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). Here, we describe multiple techniques for labeling and culturing avian embryos to study the cellular, tissue, and extracellular matrix dynamics of vascular morphogenesis.

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. Here, we show that integrin alpha(v)beta3 and matrix metalloproteinase (MMP)2 are present and active in cushion mesenchymal tissue. More importantly, functional assays show that mesenchymal invasive behavior is dependent on MMP2 activity and integrin alpha(v)beta3 binding. Inhibitors of MMP enzymatic activity and molecules that prevent integrin alpha(v)beta3 binding to MMP2, via its hemopexin domain, result in significantly reduced cellular protrusive activity and invasive behavior. Computational analyses show diminished intensity and persistence time of motility in treated invasive mesenchymal cells, but no reduction in motility of the epithelial-like cells moving over the gel surface. Thus, quantitative time-lapse data show that mesenchymal cell invasive behavior, but not epithelial cell locomotion over the gel surface, is partially regulated by the MMP2-integrin interactions.

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. An initial wide front of lateral-directed NC cells, led by NC from rhombomere 7 (r7), move as a distinct subpopulation. This is followed in time by fewer NC cells that migrate collectively from r7 to r8 in a follow-the-leader manner with extensive cellular extensions between cells. We show that post-otic migratory NC cells express RhoA, using RT-PCR on isolated, flow cytometry sorted NC cells and in neural tube culture explants. When RhoA function is altered by expression of a dominant negative or constitutively active form, or injection of C3, there are two major consequences. RhoA constitutively active expressing NC cells are less directional, slower and form fewer follow-the-leader chain assemblies. NC cells expressing RhoA-DN are less affective in retracting filopodia, migrate slower and also form fewer follow-the-leader chain assemblies. Together, these alterations to NC cell intrinsic signaling and cell-cell contact disrupt the precise spatio-temporal post-otic NC cell migratory pattern.

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. To demonstrate how the system works, we describe the protocol in use with chick embryos while following individual fluorescently labeled neural crest cells, a major migratory cell population that sorts into complex patterns of discrete streams. By combining the embryo culture directly on glass with confocal 4D time-lapse imaging, we demonstrate that individual neural crest cell migratory behaviors and cell-cell interactions may be visualized for short periods of time (<6 h). This technique can be adapted to study other migratory cell populations or developmental events in whole avian embryos.

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. The last four types of motion are quantified and found to be reduced in the presence of an alphavbeta3 integrin inhibitor. These dynamic cell motility data result in new hypotheses regarding primordial endothelial cell behavior during embryonic vasculogenesis.

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. The results show that the quail embryo provides an excellent experimental system to test reagents hypothesized to play a role in vascularization of tumors, engineered tissues, or wound sites.

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. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.

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 3p25.3. The gene is ubiquitously expressed in early development and later becomes more markedly expressed in the developing heart, limb buds, mandible and central nervous system. Expression persists in adulthood in most tissues. Sequence analysis suggests that this is a cell adhesion protein. The mouse orthologue was cloned and mapped to the syntenic region of mouse chromosome 6. Orthologues or homologues have also been identified for cow, Chinese hamster, Drosophila and Caenorhabditis elegans. The CRELD1 gene is deleted in the human cytogenetic disorder 3p- syndrome and is in the region of loss of heterozygosity for several types of cancer. A potential role for this protein in these disorders is discussed.