C XBP - 1 mRNA expression in untransfected HFL1 cells ( UT ) and in cells transfected with control siRNA ( siCtrl ) or siRNA targeting XBP - 1 ( siXBP - 1 ) . * P = 0 , 0001 .

C XBP-1 mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with control siRNA (siCtrl) or siRNA targeting XBP-1 (siXBP-1). * P = 0,0001.


D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C . * P = 0 , 005 .

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D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C. * P = 0,005.


A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL / 6mice as measured by the histological metavir score . Ctrl ( n = 8 ) ; Ctrl + 4μ8C ( n = 6 ) ; CCl4 ( n = 6 ) ; CCl4 + 4μ8C ( n = 8 ) . * P = 0 , 0077 . B Representative liver tissue sections stained with sirius red for collagen ( top ) and αSMA ( bottom ) . Scale bars = 50 μm . C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A . * P = 0 , 036 .

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL/6mice as measured by the histological metavir score. Ctrl (n = 8) ; Ctrl + 4μ8C (n = 6) ; CCl4 (n = 6); CCl4 + 4μ8C (n = 8). * P = 0,0077.B Representative liver tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars= 50 μm.C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A. * P = 0,036.


D Liver to body weight ratio from the experimental setup in A . * P = 0 , 0031 .

D Liver to body weight ratio from the experimental setup in A. * P = 0,0031.


E BiP mRNA expression in liver tissue from mice presented in A . * P = 0 , 020 .

E BiP mRNA expression in liver tissue from mice presented in A. * P = 0,020.


F Representative gel showing levels of spliced and unspliced XBP - 1 in liver tissue from control and CCl4 - induced cirrhotic mice and the effect of 4μ8C treatment .

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F Representative gel showing levels of spliced and unspliced XBP-1 in liver tissue from control and CCl4-induced cirrhotic mice and the effect of 4μ8C treatment.


G miR150 expression in liver tissue from mice in A . * P = 0 , 032 compared to untreated controls .

G miR150 expression in liver tissue from mice in A. * P = 0,032 compared to untreated controls.


H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C . * P = 0 , 031 , n = 3 .

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H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C. * P = 0,031, n = 3.


I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C . * P = 0 , 008 , n = 3 .

I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C. * P = 0,008, n = 3.


J Microscopic images of primary mouse hepatic stellate cells stained for BiP ( top ) or αSMA ( bottom ) . Scale bars = 20 μm .

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J Microscopic images of primary mouse hepatic stellate cells stained for BiP (top) or αSMA (bottom). Scale bars = 20 μm.


K Western blot showing BiP protein expression in primary mouse hepatic stellate cells .

K Western blot showing BiP protein expression in primary mouse hepatic stellate cells.


L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ± 4μ8C .

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L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ±4μ8C.


A The effect of the IRE1α inhibitor 4μ8C on TGFβ - induced skin fibrosis in C57BL / 6 mice as measured by the percentage of sirius red areas in the hypodermis . Ctrl ( n = 6 ) ; Ctrl + 4μ8C ( n = 5 ) ; TGFβ ( n = 8 ) ; TGFβ + 4μ8C ( n = 8 ) . * P = 0 , 046 . B Representative skin tissue sections stained with sirius red for collagen ( top ) and αSMA ( bottom ) . Scale bars = 50 μm . C Percentage αSMA - positive cells in the hypodermis of mice from the experimental setup in A . * P = 0 , 024 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024.


D Representative image of the area selected for laser capture microdissection ( scale bars = 100 μm ) . E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection . Ctrl ( n = 5 ) , Ctrl + 4μ8C ( n = 5 ) , TGFβ ( n = 5 ) , TGFβ + 4μ8C ( n = 5 ) . * P = 0 , 044 . F miR - 150 expression levels in the same samples as shown in E .

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D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.F miR-150 expression levels in the same samples as shown in E.


G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C . Scale bars = 20 μm .

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G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm.


H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C . * P = 0 , 049 , n = 4 .

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H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4.


I Agarose gel showing levels of spliced and unspliced XBP - 1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C .

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I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.


J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C . * P = 0 , 00007 for TGFβ vs . Ctrl and P = 0 , 00002 for TGFβ vs . TGFβ + 4μ8C . n = 4 .

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J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4.


K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C .

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K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.


L miR - 150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C . * P = 0 , 00031 compared to untreated control , n = 3 .

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L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.


A CTGF mRNA expression levels in skin ( dSSc ) and lung ( LSSc ) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C . * P = 0 , 018 for dSSc ( n = 6 ) and P = 0 , 003 for LSSc ( n = 3 ) .

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A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3).


B Collagen1α2 mRNA expression levels in the same samples as in A . * P = 0 , 0030 for dSSC and P = 0 , 0036 for LSSc .

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B Collagen1α2 mRNA expression levels in the same samples as in A. * P = 0,0030 for dSSC and P = 0,0036 for LSSc.


C Quantification of collagen protein levels in the same samples as in A . * P = 0 , 010 .

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C Quantification of collagen protein levels in the same samples as in A. * P = 0,010.


D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C .

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D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C.


E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis ( CF ) , chronic obstructive pulmonary disease ( COPD ) or asthma . * P = 0 , 00001 for asthma vs . healthy controls , and P = 0 , 00003 for ctrl vs . 4μ8C in asthma patients .

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E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients.


F Quantification of mRNA levels for BiP in same samples as in E . * P = 0 , 0023 between asthma and healthy controls and P = 0 , 0031 between 4μ8C - treated and control treatment in fibroblasts from asthma patients .

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F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.


( A ) RT - qPCR analysis of NimB1 , NimB2 , NimB3 , NimB4 and NimB5 transcripts , normalized to RpL32 , from carcass , macrophages , fat body and gut of wild type ( w1118 ) L3 wandering larvae . Data are represented as mean ± SD from three independent experiments .

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(A) RT-qPCR analysis of NimB1, NimB2, NimB3, NimB4 and NimB5 transcripts, normalized to RpL32, from carcass, macrophages, fat body and gut of wild type (w1118) L3 wandering larvae. Data are represented as mean ± SD from three independent experiments.


( B ) Representative confocal imaging of NimB4 - sGFP macrophages of embryo at stage 16 ; lateral ( left ) and ventral ( right ) views ; Tissues were stained with anti - GFP ( green , corresponding to NimB4 - sGFP ) and anti - SIMU antibodies ( red , corresponding to macrophages ) . The arrows show the presence of NimB4 - sGFP inside the embryonic macrophages . Scale bars = 20

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(B) Representative confocal imaging of NimB4-sGFP macrophages of embryo at stage 16; lateral (left) and ventral (right) views; Tissues were stained with anti-GFP (green, corresponding to NimB4-sGFP) and anti-SIMU antibodies (red, corresponding to macrophages). The arrows show the presence of NimB4-sGFP inside the embryonic macrophages. Scale bars = 20 μm


( A - C ) Representative confocal imaging of CFSE - stained apoptotic bodies ( green ) incubated with secreted NimB4 - RFP ( B , C ) or SPvkg - RFP ( A ) . Apoptotic bodies were pre - incubated in absence ( A , B ) or presence ( C ) of annexin V ( 25 μg / mL ) . Scale bar = 10 μm . ( D ) Quantification of the colocalization of NimB4 - RFP or SPvkg - RFP with the apoptotic bodies in presence or absence of Annexin V , as measured by Pearson ' s correlation coefficient . Values from at least four independent experiments are represented as mean ± SD ( * * P < 0 . 01 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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(A-C) Representative confocal imaging of CFSE-stained apoptotic bodies (green) incubated with secreted NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm. (D) Quantification of the colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and apoptosis - null NimB4 sk2 ; Df ( 3L ) H99 embryonic macrophages at stage 16 of development ; ventral and lateral view ; Tissues were co - stained with Dcp - 1 ( red , corresponding to apoptotic corpses ) and anti - SIMU ( green , corresponding to macrophages ) antibodies . The arrows show the presence of apoptotic cells inside embryonic macrophages . Scale bars = 20

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(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and apoptosis-null NimB4 sk2; Df(3L) H99 embryonic macrophages at stage 16 of development; ventral and lateral view; Tissues were co-stained with Dcp-1 (red, corresponding to apoptotic corpses) and anti-SIMU (green, corresponding to macrophages) antibodies. The arrows show the presence of apoptotic cells inside embryonic macrophages. Scale bars = 20 μm


( C ) Representative projections from confocal stacks of entire third instar wild type ( w1118 ) and NimB4 sk2 larval brains stained with anti - Draper ( green ) and anti - Dcp1 ( red ) . The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain . Scale bars = 100

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(C) Representative projections from confocal stacks of entire third instar wild type (w1118) and NimB4 sk2 larval brains stained with anti-Draper (green) and anti-Dcp1 (red). The arrows show the presence of apoptotic cells in the central area of NimB4 sk2 larval brain. Scale bars = 100 μm


( A ) Representative confocal imaging of wild type ( w1118 ) , NimB4 sk2 and draper macrophages incubated with fluorescently - labeled apoptotic cells ( red , CellTraceTM Red ) on ice for 1h and stained with AlexaFluorTM 488 phalloidin ( green ) . Scale bar : 10 μm . ( B ) Quantification of the number of apoptotic cells ( red ) binding to the macrophages ( green ) . Values from three independent experiments are represented as mean ± SD ( * * * P < 0 . 001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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(A) Representative confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained with AlexaFluorTM 488 phalloidin (green). Scale bar: 10 μm. (B) Quantification of the number of apoptotic cells (red) binding to the macrophages (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( D ) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies . Wild type , NimB4sk2 , NimB4 genomic rescue ( NimB4sk2 , [ NimB4 ] ) , draper and NimC11 , eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ns : not significant ) . ( E ) Ex vivo phagocytosis assay using AlexFluor488 S . aureus Bioparticles . Wild type , NimB4sk2 and NimC11 , eater1 macrophages from L3 wandering were incubated with bioparticles for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

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(D) Ex vivo phagocytosis assay using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11, eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant). (E) Ex vivo phagocytosis assay using AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A ) Representative fluorescence microscopy images of wild type ( w1118 ) , NimB4sk2 , draper , eater1 and croquemort △ third instar larvae macrophages stained with LysoTracker Red ( live imaging ) . Overlay of fluorescence and differential interference contrast microscopy ( DIC ) . Scale bar = 10 μm . ( B ) Mean fluorescence intensity after staining with LysoTracker Red ( live confocal imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) .

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(A) Representative fluorescence microscopy images of wild type (w1118), NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after staining with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).


( C ) Representative transmission electron micrographs of macrophages from wild type ( top , w1118 ) and NimB4sk2 ( bottom ) L3 wandering larvae . Scale bar : 2 μm ( D ) Quantification of the number of vesicles per macrophages in the electron micrographs . Values from at least three independent experiments are represented as mean ± SD ( * * * * P < 0 . 0001 by Mann - Whitney test ) .

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(C) Representative transmission electron micrographs of macrophages from wild type (top, w1118) and NimB4sk2 (bottom) L3 wandering larvae. Scale bar: 2 μm (D) Quantification of the number of vesicles per macrophages in the electron micrographs. Values from at least three independent experiments are represented as mean ± SD (**** P<0.0001 by Mann-Whitney test).


( E ) Representative confocal imaging of Rab7EYFP immunostaining in wild type ( w1118 ) and NimB4 sk2 macrophages . Tissues were stained with anti - GFP ( red , Rab7 ) , counterstained with phalloidin ( gray ) and DAPI ( blue ) . The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages . Scale bar = 10 μm .

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(E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with phalloidin (gray) and DAPI (blue). The arrows indicate the enlarged vesicles are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.


( A ) Representative confocal imaging of localization of Lamp1 - mcherry and LysoTracker Green in wild type ( w1118 ) , NimB4 sk2 and draper △ 5 macrophages ( live imaging ) . The arrows indicate the colocalization ( w1118 ) or the clustering ( NimB4sk2 and draper △ 5 ) of Lamp1 - mcherry and LysoTracker Green signals . Scale bar : 10 μm . ( B ) Quantification of the colocalization of Lamp1 - mcherry with LysoTracker Green , as measured by Pearson ' s correlation coefficient between the two signals . Values from at least five independent experiments are represented as mean ± SD ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests ) .

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(A) Representative confocal imaging of localization of Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests).


( C ) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo ™ Red . Wild type ( w1118 ) , NimB4sk2 and draper △ 5 macrophages from L3 wandering larvae were incubated with pHrodo ™ Red apoptotic cells for 60 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) . ( D ) Ex vivo phagocytosis assay using pHrodoTM Red S . aureus BioparticlesTM conjugates . Wild type , NimB4sk2 and NimC11 , eater1 Hml ∆ - Gal4 > UAS - GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S . aureus BioparticlesTM for 30 , 60 or 120 min at room temperature . Phagocytosis was quantified by flow cytometry . Data are represented as mean ± SD from three independent experiments ( * P < 0 . 05by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Ex vivo phagocytosis assay using apoptotic cells labelled with pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering larvae were incubated with pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


( A - B ) Representative fluorescence microscopy images of wild type ( A ) or NimB4sk2 ( B ) macrophages expressing Rab5 ( middle panel ) or Rab7 ( right panel ) driven by Hml ∆ - Gal4 and stained with the LysoTracker Red ( live imaging ) . Overlay of fluorescence and DIC . Scale bar = 10 μm . ( C ) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae ( confocal live imaging ) . Values from at least three independent experiments are represented as mean ± SD ( * * P < 0 . 01 , * * * * P < 0 . 0001 by ANOVA test followed by post hoc Dunnett ' s multiple comparison tests . ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5 (middle panel) or Rab7 (right panel) driven by Hml∆-Gal4 and stained with the LysoTracker Red (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of LysoTracker in macrophages from larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant).


a , MEFs of RagA + / + , RagAGTP / + and RagAGTP / GTP genotypes were deprived of amino acids for 1 h and re - stimulated for 10 min . Whole - cell protein lysates were immunoblotted for indicated proteins .

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a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min. Whole-cell protein lysates were immunoblotted for indicated proteins.


b , Total RNA was extracted from RagA + / + ( n = 3 ) , RagAGTP / + ( n = 3 ) and RagAGTP / GTP ( n = 2 ) MEFs and RagA mRNA expression determined by quantitative PCR ( mean ± s . e . m . ) .

b, Total RNA was extracted from RagA+/+ (n = 3), RagAGTP/+ (n = 3) and RagAGTP/GTP (n = 2) MEFs and RagA mRNA expression determined by quantitative PCR (mean ± s.e.m.).


c , Weights of RagA + / + ( n = 24 ) , RagAGTP / + ( n = 52 ) and RagAGTP / GTP ( n = 22 ) mice at birth ( data are scatter dots , mean ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, Weights of RagA+/+ (n = 24), RagAGTP/+ (n = 52) and RagAGTP/GTP (n = 22) mice at birth (data are scatter dots, mean).


d , Representative photographs of RagA + / + , RagAGTP / + and RagAGTP / GTP neonates . Scale bar , 1 cm .

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d, Representative photographs of RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates. Scale bar, 1 cm.


e , Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA + / + ( + / + ) , RagAGTP / + ( G / + ) and RagAGTP / GTP ( G / G ) neonates immediately after Caesarean section ( 0 h ) or after 1 h of fasting ( 1 h fast ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA+/+ (+/+), RagAGTP/+ (G/+) and RagAGTP/GTP (G/G) neonates immediately after Caesarean section (0 h) or after 1 h of fasting (1 h fast).


f , Liver and heart extracts from RagA + / + , RagAGTP / + and RagAGTP / GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins .

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f, Liver and heart extracts from RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins.


g , Survival curve of fasted neonates . Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored ( + / + : n = 13 ; G / + : n = 26 ; G / G : n = 10 ) . h , Survival curve of fasted neonates treated with rapamycin . Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored ( + / + : n = 13 ; G / + : n = 13 ; G / G : n = 6 ) . Numbers indicate the median survival for each curve . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P<0.05; **P<0.01; ***P<0.005.


a , Glycaemia drop in RagA + / + , RagAGTP / + and RagAGTP / GTP and recovery in fasted RagA + / + and RagAGTP / + but not in RagAGTP / GTP neonates ( + / + : n = 5 , 18 , 4 , 5 , 9 , 8 ; G / + : n = 10 , 26 , 10 , 13 , 26 , 21 ; G / G : n = 7 , 20 , 9 , 10 , 16 , 11 , for 0 , 1 , 2 , 3 , 6 and 10 h , respectively ; mean ± s . e . m . ) .

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a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.).


b , Rapamycin significantly increases glycaemia in RagAGTP / GTP fasted for 6 and 10 h ( mean ± s . e . m . ) . NS , not significant .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant.


c , Extension of survival by glucose injections in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; glucose : n = 5 ) .

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c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5).


d , Normal expression of genes involved in glucose metabolism in neonatal liver ( + / + : n = 2 , G / + : n = 5 ; G / G : n = 4 ; mean ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.).


e , Left : representative electron microscopy images showing abundant glycogen content in RagA + / + and RagAGTP / GTP hepatocytes before fasting ( 0 h , upper panels ) and more pronounced glycogen depletion after 10 h of fasting ( lower panels ) in RagAGTP / GTP neonates . Right : quantification of hepatic glycogen content ( + / + : n = 5 , 3 , 4 , 4 ; G / + : n = 11 , 7 , 14 , 15 ; G / G : n = 6 , 4 , 4 , 6 ; for 0 , 3 , 6 and 10 h , respectively ; mean ± s . e . m . ; AU , arbitrary units ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units).


. f , Partial rescue of hepatic glycogen content in RagAGTP / GTP neonates fasted for 10 h and treated with rapamycin ( rapa ) ( mean ± s . e . m . ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

. f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.).


g , Quantification of neonatal plasma amounts of branched - chain ( BCAA ) and essential amino acids at birth ( left ) , after 10 h fasting ( middle ) and after 10 h fasting with rapamycin treatment ( right ) ( n for + / + , G / + and G / G , respectively : n = 4 , 5 and 4 for 0 h ; n = 4 , 4 and 3 for 10 h ; n = 2 , 5 and 3 for rapamycin ; mean ± s . d . ) . Values are expressed relative to RagA + / + amounts at each time point .

O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point.


h , Extension of survival by injection of a combination of gluconeogenic amino acids ( a . a . ) in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; amino acids : n = 8 ) . * P 0 . 05 ; * * P 0 . 01 ; ns , P > 0 .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.0


a , Top : representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA + / + neonates fasted for 1 h . Typical autophagosome with a double limiting membrane ( arrows ) ; autophagosome and several autolysosomes ( arrowheads ) . Scale bars , 5 µm . Bottom : frequency of the two types of organelle ( early : autophagosomes ; late : autophagolysosomes ) detected in cell profiles of hepatocytes and skeletal myocytes from RagA + / + and RagAGTP / GTPneonates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 µm. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTPneonates.


b , Protein extracts from livers of neonates at Caesarean section ( 0 h ) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62 .

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b, Protein extracts from livers of neonates at Caesarean section (0 h) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62.


c , Protein extracts from skeletal muscle and heart from neonates at Caesarean section ( 0 h ) , fasted for 1 and 2 h , were immunoblotted for indicated proteins .

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c, Protein extracts from skeletal muscle and heart from neonates at Caesarean section (0 h), fasted for 1 and 2 h, were immunoblotted for indicated proteins.


d , Triggering of autophagy by amino - acid withdrawal in MEFs . MEFs were deprived of amino acids for the indicated time points , whole - cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting.


e , Recombinant LC3B - GFP was expressed in RagA + / + and RagAGTP / GTPMEFs and LC3B localization , in the presence and absence of amino acids , monitored by fluorescence microscopy . LC3B - GFP ( green fluorescent protein ) clustering , indicative of autophagy , was observed in amino acid - starved RagA + / + but not RagAGTP / GTPMEFs . Scale bar , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

e, Recombinant LC3B-GFP was expressed in RagA+/+ and RagAGTP/GTPMEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B-GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTPMEFs. Scale bar, 10 µm.


f , Localization of recombinant TFEB - GFP was determined in MEFs as in e . Nuclear ( active ) TFEB was observed in RagA + / + MEFs upon amino - acid withdrawal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

f, Localization of recombinant TFEB-GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.


a ) AMPKa1 / a2 double knockout ( DKO ) and wild - type ( WT ) MEFs were deprived of glucose for 1 h and re - stimulated for 10 min . Wholecell extracts were immunoblotted for the indicated proteins .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

a) AMPKa1/a2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Wholecell extracts were immunoblotted for the indicated proteins.


b , Immortalized MEFs of the indicated genotypes were deprived of growth factors , glucose , amino acids or glucose and amino acids for 1 h and re - stimulated with glucose and / or amino acids for 10 min . Whole - cell lysates were immunoblotted for the indicated proteins .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins.


c , RagA + / + and RagAGTP / GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h . Cell number is indicated relative to cell number at the start of the treatment ; mean ± s . d . ; * * * P 0 .

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c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005


d , mTOR localization as detected by immunofluorescence . In HEK - 293T cells , glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm . Re - addition of glucose leads to mTOR shuttling to the lysosomal surface , co - localizing with the lysosomal protein Lamp2 . HEK - 293T - RagBGTP cells show mTOR localized at the lysosomal surface , regardless of glucose concentrations . Scale bars , 10 μm .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 μm.


e , Glucose and amino acids affect the binding of the v - ATPase to the Ragulator complex . HEK - 293T expressing Flag - p14 was deprived of glucose or amino acids for 90 min and re - stimulated for 20 min . Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins.


f , RagA + / + and RagAGTP / GTP primary MEFs were cultured for 1 h in media with the glucose and amino - acid concentrations measured in neonates at birth ( 0 h ) or after fasting for 1 h ( 1 h ) and whole - cell protein extracts were analysed by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting.


( A ) Binding of wild type DUBm - Ubp8 to monoubiquitin . ( B ) Binding of DUBmUbp8C146A to monoubiquitin . ( C ) Binding of DUBm - Ubp8C146S to monoubiquitin . ( D ) Binding of DUBm - Ubp8C146R to monoubiquitin . ( E ) Binding of DUBm - Ubp8C146A to K48 diubiquitin . ( F ) Binding of DUBm - Ubp8C146S to K48 diubiquitin .

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(A) Binding of wild type DUBm-Ubp8 to monoubiquitin. (B) Binding of DUBmUbp8C146A to monoubiquitin. (C) Binding of DUBm-Ubp8C146S to monoubiquitin. (D) Binding of DUBm-Ubp8C146R to monoubiquitin. (E) Binding of DUBm-Ubp8C146A to K48 diubiquitin. (F) Binding of DUBm-Ubp8C146S to K48 diubiquitin.


Binding was measured by fluorescence polarization using N - terminally TAMRA - labeled monoubiquitin . The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM ( Clerici et . al , 2014 ) and 0 . 60 ± 0 . 17 nM , respectively . Error bars are s . d . calculated on five measurements per point .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Binding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM (Clerici et. al, 2014) and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point.


( A ) Equilibrium binding of OTUD1 C320A and C320R to K63 - linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63 - linked diubiquitin in which the proximal ubiquitin was fluorescently - labeled . Error bars indicate s . d . and are based on three measurements per data point . One representative experiment of two is shown .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently-labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown.


( B ) Whole cell lysates of HEK293 cells expressing HA - tagged OTUD1 WT , C320R , and C320A were immunoblotted with indicated antibodies . One representative experiment of three is shown .

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(B) Whole cell lysates of HEK293 cells expressing HA-tagged OTUD1 WT, C320R, and C320A were immunoblotted with indicated antibodies. One representative experiment of three is shown.


Polyubiquitin chains were co - immunoprecipitated with FLAG - PSMD4 , an ubiquitin receptor for the 26S proteasome , from cells expressing either USP14 wild - type , C114A , or C114R . One representative experiment of two is shown .

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Polyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown.


E Representative images of Notch activity indicated by Tg ( Tp1 : mVenus ) ( green ) and matured MG stained with GS ( magenta ) at different stages . Scale Bars : 20 μm . F Quantitative plots showing the numbers of Tp1 + ( left ) , GS + ( right ) cells in the developing retinas at different stages . We collected retinas from 60 hpf ( n = 3 dpf ( n = 12 ) , 5 dpf ( n = 12 ) , and 7 dpf ( n = 12 , Mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Representative images of Notch activity indicated by Tg(Tp1:mVenus)( green) and matured MG stained with GS (magenta) at different stages. Scale Bars: 20 μm. F Quantitative plots showing the numbers of Tp1+ (left), GS+ (right) cells in the developing retinas at different stages. We collected retinas from 60 hpf (n = 3 dpf (n = 12), 5 dpf (n = 12), and 7 dpf (n = 12, Mean ± SEM; * p<0.05, ***p<0.001, t-test).


A Representative images of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) and mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch ligands ( dla - / - / dlb - / - / dlc - / - / dld - / - / jag1b - / - / jag2b - / - ) mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative images of Notch activation labeled by Tg(Tp1:mVenus)(green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch ligands (dla-/-/dlb-/-/dlc-/-/dld-/-/jag1b-/-/jag2b-/-) mutants.


B , C Quantitative plots showing the numbers of Notch activation ( Tp1 + ) cells ( B ) and mature MG ( GS + ) cells ( C ) in A at 4 dpf . In total , we collected 11 fish for wild - type fish ; 8 fish for jag2b - / - mutants ; 15 fish for dla - / - mutants ; 19 fish for dld - / - mutants ; 19 fish for dlc - / - mutants , 11 fish for dlb - / - mutants , 11 fish for jag1b - / - mutants ( mean ± SEM ; * * * p < 0 . 001 , t - test ) .

B, C Quantitative plots showing the numbers of Notch activation (Tp1+) cells (B) and mature MG (GS+) cells (C) in A at 4 dpf. In total, we collected 11 fish for wild-type fish; 8 fish for jag2b-/- mutants; 15 fish for dla-/- mutants; 19 fish for dld-/- mutants; 19 fish for dlc-/- mutants, 11 fish for dlb-/- mutants, 11 fish for jag1b-/- mutants (mean ± SEM; ***p<0.001, t-test).


A Upper : representative images showing that GS + MGs ( magenta ) were rarely observed in 5 - dpf jag2b - / - mutants expressing H2B - CFP ( green ) driven by the crx promoter . Bottom : representative images showing that GS + MGs ( magenta ) were frequently rescued underneath PRs when jag2b expression ( green ) was driven by the crx promoter in 5 - dpf jag2b - / - mutants . The area of retina is labeled with solid line , and each layer structure is labeled with dashed lines and marked with outer nuclear layer ( ONL ) , inner nuclear layer ( INL ) , inner plexiform layer ( IPL ) , and ganglion cell layer ( GCL ) . Scale bars : 20μm . The remaining MGs are indicated by white arrowheads , and the rescued MGs are indicated by yellow arrows . B Quantification of GS + MGs ( magenta in A ) with ( green ) or without ( gray ) CFP expression driven by the crx promoter . Ctrl indicates H2B - CFP expression driven by the crx promoter ( n = 16 larvae ) ; rescue indicates jag2b - H2B - CFP expression driven by the crx promoter ( n = 21 larvae , mean ± SEM ; * p < 0 . 05 , ns , no significant difference , pair - wised t - test ) . C The proportions of jag2b - misexpressing PRs ( Green ) with underneath MGs ( GS + , magenta in A ) in 5 - dpf jag2b - / - mutants and all MGs in retina . In total , we analyzed retinae of 16 control ( Ctrl , jag2b - / - , crx : : h2b - ecfp expression ) and 21 rescue ( jag2b - / - , crx : : jag2b expression ) larvae ( Mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Upper: representative images showing that GS+ MGs (magenta) were rarely observed in 5-dpf jag2b-/- mutants expressing H2B-CFP (green) driven by the crx promoter. Bottom: representative images showing that GS+ MGs (magenta) were frequently rescued underneath PRs when jag2b expression (green) was driven by the crx promoter in 5-dpf jag2b-/- mutants. The area of retina is labeled with solid line, and each layer structure is labeled with dashed lines and marked with outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Scale bars: 20μm. The remaining MGs are indicated by white arrowheads, and the rescued MGs are indicated by yellow arrows. B Quantification of GS+ MGs (magenta in A) with (green) or without (gray) CFP expression driven by the crx promoter. Ctrl indicates H2B-CFP expression driven by the crx promoter (n = 16 larvae); rescue indicates jag2b-H2B-CFP expression driven by the crx promoter (n = 21 larvae, mean ± SEM; * p<0.05, ns, no significant difference, pair-wised t-test). C The proportions of jag2b-misexpressing PRs (Green) with underneath MGs (GS+, magenta in A) in 5-dpf jag2b-/- mutants and all MGs in retina. In total, we analyzed retinae of 16 control (Ctrl, jag2b-/- , crx::h2b-ecfp expression) and 21 rescue (jag2b-/-, crx::jag2b expression) larvae (Mean ± SEM; ***p<0.001, t-test).


D Representative image showing the GS + , but vsx1 - expression in the progeny of Tp1 + Müller - like differentiating MGs at wild - type ( jag2b + / + ) . Layer structure indicated in ( A ) . E The percentage of the progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expression GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in wild - type ( jag2b + / + ) fish ( n = 14 , Mean ± SEM ; * * * p < 0 . 001 , t - test ) . F Representative image showing vsx1 + , but GS - expression in the progeny of Tp1 + Müller - like differentiating MGs in jag2b - / - mutants . Layer structure indicated in ( A ) . G The percentage of the progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expressing GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in jag2b - / - mutants ( n = 29 , mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Representative image showing the GS+, but vsx1- expression in the progeny of Tp1+ Müller-like differentiating MGs at wild-type (jag2b+/+). Layer structure indicated in (A). E The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expression GS (gray), vsx1 (green), or both negative (magenta) in wild-type (jag2b+/+) fish (n = 14, Mean ± SEM; ***p<0.001, t-test). F Representative image showing vsx1+, but GS- expression in the progeny of Tp1+ Müller-like differentiating MGs in jag2b-/- mutants. Layer structure indicated in (A). G The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expressing GS (gray), vsx1 (green), or both negative (magenta) in jag2b-/- mutants (n = 29, mean ± SEM; ***p<0.001, t-test).


A Representative images of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) and mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch receptors ( notch1a - / - / notch1b - / - / notch3 - / - ) mutants . Scale Bars : 20 μm . B Quantitative plots showing the numbers of cells with active Notch ( Tp1 + ) and mature MG ( GS + ) cells at 4 dpf . In total , we collected 4 fish for wild - type fish ; 7 fish for notch1a - / - mutants ; 5 fish for notch1b - / - mutants ; 18 fish for notch3 - / - mutants ( Mean ± SEM ; * * p < 0 . 01 ; * * * p < 0 . 001 , t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative images of Notch activation labeled by Tg(Tp1:mVenus)( green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch receptors (notch1a-/-/notch1b-/-/notch3-/-) mutants. Scale Bars: 20 μm. B Quantitative plots showing the numbers of cells with active Notch (Tp1+) and mature MG (GS+) cells at 4 dpf. In total, we collected 4 fish for wild-type fish; 7 fish for notch1a-/- mutants; 5 fish for notch1b-/- mutants; 18 fish for notch3-/- mutants (Mean ± SEM; ** p<0.01; ***p<0.001, t-test).


C Representative images showing that GS + MGs ( magenta ) were rarely observed when H2B - CFP expression ( green ) was driven by the gfap promoter in 5 - dpf jag2b - / - mutants . The area between the upper dashed and solid lines is the outer nuclear layer ( ONL ) . The area in the middle two dash lines is the inner plexiform layer ( IPL ) . INL , inner nuclear layer ; GCL , ganglion cell layer . D Representative images showing that GS + MGs ( magenta ) were frequently rescued underneath cells with notch3 expression ( green ) driven by the gfap promoter in 5 - dpf jag2b - / - mutants . Layer structure is similar as ( C ) . E The cell number of GS + MGs ( magenta ) with or without CFP expression driven by gfap promoter . Ctrl : H2B - CFP expression ( green ) driven by the gfap promoter ( n = 5 fish ) ; rescue : notch1b ( n = 13 fish ) and notch3 ( n = 17 fish ) NICD - H2B - CFP expression ( green ) driven by the gfap promoter ( Mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , ns , no significant difference , pair - wised t - test ) . F The proportions of notch1b and notch3 NICD - misexpressing cells ( green ) with underneath MGs ( GS + , magenta ) in 5 - dpf jag2b - / - mutants , indicating that gfap promoter - driven notch1b ( n = 13 fish ) or notch3 ( n = 17 fish ) NICD expression could rescue MG loss in jag2b - / - mutants ( Mean ± SEM ; * * * p < 0 . 001 , t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Representative images showing that GS+ MGs (magenta) were rarely observed when H2B-CFP expression (green) was driven by the gfap promoter in 5-dpf jag2b-/- mutants. The area between the upper dashed and solid lines is the outer nuclear layer (ONL). The area in the middle two dash lines is the inner plexiform layer (IPL). INL, inner nuclear layer; GCL, ganglion cell layer. D Representative images showing that GS+ MGs (magenta) were frequently rescued underneath cells with notch3 expression (green) driven by the gfap promoter in 5-dpf jag2b-/- mutants. Layer structure is similar as (C). E The cell number of GS+ MGs (magenta) with or without CFP expression driven by gfap promoter. Ctrl: H2B-CFP expression (green) driven by the gfap promoter (n = 5 fish); rescue: notch1b (n = 13 fish) and notch3 (n = 17 fish) NICD-H2B-CFP expression (green) driven by the gfap promoter (Mean ± SEM; * p<0.05, ***p<0.001, ns, no significant difference, pair-wised t-test). F The proportions of notch1b and notch3 NICD-misexpressing cells (green) with underneath MGs (GS+, magenta) in 5-dpf jag2b-/- mutants, indicating that gfap promoter-driven notch1b (n = 13 fish) or notch3 (n = 17 fish) NICD expression could rescue MG loss in jag2b-/- mutants (Mean ± SEM; ***p<0.001, t-test).


Gating strategy for sorting of EpCAMhighCD24lowSca ‑ 1 + cells and rMC from lung homogenate of adult mice .

Gating strategy for sorting of EpCAMhighCD24lowSca‑1+ cells and rMC from lung homogenate of adult mice.


Time - course of epithelial stem / progenitor cell proliferation and differentiation within BALO at days 8 , 11 and 21 of co - culture with rMC .

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Time-course of epithelial stem/progenitor cell proliferation and differentiation within BALO at days 8, 11 and 21 of co-culture with rMC.


Clonal expansion of EpCAMhighCD24lowSca ‑ 1 + cells derived from either GFP - expressing mice or tdTomato - expressing ROSAmT / mG mice at day 21 of co ‑ culture .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Clonal expansion of EpCAMhighCD24lowSca‑1+ cells derived from either GFP-expressing mice or tdTomato-expressing ROSAmT/mG mice at day 21 of co‑culture.


Percentages of SCGB1A1 + SFTPC + ( BALO ) , SCGB1A1 + SFTPC - ( bronchiolospheres ) and SCGB1A1 - SFTPC + ( alveolospheres ) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca - 1 + cells isolated from Scgb1a1mCherrySftpcYFP reporter mice ( n = 4 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentages of SCGB1A1+SFTPC+ (BALO), SCGB1A1+SFTPC- (bronchiolospheres) and SCGB1A1-SFTPC+ (alveolospheres) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca-1+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice (n=4 biological replicates).


Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca - 1 + SCGB1A1 + SFTPC + cells isolated from Scgb1a1mCherrySftpcYFP reporter mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca-1+SCGB1A1+SFTPC+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice.


Heat map ( left ) and tSNE plot ( right ) ( F ) of digested day 21 BALO cultures depicting four distinct clusters ( C1 , airway , blue ; C2 , alveolar , purple ; C3 , MYO , orange , and C4 , LIF , green ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map (left) and tSNE plot (right) (F) of digested day 21 BALO cultures depicting four distinct clusters (C1, airway, blue; C2, alveolar, purple; C3, MYO, orange, and C4, LIF, green).


Violin plots of selected genes representing airway associated - genes ( G ) ( Itgb4 , Trp63 , Krt7 , and Sox2 ) and alveoli associated ‑ genes ( H ) ( Cxcl15 , Lyz2 , Sftpc , and Hopx ) . Each violin plot shows the frequency distribution of the mean transcript level ( log2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Violin plots of selected genes representing airway associated-genes (G) (Itgb4, Trp63, Krt7, and Sox2) and alveoli associated‑genes (H) (Cxcl15, Lyz2, Sftpc, and Hopx). Each violin plot shows the frequency distribution of the mean transcript level (log2).


mRNA expression analysis of epithelial cell differentiation markers Sftpc ( AEC II ) , Hopx ( AEC I ) , Foxj1 ( ciliated cells ) , Muc5ac ( secretory cells ) and p63 ( basal cells ) in BALO at days 0 , 10 , and 21 of culture ( n = 3 biological replicates with pooled cells from 4 cultures per replicate )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells) and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate)


Electron microscopy of BALO alveoli showing two cuboidal epithelial cells ( AEC II ) connected by tight junctions ( red square ) with numerous mitochondria ( M ) and lamellar bodies ( LB ) . A LIF with numerous lipid droplets ( * ) and a MYO with cisterns of rough ER ( red arrow ) are located at the basal side ( left ) . The alveolar lumen is filled with lamellar surfactant including tubular myelin ( white arrow ) ( lower right ) . Exocytosis of a lamellar body ( * ) from an AEC II ( upper right ) . Scale bars indicate 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron microscopy of BALO alveoli showing two cuboidal epithelial cells (AEC II) connected by tight junctions (red square) with numerous mitochondria (M) and lamellar bodies (LB). A LIF with numerous lipid droplets (*) and a MYO with cisterns of rough ER (red arrow) are located at the basal side (left). The alveolar lumen is filled with lamellar surfactant including tubular myelin (white arrow) (lower right). Exocytosis of a lamellar body (*) from an AEC II (upper right). Scale bars indicate 500 nm.


Electron microscopy of bronchiolar - like airway depicting columnar ciliated cells ( red arrowhead ) with basal bodies ( black arrowhead ) at the left side of the longitudinally sectioned lumen . The boxed area indicates an apical junctional complex between two ciliated cells . 1 = tight junction , 2 = adherens junction . Scale bar indicates 500 nm ( in insert : 100 nm ) .

Electron microscopy of bronchiolar-like airway depicting columnar ciliated cells (red arrowhead) with basal bodies (black arrowhead) at the left side of the longitudinally sectioned lumen. The boxed area indicates an apical junctional complex between two ciliated cells. 1 = tight junction, 2 = adherens junction. Scale bar indicates 500 nm (in insert: 100 nm).


Staining of lamellar bodies in BALO with RFP LysoTracker . Scale bars represent 100 μm .

Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm.


Western blot analysis of the surfactant proteins : pro - SPC , mature SPC , pro - SPB , mature SPB , and SPA in lung homogenate ( LH ) , AEC and day 21 BALO ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of the surfactant proteins: pro-SPC, mature SPC, pro-SPB, mature SPB, and SPA in lung homogenate (LH), AEC and day 21 BALO (n=3 biological replicates).


F - G . Alveolar diameter ( F ) and number of alveoli ( G ) in BALO at days 15 , 21 , 30 and 40 of culture was measured from n = 5 BALO in n = 3 biological replicates with tdTomato + rMC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. Alveolar diameter (F) and number of alveoli (G) in BALO at days 15, 21, 30 and 40 of culture was measured from n=5 BALO in n=3 biological replicates with tdTomato+ rMC.


H - I . Representative scheme and images of day 40 BALO alveoli ( H ) and airway ( I ) . BALO alveolar - like structures ( H ) are shown ( h ' ) ( red arrowheads ) in semithin section ( 0 . 5 µm ) stained with Toluidine blue . Scale bar indicates 100 μm ( left ) . Electron microscopy showing AEC I ( h ' ' ) ultrastructure within BALO . Scale bars indicate 2500 nm ( center ) and 1000 nm ( right ) . An airway - like structure ( i ' ) is shown with secretory and ciliated cells ( red arrowheads ) in semithin sections ( 0 . 5 µm ) longitudinally cut and stained with Toluidine blue . Scale bar indicates 50 μm ( far left ) . Electron microscopy of a bronchiolar - like airway ( I ) depicting pseudostratified epithelium ( i ' ' ) with a basal - like cell ( BC ) , not reaching the lumen in which cilia ( C ) are seen , located between a secretory ( SC ) and a ciliated cell ( Ci ) . Scale bars indicate 1000 nm ( left and right ) . Mature cilia ( i ' ' ' ) in BALO at higher magnification depicting the 9x2 + 2 structure with central doubled microtubules ( insert , red arrowhead ) , a characteristic for motile cilia . Scale bar indicates 100 nm ( far right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-I. Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar-like structures (H) are shown (h') (red arrowheads) in semithin section (0.5 µm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h'') ultrastructure within BALO. Scale bars indicate 2500 nm (center) and 1000 nm (right). An airway-like structure (i') is shown with secretory and ciliated cells (red arrowheads) in semithin sections (0.5 µm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar-like airway (I) depicting pseudostratified epithelium (i'') with a basal-like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1000 nm (left and right). Mature cilia (i''') in BALO at higher magnification depicting the 9x2+2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).


Representative images of day 21 BALO and tdTomato + rMC stained with LipidTOX ( green ) . Scale bar represents 100 μm .

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Representative images of day 21 BALO and tdTomato+ rMC stained with LipidTOX (green). Scale bar represents 100 μm.


Fluorescence images of αSMA ( green ) and neutral lipids ( LipidTOX red ) staining in WT ‑ derived rMC and BALO ( dotted lines indicate single BALO or insert ) at day 21 of culture . Scale bars represent 50 μm ( left ) and 25 μm ( right ) .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence images of αSMA (green) and neutral lipids (LipidTOX red) staining in WT‑derived rMC and BALO (dotted lines indicate single BALO or insert) at day 21 of culture. Scale bars represent 50 μm (left) and 25 μm (right).


Representative flow cytometric dot plots and histograms of PDGFRα expression in rMC ( EpCAM - CD45 - CD31 - Sca - 1 + ) isolated from the lung homogenate of PdgfraGFP reporter mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative flow cytometric dot plots and histograms of PDGFRα expression in rMC (EpCAM-CD45-CD31-Sca-1+) isolated from the lung homogenate of PdgfraGFP reporter mice.


Representative images of BALO formation at day 8 , 15 and 21 of co - culture . WT BASC were co ‑ cultivated with sorted rMC ( total PDGFRα + population ) or rMC expressing either low or high levels of PDGFRα ‑ GFP . Scale bars represent 100 μm .

Representative images of BALO formation at day 8, 15 and 21 of co-culture. WT BASC were co‑cultivated with sorted rMC (total PDGFRα+ population) or rMC expressing either low or high levels of PDGFRα‑GFP. Scale bars represent 100 μm.


tSNE plots and violin plots depicting selected genes representing MYO ( Pdgfrα , Tagln , Acta2 , Eln , and Axin2 ) ( top panels ) and LIF associated - genes ( Fgf10 , Apoe , Serpina3n , Gsn , and Gas6 ) ( bottom panels ) . Each violin plot shows the frequency distribution of the mean transcript level ( log2 ) . C3 ( MYO , orange ) and C4 ( LIF , green ) refer to the scRNA - Seq experiment in Fig . 1G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tSNE plots and violin plots depicting selected genes representing MYO (Pdgfrα, Tagln, Acta2, Eln, and Axin2) (top panels) and LIF associated-genes (Fgf10, Apoe, Serpina3n, Gsn, and Gas6) (bottom panels). Each violin plot shows the frequency distribution of the mean transcript level (log2). C3 (MYO, orange) and C4 (LIF, green) refer to the scRNA-Seq experiment in Fig. 1G.


Fluorescence image of αSMA + PDGFRαhigh MYO ( yellow arrows ) and LipidTOX + PDGFRαlow LIF ( red arrows ) from sorted PDGFRα - GFP rMC after 21 days of BALO culture . Scale bars represent 50 μm ( left ) and 25 μm ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence image of αSMA+PDGFRαhigh MYO (yellow arrows) and LipidTOX+PDGFRαlow LIF (red arrows) from sorted PDGFRα-GFP rMC after 21 days of BALO culture. Scale bars represent 50 μm (left) and 25 μm (right).


Fluorescence image containing PDGFRαhigh MYO from sorted PDGFRα - GFP rMC within BALO derived from tdTomato + mice at day 21 of culture . Scale bars represent 100 μm ( left ) and 50 μm ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence image containing PDGFRαhigh MYO from sorted PDGFRα-GFP rMC within BALO derived from tdTomato+ mice at day 21 of culture. Scale bars represent 100 μm (left) and 50 μm (right).


Gating strategy to define TR ‑ Mac from BAL of adult tdTomato + mice

Gating strategy to define TR‑Mac from BAL of adult tdTomato+ mice


Representative images of BALO after microinjection of tdTomato + TR - Mac at day 14 . TR - Mac are preferentially found in alveoli ( right ) . Scale bar represent 100 μm ( left ) and 50 μm ( right )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of BALO after microinjection of tdTomato+ TR-Mac at day 14. TR-Mac are preferentially found in alveoli (right). Scale bar represent 100 μm (left) and 50 μm (right)


Fluorescence confocal images of CD206 , Siglec - F , and isotype control staining 14 days after tdTomato + TR ‑ Mac microinjection in a day 28 BALO . Scale bars represent 100 μm ( left ) and 25 μm ( right )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence confocal images of CD206, Siglec-F, and isotype control staining 14 days after tdTomato+ TR‑Mac microinjection in a day 28 BALO. Scale bars represent 100 μm (left) and 25 μm (right)


Electron microscopy depicting filopodium of TR ‑ Mac ( white dashed lines ) with a characteristic actin filament bundle ( yellow background ) ( left panel ) in contact with AEC I within BALO alveolar - like structures . Uptake of surfactant by TR - mac is depicted by a ( * ) . Scale bar indicates 1000nm ( left , in insert : 500 nm ) and 500 nm ( right , in insert : 250 nm )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron microscopy depicting filopodium of TR‑Mac (white dashed lines) with a characteristic actin filament bundle (yellow background) (left panel) in contact with AEC I within BALO alveolar-like structures. Uptake of surfactant by TR-mac is depicted by a (*). Scale bar indicates 1000nm (left, in insert: 500 nm) and 500 nm (right, in insert: 250 nm)


Representative confocal images of Cx43 staining in tdTomato + TR - Mac monoculture in Matrigel and tdTomato + TR ‑ Mac microinjected at day 14 and analyzed in a mature day 21 BALO . Scale bars represent 50 μm ( overview ) and 5 μm ( close up )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of Cx43 staining in tdTomato+ TR-Mac monoculture in Matrigel and tdTomato+ TR‑Mac microinjected at day 14 and analyzed in a mature day 21 BALO. Scale bars represent 50 μm (overview) and 5 μm (close up)


Heat map ( left ) and tSNE plot ( right ) of the comparative analysis of digested day 23 BALO cultures with ( + ) and without ( - ) microinjected TR ‑ Mac depicting six distinct clusters ( C1 , club / secretory cells , red ; C2 , basal cells , yellow ; C3 , rMC , green ; C4 , AEC II , blue ; C5 , AEC I , purple ; and C6 , ciliated cells , pink )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map (left) and tSNE plot (right) of the comparative analysis of digested day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac depicting six distinct clusters (C1, club/secretory cells, red; C2, basal cells, yellow; C3, rMC, green; C4, AEC II, blue; C5, AEC I, purple; and C6, ciliated cells, pink)


Expression data dot plots of genes found differentially regulated in cluster C1 between day 23 BALO with ( C1 + ) and without ( C1 - ) microinjected TR - Mac . The circle size illustrates the number of cells expressing a specific gene

Expression data dot plots of genes found differentially regulated in cluster C1 between day 23 BALO with (C1+) and without (C1-) microinjected TR-Mac. The circle size illustrates the number of cells expressing a specific gene


Percentage of terminally differentiated cells ( AEC I and ciliated cells ) in day 23 BALO cultures with ( + ) and without ( - ) microinjected TR ‑ Mac

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of terminally differentiated cells (AEC I and ciliated cells) in day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac


Representative images of E11 . 5 lung explants after treatment for 48 h with 100 μM Scra or mo142 - 3p reveals reduced size and branching morphogenesis after miR142 - 3p knockdown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of E11.5 lung explants after treatment for 48 h with 100 μM Scra or mo142-3p reveals reduced size and branching morphogenesis after miR142-3p knockdown.


Organoid diameter ( n = 30 - 50 per group ) in µm before addition of 4 μM Scra or mo142 - 3p at day 6 ( control ) and 5 days after treatment ( at day 11 BALO culture ) in n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Organoid diameter (n=30-50 per group) in µm before addition of 4 μM Scra or mo142-3p at day 6 (control) and 5 days after treatment (at day 11 BALO culture) in n=3 biological replicates


Representative images of β - galactosidase staining in TOPGAL epithelium ( C ) BALO cultures before ( day 6 , control ) or 5 days after treatment with either 4 μM Scra or mo142 - 3p ( day 11 of co - culture ) . BASC and rMC were isolated form the lung homogenate of TOPGAL mice . β - galactosidase + rMC at day 11 of culture are indicated with arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of β-galactosidase staining in TOPGAL epithelium (C) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows.


Representative images of β - galactosidase staining in TOPGAL rMC ( D ) BALO cultures before ( day 6 , control ) or 5 days after treatment with either 4 μM Scra or mo142 - 3p ( day 11 of co - culture ) . BASC and rMC were isolated form the lung homogenate of TOPGAL mice . β - galactosidase + rMC at day 11 of culture are indicated with arrows .

Representative images of β-galactosidase staining in TOPGAL rMC (D) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows.


Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n = 4 BALO 15 days after treatment with 4 μM Scra or mo142 - 3p ( day 21 of co - culture ) in n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n=4 BALO 15 days after treatment with 4 μM Scra or mo142-3p (day 21 of co-culture) in n=3 biological replicates.


mRNA levels of epithelial and mesenchymal miR142 - 3p expression in Scra and mo142 - 3p - treated organoids 5 days after treatment ( n = 3 - 4 biological replicates with pooled cells from 4 cultures per replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA levels of epithelial and mesenchymal miR142-3p expression in Scra and mo142-3p-treated organoids 5 days after treatment (n=3-4 biological replicates with pooled cells from 4 cultures per replicate).


Representative images of day 21 BALO after microinjection of SC35M - GFP IAV ( green ) into the central airway - like ( * ) after 0 and 12 h pi visualizes IVA spread to the alveolar - like regions . Dotted lines illustrate BALO - borders . Scale bars represent 100 μm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of day 21 BALO after microinjection of SC35M-GFP IAV (green) into the central airway-like (*) after 0 and 12 h pi visualizes IVA spread to the alveolar-like regions. Dotted lines illustrate BALO-borders. Scale bars represent 100 μm


Quantification of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection ( n = 5 biological replicates ) . N . D . : not - detectable

Quantification of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection (n=5 biological replicates). N.D.: not-detectable


Relative NP expression in PR8 IAV infected ( HA + ) or non - infected ( HA - ) epithelial cells isolated from BALO 48 h pi , FACS - sorted according to EpCAM and HA expression ( n = 3 biological replicates with pooled cells from 4 cultures per replicate )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative NP expression in PR8 IAV infected (HA+) or non-infected (HA-) epithelial cells isolated from BALO 48 h pi, FACS-sorted according to EpCAM and HA expression (n=3 biological replicates with pooled cells from 4 cultures per replicate)


Representative flow cytometric data showing the percentage of EpCAM + NP + cells in mock - and SC35M IAV ‑ infected BALO at 48 h pi in n = 3 biological replicates with pooled cells from 4 cultures per replicate

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative flow cytometric data showing the percentage of EpCAM+NP+ cells in mock- and SC35M IAV‑infected BALO at 48 h pi in n=3 biological replicates with pooled cells from 4 cultures per replicate


Representative fluorescence images of a day 21 distal region in BALO generated from mTmG reporter mouse and infected by SC35M - Cre IAV after 0 , 10 , 14 and 25 h . Arrows indicate cell death . Scale bars represent 25 μm and 10 μm in the insert

Representative fluorescence images of a day 21 distal region in BALO generated from mTmG reporter mouse and infected by SC35M-Cre IAV after 0, 10, 14 and 25 h. Arrows indicate cell death. Scale bars represent 25 μm and 10 μm in the insert


mRNA expression of Ifnb in mock and PR8 IAV - infected BALO at 48 h pi ( n = 3 biological replicates with pooled cells from 4 cultures per replicate )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression of Ifnb in mock and PR8 IAV-infected BALO at 48 h pi (n=3 biological replicates with pooled cells from 4 cultures per replicate)


Release of TNFα ( 6 h pi ) , IL - 6 ( 48 h pi ) , IL - 1β ( 48 h pi ) detected by Bio - Plex ® Multiplex Immunoassay in the supernatant of mock and IAV infected BALO cultures with and without TR - Mac ( n = 3 biological replicates )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Release of TNFα (6 h pi), IL-6 (48 h pi), IL-1β (48 h pi) detected by Bio-Plex® Multiplex Immunoassay in the supernatant of mock and IAV infected BALO cultures with and without TR-Mac (n=3 biological replicates)


( A ) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays . Expression of different Atrophin forms with the GMR driver was induced , owing to a temperature ‐ sensitive mutant Gal80 repressor . F1 flies were allowed to develop at 18 ° C ; at this temperature the Gal80 repressor keeps transgenes silent . Newly eclosed flies ( 0 - 48 h ) were collected and killed immediately ( 0d ) or aged for 2 or 14 days at 29 ° C . This inactivates Gal80 and transgenes are switched on by GMR ‐ Gal4 . This protocol allows comparing siblings that differ exclusively in their age and transgenes expression . Control flies crossed to GMR ‐ Gal4 ; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays. Expression of different Atrophin forms with the GMR driver was induced, owing to a temperature‐sensitive mutant Gal80 repressor. F1 flies were allowed to develop at 18°C; at this temperature the Gal80 repressor keeps transgenes silent. Newly eclosed flies (0-48 h) were collected and killed immediately (0d) or aged for 2 or 14 days at 29°C. This inactivates Gal80 and transgenes are switched on by GMR‐Gal4. This protocol allows comparing siblings that differ exclusively in their age and transgenes expression. Control flies crossed to GMR‐Gal4; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated.


( B ) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points . Weak degeneration is only visible after 14 days with polyQ Atro ; in particular with Atro75QN there is an initial loss of photoreceptors ( PR , arrow ) , 30 . 7 % of the ommatidia has lost at least 1 PR , that is only 5 . 1 % of all neuronal PR have been lost at this stage ( N = 333 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points. Weak degeneration is only visible after 14 days with polyQ Atro; in particular with Atro75QN there is an initial loss of photoreceptors (PR, arrow), 30.7% of the ommatidia has lost at least 1 PR, that is only 5.1% of all neuronal PR have been lost at this stage (N=333).


( A ) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric . Upregulated genes are in red and downregulated genes are in green . The downregulating transcriptional activity increases with time . The main branch in clustering of the different Atro mutants is along time points . See also Supplementary Figure 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric. Upregulated genes are in red and downregulated genes are in green. The downregulating transcriptional activity increases with time. The main branch in clustering of the different Atro mutants is along time points. See also Supplementary Figure 4.


( A ) qPCR analysis of the fold changes ± s . d . of ft transcription . Downregulation starts at 2 days and progresses at 14 days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) qPCR analysis of the fold changes±s.d. of ft transcription. Downregulation starts at 2 days and progresses at 14 days.


( B ) qPCR analysis of the enrichment ± s . d . of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts . Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced , with respect to the amount immunoprecipitated from uninduced cell extracts according to the ( Atro - No Ab ) induced / ( Atro − No Ab ) uninduced formula . The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al , 2007 ) and has been used as a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts. Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al, 2007) and has been used as a negative control.


( C ) Histograms showing the number of ommatidia with a full complement ( 7 ) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control ( w1118 ) or different mutant backgrounds and aged at 29 ° C for 28 days . Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR . Mild overexpression of Wts via a GMR - wts transgene , which does not display any strong phenotype per se , significantly suppresses the loss of PR . No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity . N = 430 - 963 from at least four eyes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Histograms showing the number of ommatidia with a full complement (7) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control (w1118) or different mutant backgrounds and aged at 29°C for 28 days. Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR. Mild overexpression of Wts via a GMR-wts transgene, which does not display any strong phenotype per se, significantly suppresses the loss of PR. No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity. N=430-963 from at least four eyes.


( D ) Tangential eye sections and histograms showing the degeneration of GMR > Atro75QN , Ubi - Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A . UAS - EGFP is used as a negative control . Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN . Lower panels are provided as examples of quantification . A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo ; however , in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼ 5 % of ommatidia ( data not shown ) . No effect is detected with overexpression of Sav . Two ‐ tailed t ‐ test : * P0 . 05 ; * * P0 . 01 . The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5 ′ of the wts gene . Rare homozygous escapers display larger eyes , whereas in combination with GMR - GMR this line gives rise to smaller rough eyes , and , finally , GMR analysis of GMR > wtsEPG4808 indicates a 10 ‐ fold increase in the head content of wts mRNA ( data not shown ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Tangential eye sections and histograms showing the degeneration of GMR>Atro75QN, Ubi-Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A. UAS-EGFP is used as a negative control. Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN. Lower panels are provided as examples of quantification. A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo; however, in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼5% of ommatidia (data not shown). No effect is detected with overexpression of Sav. Two‐tailed t‐test: *P0.05; **P0.01. The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5′ of the wts gene. Rare homozygous escapers display larger eyes, whereas in combination with GMR-GMR this line gives rise to smaller rough eyes, and, finally, GMR analysis of GMR>wtsEPG4808 indicates a 10‐fold increase in the head content of wts mRNA (data not shown).


( A ) Tangential eye sections through ftfd clones aged 1 , 7 and 14 days , of ftfd , dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days . Clones are marked by the absence of yellow pigment . Arrows point at degenerating photoreceptors , arrowheads point at intact wt photoreceptors in mosaic ommatidia . The circled ommatidium is genetically wt but non ‐ autonomously flipped in its polarity and has not degenerated . Lower panels are masks that show clonal borders and quantification of PR number per ommatidia . After 14 days , almost all ftfd cells have degenerated , whereas almost all ftfd , dDGC13 and some ftfd , ykiB5 / + neuronalphotoreceptors have not .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Tangential eye sections through ftfd clones aged 1, 7 and 14 days, of ftfd,dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days. Clones are marked by the absence of yellow pigment. Arrows point at degenerating photoreceptors, arrowheads point at intact wt photoreceptors in mosaic ommatidia. The circled ommatidium is genetically wt but non‐autonomously flipped in its polarity and has not degenerated. Lower panels are masks that show clonal borders and quantification of PR number per ommatidia. After 14 days, almost all ftfd cells have degenerated, whereas almost all ftfd,dDGC13 and some ftfd,ykiB5/+ neuronalphotoreceptors have not.


( B ) Histograms showing the quantification of degeneration in ftfd , ftfddDGC13 and ftfdykiB5 / + clones . Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ tailed t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Histograms showing the quantification of degeneration in ftfd, ftfddDGC13 and ftfdykiB5/+ clones. Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.