Chapter 5. Avian embryos a model for the study of primary vascular assembly in warm-blooded animals.

The formation of a primary vascular bed is a dynamic process, aspects of which are readily amenable to time-lapse imaging in avian embryos. At early developmental stages, the body plan of avian embryos is very similar to mammals and has many properties that make it ideal for imaging. We devised labeling, culturing, and imaging techniques that capture high-resolution images of intact avian embryos in four dimensions over large length scales (1 to 5000 microm).

Matrix metalloproteinase 2-integrin alpha(v)beta3 binding is required for mesenchymal cell invasive activity but not epithelial locomotion: a computational time-lapse study.

Cellular invasive behavior through three-dimensional collagen gels was analyzed using computational time-lapse imaging. A subpopulation of endocardial cells, derived from explanted quail cardiac cushions, undergoes an epithelial-to-mesenchymal transition and invades the substance of the collagen gels when placed in culture. In contrast, other endocardial cells remain epithelial and move over the gel surface.

A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells.

Neural crest (NC) cells have been elegantly traced to follow stereotypical migratory pathways throughout the vertebrate embryo, yet we still lack complete information on individual cell migratory behaviors and how molecular mechanisms direct NC cell guidance. Here, we analyze the spatio-temporal migratory pattern of post-otic NC and the in vivo role of the small Rho GTPase, RhoA, using fluorescent cell labeling, molecular perturbation, and intravital 4D (3D+ time) confocal imaging in the intact chick embryo. We find that the post-otic NC cell migratory pattern is established in two phases with distinct cell migratory behaviors.

High-Resolution, Intravital 4D Confocal Time-Lapse Imaging in Avian Embryos Using a Teflon Culture Chamber Design.

INTRODUCTIONCell migration is a key aspect of many developmental processes, yet there are relatively few whole-vertebrate embryo culture systems that allow for intravital, high-resolution optical imaging of cell movements, cell-cell interactions, and cell-matrix interactions. Here, we present a protocol for 4D (3D + time), high-resolution confocal imaging of fluorescently labeled cells within living avian embryos. We discuss the culture chamber assembly and interface with a commercially available microscope-stage culture-dish heating system.

alphavbeta3 integrin-dependent endothelial cell dynamics in vivo.

A major challenge confronting developmental cell biologists is to understand how individual cell behaviors lead to global tissue organization. Taking advantage of an endothelial cell-specific marker and scanning time-lapse microscopy, we have examined the formation of the primary vascular pattern during avian vasculogenesis. Five types of distinguishable endothelial cell motion are observed during formation of a vascular plexus: (1) global tissue deformations that passively convect endothelial cells; (2) vascular drift, a sheet-like medial translocation of the entire vascular plexus; (3) structural rearrangements, such as vascular fusion; (4) individual cell migration along existing endothelial structures; and (5) cell process extension into avascular areas, resulting in new links within the plexus.

Novel approaches for the study of vascular assembly and morphogenesis in avian embryos.

Dynamic imaging of primary capillary bed formation in a warm-blooded embryo now is readily accomplished with the use of modern digital cameras, software, and instrumentation. The precise dynamic behavior of endothelial cells and their emergent vascular patterns are easily recorded and quantified in exquisite detail. As an example, we present data regarding vasculogenesis and vascular remodeling under normal and vascular endothelial growth factor-stimulated conditions, including corresponding computational analyses of endothelial cell behavior.

Culturing of avian embryos for time-lapse imaging.

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals.

Identification, genomic organization and mRNA expression of CRELD1, the founding member of a unique family of matricellular proteins.

We have isolated and characterized a unique gene that encodes a highly conserved membrane bound extracellular protein that defines a new epidermal growth factor-related gene family. The CRELD1 (Cysteine-Rich with EGF-Like Domains 1) gene (previously known as cirrin) was cloned from a human chromosome 3 BAC. Mapping of the gene confirmed its position at chromosome 3p25.