Abstract
BackgroundBone morphogenetic protein (BMP) signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders. BMP signalling output in endothelial cells is highly context- and dose-dependent. Phosphorylation of the BMP intracellular effectors, SMAD1/5/9, is routinely used to monitor BMP signalling activity. To better understand the in vivo context-dependency of BMP-SMAD signalling, we investigated differences in BMP-SMAD transcriptional activity in different vascular beds during mouse embryonic and postnatal stages. For this, we used the BRE::gfp BMP signalling reporter mouse in which the BMP response element (BRE) from the ID1-promotor, a SMAD1/5/9 target gene, drives the expression of GFP.ResultsA mosaic pattern of GFP was present in various angiogenic sprouting plexuses and in endocardium of cardiac cushions and trabeculae in the heart. High calibre veins seemed to be more BRE::gfp transcriptionally active than arteries, and ubiquitous activity was present in embryonic lymphatic vasculature. Postnatal lymphatic vessels showed however only discrete micro-domains of transcriptional activity. Dynamic shifts in transcriptional activity were also observed in the endocardium of the developing heart, with a general decrease in activity over time. Surprisingly, proliferative endothelial cells were almost never GFP-positive. Patches of transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations.ConclusionThe BRE::gfp mouse allows to investigate selective context-dependent aspects of BMP-SMAD signalling. Our data reveals the highly dynamic nature of BMP-SMAD mediated transcriptional regulation in time and space throughout the vascular tree, supporting that BMP-SMAD signalling can be a source of phenotypic diversity in some, but not all, healthy endothelium. This knowledge can provide insight in vascular bed or organ-specific diseases and phenotypic heterogeneity within an endothelial cell population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0133-x) contains supplementary material, which is available to authorized users.
Highlights
Bone morphogenetic protein (BMP) signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders
We took advantage of the unstable nature of gfp mRNA, the sensitivity and single cell resolution of in situ hybridisation (ISH) on the one hand and the direct co-observation of (enhanced) green fluorescent protein (GFP) fluorescence on the other hand to validate whether GFP protein localisation reflects well the BMP response element (BRE)::gfp transcriptional activity in endothelium
We show that there is a near to absolute correlation between the gfp mRNA expression and direct GFP fluorescence localisation in vascular beds like e.g. the cardinal vein
Summary
Bone morphogenetic protein (BMP) signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders. BMP signalling output in endothelial cells is highly context- and dose-dependent. To better understand the in vivo context-dependency of BMPSMAD signalling, we investigated differences in BMP-SMAD transcriptional activity in different vascular beds during mouse embryonic and postnatal stages. The formation of the cardiovascular and lymphatic network is crucial for development and physiology. In hypoxic environments angiogenic sprouts with tip and stalk cells emerge. From the cardinal vein some venous endothelial cells (ECs) differentiate into lymphatic ECs (LECs), that migrate to form lymphatic sacs which in turn sprout to form a lymphatic network similar to angiogenesis events [2]. Failure to establish a (lymphatic) vascular network leads to severe embryonic defects at mid-to late gestation, whereas misregulation after birth can lead to diseases such as cancer, chronic and inflammatory disorders and oedema [3,4,5]
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