Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

Dynamic endothelial cell rearrangements drive developmental vessel regression.

Vascular networks are formed and maintained through a multitude of angiogenic processes, such as sprouting, anastomosis and pruning. Only recently has it become possible to study the behavior of the endothelial cells that contribute to these networks at a single-cell level in vivo This Review summarizes what is known about endothelial cell behavior during developmental angiogenesis, focusing on the morphogenetic changes that these cells undergo.

Cell behaviors and dynamics during angiogenesis

Many studies reveal a fundamental role for extracellular matrix-mediated signaling through integrins and Rho GTPases as well as matrix metalloproteinases (MMPs) in the molecular control of vascular tube morphogenesis in three-dimensional (3D) tissue environments. Recent work has defined an endothelial cell (EC) lumen signaling complex of proteins that controls these vascular morphogenic events. These findings reveal a signaling interdependence between Cdc42 and MT1-MMP to control the 3D matrix-specific process of EC tubulogenesis. The EC tube formation process results in the creation of a network of proteolytically generated vascular guidance tunnels in 3D matrices that are utilized to remodel EC-lined tubes through EC motility and could facilitate processes such as flow-induced remodeling and arteriovenous EC sorting and differentiation. Within vascular guidance tunnels, key dynamic interactions occur between ECs and pericytes to affect vessel remodeling, diameter, and vascular basement membrane matrix assembly, a fundamental process necessary for endothelial tube maturation and stabilization. Thus, the EC lumen and tube formation mechanism coordinates the concomitant establishment of a network of vascular tubes within tunnel spaces to allow for flow responsiveness, EC-mural cell interactions, and vascular extracellular matrix assembly to control the development of the functional microcirculation.

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Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting

Transgenic animals are extensively used to study in vivo gene function as well as to model human diseases. The technology for producing transgenic animals exists for a variety of vertebrate and invertebrate species. The mouse is the most utilized organism for research in neurodegenerative diseases. The most commonly used techniques for producing transgenic mice involves either the pronuclear injection of transgenes into fertilized oocytes or embryonic stem cell-mediated gene targeting. Embryonic stem cell technology has been most often used to produce null mutants (gene knockouts) but may also be used to introduce subtle genetic modifications down to the level of making single nucleotide changes in endogenous mouse genes. Methods are also available for inducing conditional gene knockouts as well as inducible control of transgene expression. Here, we review the main strategies for introducing genetic modifications into the mouse, as well as in other vertebrate and invertebrate species. We also review a number of recent methodologies for the production of transgenic animals including retrovirus-mediated gene transfer, RNAi-mediated gene knockdown and somatic cell mutagenesis combined with nuclear transfer, methods that may be more broadly applicable to species where both pronuclear injection and ES cell technology have proven less practical.

http://biology.hunter.cuny.edu/molecularbio/Class%20Materials%20Spring%202012%20Biol302/Lecture%2014/Animal%20Transgenesis.pdf

Animal transgenesis: an overview

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.

/pdf/dynamic-analysis-of-vascular-morphogenesis-using-transgenic-37thwr0dfs.pdf

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

For the past 50 years, the Japanese quail (Coturnix japonica) has been a popular animal model in numerous fields of research. The quail's 16-d developmental period and its easily accessible embryo make C. japonica a convenient model for studies of developmental biology. Because its lifespan is relatively short and its physiology is comparable to that of humans, the adult quail is useful for studies of aging and disease. The authors describe the Japanese quail as an animal model and, drawing on their experience raising a quail colony at the California Institute of Technology, present detailed guidelines for the husbandry of the species.

Japanese quail (Coturnix japonica) as a laboratory animal model.

The ability to generate transgenic mice has been a powerful tool in studying functional genomics, and much of our knowledge about developmental biology has come from the study of chicken embryology. Unfortunately, the availability of molecular genetic techniques, such as transgenics and knockouts, has been limited for developmental biologists using avian animal models. Efforts to develop a system for the rapid production of transgenic chickens have met with many obstacles, including high animal husbandry costs and long generational times. Recently, the Japanese quail has proven to be an excellent model organism for the production of transgenic avians using lentiviral vectors. The relatively small size of the adults, short time to sexual maturity, and prodigious egg production of the Japanese quail make development of transgenic lines less labor- and space-intensive compared to chickens. The high degree of homology between chicken and quail genomes allows researchers to design highly specific DNA constructs for the production of transgenic birds. In addition, transgenic quail offer all of the advantages of the classic avian developmental model system, such as the ability to readily produce quail:chick transplant chimeras. Finally, Japanese quail are ideal for in ovo imaging of embryos expressing fluorescent reporters introduced from a transgene and/or electroporation. Here, we provide detailed methods for generating transgenic quail using high-titer lentivirus.

Japanese quail: an efficient animal model for the production of transgenic avians.

Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.

Transgenic quail as a model for research in the avian nervous system: A comparative study of the auditory brainstem

Publisher Summary This chapter presents several methods used in the laboratory for the purpose of generating transgenic avians Avian embryos are ideal for imaging because they are accessible to study at all stages of development outside of their vertebrate mother, unlike mammals The ability to combine transgenic avian technology with the use of dynamic imaging can be used to dynamically follow cell movements in living embryos Access to the avian embryo requires the researcher to cut a small opening in the eggshell After experimental manipulation, the opening is sealed with transparent adhesive tape and the egg is incubated to the desired development stage It has been possible to make transgenic chickens through the use of viral vectors and transplantation of embryonic blastodermal cells for some time Numerous techniques have been developed for isolation, culture, and reintroduction of blastoderm cells to generate chimeric animals Cell tracking studies that rely upon microinjection or micro-application of vital dyes find that their dyes can bleach from prolonged imaging or be diluted out as the labeled cells divide Using vectors that express fluorescent proteins provide both a renewable source of the fluorescent label via continued gene expression, as well as tissue-specific expression to study subsets of cells Introducing such vectors into avian embryos is typically done by either electroporation or viral vectors Lentiviral vectors are preferred to introduce foreign genes into both quail and chicken because one can recapitulate and maintain endogenous gene expression in differentiating somatic cells and in germ cells

Greg Poynter

Papers

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Japanese quail (Coturnix japonica) as a laboratory animal model.

Japanese quail: an efficient animal model for the production of transgenic avians.

Transgenic quail as a model for research in the avian nervous system: A comparative study of the auditory brainstem

Generating transgenic quail using lentiviruses.