Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Abstract

BackgroundOne of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape.Methodology/Principal FindingsWe have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally.Conclusions/SignificanceThe present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development.

Identification of emergent motion compartments in the amniote embryo

“…The protocol for generation of transgenic quails is described in detail elsewhere. [56][57][58] In vivo immunofluorescence labeling of extracellular matrix Monoclonal antibody directed against fibronectin (B3D6, DSHB, Iowa City, IA) was directly conjugated to AlexaFluor 555 (Molecular Probes) according to the manufacturer's instructions. The antibody was injected (5-25 nl boluses) bilaterally into the regions all along the length of the primitive streak using a PLI-100 (Harvard instruments) microinjector.…”

“…18,56,61 Briefly, using custom-written software, a computer-controlled wide field (10X objective) epifluorescent microscope (Leica DMR) workstation, equipped with motorized stage and cooled digital camera (Qlmaging Retiga 1300), was used to acquire 12-bit grayscale intensity images at multiple xy locations (fields) and focal planes (z-stacks). Images were uploaded to a storage server (Xserv RAID, Apple, Inc.) for archiving and subsequent processing.…”

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using Chick^GFPNeural Tube Grafting and Carbocyanine Dye DiI Injection

“…Besides all these advantages, it is important to note that vascular injection only labels luminised vessels and therefore does not identify unopened capillaries, endothelial tip cells or isolated endothelial cells. However, further progress in avian transgenesis could provide new ways to circumvent such issues, as exemplified by experiments using Tg(tie1:H2B-eYFP) quail embryos to study vascular morphogenesis 28 . Another limitation of this technique is that, for effective vessel labeling in embryos at E7.5 and beyond, larger amounts of dye need to be injected, which can make experiments expensive.…”

Non-Viral Transgenesis via Direct In Ovo Lipofection in Quail

“…By injecting a replication-defective pantropic retrovirus into quail blastoderms, Mizuarai et al (2001) reported the production of transgenic quail through germline transmission. Thereafter, the virus-mediated transgenic technique at stage X blastoderm demonstrated that transgenic quail expressed a biofunctional protein on the oviduct-specific manner and was used for studying vascular development and morphogenesis as an avian model (Kwon et al, 2010;Sato et al, 2010). The lentiviral transduced PGC-mediated transgenesis successfully produced transgenic quail although the efficiency was still too low (Shin et al, 2008).…”