Figure 2Flow 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 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 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 . 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 and Sl / Sl E12 . 5 FL , as identified by imaging flow cytometry . Scale bars : 10μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Flow 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 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 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.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 and Sl/Sl E12.5 FL, as identified by imaging flow cytometry. Scale bars: 10μm.


Figure 3Number 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 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 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 . 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 ) . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Number 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 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 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.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).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.


Figure 4Percentages 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 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Percentages 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.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 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 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.


Figure 5Confocal 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 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 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 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 . 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 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 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 . 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Confocal 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 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 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 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.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 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 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.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 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).


Figure 6Confocal 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Confocal 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 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 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 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.


Figure 7Left : 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 ∆ / ∆ 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 . 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 ∆ / ∆ 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Left: 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∆/∆ 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.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∆/∆ 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 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.


Figure 8Confocal 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Confocal 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 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).


Figure 1A - C AID expression was analyzed in colon and pancreatic human cell lines ( A ) qRT - PCR analysis of AID expression in LoVo and SW480 colon cell lines . n = 5 ( LoVo ) ; 3 ( SW480 ) . * * P - value : LoVo : 0 . 0017 ; SW480 : 0 . 0079 . ( B ) qRT - PCR analysis of AID expression in AsPC and PaTU - 8988S pancreatic cell lines ( n = 2 ) . * P - value : AsPC : 0 . 0369 ; PaTU - 8988S : 0 . 0119 . A - C AID expression was analyzed in pancreas explants from C57BL / 6 mice . Samples were treated as indicated with 50 ng / ml TNF - α . ( C ) qRT - PCR analysis of AID expression in pancreatic explants from wild - type mice ( n = 3 ) . * P = 0 . 0242 . D AID − / − or AID + / − mice were treated with 3 % DSS for 10 cycles , and colonic sections were analyzed by histology inspection after H / E staining . Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments . n = 28 ( AID − / − males ) ; 35 ( females ) ; 23 ( AID + / − males ) ; 25 ( females ) . P - value : male : 0 . 8 ; female : 0 . 246 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-C AID expression was analyzed in colon and pancreatic human cell lines (A) qRT-PCR analysis of AID expression in LoVo and SW480 colon cell lines. n = 5 (LoVo); 3 (SW480). **P-value: LoVo: 0.0017; SW480: 0.0079. (B) qRT-PCR analysis of AID expression in AsPC and PaTU-8988S pancreatic cell lines (n = 2). *P-value: AsPC: 0.0369; PaTU-8988S: 0.0119.A-C AID expression was analyzed in pancreas explants from C57BL/6 mice. Samples were treated as indicated with 50 ng/ml TNF-α. (C) qRT-PCR analysis of AID expression in pancreatic explants from wild-type mice (n = 3). *P = 0.0242.D AID−/− or AID+/− mice were treated with 3% DSS for 10 cycles, and colonic sections were analyzed by histology inspection after H/E staining. Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments. n = 28 (AID−/− males); 35 (females); 23 (AID+/− males); 25 (females). P-value: male: 0.8; female: 0.246.


Figure 2GFP immunofluorescence in colonic and pancreatic tissue from R26AID + / KIVillinCRE + / TG and R26AID + / KIp48CRE + / KI mice . Scale bar : 50 μm . qRT - PCR analysis of AID expression in colonic and pancreatic tissue from R26AID + / KIVillinCRE + / TG and R26AID + / KIp48CRE + / KI mice . n = 5 ( R26AID + / + VillinCRE + / TG ) ; 4 ( R26AID + / KIVillinCRE + / TG ) ; 2 ( R26AID + / + p48CRE + / KI ) ; 2 ( R26AID + / KIp48CRE + / KI ) . LPS + IL4 - stimulated B cells are shown as a positive control ( n = 2 ) . Bars show mean values ± SEM normalized to LPS + IL4 - treated B cells . Kaplan - Meier survival curves for R26AID + / KIVillinCRE + / TG ( left ) ( n = 47 ( R26AID + / + VillinCRE + / TG ) ; 38 ( R26AID + / KIVillinCRE + / TG ) ) and R26AID + / KI p48CRE + / KI mice ( right ) ( n = 39 ( R26AID + / + p48CRE + / KI ) ; 23 ( R26AID + / KIp48CRE + / KI ) ) .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2GFP immunofluorescence in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. Scale bar: 50 μm.qRT-PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells.Kaplan-Meier survival curves for R26AID+/KIVillinCRE+/TG (left) (n = 47 (R26AID+/+ VillinCRE+/TG); 38 (R26AID+/KIVillinCRE+/TG)) and R26AID+/KI p48CRE +/KI mice (right) (n = 39 (R26AID+/+p48CRE+/KI); 23 (R26AID+/KIp48CRE+/KI)).


Figure 3A , B Analysis of AID mutagenic activity in Elastase1 ( A ) and Elastase2a ( B ) by next - generation sequencing . Pancreatic DNA was isolated from pools of R26AID + / KIp48CRE + / KI and control R26AID + / + p48CRE + / KI 20 - week - old mice , and then PCR - amplified with specific primers and sequenced as previously described ( Perez - Duran et al , 2012 ) . Graphs show cytosine mutation frequency overall ( total ) or at AGCT hotspots . n = 2 . * P - value : Elastase1 : 0 . 0382 ; Elastase2a : 0 . 009 . C HTM - mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days . Red lines show mean values . Results of two independent experiments are shown . * * * * P < 0 . 0001 . D Representative images of γH2AX staining in pancreas explants from R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) ( 40 × magnification ) . White arrow points a representative positive cell .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B Analysis of AID mutagenic activity in Elastase1 (A) and Elastase2a (B) by next-generation sequencing. Pancreatic DNA was isolated from pools of R26AID+/KIp48CRE+/KI and control R26AID+/+p48CRE+/KI 20-week-old mice, and then PCR-amplified with specific primers and sequenced as previously described (Perez-Duran et al, 2012). Graphs show cytosine mutation frequency overall (total) or at AGCT hotspots. n = 2. *P-value: Elastase1: 0.0382; Elastase2a: 0.009.C HTM-mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days. Red lines show mean values. Results of two independent experiments are shown. ****P < 0.0001.D Representative images of γH2AX staining in pancreas explants from R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom) (40× magnification). White arrow points a representative positive cell.


Figure 4Representative images of Ki67 staining in pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) . Detail is shown on the right . Scale bar : 100 μm . Graph shows quantification of Ki67 - positive epithelial cells per mm2 of tissue ( n = 8 ) . * P = 0 . 0266 . Representative immunofluorescence staining of 20 - week - old R26AID + / KIp48CRE + / KI mice : blue , DAPI ; red , Ki67 ; green , CK8 . Scale bar : 50 μm . Detail is shown on the bottom . Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Left : Graph shows mean fluorescence intensity . Each dot represents an individual mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 8 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 077 . Right : Representative FACS staining for RAE in explants from R26AID + / + p48CRE + / KI ( red ) and R26AID + / KIp48CRE + / KI mice ( black ) . Analysis of RAE expression by ddPCR in aged ( 75 - week - old ) mice . Data are presented as the percentage of positive drops normalized to the mean control value . Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 ( R26AID + / + p48CRE + / KI ) ; 9 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 012 . Analysis of killing activity . Primary explants of pancreatic cells from R26AID + / + p48CRE + / KI or R26AID + / KIp48CRE + / KI mice were cultured with primary NK cells , and killing activity was assessed as described in Materials and Methods ( n = 4 ) . P = 0 . 19 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative images of Ki67 staining in pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom). Detail is shown on the right. Scale bar: 100 μm. Graph shows quantification of Ki67-positive epithelial cells per mm2 of tissue (n = 8). *P = 0.0266.Representative immunofluorescence staining of 20-week-old R26AID+/KIp48CRE+/KI mice: blue, DAPI; red, Ki67; green, CK8. Scale bar: 50 μm. Detail is shown on the bottom.Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Graph shows mean fluorescence intensity. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 8 (R26AID+/KIp48CRE+/KI). P = 0.077. Right: Representative FACS staining for RAE in explants from R26AID+/+p48CRE+/KI (red) and R26AID+/KIp48CRE+/KI mice (black).Analysis of RAE expression by ddPCR in aged (75-week-old) mice. Data are presented as the percentage of positive drops normalized to the mean control value. Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 (R26AID+/+p48CRE+/KI); 9 (R26AID+/KIp48CRE+/KI). *P = 0.012.Analysis of killing activity. Primary explants of pancreatic cells from R26AID+/+p48CRE+/KI or R26AID+/KIp48CRE+/KI mice were cultured with primary NK cells, and killing activity was assessed as described in Materials and Methods (n = 4). P = 0.19.


Figure 5Hematoxylin - eosin ( HE ) staining of pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Left : Representative HE staining showing an immune infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 200 μm . Right : Quantification of number of foci per mm2 of tissue . n = 13 ( R26AID + / + p48CRE + / KI ) ; 21 ( R26AID + / KIp48CRE + / KI ) . * * P = 0 . 0098 . CD3 immunohistochemistry of pancreas from 75 - week - old R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Left : Representative image of a CD3 infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 100 μm . Right : Quantification of the number of CD3 - positive cells per mm2 of tissue . n = 12 ( R26AID + / + p48CRE + / KI ) ; 13 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0149 . Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20 - week - old R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice . Scale bar : 20 μm . TNF - α expression . Total RNA was isolated from pancreas of aged ( 75 - week - old ) R26AID + / KIp48CRE + / KI and control mice , and TNF - α expression was quantified by qRT - PCR . Each dot represents an individual mouse . n = 8 ( R26AID + / + p48CRE + / KI ) ; 15 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0392 . Cell death detection . Pancreas from 75 - week - old R26AID + / + p48CRE + / KI and R26AID + / KIp48CRE + / KI mice were stained with anti - caspase - 3 . Left : Representative staining from an R26AID + / + p48CRE + / KI mouse ( top ) and an R26AID + / KIp48CRE + / KI mouse ( bottom ) . Scale bar : 50 μm . Right : Quantification of number of cells per mm2 of tissue . Each dot represents an individual mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 11 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 0545 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Hematoxylin-eosin (HE) staining of pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative HE staining showing an immune infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 200 μm. Right: Quantification of number of foci per mm2 of tissue. n = 13 (R26AID+/+p48CRE+/KI); 21 (R26AID+/KIp48CRE+/KI). **P = 0.0098.CD3 immunohistochemistry of pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative image of a CD3 infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 100 μm. Right: Quantification of the number of CD3-positive cells per mm2 of tissue. n = 12 (R26AID+/+p48CRE+/KI); 13 (R26AID+/KIp48CRE+/KI). *P = 0.0149.Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Scale bar: 20 μm.TNF-α expression. Total RNA was isolated from pancreas of aged (75-week-old) R26AID+/KIp48CRE+/KI and control mice, and TNF-α expression was quantified by qRT-PCR. Each dot represents an individual mouse. n = 8 (R26AID+/+p48CRE+/KI); 15 (R26AID+/KIp48CRE+/KI). *P = 0.0392.Cell death detection. Pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice were stained with anti-caspase-3. Left: Representative staining from an R26AID+/+p48CRE+/KI mouse (top) and an R26AID+/KIp48CRE+/KI mouse (bottom). Scale bar: 50 μm. Right: Quantification of number of cells per mm2 of tissue. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 11 (R26AID+/KIp48CRE+/KI). P = 0.0545.


figf1 ( a , b ) Multiple MBds associate with a PC3 cell ( a ) and a B - lymphoblast ( b ) . Insets : MBd labelling ( a ) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries ( b ) . MKLP1 , MBd marker ( a , b ; red ) ; CD44 , membrane ( a ; green ) ; DAPI , DNA ( a ; blue ) . Scale bar , 5 μm ( a ) and 2 μm ( b ) . ( c , d ) Three - dimensional reconstructions of polarized cells in a monolayer ( c ) and a HeLa cell ( d ) show intracellular MBds . ( c ) ZO - 1 , tight junction ; MKLP1 , MBds . Scale bar , 2 μm . Enlargement ( bottom panel ) of the box in the top panel shows five MBds ( arrows ) . ( d ) Wheat - germ agglutinin , plasma membrane ( red ) ; MKLP1 - GFP , MBds ( green ) ; DAPI , DNA ( blue ) . Scale bar , 5 μm . ( e ) Electron microscopy of an MBd in a permeabilized MCF - 7 cell showing immungold labelling with MKLP1 antibodies . Inset , lower magnification of the MBd ( boxed ) in the cell ; nucleus , right . Scale bar , 200 nm . ( f ) Time - lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds ( MKLP1 - GFP , red ) . Two MBds ( yellow arrows ) are lost on treatment , indicating digestion and / or dissociation . Time ( h : min ) post trypsin . Scale bar , 5 μm . ( g ) Two - day co - cultures of HeLa cells expressing either MKLP1 - GFP ( MBd marker ) or cytosolic RFP . Green MBds ( arrows ) associated with red cells ( asterisk ) indicate post - mitotic transfer of MBds between cells . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b).(c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1-GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm.(e) Electron microscopy of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm.(f) Time-lapse images during extracellular trypsin treatment of HeLa cells show retention of most MBds (MKLP1-GFP, red). Two MBds (yellow arrows) are lost on treatment, indicating digestion and/or dissociation. Time (h:min) post trypsin. Scale bar, 5 μm.(g) Two-day co-cultures of HeLa cells expressing either MKLP1-GFP (MBd marker) or cytosolic RFP. Green MBds (arrows) associated with red cells (asterisk) indicate post-mitotic transfer of MBds between cells. Scale bar, 10 μm.


figf2 ( a ) The CETN1 - GFP signal is brighter in the upper centrosome / spindle pole of a mitotic spindle . The merged differential interference contrast microscopy image with CETN1 - GFP labelling at two centrosomes shows a metaphase chromosome . Insets : enlargements and semi - quantitative integrated intensity profiles of centrioles . Scale bar , 5 μm . ( b ) The brighter CETN1 - GFP signal represents the older centrosome , as it co - stains more intensely for hCenexin1 and remains more intense throughout cell division ( Supplementary Fig . S1a ) . Scale bar , 5 μm . Lower left , merge . ( c , d ) Time - lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells ( c ) and hESCs ( d ) . Cells were imaged at the indicated times ( h : min ) from telophase by phase - contrast microscopy ( c ) and from metaphase by differential interference contrast microscopy ( d ) . Middle panel of ( c ) and upper left panel of ( d ) : CETN1 - GFP at centrosomes ; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome ( c , upper ; d , lower ) inherits the MBd ( lower right images in c , d ) . Mitotic midbody and MBds ( c , d ; arrows ) . MKLP1 , MBd marker ( red ) ; α - tubulin , mitotic midbody and cell - boundary marker ( green ) ; DAPI , DNA ( blue ) . Scale bars , 10 μm ( c , d ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) The CETN1-GFP signal is brighter in the upper centrosome/spindle pole of a mitotic spindle. The merged differential interference contrast microscopy image with CETN1-GFP labelling at two centrosomes shows a metaphase chromosome. Insets: enlargements and semi-quantitative integrated intensity profiles of centrioles. Scale bar, 5 μm. (b) The brighter CETN1-GFP signal represents the older centrosome, as it co-stains more intensely for hCenexin1 and remains more intense throughout cell division (Supplementary Fig. S1a). Scale bar, 5 μm. Lower left, merge.(c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1-GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).


figf3 ( a ) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1 + puncta in cells of the basal layer where stem cells reside . Scale bar , 20 μm . Inset : Enlargement of the cell marked by the arrow . ( b , c ) Electron micrographs of mitotic midbody ( b , arrow ) and multiple midbody - like structures in interphase cells with similar shapes and sizes in a juxtanuclear position ( c , arrows ) in basal cells of mouse seminiferous tubules . N , nucleus . Scale bars , 1 μm . ( d ) Representative planes of a neural progenitor cell in the ventricular zone ( Sox2 + , bottom left panel ) of an E13 . 5 mouse brain show that an intracellular MBd ( asterisk ) is associated with the ventricle - facing daughter in the asymmetrically dividing cell ( top row ) . The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell . CD133 , midbody / MBd marker ( green ) ; Na / K - ATPase , cell - border marker ( red ) ; DRAQ5 , DNA ( blue ) ; DAPI , DNA . v , ventricle . Scale bar , 5 μm . Note that abscission occurs apically in these cells . ( e ) A histological section through a hair follicle ( left , phase - contrast microscopy ) stained for the stem - cell marker keratin 15 ( K15 ) to identify the bulge region ( dotted box ) , the stem - cell niche . DNA stain ( DAPI ) and the phase - contrast microscopy image show full follicle architecture . ( f ) Upper panels show MBd - accumulating cells in the bulge region ( boxed ) co - labelled with K15 and MKLP1 . Enlargements ( lower panels ) of the boxed region highlight a cell with four MBds ( asterisks ) . N , nucleus . Scale bar , 5 μm . ( g - i ) Quantitative analysis and representative images show a decrease in MBd - accumulating cells on the differentiation of pluripotent stem cells ( g ) to fibroblast - like cells ( h ) , and an increase in MBd - accumulating cells after reprogramming differentiated cells ( h ) to induced pluripotent stem cells ( i ) . Numbers refer to mean ± s . d . , n = 3 . MKLP1 , MBds ; ZO - 1 , tight junctions ; α - tubulin , microtubules ; Aurora B , midbodies . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) A histological section through mouse seminiferous tubules labelled for MKLP1 shows several MKLP1+ puncta in cells of the basal layer where stem cells reside. Scale bar, 20 μm. Inset: Enlargement of the cell marked by the arrow.(b,c) Electron micrographs of mitotic midbody (b, arrow) and multiple midbody-like structures in interphase cells with similar shapes and sizes in a juxtanuclear position (c, arrows) in basal cells of mouse seminiferous tubules. N, nucleus. Scale bars, 1 μm.(d) Representative planes of a neural progenitor cell in the ventricular zone (Sox2+, bottom left panel) of an E13.5 mouse brain show that an intracellular MBd (asterisk) is associated with the ventricle-facing daughter in the asymmetrically dividing cell (top row). The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell. CD133, midbody/MBd marker (green); Na/K-ATPase, cell-border marker (red); DRAQ5, DNA (blue); DAPI, DNA. v, ventricle. Scale bar, 5 μm. Note that abscission occurs apically in these cells.(e) A histological section through a hair follicle (left, phase-contrast microscopy) stained for the stem-cell marker keratin 15 (K15) to identify the bulge region (dotted box), the stem-cell niche. DNA stain (DAPI) and the phase-contrast microscopy image show full follicle architecture. (f) Upper panels show MBd-accumulating cells in the bulge region (boxed) co-labelled with K15 and MKLP1. Enlargements (lower panels) of the boxed region highlight a cell with four MBds (asterisks). N, nucleus. Scale bar, 5 μm.(g-i) Quantitative analysis and representative images show a decrease in MBd-accumulating cells on the differentiation of pluripotent stem cells (g) to fibroblast-like cells (h), and an increase in MBd-accumulating cells after reprogramming differentiated cells (h) to induced pluripotent stem cells (i). Numbers refer to mean±s.d., n=3. MKLP1, MBds; ZO-1, tight junctions; α-tubulin, microtubules; Aurora B, midbodies. Scale bar, 10 μm.


figf4 ( b ) Cells pulse - chased with EdU show a decrease in EdU intensity ( x axis ) over time ( y axis ) , reflecting dilution of dye after cell divisions . ( c , d ) After a 96 h chase period , EdU levels were compared between cells with MBd numbers of > 1 , 1 and 0 ( y axis ) in HeLa ( c ) and SAOS - 2 cells ( d ) . In both cases , no significant differences were noted ( c , P = 0 . 2101 ; d , P = 0 . 5609 , one - way analysis of variance , with at least 800 cells analysed for each experiment , n = 3 ) , indicating similar cycling rates among different subpopulations of cells . ( b - d ) Each graph is a representative experiment . Cells analysed are shown by green points , the medians are depicted by vertical red lines , and horizontal red lines with ticks illustrate the interquartile range .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(b) Cells pulse-chased with EdU show a decrease in EdU intensity (x axis) over time (y axis), reflecting dilution of dye after cell divisions. (c,d) After a 96 h chase period, EdU levels were compared between cells with MBd numbers of >1, 1 and 0 (y axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, P=0.2101; d, P=0.5609, one-way analysis of variance, with at least 800 cells analysed for each experiment, n=3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analysed are shown by green points, the medians are depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.


figf5 ( a ) Depiction of FPP assay . Digitonin selectively permeabilizes the plasma membrane but not internal membranes . Proteinase K degrades cytoplasmic components but membranous compartments remain intact . Under these conditions , MKLP1 - GFP - labelled MBds ( blue ellipse ) in the cytoplasm will be degraded whereas those inside membrane - bound compartments will not . ( b ) MBds in MBd - poor hRPE - 1 cells are largely protected ( ∼ 90 % in membranous compartments , 10 cells analysed ) , whereas most MBds in HeLa cells are not ( ∼ 27 % , 11 cells analysed ) , and are thus degraded in cytoplasm . Scale bar , 5 μm . ( c ) Presence of MBds in lysosomes on chloroquine or E64d - pepstatin A ( E64d - PepA ) inhibition in hRPE - 1 and HeLa cells , but not in MCF - 7 and H9 hESCs . Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death . A representative image of hRPE - 1 cells inhibited by chloroquine is shown , depicting two MBds inside lysosomes . MKLP1 and LAMP2 are used as MBd ( red ) and lysosome ( green ) markers , respectively . DAPI , DNA ( blue ) . n = 100 MBds per treatment in each of the biological triplicates . Scale bar , 5 μm . ( d ) Percentage of MBd + cells ( MBd levels ) , percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization . MKLP1 and LAMP2 are used as markers as in c . Note that MBds are transferred into only one of the two nascent daughter cells after abscission ( Fig . 2d ) , so a 50 % maximum will be expected for MBd + cells . The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h . ( e ) Both chloroquine and E64d - PepA treatments increase the percentage of MBd + cells in hRPE - 1 cells and HeLa cells ( chloroquine , P = 0 . 0021 and P = 0 . 0187 , respectively ; E64d - PepA , P = 0 . 0022 and P = 0 . 0043 , respectively ; n = 3 for all experiments ) . In contrast , lysosomal inhibition has no detectable effect on hESCs ( H1 , H9 ) or MCF - 7 cancer cells . DMSO , dimethylsulphoxide . Data are presented as mean ± s . d . ( c - e ) , except mean ± s . e . m . in hESCs ( e ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1-GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. (b) MBds in MBd-poor hRPE-1 cells are largely protected (∼90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (∼27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm.(c) Presence of MBds in lysosomes on chloroquine or E64d-pepstatin A (E64d-PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm.(d) Percentage of MBd+ cells (MBd levels), percentage of MBds within lysosomes and percentage of cells exiting cytokinesis following synchronization. MKLP1 and LAMP2 are used as markers as in c. Note that MBds are transferred into only one of the two nascent daughter cells after abscission (Fig. 2d), so a 50% maximum will be expected for MBd+ cells. The peak of MBds transferred to cells is 3 h after plating followed by a peak of MBds entering lysosomes at 7 h.(e) Both chloroquine and E64d-PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d-PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are presented as mean±s.d. (c-e), except mean±s.e.m. in hESCs (e).


figf6 ( a ) Single - plane confocal microscopy images of MBds within LC3 - positive autophagosomes in MEFs expressing GFP - LC3 ( left ) and in hRPE - 1 cells stained for endogenous LC3 ( right ) . MBd markers : Cep55 , MKLP1 or MgcRacGAP . Autophagosomes : GFP - LC3 or LC3 . Note that MKLP1 ( blue ) and MgcRacGAP ( red ) are co - localized ( magenta ) in the autophagosome ( green ) , indicating that MBds are sorted into autophagosomes . Scale bars , 2 μm . ( b ) Decreasing autophagy levels by deletion of the Atg5 gene ( left ) or depletion of ATG7 by siRNA ( right ) significantly increases the percentage of MBd + cells ( P = 0 . 0019 and P = 0 . 021 , respectively , n = 3 ) . Immunoblots confirm loss of the Atg5 - Atg12 conjugation in mutant cells and depletion of ATG7 ( asterisk ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase . ( c ) Rapamycin ( Rapa ) and LiCl co - treatment induces autophagy and decreases the percentage of MBd + cells ( left , HeLa ; P = 0 . 0056 , n = 3 ) . Immunoblots showing increased LC3 - II levels confirm autophagy induction . Induction of autophagy by overexpression of Flag - tagged BECN1 reduces the percentage of MBd + cells ( right , MCF - 7 ; P = 0 . 0008 , n = 4 ) . Ctrl , control . ( d ) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells . Autophagic flux ( autophagic activity ) was measured by changes in the levels of LC3 - II , in the presence or absence of lysosomal inhibitors E64d - PepA . U , uninhibited . I , inhibited . Below , the average of the percentage change in LC3 - II levels after lysosomal inhibition from three experiments . α - tubulin , loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Single-plane confocal microscopy images of MBds within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MBd markers: Cep55, MKLP1 or MgcRacGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and MgcRacGAP (red) are co-localized (magenta) in the autophagosome (green), indicating that MBds are sorted into autophagosomes. Scale bars, 2 μm.(b) Decreasing autophagy levels by deletion of the Atg5 gene (left) or depletion of ATG7 by siRNA (right) significantly increases the percentage of MBd+ cells (P=0.0019 and P=0.021, respectively, n=3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of ATG7 (asterisk). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.(c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control.(d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d-PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control.


figf7 ( a ) Single - plane confocal microscopy images showing co - localization of the MBds and the autophagic receptor NBR1 in U2OS cells and p62 - deleted MEFs . MBd markers : MKLP1 or Cep55 . Scale bar , 2 μm . ( b ) The percentage of MBd + cells is significantly increased following the depletion of NBR1 ( P = 0 . 022 , n = 3 ) , but not another autophagic receptor , p62 . Co - depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone . ( c ) Deletion of the p62 gene does not affect the percentage of MBd + cells . For b and c , immunoblots verify protein loss . ( d ) Co - immunoprecipitation ( IP ) reveals that CEP55 and NBR1 form a complex . Precipitated proteins and 5 % of the input material ( Input ) were analysed by immunoblotting ( IB ) with antibodies against NBR1 or CEP55 . ( e - g ) Overexpression of CEP55 - eGFP ( enhanced green fluorescent protein ) increases the percentage of MBd + cells ( e ; P = 0 . 0007 , n = 3 ) and the percentage of NBR1 - negative MBds ( f ; P = 0 . 0568 , n = 3 ) , presumably by sequestering NBR1 ( red ) away from MBds in cells expressing CEP55 - eGFP ( green ) as shown in g , and consequently preventing MBd degradation . The dotted box in g is enlarged ( top right panel ) , and the labelling of NBR1 and CEP55 - eGFP ( middle and bottom right panels ) are also presented . DAPI , DNA ( blue ) . Scale bar , 5 μm . The data are presented as mean ± s . d . ( b , c , e , f ) . Uncropped images of blots are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Single-plane confocal microscopy images showing co-localization of the MBds and the autophagic receptor NBR1 in U2OS cells and p62-deleted MEFs. MBd markers: MKLP1 or Cep55. Scale bar, 2 μm.(b) The percentage of MBd+ cells is significantly increased following the depletion of NBR1 (P=0.022, n=3), but not another autophagic receptor, p62. Co-depletion of NBR1 and p62 does not further increase MBd levels over NBR1 depletion alone.(c) Deletion of the p62 gene does not affect the percentage of MBd+ cells. For b and c, immunoblots verify protein loss.(d) Co-immunoprecipitation (IP) reveals that CEP55 and NBR1 form a complex. Precipitated proteins and 5% of the input material (Input) were analysed by immunoblotting (IB) with antibodies against NBR1 or CEP55.(e-g) Overexpression of CEP55-eGFP (enhanced green fluorescent protein) increases the percentage of MBd+ cells (e; P=0.0007, n=3) and the percentage of NBR1-negative MBds (f; P=0.0568, n=3), presumably by sequestering NBR1 (red) away from MBds in cells expressing CEP55-eGFP (green) as shown in g, and consequently preventing MBd degradation. The dotted box in g is enlarged (top right panel), and the labelling of NBR1 and CEP55-eGFP (middle and bottom right panels) are also presented. DAPI, DNA (blue). Scale bar, 5 μm. The data are presented as mean±s.d. (b,c,e,f). Uncropped images of blots are shown in Supplementary Fig. S6.


figf8 ( a - c ) Reprogramming is more efficient after MBd enrichment . Differentiated cells ( dH1f ) and embryonic fibroblasts ( IMR90 ) are reprogrammed after stable expression of either NBR1 shRNA or shNT . Emerging iPSC colonies are scored on the basis of TRA - 1 - 60 expression . Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation ( a , b , dH1f , 3 . 1 ± 0 . 5 - fold , n = 15 , P = 0 . 00035 ; IMR90 , 3 . 4 ± 0 . 8 - fold , n = 3 , P = 0 . 02 ; data are mean ± s . e . m . ) but insignificant changes in autophagic activity ( c ) over shNT control . ( b ) Representative plates with TRA - 1 - 60 - immunostained iPSC colonies . The immunoblot ( c , top ) and densitometry ( c , bottom ; percentage of autophagic flux ) show representative results ( n = 3 ) ; α - tubulin , loading control . ( d ) MCF - 7 side - population ( SP ) cells have a significantly higher percentage of MBd + cells over the non - side - population ( MP ; P = 0 . 0015 , n = 3 ; data are mean ± s . d . ) . ( e , f ) MBd enrichment in cancer cells leads to increased anchorage - independent growth . MKLP1 - GFP - expressing HeLa cells are separated into ' MBd - high ' and ' MBd - low ' subpopulations . An increase in the ' MBd high ' over ' MBd low ' ratio is associated with an increase in soft - agar colony formation ( e ) . No significant difference was observed when the enrichment of the MBd - high subpopulation was less than threefold . More soft - agar colonies are formed when MBds are enriched by NBR1 depletion ( NBR1 shRNA ) in HeLa ( f , left ; P = 0 . 0012 , n = 3 ) and mouse 134 - 4 cells ( f , right ; P = 0 . 0086 , n = 3 ) ; control , shNT . Data are mean ± s . d . , and the colony number ( e , f ) is the sum of INT - violet - stained colonies from ten random fields .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(a-c) Reprogramming is more efficient after MBd enrichment. Differentiated cells (dH1f) and embryonic fibroblasts (IMR90) are reprogrammed after stable expression of either NBR1 shRNA or shNT. Emerging iPSC colonies are scored on the basis of TRA-1-60 expression. Cells depleted of NBR1 to increase MBd levels show an increase in iPSC colony formation (a,b, dH1f, 3.1±0.5-fold, n=15, P=0.00035; IMR90, 3.4±0.8-fold, n=3, P=0.02; data are mean±s.e.m.) but insignificant changes in autophagic activity (c) over shNT control. (b) Representative plates with TRA-1-60-immunostained iPSC colonies. The immunoblot (c, top) and densitometry (c, bottom; percentage of autophagic flux) show representative results (n=3); α-tubulin, loading control.(d) MCF-7 side-population (SP) cells have a significantly higher percentage of MBd+ cells over the non-side-population (MP; P=0.0015, n=3; data are mean±s.d.).(e,f) MBd enrichment in cancer cells leads to increased anchorage-independent growth. MKLP1-GFP-expressing HeLa cells are separated into 'MBd-high' and 'MBd-low' subpopulations. An increase in the 'MBd high' over 'MBd low' ratio is associated with an increase in soft-agar colony formation (e). No significant difference was observed when the enrichment of the MBd-high subpopulation was less than threefold. More soft-agar colonies are formed when MBds are enriched by NBR1 depletion (NBR1 shRNA) in HeLa (f, left; P=0.0012, n=3) and mouse 134-4 cells (f, right; P=0.0086, n=3); control, shNT. Data are mean±s.d., and the colony number (e,f) is the sum of INT-violet-stained colonies from ten random fields.


Figure 1E ) Representative images of prometaphase chromosomes from HeLa cells co - transfected with H2A . Z . 2 siRNA and each of the constructs in D ( green ) . Scale bar : 5μm . F ) Quantification of the percentage of cells with micronuclei from experiment ( E ) . The error bars represent the SD of three biological replicates ( Control si N = 887 ; H2A . Z . 2 si N = 1005 ; H2A . Z . 2 si + wt N = 760 ; H2A . Z . 2 si + KR N = 479 ) . Data sets were statistically analysed using Chi - square test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1E) Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and each of the constructs in D (green). Scale bar: 5μm. F) Quantification of the percentage of cells with micronuclei from experiment (E). The error bars represent the SD of three biological replicates (Control si N=887; H2A.Z.2 si N=1005; H2A.Z.2 si + wt N=760; H2A.Z.2 si + KR N=479). Data sets were statistically analysed using Chi-square test.