Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One 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 Findings: We 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/Significance: The 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

Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: Cellular model of pathology

The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials

Time-lapse microscopy of macrophages during embryonic vascular development

Macrophages are present before the onset of blood flow, but very little is known about their function in vascular development. We have developed a technique to concurrently label both endothelial cells and macrophages for time-lapse microscopy using co-injection of fluorescently conjugated acetylated low-density lipoprotein (AcLDL) and phagocytic dye PKH26-PCL.status: publishe

Embryogenesis of the first circulating endothelial cells

Prior to this study, the earliest appearance of circulating endothelial cells in warm-blooded animals was unknown. Time-lapse imaging of germ-line transformed Tie1-YFP reporter quail embryos combined with the endothelial marker antibody QH1 provides definitive evidence for the existence of circulating endothelial cells - from the very beginning of blood flow. Blood-smear counts of circulating cells from Tie1-YFP embryos showed that up to 30% of blood-borne cells are Tie1 positive; though cells expressing low levels of YFP were also positive for benzidine, a hemoglobin stain, suggesting that these cells were differentiating into erythroblasts. Electroporation-based time-lapse experiments, exclusively targeting the intra-embryonic mesoderm were combined with QH1 immunostaining. The latter antibody marks quail endothelial cells. Together the optical data provide conclusive evidence that endothelial cells can enter blood flow from vessels of the embryo proper, as well as from extra-embryonic areas. When Tie1-YFP positive cells and tissues are transplanted to wild type host embryos, fluorescent cells emigrate from such transplants and join host vessels; subsequently a few YFP cells are shed into circulation. These data establish that entering circulation is a commonplace activity of embryonic vascular endothelial cells. We conclude that in the class of vertebrates most closely related to mammals a normal component of primary vasculogenesis is production of endothelial cells that enter circulation from all vessels, both intra- and extra-embryonic.status: publishe

Convective tissue movements play a major role in avian endocardial morphogenesis

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis

Frequency-Modulated Continuous Flow Analysis Electrospray Ionization Mass Spectrometry (FM-CFA-ESI-MS) for Sample Multiplexing

A method for multiplexed sample analysis by mass spectrometry without the need for chemical tagging is presented. In this new method, each sample is pulsed at unique frequencies, mixed, and delivered to the mass spectrometer while maintaining a constant total flow rate. Reconstructed ion currents are then a time-dependent signal consisting of the sum of the ion currents from the various samples. Spectral deconvolution of each reconstructed ion current reveals the identity of each sample, encoded by its unique frequency, and its concentration encoded by the peak height in the frequency domain. This technique is different from other approaches that have been described, which have used modulation techniques to increase the signal-to-noise ratio of a single sample. As proof of concept of this new method, two samples containing up to 9 analytes were multiplexed. The linear dynamic range of the calibration curve was increased with extended acquisition times of the experiment and longer oscillation periods of the samples. Because of the combination of the samples, salt had little effect on the ability of this method to achieve relative quantitation. Continued development of this method is expected to allow for increased numbers of samples that can be multiplexed

Embryogenesis of the First Circulating Endothelial Cells

<div><p>Prior to this study, the earliest appearance of circulating endothelial cells in warm-blooded animals was unknown. Time-lapse imaging of germ-line transformed Tie1-YFP reporter quail embryos combined with the endothelial marker antibody QH1 provides definitive evidence for the existence of circulating endothelial cells – from the very beginning of blood flow. Blood-smear counts of circulating cells from Tie1-YFP embryos showed that up to 30% of blood-borne cells are Tie1 positive; though cells expressing low levels of YFP were also positive for benzidine, a hemoglobin stain, suggesting that these cells were differentiating into erythroblasts. Electroporation-based time-lapse experiments, exclusively targeting the intra-embryonic mesoderm were combined with QH1 immunostaining. The latter antibody marks quail endothelial cells. Together the optical data provide conclusive evidence that endothelial cells can enter blood flow from vessels of the embryo proper, as well as from extra-embryonic areas. When Tie1-YFP positive cells and tissues are transplanted to wild type host embryos, fluorescent cells emigrate from such transplants and join host vessels; subsequently a few YFP cells are shed into circulation. These data establish that entering circulation is a commonplace activity of embryonic vascular endothelial cells. We conclude that in the class of vertebrates most closely related to mammals a normal component of primary vasculogenesis is production of endothelial cells that enter circulation from all vessels, both intra- and extra-embryonic.</p></div

Transplantation of Tie1-YFP tissue or cells into wild type embryos show that donor cells are capable of engaging in vasculogenesis and entering circulation.

<p>The figure shows a single time point selected from two different time-lapse recordings. Both recordings are contained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s012" target="_blank">Movie S11</a>. <b>Panel (a)</b> shows Tie1-YFP tail bud tissue transplanted into a wild-type embryo (n = 6) in which fluorescent cells (green) have emigrated from the explant (red oval) and colonized host vascular elements (white arrows) in 4 out of 6 explants. The white circle indicates a pair of circulating cells that are lost from the ROI in the next frame (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s012" target="_blank">Movie S11a</a> at the 15.46 hr time point). Careful inspection of the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s012" target="_blank">Movie S11</a> reveals other Tie1-YFP endothelial cells that also entered circulation. The red oval in the diagram denotes the initial position of the transplanted tail bud tissue, before recording began. <b>Panel (b)</b> shows a wild-type embryo injected with a bolus of a disperse cell suspension isolated from HH10 Tie1-YFP embryos (n = 4). The image frame shown is extracted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s012" target="_blank">Movie S11b</a> at the 17.46 hr time point. Multiple injections were made parallel to the vertebral axis as denoted by the red stars in the DIC panel and the diagram. Most fluorescent endothelial cells (green) remained near the injection site (red stars). Many YFP cells, however, participated in vessel formation and joined the host vasculature (white arrows). Eventually, a few Tie1-YFP cells entered circulation (see the circled cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s012" target="_blank">Movie S11b</a>). These data depicting transplanted tissue or cells demonstrate that donor Tie1-YFP endothelial cells are capable of participating in primary vasculogenesis and entering circulation when transferred to a host embryo. The line drawings in Panels 6a and 6b represent an idealized HH7-8 embryo. See the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060841#pone.0060841.s012" target="_blank">Movie S11</a> regarding optics and image acquisition. Scale bars  = 100 µm.</p