a 2.5 kb vec promoter sequence was cloned between insulators from the chicken -globin gene 29 and used to drive cre expression .
r26r - lacz/+;vec - cre or r26r - yfp/+;vec - cre conceptuses were generated by crossing vec - cre/+ males with r26r - lacz/+ or r26r - yfp/+ females .
runx1;vec - cre and runx1;vec - cre conceptuses were generated by crossing runx1 ; vec - cre/+ males with runx1 females .
runx1;r26r - yfp/+;vec - cre or runx1;r26r - yfp/+;vec - cre conceptuses were generated from crossing runx1;r26r - yfp/+ ; vec - cre/+ males with runx1 females .
conceptuses were suspended in phosphate - buffered saline or in a 1:2 mixture of benzyl alcohol / benzyl benzoate and visualized with a stereomicroscope .
x - gal ( sigma ) staining was performed as described previously 4 . in some cases the conceptuses
were also incubated with rat anti - mouse pecam1 and abc reagent at 4c 30 , then treated with dab peroxidase substrate .
conceptuses were embedded in paraffin , sectioned , and counterstained with nuclear fast red . to analyze intra - aortic clusters , embryos were fixed and stained with anti - c - kit and anti - pecam1-antibodies .
a 1:2 mix of benzyl alcohol / benzyl benzoate was used to increase the transparency of tissues .
three - dimensional reconstructions were generated from z - stacks ( 6287 serial sections ) .
methylcellulose colony forming assays were performed as described previously 4 and colonies counted after 7 days .
immuno - staining was performed with phycoerytherin , allophycocyanin , or alexa fluor 647 conjugated antibodies stained cells were analyzed on a becton dickinson facscalibur flow cytometer .
a 2.5 kb vec promoter sequence was cloned between insulators from the chicken -globin gene 29 and used to drive cre expression .
r26r - lacz/+;vec - cre or r26r - yfp/+;vec - cre conceptuses were generated by crossing vec - cre/+ males with r26r - lacz/+ or r26r - yfp/+ females .
runx1;vec - cre and runx1;vec - cre conceptuses were generated by crossing runx1 ; vec - cre/+ males with runx1 females .
runx1;r26r - yfp/+;vec - cre or runx1;r26r - yfp/+;vec - cre conceptuses were generated from crossing runx1;r26r - yfp/+ ; vec - cre/+ males with runx1 females .
conceptuses were suspended in phosphate - buffered saline or in a 1:2 mixture of benzyl alcohol / benzyl benzoate and visualized with a stereomicroscope .
x - gal ( sigma ) staining was performed as described previously 4 . in some cases the conceptuses
were also incubated with rat anti - mouse pecam1 and abc reagent at 4c 30 , then treated with dab peroxidase substrate .
conceptuses were embedded in paraffin , sectioned , and counterstained with nuclear fast red . to analyze intra - aortic clusters ,
embryos were fixed and stained with anti - c - kit and anti - pecam1-antibodies .
a 1:2 mix of benzyl alcohol / benzyl benzoate was used to increase the transparency of tissues .
three - dimensional reconstructions were generated from z - stacks ( 6287 serial sections ) .
methylcellulose colony forming assays were performed as described previously 4 and colonies counted after 7 days .
immuno - staining was performed with phycoerytherin , allophycocyanin , or alexa fluor 647 conjugated antibodies stained cells were analyzed on a becton dickinson facscalibur flow cytometer .
 hscs are the founder cells of the adult hematopoietic system , and thus knowledge of the molecular program directing their generation during development is important for regenerative hematopoietic strategies . 
 runx1 is a pivotal transcription factor required for hsc generation in the vascular regions of the mouse conceptus - the aorta , vitelline and umbilical arteries , yolk sac and placenta 1 , 2 . 
 it is thought that hscs emerge from vascular endothelial cells through the formation of intra - arterial clusters 3 and that runx1 functions during the transition from  
 hemogenic endothelium to hscs 4 , 5 . here 
 we show by conditional deletion that runx1 activity in vascular endothelial cadherin ( vec ) positive endothelial cells is indeed essential for intra - arterial cluster , hematopoietic progenitor , and hsc formation . 
 in contrast , runx1 is not required in cells expressing vav , one of the first pan - hematopoietic genes expressed in hscs . 
 collectively these data show that runx1 function is essential in endothelial cells for hematopoietic progenitor and hsc formation from the vasculature , but its requirement ends once or before vav is expressed . 
Methods
Conditional deletion by VEC-Cre or Vav-Cre
Microscopic and histological analyses
Hematopoietic assays
Supplementary Material