Hematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial, and hematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a hemogenic organ akin to the dorsal aorta. Here we examine the hemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Hemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the hemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells expressing cardiac markers serve as a de novo source for transient definitive hematopoietic progenitors.

During embryonic development, smooth muscle within the ascending aorta arises from two distinct sources: second heart field progenitors and the neural crest. It has recently been hypothesized that the boundary between smooth muscle from these distinct origins may be particularly susceptible to acute aortic dissection. While the contribution of second heart field progenitors to the ascending aorta is well established, detailed mapping of the anatomical distribution of second heart field-derived smooth muscle at this smooth muscle boundary has yet to be observed using a committed cardiac progenitor Cre-lineage. Using Nkx2-5-Cre knockin mice, the anatomical distribution of second heart field derived aortic smooth muscle was mapped in detail. Specifically, Nkx2-5-Cre-labeled cells constitute the entirety of the smooth muscle layer at the aortic base and then become restricted to the adventitial side of the ascending aortic media. This distribution pattern is present by E12.5 in the embryo and persists throughout embryonic development. These data reveal previously unappreciated details regarding the anatomical distribution of second heart field-derived smooth muscle within the aorta as well as the non-cardiomyocyte fates labeled by the Nkx2-5-Cre lineage.

Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors.

Rationale During embryogenesis, hematopoietic cells appear in the myocardium prior to the initiation of coronary formation. However, their role is unknown. Objective Here we investigate whether pre-existing hematopoietic cells are required for the formation of coronary vasculature. Methods and Results As a model of for hematopoietic cell deficient animals, we used Runx1 knockout embryos and Vav1-cre; R26-DTA embryos, latter of which genetically ablates 2/3 of CD45+ hematopoietic cells. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation ex vivo. Interestingly, in both models it appears that epicardial to mesenchymal transition is adversely affected in the absence of hematopoietic progenitors. Conclusion Hematopoietic cells are not merely passively transported via coronary vessel, but substantially involved in the induction of the coronary growth. Our findings suggest a novel mechanism of coronary growth.

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