A 293FT - TetR - 3xFlag - Usp27x or 3xFlag - Usp27xC87A , a polyclonal derivative where the Bim - locus had been targeted by CRISPR / Cas9 - and four single clones obtained by serial dilution from the polyclonal line were treated as indicated ( PMA [ 16 . 2 nM ] , QVD [ 10 µM ] ) . Apoptosis was measured after 24 h of treatment by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = 14 ( 3xFlag - Usp27x line and 3xFlag - Usp27xBim2KO polyclonal line ) , n = 5 ( 3xFlag - Usp27xC87A , and 3xFlag - Usp27xBim2KO clone # 14 ) , n = 7 ( 3xFlag - Usp27xBim2KO clone # 11 ) or n = 4 ( 3xFlag - Usp27xBim2KO clone # 4 and # 7 ) separate experiments . P - values ( t - test ) for statistically significant differences are shown .

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

A 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.


B The same cells as in A ( except Usp27xC87A mutant ) were treated as in A and stained for active Bax . Data ( means / SEM ) are from n = 8 ( 3xFlag - Usp27x line and 3xFlag - Usp27xBim2KO polyclonal line ) , n = 5 ( 3xFlag - Usp27xBim2KO clone # 11 and # 14 ) or n = 4 ( 3xFlag - Usp27xBim2KO clone # 4 and # 7 ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . Expression levels of Bim , β - TrCP and Flag - Usp27x of the different cell lines are shown in Appendix Fig . S5E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The same cells as in A (except Usp27xC87A mutant) were treated as in A and stained for active Bax. Data (means/SEM) are from n=8 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xBim2KO clone #11 and #14) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown. Expression levels of Bim, β-TrCP and Flag-Usp27x of the different cell lines are shown in Appendix Fig. S5E.


A Lines based on the human melanoma cell line 1205Lu ( BRAF - V600E - positive ) were made to carry GFP - Usp27x , GFP - Usp27xC87A or GFP - Usp22 under the control of a doxycycline ( dox ) - inducible promoter . GFP - Usp27x , GFP - Usp27xC87A or GFP - Usp22 were induced with dox for 24 or 48h . Levels of Bim were determined by Western blotting . The two left panels are from the same membrane and exposure times . Similar results were obtained in n = 4 separate experiments ( left ) and n = 3 experiments ( right ) . See also Appendix Fig . S6A where 3xFlag - Usp22 is also shown to be unable to increase Bim levels .

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

A Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.


B A cell line derived from the BRAF - V600E - positive human melanoma line WM1158 was generated to carry inducible GFP - Usp27x . GFP - Usp27x was induced , and Bim was detected as in A . Similar results were obtained in n = 3 separate experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A cell line derived from the BRAF-V600E-positive human melanoma line WM1158 was generated to carry inducible GFP-Usp27x. GFP-Usp27x was induced, and Bim was detected as in A. Similar results were obtained in n=3 separate experiments.


C Derivatives of the NSCLC cell line HCC827 ( expressing constitutively active EGFR ) were generated that only express the TetR - repressor or carrying in addition either GFP - Usp27x or GFP - Usp27xC87A . Cells were treated with dox for 72h . Bim was detected by Western blotting . Similar results were obtained in n = 2 separate experiments . GFP - Usp27x , GFP - Usp27xC87A , or GFP - Usp22 expression ( A - C ) was detected using an antibody against GFP .

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

C Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.


D Caco2 cells carrying a dox inducible HA - BRAF - V600E [ 37 ] ( left two lanes ) or the same cells stably expressing 3xFlag - Usp27x were treated with dox for 48h to induce expression of HA - BRAF - V600E . Bim was detected by Western blotting . The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100 % ( normalized to the tubulin signal for each condition ) . Similar results were obtained in n = 3 separate experiments with varying induction times . See also Appendix Fig . S6D .

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

D Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.


A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF - V600ECaco2 cells carrying inducible HA - BRAF - V600E were transfected with control siRNA ( siCo3 ) or siRNA directed against Usp27x ( combination of three siRNAs targeting Usp27xmRNA ) for 24h prior to BRAF - V600E expression with dox for 2h ( left ) or 4h ( right ) . BimEL was detected by Western blotting . Similar results were also observed in n = 2 more experiments after 3h induction ( not shown , see Appendix Fig . S6E for siRNA efficacy ) .

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

A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF-V600ECaco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27xmRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). BimEL was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).


B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT - Usp27xKO ( clone 2 / 10 , generated using CRISPR - Cas - 9 ) were treated with PMA [ 16 . 2 nM ] for 16h or 24h to induce BimEL degradation . Western blots show protein levels of n = 3 independently performed experiments ( similar results were obtained in one more experiment , not shown ) . See Appendix Fig . S6G for verification of the Usp27xKO cell line by DNA sequence analysis .

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

B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce BimEL degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.


A 293FT - TetR - 3xFlagUsp27x cells were treated with PMA [ 16 . 2 nM ] plus QVD [ 10 µM ] and 3xFlag - Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide ( CHX ) . Cells were harvested at the indicated time points , and levels of BimEL were determined by Western blotting . Similar results were obtained in n = 3 separate experiments . Cut membranes shown were from one membrane with same exposure time . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point .

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

A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point.


B 1205Lu melanoma cells carrying dox - inducible GFP - Usp27x were treated with dox for 24h . Cycloheximide ( CHX , 1µg / ml ) was then added . Cells were harvested at the indicated time points , and levels of BimEL were determined by Western blotting . GFP - Usp27x was detected with anti - GFP - antibodies . Similar results were obtained in n = 3 separate experiments . A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox - inducible GFP - Usp27xC87A mutant . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point set to 100 % . The starting point for the non - Usp27x / C87A inducing cells ( - dox ) is shown in lane 1 for experiment one and two ( - dox , - CHX ) . Cut membranes shown were from one membrane with same exposure time . Same results were obtained for WM1158 melanoma cells ( data not shown ) .

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

B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).


C HCC827 cells were induced to express GFP - Usp27x with dox as indicated . 24h later cycloheximide ( CHX , 1µg / ml ) was added for the indicated time . BimEL was detected after the indicated times of treatment by Western blotting . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point . Similar results were obtained in n = 2 separate experiments . Cut membranes shown were from one membrane with same exposure time .

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

C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.


A 1205Lu melanoma cells carrying dox - inducible GFP , GFP - Usp27x or GFP - Usp27xC87A were treated with doxycycline ( dox ) as indicated . After 48 hUO126 ( 10µM ) was added for another 48 h . Apoptosis was measured by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = 3 ( GFP ) or from n ≥ 6 separate experiments ( GFP - Usp27x and GFP - Usp27xC87A ) . P - values ( t - test ) for statistically significant differences are shown . N . s . , p > 0 . 05 . The addition of QVD inhibited cells from active Caspase - 3 positive staining ( not shown ) . A stain for active Bax is shown in Appendix Fig . S7B .

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

A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 hUO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.


B HCC827 NSCLC cells carrying dox - inducible GFP - Usp27xC87A , GFP - Usp27x , or two separate lines established from these GFP - Usp27x - cells where the Bim - locus had been targeted for deletion using CRISPR / Cas9 ( Bim - KO ) , were treated with combinations of dox and the EGFR - inhibitor gefitinib as indicated . Apoptosis was measured after 72h of treatment by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = ≥ 15 ( maternal GFP - Usp27x line ) or from n ≥ 5 ( for Bim1KO ) or from n ≥ 6 ( for Bim2KO ) , or n = 4 ( for GFP - Usp27xC87A ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . Again , the addition of QVD inhibited cells from active Caspase - 3 positive staining ( not shown ) . The inset shows Bim - knock - out - efficiency ( n = 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).


Figure 8 Loss of Usp27xreduces gefitinib - induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x - locus had been targeted for deletion using CRISPR / Cas9 with two different guide RNA against Usp27x ( Usp27xKO - 1 and Usp27xKO - 4 ) , were treated with gefitinib [ 10µM ] for 48h . Apoptosis was measured by staining for active caspase - 3 ( A ) or by staining for activated Bax ( antibody : 6A7 clone ) followed by flow cytometric analysis . Data shown in A , B ( means / SEM ) are from n = 4 separate experiments . P - values ( t - test ) for statistically significant differences are shown .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8 Loss of Usp27xreduces gefitinib-induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


Flow cytometry 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 .

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

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


CFU - C numbers in wild type 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 .

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

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


Total 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 .

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

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


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 .

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

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


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

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

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


Number of EMPs in peripheral blood ( PB ) of wild type 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 . ) .

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


Representative confocal whole - mount immunofluorescence 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 ) .

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

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


Confocal whole - mount immunofluorescence 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 .

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

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


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 ) .

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

Percentage of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Percentages of macrophage populations in wild type 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 .

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

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


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

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

Percentage 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 .

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

Representative flow 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Confocal whole - mount immunofluorescence 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 .

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

Confocal whole-mount immunofluorescence analysis (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 .

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

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


Absolute numbers ( ± SD ) of E10 . 5 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 .

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

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


CFU - C numbers per wild type 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 .

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

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


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 .

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

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


Confocal WM - IF 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 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 .

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 .

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

Analysis of 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 ) .

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


Confocal whole - mount immunofluorescence ( WM - IF ) 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 .

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

Confocal whole-mount immunofluorescence (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 .

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

Confocal WM-IF analysis of 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 .

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

Confocal 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Left : number of Ter119 - Kit + CD41 + CD16 / 32 + EMPs in Tie2Cre : : Kitl ∆ / ∆ 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 .

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

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


CFU - C numbers in Tie2Cre : : Kitl ∆ / ∆ 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 .

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

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


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 ∆ / ∆ ) .

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

Analysis of long-term multi-lineage HSC potential in E11.5 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 .

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

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


Number of phenotypic LSK , HPC 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 .

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


Confocal whole - mount immunofluorescence 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 .

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

Confocal whole-mount immunofluorescence analysis of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A - 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 .

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

A-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 .

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

A-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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.


GFP immunofluorescence in colonic and pancreatic tissue from R26AID + / KIVillinCRE + / TG and R26AID + / KIp48CRE + / KI mice . Scale bar : 50 μm .

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

GFP 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 .

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

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

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)).


A , 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 .

A, 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 .

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.


Representative 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 .

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

Representative images of 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 .

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

Representative 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 ) .

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

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 .

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

Analysis of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of 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.


Hematoxylin - 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 .

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

Hematoxylin-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 .

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

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 .

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

Representative 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 .

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

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.


( 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 ) .

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

(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 .

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

(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 .

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

(e) 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 .

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

(f) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) 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.


( 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 .

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

(a) The 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(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).


( 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 .

(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 .

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

(b,c) 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.