The cardiovascular system is the first functional organ system to develop in the mammalian embryo. The blood vessels that initially comprise this organ originate by vasculogenesis, the aggregation of de novo-forming angioblasts (endothelial precursors) into simple endothelial tubes. Angioblasts in the mouse embryo first emerge from the mesoderm as Flk1+ cells around embryonic day (E) 7.0 and assemble a simple circulatory loop consisting of a heart, dorsal aorta, yolk sac plexus and sinus venosus by E8.0 
. Shortly after its formation, this early vascular circuit is remodelled by angiogenesis, the proliferation, sprouting and pruning of pre-existing vessels, transforming it into a complex network of branched endothelial tubes of varying diameter, length and identity. Such remodelling of pre-existing vessels is dependent on both genetically hardwired events and hemodynamic forces 
Given the complex nature of the vascular system and the diversity of biological processes required for its assembly and refinement, it is hardly surprising that a large number of signalling pathways are employed in its development. Mutations in pathways required for vascular development frequently manifest phenotypes that result in embryonic lethality at mid gestation. In mice, mutations affecting Notch 
, TGFβ 
, Hedgehog 
, VEGF 
, ephrin/Eph 
and angiopoietin/Tie 
signalling (among others) result in abnormal vascular development between E8.0 and E10.0 and ultimately embryonic lethality. The vascular activities of these pathways are not limited to this developmental time window, but extend to organogenesis 
, maintenance of vascular homeostasis in adulthood 
and states of pathological angiogenesis 
. Correct interpretation of how these pathways regulate vascular development between E8.0 and E10.0 would therefore improve our understanding of how they contribute to later vascularization events. Such interpretation is often impeded however, by the complex nature of the vascular phenotypes, an inability to observe the vasculature of the mutants in its entirety and an incomplete understanding of the normal sequence of vascular remodelling events that occur during this period of development. Previous studies in zebrafish 
and chick 
have provided insight into normal vascular development, but have limited applicability to the sequence of vascular remodeling events in the mammalian embryo primarily due to differences in anatomy and the increased use of plexus bed intermediates in mammals compared to zebrafish. We have sought to address this issue by generating a high resolution, three-dimensional (3D) atlas of the developing mouse vasculature between E8.0 and E10.0 (5–30 somites).
The mouse embryo grows rapidly between E8.0 and E10.0 and undergoes complex morphological and conformational changes that present significant challenges to current imaging technologies. These challenges are further complicated by the inherent properties of the vascular system as a 3D network of branched, interconnected tubes of varying length and size. Accurate assessment of vascular development at this stage therefore requires a 3D imaging modality capable of visualizing the vasculature in its un-manipulated entirety in embryos of increasing size while retaining sufficient isotropic resolution (on the order of a few microns) to capture the details of the finest capillaries. Without these properties, significant positional information about the vasculature is lost and artefacts are introduced. While confocal microscopy has been used to generate an atlas of vascular development in zebrafish embryos 
and study projections of the vasculature of dissected mouse embryos prior to E8.5 
, it does not provide sufficient specimen coverage to create the 3D images necessary to visualize the complete embryonic vasculature in mouse embryos beyond this time point. Later embryonic stages have been studied using corrosion casting and electron microscopy 
. While these investigations provided 3D representation, they did not retain information about vessels that cannot be perfused such as blind ended angiogenic sprouts, narrow vessels and vessels yet to form a complete lumen.
Recently, a new imaging modality named Optical Projection Tomography (OPT) 
was developed to obtain molecular specificity and 3D cellular level resolution over complete specimens up to 1 cubic centimetre (cc) in size. OPT has been shown to support the use of multiple molecular markers 
, and has been previously used to visualize mouse embryos 
, chick embryos 
, developing plant material 
, drosophila melanogaster 
and whole adult mouse organs 
. We have further developed OPT using frequency-distance relationship 
(FDR)-based deconvolution to obtain higher resolution images, on the order of a few microns, while still retaining the ability to image large specimen sizes 
. We set out to use this technique to study the normal sequence of mouse vascular development between E8.0 and E10.0.
Ideally one would be able to image a single living mouse embryo over an extended period of time in order to visualize the complete development of a single vasculature. 3D optical imaging of the live mouse embryo, however, is still a challenge due to the in utero
development of mouse embryos and significant light scattering caused by living tissue. This in vivo
time course can instead be approximated by static imaging of multiple genetically identical embryos collected at a range of ages throughout gestation. To this end, we performed FDR-based deconvolution OPT imaging on fixed PECAM-1-immunostained embryos ranging from 5 to 30 somites to study the normal development of the early embryonic mouse vasculature. PECAM-1 is major constituent of the endothelial cell intercellular junction and is widely used as a molecular marker for mature endothelial cells 
. With these images we are able to visualize vascular development across this age range, from vasculogenesis to capillary plexus to the development of larger vessels. The collection of all images comprises an atlas of normal development of the embryonic mouse vasculature, which is made freely available to other researchers. Analysis of this atlas has resulted in significant new information regarding the normal vascular development in the mouse embryo.