Viral nanoparticles as tools for intravital vascular imaging

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Automated sorting of live transgenic embryos

The vast selection of Drosophila mutants is an extraordinary resource for exploring molecular events underlying development and disease. We have designed and constructed an instrument that automatically separates Drosophila embryos of one genotype from a larger population of embryos, based on a fluorescent protein marker. This instrument can also sort embryos from other species, such as Caenorhabditis elegans. The machine sorts 15 living Drosophila embryos per second with more than 99% accuracy. Sorting living embryos will solve longstanding problems, including (1) the need for large quantities of RNA from homozygous mutant embryos to use in DNA microarray or gene-chip experiments, (2) the need for large amounts of protein extract from homozygous mutant embryos for biochemical studies, for example to determine whether a multiprotein complex forms or localizes correctly in vivo when one component is missing, and (3) the need for rapid genetic screening for gene expression changes in living embryos using a fluorescent protein reporter.     

The untapped potential of the GENSAT mice—A valuable resource for developmental biology

Gene Expression Nervous System Atlas (GENSAT) transgenic mice express EGFP, tdTomato, or Cre recombinase in a wide range of cell types. The mice and the bacterial artificial chromosome transgenes are available from repositories (MMRRC or CHORI), thereby making these resources readily available to the research community. This resource of 1,386 transgenic lines was developed and validated for neuroscience research. However, GENSAT mice have many potential applications in other contexts including studies of development outside of the CNS. The cell type‐specific expression of fluorescent proteins in these mice has been used to identify cells in living embryos, in living embryo explants, and in stem or progenitor cell populations in postnatal tissues. The large number of fluorescent protein driver lines generated by GENSAT greatly expands the range of cell type markers that can be used for live cell sorting. In addition, the GENSAT project has generated 278 new Cre driver lines. This review provides an overview of the GENSAT lines and information for identifying lines that may be useful for a particular application. I also provide a review of the few published cases in which GENSAT mice have been used for studies of embryonic development or analysis of stem/progenitor cells in nonneural tissues. genesis 54:245–256, 2016. © 2016 Wiley Periodicals, Inc.     

Rapid epithelial-sheet sealing in the Caenorhabditis elegans embryo requires cadherin-dependent filopodial priming.

BACKGROUND: During embryonic development, epithelia with free edges must join together to create continuous tissues that seal the interior of the organism from the outside environment; failure of epithelial sealing underlies several common human birth defects. Sealing of epithelial sheets in embryos can be extremely rapid, dramatically exceeding the rate of adherens junction formation by epithelial cells in culture or during healing of epithelial wounds. Little is known about the dynamic redistribution of cellular junctional components during such events in living embryos. RESULTS: We have used time-lapse, multiphoton laser-scanning microscopy and green fluorescent protein fusion proteins to analyze the sealing of the Caenorhabditis elegans epidermis in living embryos. Rapid recruitment of alpha-catenin to sites of filopodial contact between contralateral migrating epithelial cells, concomitant with clearing of cytoplasmic alpha-catenin, resulted in formation of nascent junctions; this preceded the formation of mature junctions. Surprisingly, upon inactivation of the entire cadherin-catenin complex, only adhesive strengthening between filopodia was reproducibly affected. Other ventral epidermal cells, which did not extend filopodia and appeared to seal along the ventral midline by coordinated changes in cell shape, successfully adhered in the absence of these proteins. CONCLUSIONS: We propose that 'filopodial priming' - prealignment of bundled actin in filopodia combined with the rapid recruitment of alpha-catenin from cytoplasmic reserves at sites of filopodial contact - accounts for the rapid rate of sealing of the embryonic epidermis of C. elegans. Filopodial priming may provide a general mechanism for rapid creation of adherens junctions during epithelial-sheet sealing in embryos.     

Live imaging and morphometric analysis of embryonic development in the ascidian Ciona intestinalis

The ascidian Ciona intestinalis is one of the model organisms of choice for comparative investigations of chordate development and for unraveling the molecular mechanisms underlying morphogenesis and cell fate specification. Taking advantage of the availability of various genetically encoded fluorescent proteins and of defined cis-regulatory elements, we combined transient transgenesis with laser scanning confocal imaging to acquire and quantitate 3D time-lapse data from living Ciona embryos. We used Ciona tissue-specific enhancers to drive expression of spectrally distinct fluorescent protein reporters to label and simultaneously visualize axially and paraxially positioned mesodermal derivatives, as well as neural precursors in individual embryos. We observed morphogenetic movements, without perturbing development, from the early gastrula throughout the larval stage, including gastrulation, neurulation, convergent extension of the presumptive notochord, and tail elongation. These multidimensional data allowed us to establish a reference system of metrics to quantify key developmental events including blastopore closure and muscle extension. The approach we describe can be used to document morphogenetic cell and tissue rearrangements in living embryos and paves the way for a live digitized anatomical atlas of Ciona.     

Retrovirus-mediated gene transfer and expression of EGFP in chicken

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)-based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.     

Localized electroporation: a method for targeting expression of genes in avian embryos

Avian embryos are a popular model for cell and developmental biologists. However, analysis of gene function in living embryos has been hampered by difficulties in targeting the expression of exogenous genes. We have developed a method for localized electroporation that overcomes some of the limitations of current techniques. We use a double-barreled suction electrode, backfilled with a solution containing a plasmid-encoding green fluorescent protein (GFP) and a neurophysiological stimulator to electroporate small populations of cells in living embryos. As many as 600 cells express GFP 24-48 h after electroporation. The number of cells that express GFP depends on the number of trains, the pulse frequency and the voltage. Surface epithelial cells and cells deep to the point of electroporation express GFP. No deformities result from electroporations, and neurons, neural crest, head mesenchyme, lens and otic placode cells have been transfected. This method overcomes some of the disadvantages of viral techniques, lipofection and in vivo electroporation. The method will be useful to biologists interested in tracing cell lineage or making genetic mosaic avian embryos.     

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780-840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms.     

New method to deliver exogenous material into developing planarian embryos

The ability to report or modify the embryological processes in living embryos is pivotal for developmental biology research. Planarian embryology has experienced renewed interest as the genetic pathways that drive adult regeneration were found to be involved in the development of embryos. The major drawback to the study of planarian embryology is the absence of methods that give access to the embryos and enable their manipulation. Herein, we report on a novel method for delivering external material into developing embryos using nanosecond laser pulses. When focused on the eggshell surface under optimal parameters, laser pulses ablate the protective case and open a pathway throughout which foreign material can be delivered. In this study, we used egg capsules from Schmidtea polychroa (Schmidt, 1861) to microinject 1 microm fluorescein isothiocyanate fluorescent beads into the live embryos. We obtained viability values ranging from 15% in early egg capsules to 100% in late developmental stages. Moreover, we measured the delivery effectiveness as the number of hatchlings containing fluorescent beads per microinjected egg capsule, reaching 100% in early stages and almost 40% in late stages. This is the first time that planarian embryos have been modified without compromising normal development. We consider that this technique will be of extreme value to future work on planarian developmental biology and regeneration, enabling the application of modern functional tools to the study of this Lophotrochozoan.     

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo. Received December 21, 1998; accepted March 31, 1999.     

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle.Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.     

Genetically encoded intracellular sensors based on fluorescent proteins

Green fluorescent protein from Aequorea victoria and its many homologs are now widely used in basic and applied research. These genetically encoded fluorescent markers can detect localization of cell proteins and organelles in living cells and also cells and tissues in living organisms. Unique instruments and methods for studies of molecular biology of a cell and high throughput drug screenings are based on fluorescent proteins. This review deals with the most intensively evolving directions in this field, the development of genetically encoded sensors. Changes in their spectral properties are used for monitoring of cell enzyme activities or changes in concentrations of particular molecules.