a segment of Chr2R. Black arrow points to a common peak in both the escort cell and cap cell populations. Orange arrows point to unique peaks seen only in the escort cell and early follicle cell population. (E) No Lsd1 binding sites are observed around the dpp locus in escort cells.
Figure 3.
Lsd1 silences engrailed in the escort cells.
(A) A Lsd1 binding peak is observed in the engrailed promoter in escort cells. (B) RT-qPCR of whole ovary extracts from bamΔ86 and bamΔ86 Lsd1ΔN double mutants. The bamΔ86 Lsd1ΔN double mutant samples exhibit a relative 6-fold increase of engrailed transcripts when compared to bamΔ86 mutants. (C and C′) w1118 and (D–E′) Lsd1ΔN homozygous germaria stained for Engrailed (En) (green) and VASA (red) and DNA (blue). Arrows point to Engrailed positive cells. In w1118 control germaria, Engrailed is expressed in the terminal filament and cap cells, whereas Lsd1 mutants display engrailed expression in the anterior escort cells. Scale bars = 10 µM.
Figure 4.
engrailed mutations suppress the Lsd1 RNAi phenotype.
Ovarioles from (A) c587-gal4>UAS-Lsd1RNAi (B) c587-gal4>UAS-Lsd1RNAi; UAS enRNAi-1/+ (C) c587-gal4>UAS-Lsd1RNAi; en7/+ and (D) c587-gal4>UAS-Lsd1RNAi; en4/+ stained for Hts (green), VASA (red) and DNA (blue). (E) Graph showing the percentage of germaria that contain the indicated number of single germ cells with round fusomes for each genotype. (F) Graph showing the percentage of ovarioles that have a given number of developing egg chambers for each genotype. c587-gal4>UAS-Lsd1RNAi (n = 171); c587-gal4>UAS-Lsd1RNAi; en7/+ (n = 90); c587-gal4>UAS-Lsd1RNAi; enspt/+ (n = 95); c587-gal4>UAS-Lsd1RNAi; en4/+ (n = 115); c587-gal4>UAS-Lsd1RNAi; enRNAi-1/+ (n = 108); c587-gal4>UAS-Lsd1RNAi; enRNAi-2/+ (n = 96). (G) c587-gal4>UAS-Lsd1RNAi; UAS hhRNAi/+ (H) c587-gal4>UAS-Lsd1RNAi; hhAC/+ and (I) c587-gal4>UAS-Lsd1RNAi; hh2/+ stained for Hts (green), VASA (red) and DNA (blue). (J) Graph showing the percentage of germaria that contain the indicated number of single germ cells with round fusomes for each genotype. c587-gal4>UAS-Lsd1RNAi (n = 307); c587-gal4>UAS-Lsd1RNAi; UAS hhRNAi/+ (n = 222) c587-gal4>UAS-Lsd1RNAi; hhAC/+ (n = 88) and c587-gal4>UAS-Lsd1RNAi; hh2/+ (n = 184). Scale bars = 10 µM.
Figure 5.
Ectopic expression of engrailed in the escort cells results in an expansion of stem cell-like cells in the germline.
(A) c587-gal4>UAS-en::GFP germaria stained for Hts (green), VASA (red) and DNA (blue). (B) Control and (C) c587-gal4>UAS-en::GFP germaria stained for Dad-LacZ (Dad) (green), VASA (red) and DNA (blue). In control germaria, Dad-LacZ is expressed at high levels in the germline stem cells and its expression is reduced in the early differentiating cells. Germaria overexpressing engrailed display an expansion of Dad-LacZ positive cells. (D) Germaria from c587-gal4>UAS-en::GFP, UAS-dppRNAi-1 and (E) c587-gal4>UAS-en::GFP, UAS-dppRNAi-3 ovaries stained for Hts (green), VASA (red) and DNA (blue). Reducing the levels of dpp in the escort cells suppressed the engrailed overexpression phenotype. (F) Graph showing the percentage of germaria that contained a given number of cells with round fusomes for each genotype. (G) c587-gal4>UAS-hh(III) and (H) c587-gal4>UAS-hh(II) stained for Hts (green), VASA (red) and DNA (blue). Scale bars = 10 µM.
Figure 6.
engrailed mis-expression in adult escort cells results in delayed germ cell differentiation.
c587-gal4>UAS-en::GFP females were kept at (A) 18°C during both development and in adulthood or (B) moved to 29°C after eclosion. (A,B) The germaria were stained for alpha Spectrin (Spec) (green), VASA (red) and DNA (blue) and (A′,B′) Engrailed (En) (grayscale). (C) Graph showing the percentage of germaria with the indicated number of single germ cells with round fusomes when subjected to different temperatures. c587-gal4>UAS-en::GFP 18°C>18°C (n = 100), c587-gal4>UAS-en::GFP 18°C>29°C (n = 80). Scale bars = 10 µM.
Figure 7.
raw represents another potential functional Lsd1 target gene.
(A–C) c587-gal4 and (D–F) c587-gal4>UAS-Lsd1RNAi germaria stained for Rho1 (A,D), APC1 (B,E), and Broad (Br) (C,F). Alpha-Spectrin (Spec) or Hts (green), Vasa (red) and DNA (blue) staining of the same germaria are shown in color. (G) Screen shot of raw locus showing a peak of Lsd1 binding in the c587-gal4>UASt-HA-Lsd1 sample relative to the input DNA. (H) c587-gal4>UAS-Lsd1RNAi, (I) c587-gal4>UAS-Lsd1RNAi; UAS raw135/+ (J) c587-gal4>UAS-Lsd1RNAi; raw155/+ and (K) c587-gal4>UAS-Lsd1RNAi; rawRNAi/+ stained for Hts (green), VASA (red) and DNA (blue). (L) Graph showing the percentage of germaria that contain the indicated number of single germ cells with round fusomes for each genotype. c587-gal4>Lsd1RNAi (n = 351); c587-gal4>UAS-Lsd1RNAi; UAS rawRNAi/+ (n = 155); c587-gal4>UAS-Lsd1RNAi; raw135.47/+ (n = 224); and c587-gal4>UAS-Lsd1RNAi; raw155.27/+ (n = 229). Scale bars = 10 µM.
Figure 8.
Further genetic analysis shows that reduced levels of several genes suppress the Lsd1 mutant phenotype.
(A) The graph shows the percentage of germaria that contain the indicated number of single germ cells with round fusomes for each genotype. Control 1 represents c587-gal4>UAS-Lsd1RNAi; +/+ and the rest represent c587-gal4>UAS-Lsd1RNAi; gene specific RNAi/+. The resulting females were shifted to 29°C for seven days. The number (n) of germaria counted for each sample is indicated above the bar. The yellow dotted line marks the 50% threshold. All the indicated lines were crossed on their own to the c587-gal4 driver. None of the resulting ovaries displayed phenotypes except for CG13192HMS01384 (egg chamber defects) and par-1HMS00405 (follicle cell defects). (B) Model of Lsd1 function in escort cells.