I really like those experiments. The power of clonal analysis!
Clonal analysis. VE-cadherin-CreER mice were crossed to either the Rosa-lacZ or the multicolour Cre reporters. At E7.5, E8.5 or E9.5, dams received an intra-peritoneal injection (25G needle) of tamoxifen (Sigma), generally 0.25–0.5 mg, dissolved in 100 ml corn oil. These low doses of tamoxifen were selected to induce rare recombination events and generate single or well-separated clones. Embryos were dissected at E13.5 or E14.5. For lacZ-labelled clones, embryos were fixed and stained with X-gal as described earlier, and those containing sparsely labelled cells were further analysed. Hearts with just a single cluster or two or three well-isolated clusters of cells were sectioned, stained with an endothelial-specific antibody (anti-CD31) before cells were counted and identities assigned by marker (CD31) expression and their location and morphology. Clones marked with multicolour reporters were fixed, sectioned and stained with either DAPI or CD31 as described earlier and analysed using fluorescent filters specific to each fluorophore. Endothelial cell division rate for each clone was estimated from final cell counts, assuming that recombination occurred between 6 and 48 h 44,45 after tamoxifen injection.
Their organ culture method is worthy noticing as well.
Organ cultures. Heart cultures were carried out as described 26 with the following modifications. Embryonic hearts were from either wild-type or apelin-nlacZ embryos. The atria and attached sinus venosus were left intact or dissected from the ventricle with fine-point forceps. Intact hearts or separated SV/A or ventricles were cultured dorsal side up at the air–liquid interface on 8-mm Millicell Cell Culture Insert Filters (Millipore). For tissue recombination experiments, SV/A tissue was placed adjacent to the ventricle at the position where the original SV/A was removed. Cultures were maintained at 37C and 5% CO2 in DMEM media supplemented with 2 ug/ml heparin, 10% fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 uM L-glutamine. After 72 h, explants were fixed with 4% paraformaldehyde and subjected to whole-mount X-gal staining. Some stained explants were then sectioned and immunostained with anti-CD31 antibodies to confirm that X-gal1 nuclei were coronary endothelial cells. Control littermates were fixed immediately after dissection but before culturing, and then immunostained to assess coronary vessel (anti-CD31) and epicardial (anti-a4 integrin) coverage at the beginning of the culture period.
The significance of this work, not just "the century-old enigma resolved", as Mark said, "If we can learn how to reprogram cells to build a new coronary artery just like the original, bypass grafts could last the rest of a lifetime.".
Nature 464, 549-553 (25 March 2010)
DOI: 10.1038/nature08873
Received 30 September 2009; Accepted 4 February 2010Coronary arteries form by developmental reprogramming of venous cells
Kristy Red-Horse 1, Hiroo Ueno 2, Irving L. Weissman 2 & Mark A. Krasnow 1
1. Department of Biochemistry and Howard Hughes Medical Institute,
2. Institute of Stem Cell Biology and Regenerative Medicine,
Stanford University School of Medicine, Stanford, California 94305-5307, USACorrespondence to: Mark A. Krasnow 1 (Email: krasnow@stanford.edu).
Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus—the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.
This data supports our experiments in avian embryos, however the exuberance of scientists in the field made them really want to find endothelial cells in the proepicardium, so they did. The real finding here is that it takes two cell types to make a coronary vessel: endothelial and smooth muscle, and they come from different sources.