Cytokine and cytolytic granule production in CD8 + T cells . Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg / ml of phytohaemagglutinin for 2 days and then rested for 1 day . Cells were pretreated with DMSO , MB , 25 μg / ml of pembrolizumab ( Pem ) or 20 μg / ml of nivolumab ( Niv ) for 1 h and then seeded in a 96 - well precoated with 10 μg / ml aCD3 / aCD28 , with media supplemented with 10 μg / ml of human PD - L1 Protein . Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8 + populations . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell . JP - luc cells were stimulated with Raji - L1 or parental Raji cells preloaded with 1 μg / ml of superantigen ( SEE ) in the presence of MB at indicated concentration or 25μg / ml of pembrolizumab ( Pem ) or 20μg / ml of nivolumab ( Niv ) for 6 hours . Luciferase activity was measured by luminometer . Data are representative of three independent experiments , and were analyzed by unpaired t - test . JP - luc : Jurkat cell harboring NFAT - luciferase transgene and overexpressing PD - 1 ; Raji - L1 : Raji overexpressing PD - L1 . Data information : Data are representative of three independent experiments , and were analyzed by unpaired t - test . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Curated heatmaps of top 76 up and 72 down regulated genes . Highlighted genes were chosen based on biological interest . The coloured bar represents the expression level from low ( green ) to high ( red ) .

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Curated heatmaps of top 76 up and 72 down regulated genes. Highlighted genes were chosen based on biological interest. The coloured bar represents the expression level from low (green) to high (red).


Real - time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1 , B Blimp - 1 , C FcRn and D Lct and increase of the adult markers E Sis , F Treh and G Arg2 ( n = 3 individual wells from single organoid culture ( see material and methods ) ; experiment was repeated in four to eight independent organoid cultures with similar results ) .

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Real-time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct and increase of the adult markers E Sis, F Treh and G Arg2 (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four to eight independent organoid cultures with similar results).


Enzyme activity assay of fetal organoids for H Lactase , I sucrase , J maltase , K trehalase and L arginase . Activity is given in µM glucose / µg protein · min - 1 ( experiment was generated from single organoid culture ( see material and methods ) and repeated in 3 independent organoid cultures with similar results ) .

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Enzyme activity assay of fetal organoids for H Lactase, I sucrase, J maltase, K trehalase and L arginase. Activity is given in µM glucose/µg protein·min-1 (experiment was generated from single organoid culture (see material and methods) and repeated in 3 independent organoid cultures with similar results).


Relative expression detected by Real - time qPCR in fetal organoids ( ) and adult organoids ( ) . Neonatal markers A Ass1 , B Blimp - 1 , C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids . Mature markers E Sis , F Treh and G Arg2 increase in fetal organoids to adult levels ( n = 3 individual wells from single organoid culture ( see material and methods ) ; experiment was repeated in four independent organoid cultures with similar results ) .

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Relative expression detected by Real-time qPCR in fetal organoids ( ) and adult organoids ( ). Neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids. Mature markers E Sis, F Treh and G Arg2 increase in fetal organoids to adult levels (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four independent organoid cultures with similar results).


Enzyme activity assay of fetal ( ) and adult ( ) organoids . H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28 , while I sucrase , J maltase , K trehalase and L arginase increase to adult levels . Activity is given in µM glucose / µg protein · min - 1 ( n = 3 independent organoid cultures ) .

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Enzyme activity assay of fetal ( ) and adult ( ) organoids. H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28, while I sucrase, J maltase, K trehalase and L arginase increase to adult levels. Activity is given in µM glucose/µg protein·min-1 (n=3 independent organoid cultures).


Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ) , as revealed by real - time qPCR . Decrease in relative expression of neonatal marker A Blimp - 1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated

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Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. Decrease in relative expression of neonatal marker A Blimp-1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated organoids


Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture . Activity is given in µM glucose / µg protein · min - 1 ( n = 3 independent organoid cultures ) .

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Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).


Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ) , as revealed by real - time qPCR . expression of adult marker E Treh and F Arg2 remains unchanged ( n = 3 individual wells from single organoid

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Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. expression of adult marker E Treh and F Arg2 remains unchanged (n=3 individual wells from single organoid culture


Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture . Activity is given in µM glucose / µg protein · min - 1 ( n = 3 independent organoid cultures ) .

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Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).


Microscopic images of fetal organoids at day 3 , 13 , 20 and 30 of culture . Scale bars : 500 µm .

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Microscopic images of fetal organoids at day 3, 13, 20 and 30 of culture. Scale bars: 500 µm.


Percentage of fetal spheroids decreases during culture ( n = 3 individual wells from single organoid culture ( see material and methods ) ; experiment was repeated in ten independent organoid cultures with similar results ) .

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Percentage of fetal spheroids decreases during culture (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in ten independent organoid cultures with similar results).


Immmunohistochemistry of fetal organoids for C Ass1 D Sis and E Arg2 . Quantification of 3 slides per staining , 7 to 10 organoids / spheroids per slide . Scale bars : 50µm .

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Immmunohistochemistry of fetal organoids for C Ass1 D Sis and E Arg2. Quantification of 3 slides per staining, 7 to 10 organoids / spheroids per slide. Scale bars: 50µm.


( C ) All eight subunits of the CCT chaperonin complex were enriched in U2OS - A3A samples . Proteins detected by MS are displayed by fold enrichment ( log2 ) from U2OS - A3A cells compared to parental cells ( x - axis ) , and by protein abundance within the U2OS - A3A samples ( y - axis ) . Significantly enriched proteins ( p < 0 . 05 by one - tailed t - test , n = 3 ) are denoted in dark blue . Components of the CCT complex are highlighted and labeled .

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(C) All eight subunits of the CCT chaperonin complex were enriched in U2OS-A3A samples. Proteins detected by MS are displayed by fold enrichment (log2) from U2OS-A3A cells compared to parental cells (x-axis), and by protein abundance within the U2OS-A3A samples (y-axis). Significantly enriched proteins (p<0.05 by one-tailed t-test, n=3) are denoted in dark blue. Components of the CCT complex are highlighted and labeled.


( A ) A3A co - precipitates with CCT complex subunits . U2OS and HepaRG cells with dox - inducible A3A - HA transgenes were treated with dox prior to immunoprecipitation of HA . HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex .

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(A) A3A co-precipitates with CCT complex subunits. U2OS and HepaRG cells with dox-inducible A3A-HA transgenes were treated with dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex.


( C ) A3A interacts with CCT in leukemia cells . HA immunoprecipitation in hematopoietic cell lines with inducible A3A - HA was analyzed by immunoblot . HA , CCT1 , and CCT5 antibodies were used to evaluate co - precipitation of CCT subunits with A3A .

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(C) A3A interacts with CCT in leukemia cells. HA immunoprecipitation in hematopoietic cell lines with inducible A3A-HA was analyzed by immunoblot. HA, CCT1, and CCT5 antibodies were used to evaluate co-precipitation of CCT subunits with A3A.


( A ) CCT co - precipitates with A3A but not A3B or A3G . 293T cells were transfected with empty vector ( V ) , A3A - HA , A3B - HA , or A3G - HA . CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA .

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(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA.


( B ) A3A , but not other APOBEC3 enzymes , interacts with CCT complex subunits . After transfection of 293T cells with HA - tagged APOBEC3 expression plasmids , IP was performed with anti - HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits . Arrows mark location of each APOBEC3 protein on HA immunoblot .

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(B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot.


( F ) CCT co - precipitates with endogenous A3A . Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1 . Chemiluminescent exposure times described as short and long are indicated beside the blot . IgG antibody was used as a control throughout .

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(F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.


( A ) Knockdown of one CCT complex member results in depletion of other CCT subunits . Following siCCT4 or siCCT7 transfection , cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1 , CCT4 , CCT5 , and CCT7 . GAPDH was used as a loading control .

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(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control.


( B ) Decreased cell viability upon CCT depletion and A3A expression . CCT4 ( left ) and CCT7 ( right ) subunits were depleted by siRNA transfection in U2OS cells with dox - inducible A3A transgenes . Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit , but no alteration in A3A expression . Scr indicates non - targeted siRNA , used as control . Ku - 86 is a loading control . Following siRNA transfection to deplete CCT subunits , U2OS - A3A cells were treated with dox . Viability was determined by colorimetric change after addition of a water - soluble tetrazolium salt . Percent viability was normalized to untreated controls . Statistical analysis was performed using a paired two - tailed t - test , n = 3 biological replicates ; error bars , SEM .

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(B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.


( D ) Viability of cells with catalytically inactive A3A . U2OS - A3A - C106S cells were induced with dox to express the catalytically inactive A3A mutant ( C106S ) , depleted of CCT4 by siRNA , or combination . Cells were subsequently evaluated by live - dead staining assessed by FACS . Bar chart shows quantitation of FACS results averaged over three biological replicates . Statistical analysis was performed using a paired two - tailed t - test , n = 3 ; error bars , SEM . Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting . Ku - 86 is a loading control .

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(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.


( E ) Increased DNA damage signaling upon CCT depletion and A3A expression . Depletion of CCT7 in K562 - A3A cells was achieved by siRNA targeting . Expression of A3A was induced by dox treatment . Intracellular staining with anti - γH2AX antibody was analyzed by flow cytometry . Bar chart shows quantification of FACS results obtained over three biological replicates . Statistical analysis was performed using a two - tailed t - test , n = 3 ; error bars , SEM . Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment . Ku - 86 is a loading control . * * p - value < 0 . 01 , * * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 , n . s . non - significant .

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(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.


( C ) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity . Breast , bladder , and cervical cancer samples from the TCGA database were divided into CCT - mutated and non - mutated genomes , as above . The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average . Comparison by Wilcoxon rank sum test yielded p - values denoted .

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(C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted.


Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . Immunodetection of the indicated proteins after

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Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE


Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . ( C ) in - gel activity for CI and ( D ) CIV ( shown is a representative gel from two technical and two biological replicates ) .

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Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).


BNGE of whole - body zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) and its respective scaf1 + / + counterpart . Immunodetection of the indicated

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BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteins


BNGE of whole - body zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) and its respective scaf1 + / + counterpart . ( G ) in - gel activity of CI and ( H ) CIV ( representative gel from two technical and three biological replicates ) .

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BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).


I , 2D BNGE / SDS electrophoresis : 1st dimension with digitonin ( Dig ) and 2nd dimension with SDS , followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti - SCAF1 antibody . Asterisks indicate missing bands in scaf1Δ1 / Δ1 .

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I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.


A , Quantitative data - independent scanning ( DiS ) mass spectrometry protein profiles for CI , CIII and CIV . Vertical numbers indicate the BNGE gel slices . Left and right profiles correspond to scaf1 + / + and scaf1Δ1 / Δ1 animals , respectively . Red heatmap corresponds to the E - score from two proteotypic Scaf1 - derived tryptic peptides spanning sequences ascribed to the CIII interacting site ( in green ) and to the CIV interacting site ( in yellow ) . Thick blue line , marked with an asterisk , indicates the putative proteolytic site in Scaf1 .

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

A, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1.


A , B , Representative images from scaf1 + / + and scaf1 - / - ( A ) female and ( B ) male adult zebrafish .

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

A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish.


c - f , Size of scaf1Δ1 / Δ1 and scaf1Δ2 / Δ2 ( scaf1 - / - ) fish in comparison with their respective scaf1 + / + lines , ( C ) length and ( E ) weight of females ( Δ1 + / + n = 10 , Δ1 - / - n = 12 , Δ2 + / + n = 24 , Δ2 - / - n = 18 ) ; ( D ) length and ( F ) weight of males ( Δ1 + / + n = 16 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 23 ) .

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

c-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23).


G - K , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( G , I ) Adipose tissue area per total section area ( average of 3 sections / biological replicate ) and ( H , J ) adipocyte size ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) of females ( G , H ) ( Δ1 n = 8 , Δ2 n = 8 , same number of animals for homozygous mutants and controls ) and males ( I , J ) ( Δ1 n = 8 , Δ2 n = 7 , same number of animals for homozygous mutants and controls ) . K , Representative images of ventral fat deposits in females ( dotted lines ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).


L - N , Effect of Scaf1 loss of function on female fertility . ( L ) Number of eggs per clutch ( Δ1 + / + n = 12 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 10 ) . ( M ) Quantification of mature ovary follicles per ovary section ( average of three sections / biological replicate ) ( Δ1 n = 5 , Δ2 n = 8 ; same number of animals for homozygous scaf1 + / + and scaf1 - / - ) . ( N ) Representative images of H & E - stained ovaries . Dotted lines delineate adipose tissue .

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

L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.


Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) and scaf1 + / + fish ( n = 3 ) . ( A ) Representative images showing mitochondria .

O O O O O O O O B-CONTROLLED_VAR 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

Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (A) Representative images showing mitochondria.


Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) and scaf1 + / + fish ( n = 3 ) . ( B ) Mitochondria size ( 100 - 150 mitochondria per biological sample ) . ( C ) Cristae lumen width ( average of three cristae per mitochondria , 20 mitochondria per biological sample ) . Different biological replicates are represented with different color tones .

O O O O O O O O B-CONTROLLED_VAR 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

Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (B) Mitochondria size (100-150 mitochondria per biological sample). (C) Cristae lumen width (average of three cristae per mitochondria, 20 mitochondria per biological sample). Different biological replicates are represented with different color tones.


D , E , Mitochondrial DNA copy number per nuclear copy number in muscle in females ( D ) and males ( E ) ( Δ1 n = 6 , Δ2 n = 6 and same number for their respective controls ) .

O O O O O O O O O O O O O O O O O O O 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, E, Mitochondrial DNA copy number per nuclear copy number in muscle in females (D) and males (E) (Δ1 n=6, Δ2 n=6 and same number for their respective controls).


F , Survival curve of 4 days post - fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors ( three experimental replicates per biological replicate and three biological replicates ) .

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F, Survival curve of 4 days post-fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors (three experimental replicates per biological replicate and three biological replicates).


G , H , Oxygen consumption of 48 hours post - fertilization embryos using the XFe24 Seahorse analyzer , ( G ) Representative oxygen consumption rate ( OCR ) profile along time and ( H ) maximum OCR ( Δ1 n = 11 , Δ2 n = 11 , n = 10 , n = 11 respectively for their controls ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H, Oxygen consumption of 48 hours post-fertilization embryos using the XFe24 Seahorse analyzer, (G) Representative oxygen consumption rate (OCR) profile along time and (H) maximum OCR (Δ1 n=11, Δ2 n=11, n=10, n=11 respectively for their controls).


I , Maximum uncoupled ( FCCP ) OCR in isolated mitochondria from adult fish ( male and females Δ1 n = 4 and Δ2 n = 4 , and same number for their respective controls ) with the indicated site I [ pyruvate ( Pyr ) , glutamate ( Glu ) , malate ( Mal ) ] or site II [ succinate ( Succ ) ] substrates .

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

I, Maximum uncoupled (FCCP) OCR in isolated mitochondria from adult fish (male and females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with the indicated site I [pyruvate (Pyr), glutamate (Glu), malate (Mal)] or site II [succinate (Succ)] substrates.


J , K , Respiratory control ratio ( RCR ) ( State 3 / State4 ) ( J ) and P / O ratio ( K ) in isolated mitochondria from adult fish ( male and females Δ1 n = 4 , and same number for their respective controls ) with the indicated substrates .

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

J, K, Respiratory control ratio (RCR)(State 3/State4) (J) and P/O ratio (K) in isolated mitochondria from adult fish (male and females Δ1 n=4, and same number for their respective controls) with the indicated substrates.


A , Representative images of scaf1 - / - and scaf1 + / + fish fed with the indicated diets .

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

A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets.


B , C , Changes in ( B ) length and ( C ) weight over time ( Δ1 + / + n = 10 , Δ1 - / - n = 10 , Δ2 + / + n = 10 , Δ2 - / - n = 12 - 13 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13.


D - F , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( D ) Adipose tissue area per total section area ( average of three sections / biological replicate ) and ( E ) adipocyte size ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) ( Δ1 + / + n = 3 , Δ2 n = 8 ) . scaf1Δ1 and scaf1Δ2 are represented with circles and squares , respectively .

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

D-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively.


Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( F ) Representative images of ventral fat deposits ( dotted lines ) .

Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (F) Representative images of ventral fat deposits (dotted lines).


G , Number of eggs per clutch ( standard diet Δ1 + / + n = 8 , Δ1 - / - n = 8 , Δ2 + / + n = 7 , Δ2 - / - n = 12 , double diet Δ1 + / + n = 7 , Δ1 - / - n = 8 , Δ2 + / + n = 8 , Δ2 - / - n = 9 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Number of eggs per clutch (standard diet Δ1 +/+ n=8, Δ1 -/- n=8, Δ2 +/+ n=7, Δ2 -/- n=12, double diet Δ1 +/+ n=7, Δ1 -/- n=8, Δ2 +/+ n=8, Δ2 -/- n=9).


H , Representative images of H & E - stained ovaries . Black dotted lines outline the ovaries , blue dotted line indicates a mature follicle . I , Quantification of mature ovary follicles per ovary section ( average of three sections / biological sample ) ( Δ1 n = 2 - 3 , Δ2 n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of H&E-stained ovaries. Black dotted lines outline the ovaries, blue dotted line indicates a mature follicle. I, Quantification of mature ovary follicles per ovary section (average of three sections/biological sample) (Δ1 n=2-3, Δ2 n=6).


A , B , Representative images of scaf1 - / - and scaf1 + / + females ( A ) and males ( B ) fed with the indicated diets .

O O O O O O O B-CONTROLLED_VAR 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

A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.


C , D , Length of females ( C ) and males ( D ) after the indicated diets ( Δ1 + / + n = 5 , Δ1 - / - n = 5 , Δ2 + / + n = 5 , Δ2 - / - n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D, Length of females (C) and males (D) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).


E , F , Weight of females ( E ) and males ( F ) after the indicated diets ( Δ1 + / + n = 5 , Δ1 - / - n = 5 , Δ2 + / + n = 5 , Δ2 - / - n = 5 ) .

E, F, Weight of females (E) and males (F) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).


G , number of eggs per clutch ( standard diet : Δ1 + / + n = 4 , Δ1 - / - n = 3 , Δ2 + / + n = 3 , Δ2 - / - n = 2 , double diet : Δ1 + / + n = 5 , Δ1 - / - , n = 5 Δ2 + / + n = 1 , Δ2 - / - n = 2 ) . H , Quantification of mature ovary follicles per ovary section in haematoxylin - eosin ( H & E ) histological sections ( average of three sections / biological sample ) ( Δ1 n = 3 , Δ2 n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2). H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3).


adipose tissue quantification in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in females and males : adipose tissue area ( I , J )

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

adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males : adipose tissue area (I, J)


adipose tissue quantification in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in females and males adipocyte size ( K , L ) .

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

adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males adipocyte size (K, L).


A , Immunoblot of the indicated proteins of BNGE from female scaf1 + / + and scaf1 - / - whole zebrafish mitochondria for the indicated diet ( representative of n = 2 ) .

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

A, Immunoblot of the indicated proteins of BNGE from female scaf1+/+ and scaf1-/- whole zebrafish mitochondria for the indicated diet (representative of n=2).


B , Maximum uncoupled ( FCCP ) oxygen consumption rate in whole zebrafish mitochondria ( females Δ1 n = 4 and Δ2 n = 4 , and same number for their respective controls ) with glutamate ( Glu ) , malate ( Mal ) and succinate ( Succ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ).


RNAseq data from scaf1 + / + and scaf1 - / - skeletal muscle for the indicated diet ( standard diet scaf1 + / + and scaf1 - / - n = 4 , double diet scaf1 + / + n = 3 , scaf1 - / - n = 4 ) . ( C - F ) Volcano plots of differentially expressed genes ( DEGs ) . ( C ) Comparison between scaf1 - / - and scaf1 + / + zebrafish in standard diet . ( D ) Comparison between scaf1 - / - and scaf1 + / + zebrafish in double diet . ( E ) Comparison of scaf1 + / + zebrafish in double diet and standard diet . ( F ) Comparison of scaf1 - / - zebrafish in double diet and standard diet . In blue , significant DEGs ( p - adj < 0 . 05 , log2FC > | 1 | ) , in grey not significant DEGs , red circles represent non - significant differentially regulated OXPHOS genes , green circles represent significant differentially regulated OXPHOS genes , purple scaf1 ( cox7a2l ) .

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

RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (C-F) Volcano plots of differentially expressed genes (DEGs). (C) Comparison between scaf1-/- and scaf1+/+ zebrafish in standard diet. (D) Comparison between scaf1-/- and scaf1+/+ zebrafish in double diet. (E) Comparison of scaf1+/+ zebrafish in double diet and standard diet. (F) Comparison of scaf1-/- zebrafish in double diet and standard diet. In blue, significant DEGs (p-adj < 0.05, log2FC > |1|), in grey not significant DEGs, red circles represent non-significant differentially regulated OXPHOS genes, green circles represent significant differentially regulated OXPHOS genes, purple scaf1 (cox7a2l).


HMRs express RNAs . Heat maps show RNA expression levels in 16 . 5 dpc prospermatogonia . Only uniquely mapped RNA - seq reads were used for the analyses .

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

HMRs express RNAs. Heat maps show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses.


metaplots ( B ) show RNA expression levels in 16 . 5 dpc prospermatogonia . Only uniquely mapped RNA - seq reads were used for the analyses . Positions ( 1 ) - ( 4 ) in ( A and B ) ( green arrows ) indicate the same positions ( ± 2 kbp from the centers ) . The lower chart in ( B ) represents mappability defined as the percentage of unique sequences in all possible 50 - nt sequences ( see Appendix Figure S2B ) . TSS isoforms are included in 55798 TSSs . Low mappability at the centers of HMR _ - L1s is due to high occurrence of repeat ( retrotransposon ) sequences . RPM , reads per million mapped reads .

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

metaplots (B) show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. Positions (1)-(4) in (A and B) (green arrows) indicate the same positions (±2 kbp from the centers). The lower chart in (B) represents mappability defined as the percentage of unique sequences in all possible 50-nt sequences (see Appendix Figure S2B). TSS isoforms are included in 55798 TSSs. Low mappability at the centers of HMR_-L1s is due to high occurrence of repeat (retrotransposon) sequences. RPM, reads per million mapped reads.


Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons ( HMR _ - L1s ) . Metaplots show the average ChIP - seq signals across HMR _ - L1s , transcription start sites ( TSSs ) , and randomly selected sites . The signals were normalized by either H3 or input signal . The results shown in heat maps were not normalized by H3 or input .

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Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_-L1s). Metaplots show the average ChIP-seq signals across HMR_-L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heat maps were not normalized by H3 or input.


The initiation form of Pol2 with an unphosphorylated carboxy terminal domain ( Pol2 null - P CTD ) is highly enriched in HMR _ - L1s .

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

The initiation form of Pol2 with an unphosphorylated carboxy terminal domain (Pol2 null-P CTD) is highly enriched in HMR_-L1s.


RNA - immunoprecipitation ( RIP ) analyses of EGFP - MIWI2 and EGFP . EGFP - MIWI2 and EGFP were immunoprecipitated from 16 . 5 dpc prospermatogonia , and long RNAs bound to these proteins and input RNAs were sequenced . RNAs expressed from HMR _ - L1s and piRNA clusters , but not Ensembl and prospermatogonium - expressed mRNAs , are overall enriched by immunoprecipitation ( IP ) of EGFP - MIWI2 ( left ) . This enrichment is not observed when EGFP is immunoprecipitated ( right ) . The numbers of mRNAs , piRNA clusters , and HMR _ - L1s analyzed were shown in parentheses . Prospermatogonium - expressed mRNAs are those that are highly and specifically expressed in prospermatogonia . The bottom and top of boxes represent 25 % percentile and 75 % percentile , respectively . The line in the box represents the median . Circles represent outliers , and the bottom ( top ) of whiskers represents the minimum ( maximum ) values except for outliers .

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

RNA-immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_-L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_-L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers.


RIP analyses of EGFP - MIWI2 in newborn testes from WT and Mili KO mice . RNA bound to EGFP - MIWI2 and input RNA were sequenced . All RNAs derived from each class was counted for this analysis .

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RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA were sequenced. All RNAs derived from each class was counted for this analysis.


HMR _ - L1s preferentially generate piRNAs . X - and Y - axes represent expression levels of long RNAs ( 16 . 5 dpc prospermatogonia ) as determined by RNA - seq and MIWI2 - bound piRNAs ( 18 . 5 dpc testes ) as determined by small RNA - seq , respectively . Circles in the charts represent individual mRNAs , piRNA clusters and HMR _ - L1s . More than half of the HMR _ - L1s ( right ) are located in the area of y > x ( upper part of the diagonal ) , whereas most mRNAs ( left ) are located in the area of y < x ( lower part of the diagonal ) . RPKM , reads per kilobase per million mapped reads .

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HMR_-L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters and HMR_-L1s. More than half of the HMR_-L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads.


Frequent 10 - nt overlap between sense and antisense piRNAs derived from HMR _ - L1s . Top 100 HMR _ - L1s that generate the largest number of unique piRNAs were analyzed . Frequencies of 1 − 18 - nt overlaps were analyzed for each HMR _ - L1 .

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Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_-L1s. Top 100 HMR_-L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1−18-nt overlaps were analyzed for each HMR_-L1.


piRNAs derived from HMR _ - L1s are enriched in adenine at their 10th nucleotide . HMR _ - L1s generating more than 40 unique piRNAs were analyzed ( 678 HMR _ - L1s ) . The proportions of piRNAs with adenine ( left ) and uridine ( right ) at the indicated positions were analyzed for each HMR _ - L1 . See A for the explanation of box plots .

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piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.


DNA methylation status of a portion of the Rasgrf1 differentially methylated region ( DMR ) in 0 − 2 - dpp Mili + / − and Mili − / − prospermatogonia . Regions corresponding to B , C , D , E , and F are demethylated in Mili − / − prospermatogonia . Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing ( see below ) . The top dark gray bar represents a portion of the Rasgrf1 gametic DMR ( Tomizawa et al , 2011 ) . The CpG cluster is shown . The deleted RMER4B sequence is indicated .

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DNA methylation status of a portion of the Rasgrf1 differentially methylated region (DMR) in 0−2-dpp Mili+/− and Mili−/− prospermatogonia. Regions corresponding to B, C, D, E, and F are demethylated in Mili−/− prospermatogonia. Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing (see below). The top dark gray bar represents a portion of the Rasgrf1 gametic DMR (Tomizawa et al, 2011). The CpG cluster is shown. The deleted RMER4B sequence is indicated.


DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B / ΔRMER4B and control spermatogonia are shown . The regions examined are shown in ( A ) . Black ( white ) circles represent methylated ( unmethylated ) CpGs .

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DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B/ΔRMER4B and control spermatogonia are shown. The regions examined are shown in (A). Black (white) circles represent methylated (unmethylated) CpGs.


Rasgrf1 RMER4B functions in cis . Wild - type and mutant alleles of the Rasgrf1 DMR in Rasgrf1 + / ΔRMER4B spermatogonia were analyzed separately .

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Rasgrf1 RMER4B functions in cis. Wild-type and mutant alleles of the Rasgrf1 DMR in Rasgrf1+/ΔRMER4B spermatogonia were analyzed separately.


Structure of the chr7 piRNA cluster . Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP - seq , RNA - seq , and small RNA - seq . Only uniquely mapped reads were used for displaying the sequencing result . For RNA - seq and small RNA - seq , only the results of minus strand are shown , because plus strand does not express appreciable level of RNA . A promoter region deleted is shown above the putative piRNA precursor . RPM , Read Per Million .

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Structure of the chr7 piRNA cluster. Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP-seq, RNA-seq, and small RNA-seq. Only uniquely mapped reads were used for displaying the sequencing result. For RNA-seq and small RNA-seq, only the results of minus strand are shown, because plus strand does not express appreciable level of RNA. A promoter region deleted is shown above the putative piRNA precursor. RPM, Read Per Million.


Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter / Δpromoter testes . Unchanged expression of piRNAs from chr10 cluster ( bottom ) shows chr7 cluster is specifically affected . Only uniquely mapped piRNAs are shown .

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Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter/Δpromoter testes. Unchanged expression of piRNAs from chr10 cluster (bottom) shows chr7 cluster is specifically affected. Only uniquely mapped piRNAs are shown.


DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔpromoter / Δpromoter . See Figures 4A and B for information about the region examined .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔpromoter/Δpromoter. See Figures 4A and B for information about the region examined.


Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B / ΔRMER4B mice . Top panel shows overall view of chr7 piRNA cluster , and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster . Only uniquely mapped piRNAs are shown .

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Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. Top panel shows overall view of chr7 piRNA cluster, and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster. Only uniquely mapped piRNAs are shown.


DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.


DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatocytes , spermatids , and sperms ( D ) from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatocytes, spermatids, and sperms (D) from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.


DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B / ΔRMER4B mice . DNA methylation status of the Rasgrf1 DMR in E12 . 5 whole embryos is shown . E12 . 5 whole embryos were obtained by the crosses shown in the panel . Two independent bisulfite - treated DNA sample were examined for Homo13 embryo .

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DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.


A Domain architecture of S . pombe bromodomain AAA - ATPases . The D1 and D2 ATPase domains ( AAA ) are shaded blue and orange , respectively , and the bromodomain ( BD ) is grey . The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [ 74 ] using the scoring matrix BLOSUM50 .

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A Domain architecture of S. pombe bromodomain AAA-ATPases. The D1 and D2 ATPase domains (AAA) are shaded blue and orange, respectively, and the bromodomain (BD) is grey. The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [74] using the scoring matrix BLOSUM50.


B Venn diagrams showing overlap between genes up - regulated ( 1 . 5 fold ) in abo1Δ mutants with genes up - regulated under the indicated condition , along with the significance of the overlaps ( based on hypergeometric distribution ) .

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B Venn diagrams showing overlap between genes up-regulated (1.5 fold) in abo1Δ mutants with genes up-regulated under the indicated condition, along with the significance of the overlaps (based on hypergeometric distribution).


A Normalised cumulative nucleosome ( 150 ± 30 bp size class ) position frequency profiles for 4013 S . pombe genes aligned at the transcription start site ( TSS ) plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values ( calculated using a two - tailed unpaired t - test ) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ( " - 4 " nucleosome and " + 4 " nucleosome ) are shown .

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A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ("-4" nucleosome and "+4" nucleosome) are shown.


B Nucleosome position frequency values for the coding regions of 4013 S . pombe genes were k - means clustered ( k = 9 ) using the abo1Δ data from biorep 1 ( low MNase ) and displayed with positive values coloured yellow and other values coloured blue ( left - hand panel ) . The cluster order was then used to display the equivalent wild type frequency values in the right - hand panel .

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B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.


C Level of histone gene mRNAs was determined by qRT - PCR . Data are the mean of four independent biological repeats and error bars are ± SEM . Two - tailed unpaired t - tests showed no significant differences ( P > 0 . 05 ) between wild type and abo1Δ cells .

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C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells.


D Whole cell extracts were subjected to western blotting with histone H3 ( Abcam ) , histone H2A ( Abcam ) and tubulin antibodies . Examples of the primary data are shown ( left ) along with a quantification of histone H2A and H3 levels normalized to tubulin ( right ) . Data are the mean of at least three independent repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test .

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D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.


A Whole cell extracts ( WCE ) were prepared from the indicated strains , immunoprecipitated ( IP ) with anti - GFP antibody and subjected to western blotting with anti - V5 - Pk ( Serotec ) and anti - GFP ( Life Technologies ) antibodies . Data are representative of three independent biological repeats . B As for A .

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A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.B As for A.


C RNA purified from wild type , abo1Δ and spt16 - 18 cells was analysed by northern blotting using a probe to the 3 ′ end of spbc19c7 . 11 ( top panel ) . RNA ( 5 µg ) used for northern blotting was analysed on an ethidium bromide stained 1 % TAE agarose gel ( bottom panel ) . Data are representative of two independent biological repeats .

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C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.


D RNA purified from wild type , and abo1Δ cells was analysed by strand specific RT - PCR . RNA from hip1Δ cells was analysed as a control . One primer , complementary to either the forward or reverse transcripts , was included during the reverse transcription step the second primer was then added during PCR amplification . Control reactions omitting the reverse transcription step ( - RT ) were included to demonstrate the absence of contaminating genomic DNA . Data are representative of two independent biological repeats .

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D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.


E The indicated strains were subjected to ChIP analysis with anti - GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - values were calculated using a two - tailed unpaired t - test . Significant ( P < 0 . 05 ) values are indicated .

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E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated.


F ChIP analysis of histone H3 levels at act1 + was determined by qPCR . Data are the mean of three independent biological repeats and error bars represent ± SEM . P - value was calculated using a two - tailed unpaired t - test .

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F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test.


G The indicated strains were subjected to ChIP analysis as described for E .

G The indicated strains were subjected to ChIP analysis as described for E.


H Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with MMS ( 0 . 006 % ) . Plates were incubated for 3 - 5 days at 30 ° C or 7 days at 20 ° C . Images are representative of three independent biological repeats .

H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.


A The position of ura4 + reporter alleles in centromere 1 is shown in the top panel . Strains containing the imr : : ura4 + allele were grown to log phase in YE5S medium , subjected to 5 - fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5 - FOA ( 1 mg / ml ) . Data are representative of three independent biological repeats .

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A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.


B Strains carrying the otr : : ura4 + were analysed as described for A .

B Strains carrying the otr::ura4+ were analysed as described for A.


C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT - PCR . Data is the mean of three independent repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired t - test .

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C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.


D RNA was purified from the indicated strains and subjected to qRT - PCR for the subtelomeric gene , tlh1 + . Data is the mean of three biological repeats and error bars indicate ± SEM . P - value was calculated using a two - tailed unpaired Student ' s t - test .

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D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test.


E The position of insertion of ade6 + reporter allele in the silent mat locus is shown in the top panel . Log phase cells containing the mat3 - M : : ade6 + allele were subjected to five - fold serial dilution and spotted onto YE5S agar plates lacking adenine ( Low Ade ) were incubated for 4 days at 30 ° C . Data are representative of at least three independent biological repeats . Note , microarray analysis indicates that deletion of abo1 + does not influence the expression of ade6 + ( or ura4 + ) when these genes are present at their normal genomic loci .

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E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.


A ChIP analysis was performed on wild type ( untagged ) and abo1 - GFP cells and the resulting DNA analysed by qPCR for centromeric ( dh , dg and imr ) repeat sequences . Data are the mean of four independent biological repeats and error bars represent ± SEM . P - values calculated using a two - tailed unpaired t - test indicates that all loci are significantly enriched ( P < 0 . 05 ) relative the untagged control .

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A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.


B The indicated strains expressing GFP - Swi6 were subjected to ChIP analysis . The level of centromeric ( dg ) repeat sequences relative to the euchromatic control locus , adh1 + was determined by qPCR and scaled to a clr4Δ ( - H3 K9me control ) mutant . Data are the mean of two independent ChIP experiments and error bars represent the range of the data .

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B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.


C ChIP analysis of histone H3 levels at centromeric ( dg ) repeat sequences was determined by qPCR . Data are the mean of three independent ChIP experiments and error bars are ± SEM . P - values were calculated using a two - tailed unpaired t - test .

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C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.


D . ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences regions in wild type and pob3Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant difference ( P > 0 . 05 ) for wild type and pob3Δ cells .

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D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.


E ChIP analysis of Abo1 - GFP at centromeric ( dh and imr ) repeat sequences in wild type and pob3Δ cells . Data are the mean of duplicate experiments and error bars represent the range of the data .

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E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.


F ChIP analysis of Abo1 - GFP over the dh and imr regions in wild type and swi6Δ cells . Data are the mean of three independent experiments and error bars represent ± SEM . P - values , calculated using a two - tailed unpaired t - test , indicated no significant differences ( P > 0 . 05 ) for wild type and swi6Δ cells .

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F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.


A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT - PCR , normalised to act1 + mRNA and scaled relative to the wild type level . Data are the mean of three independent biological repeats and error bars represent ± SEM .

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A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT-PCR, normalised to act1+ mRNA and scaled relative to the wild type level. Data are the mean of three independent biological repeats and error bars represent ±SEM.


B Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β - galactosidase assays . Data are the mean of three independent biological repeats and error bars represent ± SEM . C As for

O O O O O O O O O O O O O O O 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 Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β-galactosidase assays. Data are the mean of three independent biological repeats and error bars represent ±SEM.C As for B


D ChIP DNA samples from wild type ( untagged ) and abo1 - GFP cells were analysed by qPCR for Tf2 LTR . Data are the mean of four independent biological repeats and error bars represent ± SEM .

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D ChIP DNA samples from wild type (untagged) and abo1-GFP cells were analysed by qPCR for Tf2 LTR. Data are the mean of four independent biological repeats and error bars represent ±SEM.


E Normalised cumulative nucleosome ( 150 ± 30 bp ) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase ( biorep1 ) and high MNase ( biorep 2 ) datasets . P - values were calculated using a two - tailed unpaired t - test .

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E Normalised cumulative nucleosome (150 ± 30 bp) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase (biorep1) and high MNase (biorep 2) datasets.P-values were calculated using a two-tailed unpaired t-test.


A Log phase cells were subjected to five - fold serial dilution and spotted onto rich ( YE5S ) agar or agar supplemented with TBZ ( 15 μg / ml ) and incubated for 3 - 5 days 30 ° C . Data are representative of at three independent biological repeats .

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A Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with TBZ (15 μg/ml) and incubated for 3-5 days 30°C. Data are representative of at three independent biological repeats.


B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored ( red - white ) colonies . At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis .

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B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored (red-white) colonies. At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis.


C Heterothallic ( h90 ) strains were cultured on nitrogen limiting ( EMMG ) medium for 3 days at 25 ° C to induce mating and meiosis . The resulting asci were stained with DAPI and visualised by fluorescence microscopy . Data are representative of three 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 O

C Heterothallic (h90) strains were cultured on nitrogen limiting (EMMG) medium for 3 days at 25°C to induce mating and meiosis. The resulting asci were stained with DAPI and visualised by fluorescence microscopy. Data are representative of three biological repeats


D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4 - 5 days at 30 ° C . Data are the mean of three independent biological repeats and error bars are ± SEM . P - value was calculated using a two - tailed unpaired 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

D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4-5 days at 30°C. Data are the mean of three independent biological repeats and error bars are ±SEM. P-value was calculated using a two-tailed unpaired t-test.


H & amp ; amp ; E staining of adult head sections of ( A ) repo > GFP . nls and ( B ) repo > Shits1 at 29 ° C for 3 days . Lamina degeneration was identified as vacuoles in ( B ) . Cryosection of adult ( C ) repo > H2B - RFP exhibited the expression of the nuclear red fluorescent protein ( RFP ) in the glia ( epithelialglialnucleus : arrowhead ) and ( D ) repo > Shits1 at 28 ° C for 12 days . Vacuoles were identified in the lamina neuropile ( D ) . Epithelial ( arrowhead ) ( E ) , marginal ( arrow ) and distal satellite ( arrowhead ) glianuclei ( F ) are labeled by HisCl - GAL4 and NP2109 - GAL4 , respectively . Note that HisCl - GAL4 is not expressed in all epithelialglia . ( G , H ) Weak lamina vacuolization was identified in HisCl > Shits1 ( G , 0 . 92 % ) and NP2109 > Shits1 ( H , 1 . 46 % ) at 29 ° C for 14 days . ( I , J ) A single MARCMglia clone ( GFP , green ) expressed Shits1 at 21 ° C ( I ) and 29 ° C ( J ) . ( J ) A vacuole occurred within a glial clone . DAPI ( white ) stains the nuclei ( C , D , and G - J ) . ( K ) The percentage of the vacuole area in the lamina of the repo > Shits1flies at 28 ° C progressively increased . n indicated in each column . P - values were calculated using one - way ANOVA with Bonferroni ' s post - test . ( L ) When repo > Shits1flies were shifted to 28 ° C for 12 days and then shifted to 17 ° C for 9 additional days , the vacuolization was not alleviated . P - values were calculated using one - way ANOVA with Tukey ' s post - test . n indicated in each column .

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

H&amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.


( A - E ) Horizontal sections of the adult head lamina cartridge . ( A ) In repo > GFP . nls flies , two lamina neurons L1 , L2 axons ( L ) and R1 - 6 axons ( blue area ) were surrounded by the electron - dense cytoplasm of the epithelial glia ( G ) . The section was examined at three different depths , and the size of a single cartridge is not significantly different at different depths . ( B - D ) repo > Shits1 adults maintained at 29 ° C for 4 days . Axons were enlarged but intact . A large number of small and medium vacuoles were identified in the epithelial glia . ( C ) Small vacuoles in the glia that contained a double - membrane structure ( arrowhead ) . ( C ' ) Higher magnification of the boxed area in ( C ) . ( D ) A double - membrane autophagosome - like structure ( arrowhead ) in the glialcytoplasm . ( E ) In repots > DERDN adults maintained at 29 ° C for 2 days , large vacuoles were identified in between the lamina cartridges . N : glialnuclei . ( E ' ) Higher magnification of the boxed area in ( E ) , which shows autophagosome - like vesicles ( arrowhead ) in the gliacytoplasm .

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

(A-E) Horizontal sections of the adult head lamina cartridge. (A) In repo>GFP.nls flies, two lamina neurons L1, L2 axons (L) and R1-6 axons (blue area) were surrounded by the electron-dense cytoplasm of the epithelial glia (G). The section was examined at three different depths, and the size of a single cartridge is not significantly different at different depths. (B-D) repo>Shits1 adults maintained at 29°C for 4 days. Axons were enlarged but intact. A large number of small and medium vacuoles were identified in the epithelial glia. (C) Small vacuoles in the glia that contained a double-membrane structure (arrowhead). (C') Higher magnification of the boxed area in (C). (D) A double-membrane autophagosome-like structure (arrowhead) in the glialcytoplasm. (E) In repots>DERDN adults maintained at 29°C for 2 days, large vacuoles were identified in between the lamina cartridges. N: glialnuclei. (E') Higher magnification of the boxed area in (E), which shows autophagosome-like vesicles (arrowhead) in the gliacytoplasm.


( A ) Rh1 > lacZ + H2B - RFP exhibited nuclearRFP expression in the R1 - 6 photoreceptors ( red ) . ( B ) Rh1 > lacZ + Shits1 maintained at 29 ° C for 14 days resulted in lamina vacuolization . ( C ) Pan - Rh7 > H2B - RFP exhibited expression in R7 ( arrow ) and R8 ( arrowhead ) ( red ) . ( D ) Pan - Rh7 > Shits1 incubated at 29 ° C for 14 days did not exhibit lamina degeneration . ( E ) Lamina monopolar ( L ) neurons ( red ) were selectively labeled by the Ln - GAL4 driven H2B - RFP with repo - GAL80 . ( F ) Ln > Shits1 , repoGAL80 shifted to 29 ° C for 14 days did not exhibit lamina degeneration . ( G ) R1 - 6 photoreceptors were killed in Rh1ts > lacZ + Hid shifted to 29 ° C for 14 days . Rh1ts indicates tubGAL80ts ; Rh1 - GAL4 . Degeneration was induced in the lamina in addition to the retina . ( H ) rdgC306 / + mutant exposed to constant light for 14 days exhibited degeneration in the retina , as well as the lamina . DAPI : nuclei ( white in A - H ) . ( I , K ) Rh1 > lacZ + Shits1 at 29 ° C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus . Arrow : vacuole with internal debris . Arrowhead : large vacuole . ( J ) Lamina cartridge of Rh1 > lacZ + H2B - RFP . N indicates glialnuclei . ( L ) The percentage of the vacuole area in the lamina at 29 ° C for 14 days was examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . Scale bar : 20 μm .

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

(A) Rh1>lacZ+H2B-RFP exhibited nuclearRFP expression in the R1-6 photoreceptors (red). (B) Rh1>lacZ+Shits1 maintained at 29°C for 14 days resulted in lamina vacuolization. (C) Pan-Rh7>H2B-RFP exhibited expression in R7 (arrow) and R8 (arrowhead) (red). (D) Pan-Rh7>Shits1 incubated at 29°C for 14 days did not exhibit lamina degeneration. (E) Lamina monopolar (L) neurons (red) were selectively labeled by the Ln-GAL4 driven H2B-RFP with repo-GAL80. (F) Ln>Shits1, repoGAL80 shifted to 29°C for 14 days did not exhibit lamina degeneration. (G) R1-6 photoreceptors were killed in Rh1ts>lacZ+Hid shifted to 29°C for 14 days. Rh1ts indicates tubGAL80ts; Rh1-GAL4. Degeneration was induced in the lamina in addition to the retina. (H) rdgC306/+ mutant exposed to constant light for 14 days exhibited degeneration in the retina, as well as the lamina. DAPI: nuclei (white in A-H). (I, K) Rh1>lacZ+Shits1 at 29°C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus. Arrow: vacuole with internal debris. Arrowhead: large vacuole. (J) Lamina cartridge of Rh1>lacZ+H2B-RFP. N indicates glialnuclei. (L) The percentage of the vacuole area in the lamina at 29°C for 14 days was examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( A ) repots > Shits1 + H2B - RFP , ( B ) repots > Shits1 + Egfrλtop and ( C ) repots > Shits1 + RlSem incubated for 12 days at 28 ° C . ( D ) repots > DERDN for 7 days at 28 ° C . ( E ) Epithelial glia MARCM clone and ( F ) Egfrco mutant glia MARCM clone ( labeled with Tomato , green ) . The penetrance is indicated as the number of samples with vacuoles over the total number of samples . DAPI : nuclei ( white in A - F ) . ( G ) Percentage of vacuole area in the lamina neuropile of ( A - D ) . The P - values were calculated using one - way ANOVA with Dunnett ' s post - test . ( H ) Percentage of vacuole area in the lamina of repots > Shits1 flies in the indicated genetic background . The adults were shifted to 28 ° C for 12 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . Scale bar : 20 μm .

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

(A) repots>Shits1+H2B-RFP, (B) repots>Shits1+Egfrλtop and (C) repots>Shits1+RlSem incubated for 12 days at 28°C. (D) repots>DERDN for 7 days at 28°C. (E) Epithelial glia MARCM clone and (F) Egfrco mutant glia MARCM clone (labeled with Tomato, green). The penetrance is indicated as the number of samples with vacuoles over the total number of samples. DAPI: nuclei (white in A-F). (G) Percentage of vacuole area in the lamina neuropile of (A-D). The P-values were calculated using one-way ANOVA with Dunnett's post-test. (H) Percentage of vacuole area in the lamina of repots>Shits1 flies in the indicated genetic background. The adults were shifted to 28°C for 12 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.