Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic
mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in
heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel
remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that
proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical
cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of
fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in
normal embryos and in embryos with impaired contractility (Mlc2a^(–/–)). We found abnormal plasma and erythroblast circulation in
these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel
remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear
stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued
remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that
hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac

/pdf/vascular-remodeling-of-the-mouse-yolk-sac-requires-lc5394mcma.pdf

Vascular remodeling of the mouse yolk sac requires hemodynamic force

1. Lucitti et al. 2007. Development doi:10.1242/dev.02883
 [OpenUrl][1][Abstract/FREE Full Text][2]

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The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.

/pdf/temporal-transcriptional-profiling-of-somatic-and-germ-cells-4ini26c94k.pdf

Temporal transcriptional profiling of somatic and germ cells reveals biased lineage priming of sexual fate in the fetal mouse gonad.

Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1

We report the first endothelial lineage-specific transgenic mouse allowing live imaging at subcellular resolution. We generated an H2B-EYFP fusion protein which can be used for fluorescent labeling of nucleosomes and used it to specifically label endothelial cells in mice and in differentiating embryonic stem (ES) cells. A fusion cDNA encoding a human histone H2B tagged at its C-terminus with enhanced yellow fluorescent protein (EYFP) was expressed under the control of an Flk1 promoter and intronic enhancer. The Flk1::H2B-EYFP transgenic mice are viable and high levels of chromatin-localized reporter expression are maintained in endothelial cells of developing embryos and in adult animals upon breeding. The onset of fluorescence in differentiating ES cells and in embryos corresponds with the beginning of endothelial cell specification. These transgenic lines permit real-time imaging in normal and pathological vasculogenesis and angiogenesis to track individual cells and mitotic events at a level of detail that is unprecedented in the mouse.

/pdf/using-a-histone-yellow-fluorescent-protein-fusion-for-comn3fe9yo.pdf

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice.

The development of the cardiovascular system is a highly dynamic process dependent on multiple signaling pathways regulating proliferation, differentiation, migration, cell-cell and cell-matrix interactions. To characterize cell and tissue dynamics during the formation of the cardiovascular system in mice, we generated a novel transgenic mouse line, Tg(Flk1::myr-mCherry), in which endothelial cell membranes are brightly labeled with mCherry, a red fluorescent protein. Tg(Flk1::myr-mCherry) mice are viable, fertile, and do not exhibit any developmental abnormalities. High levels of mCherry are expressed in the embryonic endothelium and endocardium, and expression is also observed in capillaries in adult animals. Targeting of the fluorescent protein to the cell membrane allows for subcellular imaging and cell tracking. By acquiring confocal time lapses of live embryos cultured on the microscope stage, we demonstrate that the newly generated transgenic model beautifully highlights the sprouting behaviors of endothelial cells during vascular plexus formation. We have also used embryos from this line to imaging the endocardium in the beating embryonic mouse heart, showing that Tg(Flk1::myr-mCherry) mice are suitable for the characterization of cardio dynamics. Furthermore, when combined with the previously described Tg(Flk1::H2B-EYFP) line, cell number in addition to cell architecture is revealed, making it possible to determine how individual endothelial cells contribute to the structure of the vessel.

/pdf/a-membrane-associated-mcherry-fluorescent-reporter-line-for-g6ky8pwxab.pdf

Olivia G. Kelly

Papers

Using a histone yellow fluorescent protein fusion for tagging and tracking endothelial cells in ES cells and mice.

A membrane associated mCherry fluorescent reporter line for studying vascular remodeling and cardiac function during murine embryonic development