( f ) U937 were treated with indicated siRNA infected with virulent M . tuberculosis H37Rv . Autophagy was induced by starvation , or were kept in full medium , as indicated . Colony forming units were counted , expressed as a of number of plated colonies from untreated , to assess survival . Data are means ± s . e . m . ( n = 6 ; two independent transfections , 6 independent infections ) . Asterisk indicates P 0 . 05 , dagger indicates P > 0 . 05 ( t - ) for indicated data .

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(f) U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data.


( b ) HeLa were transfected with plasmids encoding - tagged IRGMb , IRGMd WT ( wild - type IRGMd ) or IRGMS47N mutant ( control ; vector expressing GFP only ) , after 48 h were labelled with MTR imaged by live - confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm.


( c ) of green red channels along a drawn through two adjacent , one GFP - IRGMd positive one GFP - IRGMd negative , as indicated in inset .

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(c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative, as indicated in the inset.


( d ) of GFP + MTR + from an experiment performed as in b .

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(d) Quantification of GFP+ MTR+ cells from an experiment performed as in b.


( e ) Nitrocellulose filters spotted with indicated lipids were incubated with GST or GST - IRGM . Binding was detected by immunoblotting .

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(e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting.


( f ) Agarose beads ( control beads ) cardiolipin - bound agarose beads ( CL beads ) were incubated with GST or GST - IRGMd . were eluted from beads immunoblotted . Two lanes on left indicate immunoblotting of samples used to incubate beads .

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(f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads.


( g ) Nitrocellulose filters spotted with indicated lipids ( defined in e ) were incubated with wild - type IRGMd , IRGMdS47N , IRGM isoform IRGMb at indicated concentrations . GST control is shown in Supplementary Information , Figure S1d . Membranes in e - g were probed with anti - GST antibody .

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(g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.


( a ) HeLa transfected with plasmids encoding GFP or GFP - IRGMd were stained with MTR imaged by live - confocal microscopy . Scale bars , 5 μm . ( b - d ) of mitochondrial morphologies in transfected with IRGMd , IRGMdS47N or IRGMb GFP fusions ( control express GFP only ) .

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(a) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b-d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only).


( e ) HeLa were transfected with indicated siRNAs vector encoding GFP - IRGMd , labelled with MTR . of with mitochondrial morphologies ranging from normal , punctiform ( dots ) elongated were quantified .

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(e)HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified.


( f ) HeLa were transfected with indicated siRNAs vector encoding GFP - IRGMd ( or vector encoding GFP only , as a control ) labelled with MTR . Percentages of GFP + with MTR at 48 h were quantified .

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(f) HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified.


( g ) Confocal microscopy of HeLa transfected with vector encoding GFP - DRP1 ( A ) or GFP - IRGMd ( B - G ) labelled with MTR ( A - D ) or antibodies against COX - IV ( F ) . A , steady - state DRP1 distribution ; B - D , translocation of IRGMd from cytosol to mitochondria at indicated times post - transfection ; E - G , co - localization of IRGMd COX - IV ( G is merged from E F ) . Scale bars , 5 μm .

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(g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP-DRP1 (A) or GFP-IRGMd (B-G) and labelled with MTR (A-D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B-D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E-G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm.


( h ) HeLa , co - transfected with GFP - IRGMd , control , BECN1 or ATG7 siRNA , were labelled with MTR 48 h post - transfection , imaged by live - microscopy , percentages of GFP + that were also MTR + were quantified .

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(h) HeLa cells, co-transfected with GFP-IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.


( i ) from Atg7 wild - type ( WT ) or Atg7 − / − MEFs transfected with vectors encoding GFP - IRGMd for 48 h were stained with MTR , imaged , of GFP + that were also MTR + was quantified .

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(i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP-IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified.


( j ) Wild - type W2 ( Bax / Bak + / + ) or mutant D3 ( Bax / Bak − / − ) BMK were transfected with vectors encoding GFP - IRGMd after 48 h were labelled with MTR . were imaged by live - microscopy , percentages of GFP + that were also MTR + were quantified .

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(j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP-IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.


( k ) HeLa were transfected with vectors encoding GFP - IRGMd , treated with z - VAD as indicated , after 48 h stained with MTR . were imaged by live - microscopy , percentages of GFP + that were also MTR + were quantified . Data are means ± s . e . m . ( n = 3 ) . Dagger indicates P ≥ 0 . 05 , asterisk indicates P 0 . 05 double asterisks indicate P 0 . 01 ( t - ) for indicated data .

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(k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.


( a ) of HeLa transfected with vectors encoding GFP or GFP - IRGMd that were rounded 48 h post - transfection .

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(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP-IRGMd that were rounded 48 h post-transfection.


( b ) HeLa transfected with plasmids encoding GFP or GFP - IRGMd were stained with 7 - AAD after 48 h percentages of GFP + that were also 7 - AAD + were quantified .

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(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.


( c ) HeLa transfected with vectors encoding GFP - IRGMd versus GFP - IRGMdS47N YFP - IRGMb ( vector encoding GFP only was transfected as a control ) were stained with 7 - AAD after 48 h , percentages of GFP + ( for IRGMd IRGMS47N ) or YFP + ( for IRGMb ) that were also 7 - AAD + were quantified .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

(c) HeLa cells transfected with vectors encoding GFP-IRGMd versus GFP-IRGMdS47N and YFP-IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified.


( d ) HeLa were transfected with plamsids encoding GFP - IRGMd , GFP - IRGMDS47N or YFP - IRGMb ( vector encoding GFP only was transfected as a control ) , after 48 h were stained with propidium iodide ( PI ) . Percentages of GFP + or YFP + that were also PI + were quantified .

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(d) HeLa cells were transfected with plamsids encoding GFP-IRGMd, GFP-IRGMDS47N or YFP-IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified.


( e ) of HeLa transfected with vectors encoding GFP - IRGMd ( bottom ) were stained for active caspases 3 7 using FLICA dye . Staurosporine ( STS ; middle ) was used as a positive control . were transfected with vectors encoding GFP only as a control ( top ) .

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(e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top).


( f ) HeLa were transfected with plasmids encoding GFP - IRGMd were stained with antibodies against HMGB1 ( absence of HMGB1 stain is a marker of HMGB1 release ) or DAPI . Top : indicate GFP - IRGMd ( left ) , HMGB1 ( middle ) DAPI ( right ) localization . Bottom : phase - contrast microscopy image of ( left ) image merged from other panels ( right ) . Scale bars , 5 μm .

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(f) HeLa cells were transfected with plasmids encoding GFP-IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP-IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm.


( g - i ) were transfected with plasmids encoding GFP or GFP - IRGMd , control or DRP1 siRNA , as indicated . After 48 h , ( g ) were stained with 7 - AAD percentages of GFP + that were also 7 - AAD + were quantified , ( h ) rounded were quantified , or ( i ) were stained with HMGB1 HMGB1 + nuclei were quantified . Data are means ± s . e . m . ( n = 3 ) . Asterisk indicates P 0 . 05 double asterisk indicate P 0 . 01 ( t - ) .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O 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-i) Cells were transfected with plasmids encoding GFP or GFP-IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).


( a , b ) HeLa were transfected with plasmids encoding GFP , CFP - IRGMa , GFP - IRGMc or GFP - IRGMd , after 24 h ( a ) or 48 h ( b ) , stained with MTR . Percentages of positively transfected that were also MTR + were then quantified . ( c , d ) were transfected as in a , b , after 24 h ( c ) or 48 h ( d ) percentages of positively transfected that were also rounded by were quantified .

O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with plasmids encoding GFP, CFP-IRGMa, GFP-IRGMc or GFP-IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified.


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Number of EMPs per equivalent ( e . e . ) of wild type Sl / Sl E9 . 5 E11 . 5 YS , determined by flow cytometry ( panels in B , D ) . E9 . 5 Kit + CD41 + CD16 / 32 + EMP numbers are mean ± SD from 4 wild type 3 Sl / Sl biological replicates , with each replicate consisting of single or 2 pooled YS of same genotype . Total number of analyzed : 7 + / + ( 14 - 23sp ) , 5 Sl / Sl ( 17 - 25sp ) . E11 . 5 Kit + CD41 + EMP numbers are mean ± SD of 5 + / + 6 Sl / Sl YS analyzed individually over 2 independent experiments . Total live per YS : 1 . 7 ± 0 . 3x105 ( wt ) , 1 . 7 ± 0 . 2x105 ( Sl / Sl ) at E9 . 5 ; 4 . 2 ± 1 . 4x105 ( wt ) , 4 . 0 ± 2 . 2x105 ( Sl / Sl ) at E11 . 5 .

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Number of EMPs per embryo equivalent (e.e.) of wild type and Sl/Sl E9.5 and E11.5 YS, determined by flow cytometry (panels in B,D). E9.5 Kit+ CD41+ CD16/32+ EMP numbers are the mean±SD from 4 wild type and 3 Sl/Sl biological replicates, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of embryos analyzed: 7 +/+ (14-23sp), 5 Sl/Sl (17-25sp). E11.5 Kit+ CD41+ EMP numbers are the mean±SD of 5 +/+ and 6 Sl/Sl YS analyzed individually over 2 independent experiments. Total live cells per YS: 1.7±0.3x105 (wt), 1.7±0.2x105 (Sl/Sl) at E9.5; 4.2±1.4x105 (wt), 4.0±2.2x105 (Sl/Sl) at E11.5.


Example of flow cytometry of E9 . 5 YS EMPs quantified in panel ( A ) . Analyses were performed on Ter119 - live .

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Example of flow cytometry analysis of E9.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.


Number of CFU - C in wild type Sl / Sl E9 . 5 E11 . 5 YS . E9 . 5 data ( mean ± SD ) are from 3 wild type 2 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of single or 2 pooled YS of same genotype . Total number of analyzed : 5 + / + ( 17 - 23sp ) , 3 Sl / Sl ( 17 - 25sp ) over 2 independent experiments . For E11 . 5 , data are from 4 wild type 6 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of single or 2 pooled YSs of same genotype . Total number of analyzed : 6 + / + , 7 Sl / Sl over 4 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid .

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Number of CFU-C in wild type and Sl/Sl E9.5 and E11.5 YS. E9.5 data (mean±SD) are from 3 wild type and 2 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of analyzed embryos: 5 +/+ (17-23sp), 3 Sl/Sl (17-25sp) over 2 independent experiments. For E11.5, data are from 4 wild type and 6 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YSs of the same genotype. Total number of embryos analyzed: 6 +/+, 7 Sl/Sl over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.


Example of flow cytometry of E11 . 5 YS EMPs quantified in panel ( A ) . Analyses were performed on Ter119 - live .

Example of flow cytometry analysis of E11.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.


Confocal WM - IF of CD31 + pHH3 + Kit + proliferating EMPs ( arrowheads ) in E9 . 5 wild type Sl / Sl YS . A single 2 . 5μm - thick Z section is shown . For each YS , CD31 + Kit + pHH3 + were counted in 3 to 5 fields using a 25x objective of pHH3 + proliferating EMPs was calculated . Bar graphs represent mean ± SD from 3 wild type ( 21 - 24sp ) 3 Sl / Sl ( 22 - 23sp ) YS . Scale bars : 50μm .

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Confocal WM-IF analysis of CD31+ pHH3+ Kit+ proliferating EMPs (arrowheads) in E9.5 wild type and Sl/Sl YS. A single 2.5μm-thick Z section is shown. For each YS, CD31+ Kit+ pHH3+ cells were counted in 3 to 5 fields using a 25x objective and the percentage of pHH3+ proliferating EMPs was calculated. Bar graphs represent the mean±SD from 3 wild type (21-24sp) and 3 Sl/Sl (22-23sp) YS. Scale bars: 50μm.


cycle of wild type Sl / Sl E11 . 5 YS EMPs . of EMPs ( Ter119 - Kit + CD41 + ) in G0 / G1 or S / G2 / M was determined by flow cytometry on basis of BrdU 7 - AAD incorporation . a : apoptotic ( not shown ) . were analyzed individually . N = 6 ( + / + ) , N = 5 ( Sl / Sl ) over 3 independent experiments . E11 . 5 were staged according to tail somite as described in [ 76 ] were : 12 - 17 ( + / + ) ; 12 - 17 ( Sl / Sl ) . Bar graphs show mean ± SD , FACS plots representative results .

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Cell cycle analysis of wild type and Sl/Sl E11.5 YS EMPs. The percentage of EMPs (Ter119- Kit+ CD41+) in G0/G1 or S/G2/M was determined by flow cytometry on the basis of BrdU and 7-AAD incorporation. a: apoptotic (percentage not shown). Embryos were analyzed individually. N=6 (+/+), N=5 (Sl/Sl) over 3 independent experiments. E11.5 embryos were staged according to tail somite counts as described in [76] and were: 12-17 (+/+); 12-17 (Sl/Sl). Bar graphs show mean±SD, and FACS plots representative results.


Flow cytometry of EMPs ( Ter119 - Kit + CD41 + CD16 / 32 + ) early erythroid progenitors ( Early E : Ter119 - Kit + CD41 - CD71 + CD44 + ) in wild type Sl / Sl E11 . 5 FL . EMP early E data are mean ( ± SD ) of 6 wild type 4 Sl / Sl biological replicates , with a total of 10 wild type 6 Sl / Sl analyzed in pools of 1 - 3 FL per genotype . Bar graphs on right show corresponding absolute numbers ( nr ) per FL . Total FL are mean ( ± SD ) of 12 wild type 13 Sl / Sl . Tail somite range : 10 - 16 ( wild type ) , 12 - 15 ( Sl / Sl ) . Note that although EMP frequency in Sl / Sl FL is increased , their absolute number is severely decreased .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O 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-TISSUE O O O O O B-EXP_ASSAY O O O O

Flow cytometry analysis of EMPs (Ter119- Kit+ CD41+ CD16/32+) and early erythroid progenitors (Early E: Ter119- Kit+ CD41- CD71+ CD44+) in wild type and Sl/Sl E11.5 FL. EMP and early E data are the mean (±SD) of 6 wild type and 4 Sl/Sl biological replicates, with a total of 10 wild type and 6 Sl/Sl embryos analyzed in pools of 1-3 FL per genotype. Bar graphs on the right show corresponding absolute cell numbers (nr) per FL. Total FL cell counts are the mean (±SD) of 12 wild type and 13 Sl/Sl embryos. Tail somite range: 10-16 (wild type), 12-15 (Sl/Sl). Note that although EMP frequency in Sl/Sl FL is increased, their absolute number is severely decreased.


CFU - C numbers in wild type Sl / Sl E11 . 5 FL . Data are mean ( ± SD ) of 10 wild type 10 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of from 1 - 2 FLs . A total of 12 wild type 11 Sl / Sl FL were plated over 6 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O 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-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O

CFU-C numbers in wild type and Sl/Sl E11.5 FL. Data are the mean (±SD) of 10 wild type and 10 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of cell from 1-2 FLs. A total of 12 wild type and 11 Sl/Sl FL were plated over 6 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.


Total numbers ( nr ) of E12 . 5 wild type ( n = 7 ) Sl / Sl ( n = 8 ) FL . Right : CFU - E numbers per wild type Sl / Sl E12 . 5 FL . Data are mean ( ± SD ) of 3 biological replicates analyzed over 2 independent experiments . Each biological replicate consisted of 1 - 2 FL samples pooled according to genotype was plated in duplicate into a methylcellulose medium containing EPO ; colonies were scored after 2 days . Total number of FLs analyzed : 4 wild type 6 Sl / Sl .

O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O

Total cell numbers (nr) of E12.5 wild type (n=7) and Sl/Sl (n=8) FL. Right: CFU-E numbers per wild type and Sl/Sl E12.5 FL. Data are the mean (±SD) of 3 biological replicates analyzed over 2 independent experiments. Each biological replicate consisted of 1-2 FL samples pooled according to genotype and was plated in duplicate into a methylcellulose based medium containing EPO; colonies were scored after 2 days. Total number of FLs analyzed: 4 wild type and 6 Sl/Sl.


of erythroid lineage in E12 . 5 wild type Sl / Sl FL . Absolute ( top ) percentages ( bottom ) of proerythroblasts ( ProE ) , basophilic erythroblasts ( BasoE ) , polychromatic erythroblasts ( PolyE ) orthochromatic erythroblasts ( OrthoE ) were determined by imaging flow cytometry . number ( nr ) of per FL was on total cellularity their relative proportions . Data are mean ( ± SD ) of 3 biological replicates for each genotype , with replicates consisting of single or 2 pooled FL samples . A total of 4 wild type , 5 Sl / + 6 Sl / Sl FLs were analyzed .

O B-CELL_TYPE O O O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O

Quantification of erythroid lineage cells in E12.5 wild type and Sl/Sl FL. Absolute cell counts (top) and percentages (bottom) of proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE) and orthochromatic erythroblasts (OrthoE) were determined by imaging flow cytometry analysis. The number (nr) of cells per FL was based on total cellularity and their relative proportions. Data are the mean (±SD) of 3 biological replicates for each genotype, with replicates consisting of single or 2 pooled FL samples. A total of 4 wild type, 5 Sl/+ and 6 Sl/Sl FLs were analyzed.


Representative examples of maturing erythroblast series in wild type Sl / Sl E12 . 5 FL , as identified by imaging flow cytometry . Scale bars : 10μm .

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Representative examples of maturing erythroblast series in wild type and Sl/Sl E12.5 FL, as identified by imaging flow cytometry. Scale bars: 10μm.


Number of EMPs in peripheral blood ( PB ) of wild type Sl / Sl midgestation , calculated from of PB EMPs ( Ter119 - Kit + CD41 + CD16 / 32 + ) determined by flow cytometry ( Figure EV1E , F ) total ( 4 . 2 ± 1 . 6x105 ( wt ) , 3 . 1 ± 1 . 1x105 ( Sl / Sl ) at E9 . 75 ; 2 . 6 ± 0 . 8x106 ( wt ) , 2 . 3 ± 1 . 4x106 ( Sl / Sl ) at E11 . 5 ) . For E9 . 75 - E10 , PB from 2 wild type ( 28 - 30sp ) 5 Sl / Sl ( 27 - 31 ) was analyzed . For E11 . 5 , PB from 1 - 3 was pooled 6 wild type 4 Sl / Sl biological replicates were analyzed , with 10 wild type 6 Sl / Sl analyzed in total . Graphs show mean ( ± SD ) of PB EMPs per equivalent ( e . e . ) .

B-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O

Number of EMPs in peripheral blood (PB) of wild type and Sl/Sl midgestation embryos, calculated from the percentage of PB EMPs (Ter119- Kit+ CD41+ CD16/32+) determined by flow cytometry (Figure EV1E,F) and the total cell count (4.2±1.6x105 (wt), 3.1±1.1x105 (Sl/Sl) at E9.75; 2.6±0.8x106 (wt), 2.3±1.4x106 (Sl/Sl) at E11.5). For E9.75-E10, PB from 2 wild type (28-30sp) and 5 Sl/Sl (27-31) individual embryos was analyzed. For E11.5, PB from 1-3 embryos was pooled and 6 wild type and 4 Sl/Sl biological replicates were analyzed, with 10 wild type and 6 Sl/Sl embryos analyzed in total. Graphs show the mean (±SD) of PB EMPs per embryo equivalent (e.e.).


Representative confocal whole - mount immunofluorescence of wild type Sl / Sl FL at onset of hematopoietic colonization . Kit + EMPs are shown in , CD31 + vasculature in green . Maximum 3D projections from 350μm - thick Z stacks are shown . Scale bars : 100μm . Number of analyzed : 3 wild type 3 Sl / Sl for E9 . 75 ( 27 - 29sp ) , 7 wild type 8 ( Sl / Sl ) for E10 . 5 ( 34 - 36sp ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O B-CELL_TYPE O O B-GENEPROD O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 whole-mount immunofluorescence images of wild type and Sl/Sl FL at the onset of hematopoietic colonization. Kit+ EMPs are shown in white, CD31+ vasculature in green. Maximum intensity 3D projections from 350μm-thick Z stacks are shown. Scale bars: 100μm. Number of embryos analyzed: 3 wild type and 3 Sl/Sl for E9.75 (27-29sp), 7 wild type and 8 (Sl/Sl) for E10.5 (34-36sp).


Confocal whole - mount immunofluorescence of proliferating hematopoietic progenitors in wild type Sl / Sl E10 . 5 ( 34 - 36sp ) FL . Very few CD31 + pHH3 + Kit + proliferating hematopoietic ( arrowheads ) were detected in Sl / Sl FL . A single 2 . 5μm - thick slice is shown . Scale bars : 20μm . A total of 7 wild type 8 Sl / Sl were analyzed .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal whole-mount immunofluorescence analysis of proliferating hematopoietic progenitors in wild type and Sl/Sl E10.5 (34-36sp) FL. Very few CD31+ pHH3+ Kit+ proliferating hematopoietic cells (arrowheads) were detected in the Sl/Sl FL. A single 2.5μm-thick slice is shown. Scale bars: 20μm. A total of 7 wild type and 8 Sl/Sl embryos were analyzed.


of cycling Ter119 - Kit + CD41 + CD16 / 32 + EMPs Ter119 - Kit + CD41 - CD71 + CD44 + early erythroid ( Early E ) in E11 . 5 FL , determined by flow cytometric of BrdU 7 - AAD incorporation . Data are mean ( ± SD ) of 6 wild type 5 Sl / Sl FLs analyzed individually over 3 independent experiments . Tail somite range : 12 - 17 ( + / + ) ; 12 - 17 ( Sl / Sl ) .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O

Percentage of cycling Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid (Early E) cells in the E11.5 FL, determined by flow cytometric analysis of BrdU and 7-AAD incorporation. Data are the mean (±SD) of 6 wild type and 5 Sl/Sl FLs analyzed individually over 3 independent experiments. Tail somite range: 12-17 (+/+); 12-17 (Sl/Sl).


of early apoptotic EMPs Early E in wild type Sl / Sl E11 . 5 FL , determined by flow cytometric of AnnexinV among 7 - AAD - Ter119 - CD41 + Kit + CD16 / 32 + EMPs 7 - AAD - Ter119 - Kit + CD41 - CD71 + CD44 + Early E . Data are mean ( ± SD ) of 9 wild type 7 Sl / Sl biological replicates analyzed over 3 independent experiments , with each replicate consisting of 1 - 3 FLs of identical genotypes . A total number of 13 wild type 9 Sl / Sl were analyzed . Tail somite range : 11 - 17 for both genotypes .

O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O 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-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O

Percentage of early apoptotic EMPs and Early E in wild type and Sl/Sl E11.5 FL, determined by flow cytometric analysis of AnnexinV staining among 7-AAD- Ter119- CD41+ Kit+ CD16/32+ EMPs and 7-AAD- Ter119- Kit+ CD41- CD71+ CD44+ Early E. Data are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments, with each replicate consisting of 1-3 FLs of identical genotypes. A total number of 13 wild type and 9 Sl/Sl embryos were analyzed. Tail somite range: 11-17 for both genotypes.


Percentages of macrophage populations in wild type Sl / Sl E14 . 5 brain , skin ( dissected from back ) , lungs limb buds , as determined by flow cytometry . were gated as Ter119 - CD45 + further gated into F4 / 80hi CD11blo or F4 / 80lo CD11bhi populations . Representative dot plots of skin are shown in Appendix Figure S1A . Data are mean ( ± SD ) of 4 biological replicates for either genotype , with each replicate consisting of or pooled samples ( from up to 3 ) . A total of 8 wild type 5 Sl / Sl were analyzed over 2 independent experiments .

O O B-CELL_TYPE O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 macrophage populations in wild type and Sl/Sl E14.5 brain, skin (dissected from the back), lungs and limb buds, as determined by flow cytometry. Cells were gated as Ter119- CD45+ and further gated into F4/80hi CD11blo or F4/80lo CD11bhi populations. Representative dot plots of skin analysis are shown in Appendix Figure S1A. Data are the mean (±SD) of 4 biological replicates for either genotype, with each replicate consisting of individual or pooled samples (from up to 3 embryos). A total of 8 wild type and 5 Sl/Sl embryos were analyzed over 2 independent experiments.


absolute numbers of tissue macrophages per E14 . 5 FL . Data are mean ( ± SD ) . Number of analyzed : as in ( A ) .

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Percentage and absolute numbers of tissue macrophages per E14.5 FL. Data are mean (±SD). Number of embryos analyzed: as in (A).


Representative flow cytometry of macrophages in wild type Sl / Sl E10 . 5 AGM + VU . were gated as shown , with percentages in gates as indicated . Data are mean ( ± SD ) of 4 wild type ( 32 - 35 sp ) or 3 Sl / Sl ( 34 - 35 sp ) biological replicates , analyzed over 2 independent experiments . were analyzed individually . of flow cytometry in ( C ) , showing of F4 / 80 + CD11b + macrophages within live . E11 . 5 data are mean ( ± SD ) of 4 wild type ( 10 - 13 tail somites ) or 3 Sl / Sl ( 10 - 13 tail somites ) biological replicates , analyzed over 3 independent experiments . were analyzed individually .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O

Representative flow cytometry analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. Cells were gated as shown, with percentages in individual gates as indicated. Data are the mean (±SD) of 4 wild type (32-35 sp) or 3 Sl/Sl (34-35 sp) biological replicates, analyzed over 2 independent experiments. Embryos were analyzed individually. Quantification of flow cytometry analysis in (C), showing percentage of F4/80+CD11b+ macrophages within live cells. E11.5 data are the mean (±SD) of 4 wild type (10-13 tail somites) or 3 Sl/Sl (10-13 tail somites) biological replicates, analyzed over 3 independent experiments. Embryos were analyzed individually.


Confocal whole - mount immunofluorescence of macrophages in wild type Sl / Sl E10 . 5 AGM + VU . 3D panels show maximum projections from 400μm - thick Z stacks . Slice panels show single 2 . 5μm - thick Z slices of clusters in dorsal aorta ( da ) . A total of 4 wild type ( 31 - 34 sp ) 3 Sl / Sl ( 31 - 33 sp ) were analyzed . Scale bars : 50μm ( 3D ) , 10μm ( slice ) . of whole - mount immunofluorescence in ( E ) , showing absolute numbers ( mean ± SD ) of F4 / 80 + macrophages within hematopoietic clusters in dorsal aorta .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O B-TISSUE O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O B-TISSUE I-TISSUE O

Confocal whole-mount immunofluorescence analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. 3D panels show maximum intensity projections from 400μm-thick Z stacks. Slice panels show single 2.5μm-thick Z slices of clusters in the dorsal aorta (da). A total of 4 wild type (31-34 sp) and 3 Sl/Sl (31-33 sp) embryos were analyzed. Scale bars: 50μm (3D), 10μm (slice). Quantification of whole-mount immunofluorescence in (E), showing absolute numbers (mean ± SD) of F4/80+ macrophages within hematopoietic clusters in the dorsal aorta.


Confocal whole - mount immunofluorescence ( WM - IF ) of Kit + in wild type Sl / Sl E10 . 5 AGM ( 32 - 36sp ) . are 3D projections from 400μm - thick Z stacks , are representative of 14 wild type 11 Sl / Sl analyzed . Total numbers of Kit + ( ) in dorsal aorta were quantified showed a significant reduction in Sl / Sl compared to wild type ( p = 0 . 008 ) . CD31 + vasculature is shown in green . Arrowheads : hematopoietic clusters . Scale bars : 100μm . of WM - IF in ( A ) , showing absolute numbers ( ± SD ) of clusters of indicated separately shown relative to location . A total of 7 wild type 7 Sl / Sl were counted .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O 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-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O 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-EXP_ASSAY O

Confocal whole-mount immunofluorescence analysis (WM-IF) of Kit+ cells in wild type and Sl/Sl E10.5 AGM (32-36sp). Images are 3D projections from 400μm-thick Z stacks, and are representative of 14 wild type and 11 Sl/Sl embryos analyzed. Total numbers of Kit+ cells (white) in the dorsal aorta were quantified and showed a significant reduction in Sl/Sl compared to wild type (p=0.008). CD31+ vasculature is shown in green. Arrowheads: hematopoietic cell clusters. Scale bars: 100μm. Quantification of WM-IF in (A), showing absolute numbers (±SD) of clusters of indicated size and separately shown relative to location. A total of 7 wild type and 7 Sl/Sl embryos were counted.


Flow cytometry of HSC lineage in wild type Sl / Sl E11 . 5 AGM + VU . were Ter119 - VE - Cadherin + Kit + gated percentages of within pre - HSC type I gate ( red ) pre - HSC type II / HSC gate ( green ) are shown . Percentages are mean ( ± SD ) of 10 wild type 9 Sl / Sl biological replicates , with each replicate consisting of 1 - 3 pooled AGM + VU . A total of 15 wild type 11 Sl / Sl were analyzed over 5 independent experiments .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O

Flow cytometry analysis of the HSC lineage in wild type and Sl/Sl E11.5 AGM+VU. Cells were Ter119- VE-Cadherin+ Kit+ gated and percentages of cells within the pre-HSC type I gate (red) and the pre-HSC type II/HSC gate (green) are shown. Percentages are the mean (±SD) of 10 wild type and 9 Sl/Sl biological replicates, with each replicate consisting of 1-3 pooled AGM+VU. A total of 15 wild type and 11 Sl/Sl embryos were analyzed over 5 independent experiments.


Absolute numbers ( ± SD ) of E10 . 5 E11 . 5 pre - HSC type I ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 - ) pre - HSC type II / HSC ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 + ) , as determined by flow cytometry in ( C ) . Number of E11 . 5 analyzed : as in ( A ) . For E10 . 5 , 5 biological replicates were analyzed for either genotype , with each replicate consisting of 1 - 4 pooled AGM + VU . A total of 12 wild type ( 31 - 35sp ) 9 Sl / Sl ( 30 - 34sp ) were analyzed over 4 independent experiments . Total live per AGM + VU : 2 . 8 ± 1 . 0x105 ( wt ) , 2 . 3 ± 0 . 6x105 ( Sl / Sl ) at E10 . 5 ; 3 . 9 ± 1 . 1x105 ( wt ) , 3 . 8 ± 1 . 4x105 ( Sl / Sl ) at E11 . 5 .

O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-TISSUE O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Absolute numbers (±SD) of E10.5 and E11.5 pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-) and pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+), as determined by flow cytometry in (C). Number of E11.5 embryos analyzed: as in (A). For E10.5, 5 biological replicates were analyzed for either genotype, with each replicate consisting of 1-4 pooled AGM+VU. A total of 12 wild type (31-35sp) and 9 Sl/Sl (30-34sp) embryos were analyzed over 4 independent experiments. Total live cells per AGM+VU: 2.8±1.0x105 (wt), 2.3±0.6x105 (Sl/Sl) at E10.5; 3.9±1.1x105 (wt), 3.8±1.4x105 (Sl/Sl) at E11.5.


CFU - C numbers per wild type Sl / Sl E11 . 5 AGM + VU . are mean ( ± SD ) of 4 wild type 6 Sl / Sl biological replicates plated in duplicate . Biological replicates consisted of from or 2 pooled AGM + VU . A total of 6 wild type 7 Sl / Sl AGM + VU were analyzed over 4 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O 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-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O

CFU-C numbers per wild type and Sl/Sl E11.5 AGM+VU. Counts are the mean (±SD) of 4 wild type and 6 Sl/Sl biological replicates plated in duplicate. Biological replicates consisted of cells from individual or 2 pooled AGM+VU. A total of 6 wild type and 7 Sl/Sl AGM+VU embryos were analyzed over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.


of long - term multi - lineage HSC potential in E11 . 5 Sl / Sl AGM + VU . Irradiated CD45 . 1 syngeneic mice were transplanted with 1 e . e . of wild type , Sl / + or Sl / Sl CD45 . 2 + AGM + VU . PB chimerism is represented as of donor CD45 . 2 + among total CD45 + , 16 after transplant . A total of 14 recipients were transplanted with wild type or Sl / + 8 with Sl / Sl , over 4 independent experiments . Wild type is represented with a circle , Sl / + a triangle Sl / Sl with a square . Tail somite range : 9 - 17 ( + / + , Sl / + ) ; 10 - 17 ( Sl / Sl ) . * : p = 0 . 027 , one - Mann - Whitney ' s U ; p = 0 . 055 , two - Mann - Whitney ' s U .

O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of long-term multi-lineage HSC potential in E11.5 Sl/Sl AGM+VU. Irradiated CD45.1 syngeneic mice were transplanted with 1 e.e. of wild type, Sl/+ or Sl/Sl CD45.2+ AGM+VU cells. PB chimerism is represented as the percentage of donor CD45.2+ cells among total CD45+ cells, 16 weeks after transplant. A total of 14 recipients were transplanted with wild type or Sl/+ cells and 8 with Sl/Sl cells, over 4 independent experiments. Wild type is represented with a circle, Sl/+ a triangle and Sl/Sl with a square. Tail somite range: 9-17 (+/+, Sl/+); 10-17 (Sl/Sl). *:p=0.027, one-tailed Mann-Whitney's U test; p=0.055, two-tailed Mann-Whitney's U test.


Confocal WM - IF of pHH3 expression in Kit + of wild type Sl / Sl E10 . 5 dorsal aorta ( 33 - 36sp ) . are 2 . 5μm - thick single longitudinal Z slices ; dorsal up . Arrowheads indicate examples of proliferating CD31 + pHH3 + Kit + hematopoietic ( magnified in insets ) ; total number of these per dorsal aorta was quantified showed reduced proliferation in mutant ( p = 0 . 01 ) . A total of 4 per genotype were analyzed . Scale bars : 50μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE 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-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

Confocal WM-IF analysis of pHH3 expression in Kit+ cells of wild type and Sl/Sl E10.5 dorsal aorta (33-36sp). Images are 2.5μm-thick single longitudinal Z slices; dorsal up. Arrowheads indicate examples of proliferating CD31+ pHH3+ Kit+ hematopoietic cells (magnified in insets); the total number of these cells per dorsal aorta was quantified and showed reduced proliferation in the mutant (p=0.01). A total of 4 embryos per genotype were analyzed. Scale bars: 50μm.


Confocal WM - IF of cCasp3 expression in Kit + of wild type Sl / Sl E10 . 5 dorsal aorta ( 33 - 36sp ) . 2 . 5μm - thick single longitudinal Z slices ; dorsal up . A higher number of Kit + cCasp3 + apoptotic cluster were detected in Sl / Sl dorsal aorta ( p = 0 . 04 ) . Arrowheads indicate a CD31 + Kit + cCasp3 - hematopoietic cluster for wild type an apoptotic CD31 + Kit + cCasp3 + hematopoietic for Sl / Sl ( both magnified in insets ) . A total of 4 wild type 3 Sl / Sl were analyzed . Scale bars : 50μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal WM-IF analysis of cCasp3 expression in Kit+ cells of wild type and Sl/Sl E10.5 dorsal aorta (33-36sp). 2.5μm-thick single longitudinal Z slices; dorsal up. A higher number of Kit+ cCasp3+ apoptotic cluster cells were detected in the Sl/Sl dorsal aorta (p=0.04). Arrowheads indicate a CD31+ Kit+ cCasp3- hematopoietic cluster for wild type and an apoptotic CD31+ Kit+ cCasp3+ hematopoietic cell for Sl/Sl (both magnified in insets). A total of 4 wild type and 3 Sl/Sl embryos were analyzed. Scale bars: 50μm.


of apoptosis among pre - HSC type II / HSCs freshly isolated from wild type Sl / Sl E11 . 5 AGM . Ter119 - VE - Cadherin + Kit + CD41 + CD45 + were analyzed by flow cytometry for Annexin V binding to detect early apoptotic . Percentages are mean ( ± SD ) of 9 wild type 7 Sl / Sl biological replicates analyzed over 3 independent experiments . replicates consisted of 1 - 3 AGMs . A total of 13 wild type 9 Sl / Sl ( 11 - 17 tail sp ) were analyzed . of apoptotic pre - HSC type II / HSCs in mutant AGM was significantly increased .

O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O 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-TISSUE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-TISSUE O O O O

Analysis of apoptosis among pre-HSC type II/HSCs freshly isolated from wild type and Sl/Sl E11.5 AGM. Ter119- VE-Cadherin+ Kit+ CD41+ CD45+ cells were analyzed by flow cytometry for Annexin V binding to detect early apoptotic cells. Percentages are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments. Individual replicates consisted of 1-3 AGMs. A total of 13 wild type and 9 Sl/Sl embryos (11-17 tail sp) were analyzed. The percentage of apoptotic pre-HSC type II/HSCs in the mutant AGM was significantly increased.


Multiplex Fluidigm qRT - PCR of pre - HSC type I II , sorted as shown in ( C ) from E11 . 5 wild type ( n = 4 biological replicates ; total number of : 9 ; 11 - 17 tail somites ) Sl / Sl ( n = 5 biological replicates ; total number of : 9 ; 11 - 17 tail somites ) . Data are mean ( ± SD ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O

Multiplex Fluidigm qRT-PCR analysis of pre-HSC type I and II, sorted as shown in (C) from E11.5 wild type (n=4 biological replicates; total number of embryos: 9; 11-17 tail somites) and Sl/Sl (n=5 biological replicates; total number of embryos: 9; 11-17 tail somites). Data are mean (±SD).


Confocal whole - mount immunofluorescence ( WM - IF ) of Kitl - tdTomato expression in E8 . 5 ( 6 - 7sp ) conceptus ( maximum 3D projection from a 500μm - thick Z - stack ) . Kitl - tdTomato expression ( red ) is seen in YS , allantois ( a ; from which umbilical artery derives ) , para - aortic splanchnopleura ( PAS ) of Kitl - tdTomato : : 23GFP transgenic concepti ( proper , from which head has been removed , is outlined with a purple dashed ) . 23GFP transgene ( green ) labels YS blood islands ( bi ; yellow dashed ) ; this GFP reporter is transcribed from a heterologous hsp68 promoter under spatiotemporal control of Runx1 hematopoietic + 23 enhancer , resulting in GFP expression in primitive erythroid ( - fated ) , hemogenic endothelium , emerging HSPCs [ 2 , 74 ] . Endothelial ( CD31 + ) are in . Arrowheads indicate paired aortae ( pa ) . Purple boxed is magnified in ( B ) ; yellow boxed is magnified in ( C ) . Scale bar : 100μm . N = 2 analyzed . Single 2 . 5μm - thick Z slice from region highlighted in purple box in ( A ) . Arrowheads indicate Kitl - tdTomato + CD31 + endothelial in one of paired aortae ( pa ) . Scale bar : 25μm . Single 2 . 5μm - thick Z slice from region highlighted in yellow box in ( A ) , showing Kitl - tdTomato expression in E8 . 5 YS BI . Arrowheads indicate Kitl - tdTomato + CD31 + endothelial ; arrow indicates a Kitl - tdTomato + perivascular . Scale bar : 25μm . Single 2 . 5μm - thick Z slice of 10 . 5 ( 35sp ) YS ( WM - IF ) showing Kitl - tdTomato ( red ) , Kit ( ) CD31 ( green ) expression . Arrowheads indicate Kitl - tdTomato + CD31 + endothelial . N = 3 analyzed . Scale bar : 50μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-TISSUE O O B-CELL_TYPE O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE O O O O O 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-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O

Confocal whole-mount immunofluorescence (WM-IF) analysis of Kitl-tdTomato expression in the E8.5 (6-7sp) conceptus (maximum intensity 3D projection from a 500μm-thick Z-stack). Kitl-tdTomato expression (red) is seen in the YS, allantois (a; from which the umbilical artery derives), and para-aortic splanchnopleura (PAS) of Kitl-tdTomato::23GFP transgenic concepti (the embryo proper, from which the head has been removed, is outlined with a purple dashed line). The 23GFP transgene (green) labels the YS blood islands (bi; yellow dashed line); this GFP reporter is transcribed from a heterologous hsp68 promoter under the spatiotemporal control of the Runx1 hematopoietic +23 enhancer, resulting in GFP expression in all primitive erythroid(-fated) cells, hemogenic endothelium, and emerging HSPCs[2,74]. Endothelial cells (CD31+) are in white. Arrowheads indicate the paired aortae (pa). Purple boxed area is magnified in (B); yellow boxed area is magnified in (C). Scale bar: 100μm. N=2 embryos analyzed. Single 2.5μm-thick Z slice from the region highlighted in the purple box in (A). Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells in one of the paired aortae (pa). Scale bar: 25μm. Single 2.5μm-thick Z slice from the region highlighted in the yellow box in (A), showing Kitl-tdTomato expression in the E8.5 YS BI. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells; the arrow indicates a Kitl-tdTomato+ perivascular cell. Scale bar: 25μm. Single 2.5μm-thick Z slice of 10.5 (35sp) YS (WM-IF) showing Kitl-tdTomato (red), Kit (white) and CD31 (green) expression. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells. N=3 embryos analyzed. Scale bar: 50μm.


Confocal WM - IF of Kitl - tdTomato expression ( red ) in a E10 . 5 ( 36sp ) AGM region . Top panels ( 3D view ) show a maximum 3D projection from a 400μm - thick Z stack . Bottom panels ( slice view ) show a single 2 . 5μm - thick Z slice of dorsal aorta , from boxed region in top panels . CD31 ( vasculature ; green ) ; Kit ( hematopoietic progenitors ; ) . Scale bars : 100μm ( top ) ; 25μm ( bottom ) . fl : liver ; va : vitelline artery plexus ; da : dorsal aorta ; fp : floor plate ; nt : neural tube . N = 6 analyzed .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O B-TISSUE O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O

Confocal WM-IF analysis of Kitl-tdTomato expression (red) in a E10.5 (36sp) AGM region. Top panels (3D view) show a maximum intensity 3D projection from a 400μm-thick Z stack. Bottom panels (slice view) show a single 2.5μm-thick Z slice of the dorsal aorta, from the boxed region in the top panels. CD31 (vasculature; green); Kit (hematopoietic progenitors; white). Scale bars: 100μm (top); 25μm (bottom). fl: fetal liver; va: vitelline artery plexus; da: dorsal aorta; fp: floor plate; nt: neural tube. N=6 embryos analyzed.


Confocal immunofluorescence of cryosections through AGM region of E10 . 5 ( 34 - 36sp ) Kitl - tdTomato transgenic . Arrowheads indicate Kitl - tdTomato + CD31 + endothelial ( top ) , Kitl - tdTomato + PDGFR - β + ( middle ) or Kitl - tdTomato + α - SMA + ( bottom ) perivascular smooth muscle . Scale bars : 25μm or 50μm ( PDGFR - β panel ) . N > 6 analyzed .

B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

Confocal immunofluorescence analysis of cryosections through the AGM region of E10.5 (34-36sp) Kitl-tdTomato transgenic embryos. Arrowheads indicate Kitl-tdTomato+ CD31+ endothelial cells (top), Kitl-tdTomato+ PDGFR-β+ (middle) or Kitl-tdTomato+ α-SMA+ (bottom) perivascular smooth muscle cells. Scale bars: 25μm or 50μm (PDGFR-β panel). N>6 embryos analyzed.


Confocal immunofluorescence of Kitl - tdTomato expression in E10 . 5 FL . A cryosection through a E10 . 5 ( 35 - 36sp ) Kitl - tdTomato transgenic FL is shown . Arrowheads indicate Kitl - tdTomato + CD31 + Kit - endothelial . Asterisks indicate Kitl - tdTomato + CD31 - Kit - mesenchymal or hepatoblasts . Note proximity of Kitl - tdTomato + Kit + hematopoietic . Scale bars : 50μm ( left panel ) , 25μm ( magnified inset ) . N > 5 analyzed .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

Confocal immunofluorescence analysis of Kitl-tdTomato expression in the E10.5 FL. A cryosection through a E10.5 (35-36sp) Kitl-tdTomato transgenic FL is shown. Arrowheads indicate Kitl-tdTomato+ CD31+ Kit- endothelial cells. Asterisks indicate Kitl-tdTomato+ CD31- Kit- mesenchymal cells or hepatoblasts. Note proximity of Kitl-tdTomato+ cells and Kit+ hematopoietic cells. Scale bars: 50μm (left panel), 25μm (magnified inset). N>5 embryos analyzed.


Left : number of Ter119 - Kit + CD41 + CD16 / 32 + EMPs in Tie2Cre : : Kitl ∆ / ∆ control Kitlf / f or f / + E11 . 5 YS , determined by flow cytometry . Data are mean ( ± SD ) of 8 control 7 Tie2Cre : : Kitl ∆ / ∆ YS analyzed individually over 4 independent experiments . Tail somite range : 13 - 16 . Middle : number of pre - HSC type I ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 - ) pre - HSC type II / HSC ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 + ) in Tie2Cre : : Kitl ∆ / ∆ control Kitlf / f or f / + E11 . 5 AGM + VU , determined by flow cytometry . Data are mean ( ± SD ) of 7 control 5 Tie2Cre : : Kitl ∆ / ∆ AGM + VU analyzed individually over 3 independent experiments . Tail somite range : 14 - 15 ( Kitlf / f , f / + ) ; 14 - 16 ( Tie2Cre : : Kitl ∆ / ∆ ) . Right : number of Ter119 - Kit + CD41 + CD16 / 32 + EMPs Ter119 - Kit + CD41 - CD71 + CD44 + early erythroid in Tie2Cre : : Kitl ∆ / ∆ control Kitlf / f or f / + E11 . 5 FL , determined by flow cytometry . Data are mean ( ± SD ) of 7 control 5 Tie2Cre : : Kitl ∆ / ∆ FL analyzed individually over 3 independent experiments . Tail somite range : 13 - 16 .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O

Left: number of Ter119- Kit+ CD41+ CD16/32+ EMPs in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 YS, determined by flow cytometry. Data are the mean (±SD) of 8 control and 7 Tie2Cre::Kitl∆/∆ YS analyzed individually over 4 independent experiments. Tail somite range: 13-16. Middle: number of pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-) and pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+) in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 AGM+VU, determined by flow cytometry. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ AGM+VU analyzed individually over 3 independent experiments. Tail somite range: 14-15 (Kitlf/f, f/+); 14-16 (Tie2Cre::Kitl∆/∆). Right: number of Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid cells in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 FL, determined by flow cytometry. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ FL analyzed individually over 3 independent experiments. Tail somite range: 13-16.


CFU - C numbers in Tie2Cre : : Kitl ∆ / ∆ control Kitlf / f or f / + E11 . 5 YS . Data are mean ( ± SD ) of 6 control 3 Tie2Cre : : Kitl ∆ / ∆ YS plated in duplicate in 2 independent experiments . Tail somite range : 13 - 16 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O O O

CFU-C numbers in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 YS. Data are the mean (±SD) of 6 control and 3 Tie2Cre::Kitl∆/∆ YS plated in duplicate in 2 independent experiments. Tail somite range: 13-16.


of long - term multi - lineage HSC potential in E11 . 5 Tie2Cre : : Kitl ∆ / ∆ control Tie2Cre : : Kitl ∆ / + or Kitlf / f or f / + AGM + VU . Irradiated CD45 . 1 syngeneic mice were transplanted with 1 e . e . of AGM + VU . PB chimerism is represented as of donor CD45 . 2 + among total CD45 + , 16 after transplant . A total of 29 recipients were transplanted with Tie2Cre : : Kitl ∆ / + or Kitlf / f or f / + 8 with Tie2Cre : : Kitl ∆ / ∆ , over 7 independent experiments . Kitlf / f or f / + is represented with a circle , Tie2Cre : : Kitl ∆ / + a triangle Tie2Cre : : Kitl ∆ / ∆ with a square . Tail somite range : 12 - 17 ( control ) ; 12 - 17 ( Tie2Cre : : Kitl ∆ / ∆ ) .

O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O

Analysis of long-term multi-lineage HSC potential in E11.5 Tie2Cre::Kitl∆/∆ and control Tie2Cre::Kitl∆/+ or Kitlf/f or f/+AGM+VU. Irradiated CD45.1 syngeneic mice were transplanted with 1 e.e. of AGM+VU cells. PB chimerism is represented as the percentage of donor CD45.2+ cells among total CD45+ cells, 16 weeks after transplant. A total of 29 recipients were transplanted with Tie2Cre::Kitl∆/+ or Kitlf/f or f/+ cells and 8 with Tie2Cre::Kitl∆/∆ cells, over 7 independent experiments. Kitlf/f or f/+ is represented with a circle, Tie2Cre::Kitl∆/+ a triangle and Tie2Cre::Kitl∆/∆ with a square. Tail somite range: 12-17 (control); 12-17 (Tie2Cre::Kitl∆/∆).


CFU - C numbers in Tie2Cre : : Kitl ∆ / ∆ control Kitlf / f or f / + E11 . 5 Fl . Data are mean ( ± SD ) of 7 control 5 Tie2Cre : : Kitl ∆ / ∆ FL plated in duplicate in 3 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid . Tail somite range : 13 - 16 .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O

CFU-C numbers in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 Fl. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ FL plated in duplicate in 3 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid. Tail somite range: 13-16.


Number of phenotypic LSK , HPC HSC per FL in E12 . 5 Tie2Cre : : Kitl ∆ / ∆ control ( Kitlf / f or f / + ) FL , determined by flow cytometry total FL . were gated as 7 - AAD - Lin - ( F4 / 80 - CD3e - Nk1 . 1 - Ter119 - Gr - 1 - B220 - CD19 - ) . LSK : Lin - Sca1 + Kit + ; HPC : Lin - Sca1 + Kit + CD48 + CD150 - HSC : Lin - Sca1 + Kit + CD48 - CD150 + . Gating strategy is shown in Figure EV4E . Data are mean ( ± SD ) of 12 control 6 Tie2Cre : : Kitl ∆ / ∆ FLs analyzed individually over 3 independent experiments . Total cellularity of E12 . 5 Kitlf / f or f / + ( n = 14 ) Tie2Cre : : Kitl ∆ / ∆ ( n = 11 ) FL . Error bars represent SD .

B-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O

Number of phenotypic LSK, HPC and HSC per FL in E12.5 Tie2Cre::Kitl∆/∆and control (Kitlf/f or f/+) FL, determined by flow cytometry and total FL cell counts. Cells were gated as 7-AAD- Lin- (F4/80- CD3e- Nk1.1- Ter119- Gr-1- B220- CD19-). LSK: Lin- Sca1+ Kit+; HPC: Lin- Sca1+ Kit+ CD48+ CD150- and HSC: Lin- Sca1+ Kit+ CD48- CD150+. Gating strategy is shown in Figure EV4E. Data are the mean (±SD) of 12 control and 6 Tie2Cre::Kitl∆/∆ FLs analyzed individually over 3 independent experiments. Total cellularity of E12.5 Kitlf/f or f/+ (n=14) and Tie2Cre::Kitl∆/∆ (n=11) FL. Error bars represent SD.


Confocal whole - mount immunofluorescence of phosphorylated Akt ( pAkt ) ( A ) phosphorylated p44 / 42 MAPK ( pErk1 / 2 ) ( B ) in Kit + of wild type ( 32 - 36sp ) Sl / Sl ( 31 - 35sp ) E10 . 5 dorsal aorta , yolk sac liver . are 2 . 5μm - thick single longitudinal Z slices ; dorsal up ( DA ) . Arrowheads point to Kit + with varying of pAkt pErk1 / 2 expression . A total of 2 wild type 2 Sl / Sl for each were analyzed . Scale bars : 20μm . of immunofluorescence in ( A , B ) , showing mean ( MFI ) of pAkt p44 / 42 MAPK ( pErk1 / 2 ) within wild type ( + / + ) Sl / Sl CD31 + Kit + dorsal aorta ( DA ) , yolk sac ( YS ) liver ( FL ) hematopoietic . MFI values were normalized to background in adjacent ventral mesenchyme for DA , to erythrocytes in YS or stroma in FL . Additional was performed on sections from 2 wild type 2 Sl / Sl of similar range . MFI measurements were taken using ImageJ . Number of measured for pAkt : 100 hematopoietic 73 background ( + / + ) ; 79 hematopoietic 52 background ( Sl / Sl ) in AGM ; 127 hematopoietic 95 background ( + / + ) ; 110 hematopoietic 82 background ( Sl / Sl ) in YS ; 102 hematopoietic 78 background ( + / + ) ; 78 hematopoietic 83 background ( Sl / Sl ) in FL . Number of measured for pErk1 / 2 : 94 hematopoietic 82 background ( + / + ) ; 114 hematopoietic 91 background ( Sl / Sl ) in AGM ; 174 hematopoietic 109 background ( + / + ) ; 123 hematopoietic 82 background ( Sl / Sl ) in YS ; 100 hematopoietic 70 background ( + / + ) ; 63 hematopoietic 57 background ( Sl / Sl ) in FL . N = 4 ( + / + ) , 4 ( Sl / Sl ) analyzed for DA YS ; N = 3 ( + / + ) , 2 ( Sl / Sl ) for FL . Error bars represent SD .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O B-CELL_TYPE O B-TISSUE O B-TISSUE O B-TISSUE O 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-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O

Confocal whole-mount immunofluorescence analysis of phosphorylated Akt (pAkt) (A) and phosphorylated p44/42 MAPK (pErk1/2) (B) in Kit+ cells of wild type (32-36sp) and Sl/Sl (31-35sp) E10.5 dorsal aorta, yolk sac and fetal liver. Images are 2.5μm-thick single longitudinal Z slices; dorsal up (DA). Arrowheads point to Kit+ cells with varying levels of pAkt and pErk1/2 expression. A total of 2 wild type and 2 Sl/Sl embryos for each staining were analyzed. Scale bars: 20μm. Quantification of the immunofluorescence in (A, B), showing mean fluorescence intensity (MFI) ratio of pAkt and p44/42 MAPK (pErk1/2) within wild type (+/+) and Sl/Sl CD31+ Kit+ dorsal aorta (DA), yolk sac (YS) and fetal liver (FL) hematopoietic cells. MFI values were normalized to background in the adjacent ventral mesenchyme for DA, to erythrocytes in the YS or stroma in the FL. Additional quantification was performed on sections from 2 wild type and 2 Sl/Sl embryos of similar age range. MFI measurements were taken using ImageJ. Number of cells measured for pAkt: 100 hematopoietic cells and 73 background (+/+); 79 hematopoietic and 52 background (Sl/Sl) in the AGM; 127 hematopoietic cells and 95 background (+/+); 110 hematopoietic and 82 background (Sl/Sl) in the YS; 102 hematopoietic cells and 78 background (+/+); 78 hematopoietic and 83 background (Sl/Sl) in the FL. Number of cells measured for pErk1/2: 94 hematopoietic cells and 82 background (+/+); 114 hematopoietic and 91 background (Sl/Sl) in the AGM; 174 hematopoietic cells and 109 background (+/+); 123 hematopoietic and 82 background (Sl/Sl) in the YS; 100 hematopoietic cells and 70 background (+/+); 63 hematopoietic and 57 background (Sl/Sl) in the FL. N=4 (+/+), 4 (Sl/Sl) embryos analyzed for DA and YS; N=3 (+/+), 2 (Sl/Sl) for FL. Error bars represent SD.


Multiplex Fluidigm qRT - PCR of pre - HSC type I ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 - ) , pre - HSC type II / HSC ( Ter119 - VE - Cadherin + Kit + CD41 + CD45 + ) , YS FL EMP ( Ter119 - Kit + CD41 + CD16 / 32 + ) , FL Early E ( Ter119 - Kit + CD41 - ) peripheral blood LMPP ( Lin - CD19 - B220 - CD45 + Kit + Flt3 + IL7Rα + ) , isolated from E11 . 5 ( 11 - 17 tail somites ) wild type Sl / Sl . 4 biological replicates for wild type ( 9 total ) 5 biological replicates ( 9 total ) for Sl / Sl were analyzed . Data are mean ( ± SD ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Multiplex Fluidigm qRT-PCR analysis of pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-), pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+), YS and FL EMP (Ter119- Kit+ CD41+ CD16/32+), FL Early E (Ter119- Kit+ CD41-) and peripheral blood LMPP (Lin- CD19- B220- CD45+ Kit+ Flt3+ IL7Rα+), isolated from E11.5 (11-17 tail somites) wild type and Sl/Sl embryos. 4 biological replicates for wild type (9 total embryos) and 5 biological replicates (9 total embryos) for Sl/Sl were analyzed. Data are mean (±SD).


A . Confocal microscopy image of PD myotubes ( cl . 3LE8 ) infected for 72 h with adenovirus containing TFEB ( PD + Ad - TFEB 72 h ) shows a dramatic reduction in number of large LAMP1 - positive lysosomes ( red ) compared to that in untreated ( PD ) or adenovirus ( PD + Ad - null ) - treated PD myotubes . WT myotubes are shown on left panel . Nuclei are stained with Hoechst ( blue ) . TFEB was detected with anti - Flag antibody ( green ) . B . Distribution of lysosomal differs significantly in Ad - null Ad - TFEB PD myotubes ( p = 6 . 32 × 10 − 8 ; Kolmogorov - Smirnov ) . Lysosomal is expressed as number of pixels representing lysosomal ( LAMP1 - positive structures ) . median lysosomal of Ad - TFEB infected myotubes ( m = 367 . 13 pixels , n = 703 , range 208 - 2659 ) was significantly lower than that of Ad - null infected myotubes ( m = 491 . 16 pixels , n = 1395 , range 200 - 2857 ; p = 6 . 5 × 10 − 12 ; Wilcoxon rank sum ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-SUBCELLULAR O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O 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-ORGANISM O O O B-CELL_TYPE O O O O O O O O O O O O O O O O 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. Confocal microscopy image of PD myotubes (cl. 3LE8) infected for 72 h with adenovirus containing TFEB (PD + Ad-TFEB 72 h) shows a dramatic reduction in the number of large LAMP1-positive lysosomes (red) compared to that in untreated (PD) or adenovirus (PD + Ad-null)-treated PD myotubes. WT myotubes are shown on the left panel. Nuclei are stained with Hoechst (blue). TFEB was detected with anti-Flag antibody (green).B. Distribution of lysosomal size differs significantly in Ad-null and Ad-TFEB PD myotubes (p = 6.32 × 10−8; Kolmogorov-Smirnov test). Lysosomal size is expressed as number of pixels representing lysosomal area (LAMP1-positive structures). The median lysosomal size of Ad-TFEB infected myotubes (m = 367.13 pixels, n = 703, range 208-2659) was significantly lower than that of Ad-null infected myotubes (m = 491.16 pixels, n = 1395, range 200-2857; p = 6.5 × 10−12; Wilcoxon rank sum test).


C . Confocal microscopy image of PD myotubes infected for 24 h with Ad - TFEB shows relocation of lysosomes to plasma ( top ) . showing LAMP1 ( red ) on plasma in a PD myotube infected with Ad - TFEB ( bottom ; arrows ) but not in a non - infected ( middle ) . Non - permeabilized were incubated with anti - LAMP1 antibody at 4 ° C for 40 min , followed by fixation with secondary antibody .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O O O B-ORGANISM O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O O O O O O

C. Confocal microscopy image of PD myotubes infected for 24 h with Ad-TFEB shows relocation of lysosomes to the plasma membrane (top). Images showing LAMP1 staining (red) on plasma membrane in a PD myotube infected with Ad-TFEB (bottom; arrows) but not in a non-infected cell (middle). Non-permeabilized cells were incubated with anti-LAMP1 antibody at 4°C for 40 min, followed by fixation and staining with secondary antibody.


D . Confocal microscopy of live non - infected PD myotubes ( left ) or PD myotubes infected for 72 h with Ad - TFEB ( right ) show a dramatic reduction in amount of accumulated glycogen in TFEB - treated . were incubated with glucose ( 2 - NBDG ; green ) , extensively washed , analysed by confocal microscopy . Bar : 10 µm for panels .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.


Confocal microscopy of live unstained fibres from a GFP - LC3 : WT mouse ( WT ) from untreated ( GAA − / − ) or ERT - treated ( GAA − / − ; + ERT ) GFP - LC3 : GAA − / − mice . show typical centrally located autophagic build - up with multiple clusters of LC3 - positive autophagosomes . Structures like these are never seen in WT muscle . Autophagic build - up is present virtually in fibres derived from EDL or gastrocnemiusmuscles ( not shown ) , but only in 60 - 70 % of FDBfibres . Labelled recombinant human GAA ( red ) was administered i . v . into 3 - 4 month - old GFP - LC3 : GAA − / − mice ( n = 5 ) at a dose of 100 mg / kg twice with a 24 h interval . Mice were sacrificed next day , live fibres ( shown for EDLmuscle ) were analysed by confocal microscopy . labelled recombinant GAA was detected almost exclusively in autophagic vesicles . At least 500 fibres were analysed for each condition . Bar : 10 µm .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-TISSUE O O O O O O O O O B-CELL_TYPE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM 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-ORGANISM O O O O O O B-CELL_TYPE O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_TYPE O O O O O O O O O O O O

Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.


A . Immunostaining of non - infected ( day 13 in differentiation medium ) infected with a mutant form of TFEB ( TFEBmt ) with LAMP1 ( red ) Flag ( green ) . Ad - TFEBmt was added to myotubes for 72 h on day 10 in differentiation medium . Ad - TFEBmt - infected show massive accumulation of TFEB in nuclei significant reduction in lysosomal , similar to that seen with TFEB .

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-GENEPROD O

A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB.


B . Western blot of lysates confirms presence of TFEB in fraction ; different intensities of two bands corresponding to LAMP1 in untreated TFEBmt - treated samples may reflect differences in glycosylation pattern .

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B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern.


C . Western blot of lysates from untreated , Ad - null , TFEB , TFEBmt - treated myotubes with anti - caspase - 3 antibody . No activated ( ) products are detected in any condition . α - Tubulin was used as a loading control .

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C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control.


D . TUNEL shows presence of some apoptotic in Ad - TFEB - infected cultures , but not in control cultures infected with adenovirus alone ( n = 3 ) . Bar : 10 µm for panels .

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D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.


A . Confocal microscopy of live fibres derived from 3 to 4 month - old GFP - LC3 : WT ( top left ) , untreated GFP - LC3 : GAA − / − ( bottom left ) or TFEB - treated GFP - LC3 : GAA − / − ( right ) mice . fibres were transfected with mCherry - LAMP1 to visualize lysosomes ( red ) . effects of TFEB are clearly visible - overall reduction in lysosomal , appearance of normal lysosomes ( similar to those in WT ) , lysosomal docking to plasma ( inset ) . Bar : 10 µm .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O

A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible - overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.


B . FDBmuscle of a GAA − / − mouse was transfected with both GFP - TFEB mCherry - LAMP1 ( GAA − / − + GFP - TFEB ) . were taken before ( left ) after 4 h ( right ) of time - lapse microscopy . Lysosomal clearance is visible in TFEB - transfected fibre at both time points . Lysosomes appear to " exit " fibre ( inset arrow ) at 4 h time point when TFEB is activated as evidenced by its translocation ( green nuclei ) . Bar : 10 µm . C . mean maximum velocity of lysosomes ( top ) number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated TFEB - treated PD fibres ( note : data for Flag - GFP - TFEB - treated fibres are pooled ) . Lysosomal velocities were calculated from time - lapse using ImageJ software with manual tracking plug - in . For each condition trajectories of multiple lysosomes were followed ( n = 26 untreated ; n = 43 TFEB - treated ) three highest velocity measurements per lysosome were recorded . In TFEB - treated fibres , maximum velocity of lysosomes was significantly increased ( p = 2 . 07 × 10 − 17 ) number of large lysosomes was significantly decreased ( p = 1 . 0 × 10 − 3 ) . Ten untreated 24 TFEB - treated fibres were analysed for calculations . * indicates statistically significant differences ( p ≤ 0 . 001 ; Student ' s t - ) . Error bars represent 95 % confidence intervals .

O O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O 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. FDBmuscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to "exit" the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.


Confocal microscopy of live fibres from GFP - LC3 : GAA − / − mice . Muscle was transfected with mCherry - LAMP1 only . image ( a single frame from time - lapse series presented in Movie 5 ) shows lysosomes ( red ) , LC3 - positive autophagosomes ( green ) a number of autolysosomes ( yellow ) . Muscle was transfected with Flag - TFEB mCherry - LAMP1 . Massive formation of autolysosomes is indicated by yellow structures ; three lower panels provide a snapshot of process of exocytosis . structures at plasma are labelled with both LC3 LAMP1 , indicating that they represent amphisomes ( a product of fusion between autophagic vesicles late endosomes ) or autolysosomes ( a product of fusion between autophagic vesicles lysosomes ) . Bar : 10 µm for panels .

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Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.