bioimagesUnknownTiffiney R. HartmanThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201212094JCB

boie mutant flies on normal food lacked apoptosis (nuclei (FC-NA, green), germ cells (vasa, blue), and apoptosis (ApopTag, red)).

Expression of BoiS983A in boie mutant flies (boie;UAS-Boi983A/babGal4) rescues Hh sequestration in apical cells.Hh (green) and Fas3 (blue) are shown.

Nutrient-restricted wild-type flies were re-fed yeast for 1h and endogenous Hh localization was<br />analyzed using a Hh antibody. Hh (red) and follicle cells (Fas3, blue) are shown.

Active HhN-GFP is not modified by cholesterol, but is retained in the apical cells in nutrient-restricted flies and released in re-fed flies. Nutrient-restricted flies were imaged for Hh-N-GFP (green), vasa (red), and fas3 and lamC (blue).

Germaria from nutrient restricted WT females were immunostained for follicle cells (1B1, green), germ cells (Vasa, blue) and apoptosis (ApopTag, red).

Wild-type flies expressing Hh-GFP in the apical cells (Hh-GFP/babGal4) were fed normal food for 3 days. Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh localization. Hh-GFP (green) and germ cells labeled with Vasa (red) are also shown.

S6KRNAi flies expressing HhGFP in apical cells (S6KRNAi;HhGFP/babGal4 ) in nutrient-restricted flies. Hh-GFP (green), follicle cells (Fas3, blue), apical cells (LamC, blue) and germ cells (Vasa, red).

Nutrient-restricted WT flies expressing Hh-GFP in apical cells (Hh-GFP/babGal4) were re-fed for 6 hours with yeast and stained for Hh-GFP (green). Follicle cells and apical cells are both labeled in blue (Fas3 and LamC, respectively), germ cells are red (Vasa).

Boi (green) localization in apical cells in nutrient-restricted flies re-fed yeast for 1h.

Boi (green) localization in apical cells in nutrient-restricted flies re-fed yeast for 3h.

Nutrient-restricted WT flies were analyzed for endogenous Hh localization using a Hh antibody. Hh (green), germ cells (Vasa, red), and epithelial cells (Fas3, blue) are shown.

Germaria from nutrient-restricted boie mutant females were immunostained for follicle cells (1B1, green), germ cells (Vasa, blue) and apoptosis (ApopTag, red).

boie mutant flies expressing HhGFP and WT Boi (boie;HhGFP/+;UAS-Boi/babGal4 ) accumulate Hh-GFP in FSCs after re-feeding. Hh-GFP (green), follicle cells (Fas3, blue), apical cells (LamC, blue) and germ cells (Vasa, red).

Nutrient-restricted wild-type flies were re-fed yeast extract for 6 hours and endogenous Hh localization was analyzed using a Hh antibody. Hh (green), germ cells (Vasa, red), and epithelial cells (Fas3, blue) are shown.

Re-feeding nutrient-restricted flies stimulates Hh release and FSC proliferation. A) Wild-type flies expressing Hh-GFP in the apical cells (Hh-GFP/babGal4) were nutrient-restricted for 3 days. Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh localization. Hh-GFP (green) and germ cells labeled with Vasa (red) are also shown.

S6KRNAi flies expressing HhGFP in apical cells (S6KRNAi;HhGFP/babGal4 ) fail to accumulate Hh-GFP in FSCs after re-feeding. Hh-GFP (green), follicle cells (Fas3, blue), apical cells (LamC, blue) and germ cells (Vasa, red).

Active HhN-GFP is not modified by cholesterol. Flies were fed normal food for 3 days and imaged for Hh-N-GFP (green), vasa (red), and fas3 and lamC (blue).

Active HhN-GFP is not modified by cholesterol. Nutrient-restricted flies were re-fed yeast for 6h and imaged for Hh-N-GFP (green), vasa (red), and fas3 and lamC (blue).

Active HhN-GFP is not modified by cholesterol. Nutrient-restricted flies were re-fed yeast for 3h and imaged for Hh-N-GFP (green), vasa (red), and fas3 and lamC (blue).

Re-feeding nutrient-restricted flies stimulates Hh release and FSC proliferation. B) Nutrient-restricted wild-type flies expressing Hh-GFP in the apical cells (Hh-GFP/babGal4) were re-fed yeast for 6h. Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh localization. Hh-GFP (green) and germ cells labeled with Vasa (red) are also shown.

Boi (green) localization in apical cells in flies fed normal food for 3 days.

A-D) Flies bearing Gal4 transgenes were crossed to flies bearing a UAS-GFP-nls reporter transgene. GFP indicates the expression pattern of the promoter driving Gal4 expression. A,B) bab-Gal4 is expressed predominantly in apical cells in both normal food and nutrient-restricted conditions.

Active HhN-GFP is not modified by cholesterol. Nutrient-restricted flies were re-fed yeast for 1h and imaged for Hh-N-GFP (green), vasa (red), and fas3 and lamC (blue).

Loss of Boi stimulates FSC proliferation in nutrient-restricted flies. boie mutant flies expressing HhGFP in apical cells (boie;HhGFP/babGal4tubGal80ts) were nutrient-restricted and stained for Hh-GFP (green). Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh-GFP localization, and germ cells are red (Vasa).

WT flies on normal food lacked apoptosis (nuclei, (FC-NA, green), germ cells (vasa, blue), and apoptosis (ApopTag, red)).

Nutrient-restricted wild-type flies were re-fed yeast for 24h and endogenous Hh localization was<br />analyzed using a Hh antibody. Hh (red) and follicle cells (Fas3, blue) are shown.

In the presence of human S6K, Boi S983 is phosphorylated (lane 3, lower band). Mutation of S983 to A abrogates phosphorylation (lane 4), indicating that S983 is the primary site of phosphorylation. Autophosphorylation of S6K also is observed (lanes 2-4, upper band). No signal is observed in the absence of S6K (lane 1) or when GST alone is used as a substrate (lane 2).

Dividing follicle stem cells are identified by PH3 positive staining (red) of the triangular shaped nucleus in a FC-NA positive cell (green) and low Fas3 (blue).

Flies bearing the 109-53-Gal4 transgene was crossed to flies bearing a UAS-tau-GFP reporter transgene. 109-53-Gal4 is expressed predominantly in apical cells. Arrowheads indicate FSCs. Brackets indicate apical cells.

Follicle stem cells can be identified by their position before the first flattened egg chamber (germ cells (Vasa), blue) in a MARCM clone (green) stained for lamC (red)

Follicle stem cells can be identified by triangular nuclear staining of FC-NA (red), low Fas3 (blue) and FSC position as the first marked cell in a MARCM clone (green)

Flies expressing BoiS983 (boie;HhGFP/+;UAS-Boi983A/babGal4) do not accumulate HhGFP in FSCs after re-feeding. Hh-GFP (green), follicle cells (Fas3, blue), apical cells (LamC, blue) and germ cells (Vasa, red).

Loss of Boi stimulates FSC proliferation in nutrient-restricted flies. boie mutant flies expressing HhGFP in apical cells (boie;HhGFP/babGal4tubGal80ts) were nutrient-restricted and re-fed yeast and stained for Hh-GFP (green). Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh-GFP localization, and germ cells are red (Vasa).

Hh (green) was released from re-fed fllies with reduced insulin receptor in the apical cells (InRRNAiJF01482/babGal4). Apical cells are labeled in blue (LamC) and germ cells are red (Vasa). Brackets indicate apical cells.

Boi (green) localization in apical cells in nutrient-restricted flies re-fed yeast for 6h.

Re-feeding nutrient-restricted flies stimulates Hh release and FSC proliferation. B) Nutrient-restricted wild-type flies expressing Hh-GFP in the apical cells (Hh-GFP/babGal4) were re-fed yeast for 1h. Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh localization. Hh-GFP (green) and germ cells labeled with Vasa (red) are also shown.

FSCs can be identified by triangular nuclear FC-NA staining (red), low Fas3 (blue) and position before the first flat egg chamber (Vasa, green).

Nutrient-restricted wild-type flies were re-fed yeast for 6h and endogenous Hh localization was<br />analyzed using a Hh antibody. Hh (red) and follicle cells (Fas3, blue) are shown.

Nutrient-restricted WT flies expressing Hh-GFP in apical cells (Hh-GFP/babGal4) were re-fed for 6 hours with yyeast extract (y.e.) and stained for Hh-GFP (green). Follicle cells and apical cells are both labeled in blue (Fas3 and LamC, respectively), germ cells are red (Vasa).

Boi localization (green) in apical cells in flies nutrient-restricted for 3 days.

Expression of BoiS983A in Boi mutant flies (boie;UAS-Boi983A/babGal4) that were fed normal food rescues Hh sequestration in apical cells. Boi (green), Vasa (blue), and LamC (red) are shown.

Boi (green) localization in apical cells in nutrient-restricted flies re-fed yeast for 24h.

Coomassie stained gel showing levels of GST (lane 2) or GST-Boi (lanes 1, 3, 4) used in the assay.

C,D) 109-30-Gal4 is expressed in FSCs and their daughter cells in the germarium through stage 3 in both normal food and nutrient-restricted conditions.

Nutrient-restricted wild-type flies were re-fed yeast extract + 0.2 mg/g cholesterol for 6 hours and endogenous Hh localization was analyzed using a Hh antibody. Hh (green), germ cells (Vasa, red), and epithelial cells (blue) are shown.

Nutrient-restricted WT flies expressing Hh-GFP in apical cells (Hh-GFP/babGal4) were re-fed with ethanol vehicle control and stained for Hh-GFP (green). Follicle cells and apical cells are both labeled in blue (Fas3 and LamC, respectively), germ cells are red (Vasa).

Active HhN-GFP is not modified by cholesterol. Nutrient-restricted flies were re-fed yeast for 24h and imaged for Hh-N-GFP (green), vasa (red), and fas3 and lamC (blue).

Hh (green) was observed in over 60% of apical cells of nutrient-restricted InRRNAiJF01482/babGal4 flies. Apical cells are labeled in blue (LamC) and germ cells are red (Vasa). Brackets indicate apical cells.

Wild-type flies were nutrient-restricted and endogenous Hh localization was analyzed using a Hh antibody. Hh (red) and follicle cells (Fas3, blue) are shown.

Nutrient-restricted WT flies expressing Hh-GFP in apical cells (Hh-GFP/babGal4) were re-fed for 6 hours with yeast extract (y.e.) + 0.2 mg/g cholesterol and stained for Hh-GFP (green). Follicle cells and apical cells are both labeled in blue (Fas3 and LamC, respectively), germ cells are red (Vasa).

Re-feeding nutrient-restricted flies stimulates Hh release and FSC proliferation. B) Nutrient-restricted wild-type flies expressing Hh-GFP in the apical cells (Hh-GFP/babGal4) were re-fed yeast for 3h. Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh localization. Hh-GFP (green) and germ cells labeled with Vasa (red) are also shown.

Re-feeding nutrient-restricted flies stimulates Hh release and FSC proliferation. B) Nutrient-restricted wild-type flies expressing Hh-GFP in the apical cells (Hh-GFP/babGal4) were re-fed yeast for 24h. Follicle cells (Fas3) and apical cells (LamC) are both labeled in blue to enable mapping of Hh localization. Hh-GFP (green) and germ cells labeled with Vasa (red) are also shown.

bioimagesFigure 8 - A bottom panelImage 139536 (Figure 8 - C bottom panel)Image 139508 (Figure 2 - E)Image 147069 (Supplemental Figure 3 - E)Image 139512 (Figure 2 - I)Supplemental Figure 2 - EImage 139530 (Figure 6 - C)Supplemental Figure 2 - ASupplemental Figure 2 - BSupplemental Figure 2 - CSupplemental Figure 2 - DImage 139535 (Figure 8 - A bottom panel)Image 147072 (Supplemental Figure 3 - H)Image 147068 (Supplemental Figure 2 - E)Image 139524 (Figure 4 - C)Image 139534 (Figure 8 - A bottom panel)Supplemental Figure 3 - A-DImage 139529 (Figure 6 - B )Image 139533 (Figure 7 - E)Image 139517 (Figure 3 - B )Image 139506 (Figure 2 - C)Figure 3 - BImage 139532 (Figure 7 - D)Image 139523 (Figure 4 - C)Image 139541 (Supplemental Figure 3 - A-D)Supplemental Figure 5 - F2Image 139542 (Supplemental Figure 3 - A-D)Supplemental Figure 5 - F1Figure 5 - AImage 139516 (Figure 3 - B )Supplemental Figure 3 - FSupplemental Figure 3 - GSupplemental Figure 3 - EImage 139514 (Figure 2 - K)Image 147077 (Supplemental Figure 5 - F2)Supplemental Figure 3 - HSupplemental Figure 3 - IImage 139504 (Figure 2 - A)Figure 5Figure 4Figure 7Figure 6Image 139537 (Figure 8 - C bottom panel)Image 139521 (Figure 4 - C)Figure 8Figure 3Figure 2Figure 8 - C bottom panelSupplemental Figure 4Image 139522 (Figure 4 - C)Supplemental Figure 5Supplemental Figure 2Supplemental Figure 3Image 147076 (Supplemental Figure 5 - F1)Figure 7 - DFigure 7 - EImage 139544 (Supplemental Figure 4 - A)Image 139509 (Figure 2 - F)Image 147067 (Supplemental Figure 2 - D)Image 139519 (Figure 3 - B )Image 139513 (Figure 2 - J)Image 139507 (Figure 2 - D)Image 139531 (Figure 6 - C)Image 139526 (Figure 5 - A)Figure 2 - GFigure 2 - HImage 139527 (Figure 5 - A)Figure 2 - EFigure 2 - FFigure 2 - KFigure 2 - LFigure 2 - IFigure 2 - JImage 139525 (Figure 4 - C)Image 139543 (Supplemental Figure 3 - A-D)Figure 2 - CImage 139511 (Figure 2 - H)Figure 2 - DFigure 2 - AImage 139540 (Supplemental Figure 3 - A-D)Figure 2 - BImage 139510 (Figure 2 - G)Image 147066 (Supplemental Figure 2 - C)Image 147073 (Supplemental Figure 3 - I)Image 139528 (Figure 6 - B )Image 139505 (Figure 2 - B)Figure 4 - CImage 139545 (Supplemental Figure 4 - B)Image 147064 (Supplemental Figure 2 - A)Image 139520 (Figure 4 - C)Image 139518 (Figure 3 - B )Image 147071 (Supplemental Figure 3 - G)Image 139546 (Supplemental Figure 4 - C)Supplemental Figure 4 - ASupplemental Figure 4 - BSupplemental Figure 4 - CSupplemental Figure 4 - DFigure 6 - CImage 147065 (Supplemental Figure 2 - B)Figure 6 - BImage 139515 (Figure 2 - L)Image 147070 (Supplemental Figure 3 - F)Image 139547 (Supplemental Figure 4 - D)Daniel ZinshteynAlana M. O’ReillyYingbiao JiTiffiney R. HartmanTodd I. StrochlicfalseDiet controlsDrosophilafollicle stem cell proliferation via Hedgehog sequestration and release2013-05-20T11:24:30Z2018-11-29T11:24:30Z2018-11-29T11:24:30ZS-JCBD-20121209410.1083/jcb.201212094