Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.     

Higher harmonic generation microscopy for developmental biology

Optical higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to its interacted matters due to an energy-conservation characteristic, providing the "noninvasiveness" nature desirable for biological studies. Combined with its nonlinearity, higher harmonic generation microscopy provides excellent three-dimensional (3D) sectioning capability, offering new insights into the studies of embryonic morphological changes and complex developmental processes. By choosing a laser working in the biological penetration window, here we present a noninvasive in vivo light microscopy with sub-micron 3D resolution and millimeter penetration, utilizing endogenous higher harmonic generation signals in live specimens. Noninvasive imaging was performed in live zebrafish (Danio rerio) embryos. The complex developmental processes within > 1-mm-thick zebrafish embryos can be observed in vivo without any treatment. No optical damage was found even with high illumination after long-term observations and the examined embryos all developed normally at least to the larval stage. The excellent 3D resolution of the demonstrated technology allows us to capture the subtle developmental information on the cellular or sub-cellular levels occurring deep inside the live embryos and larvae. This technique can not only provide in vivo observation of the cytoarchitecture dynamics during embryogenesis with submicron resolution and millimeter penetration depth, but would also make strong impact in developmental and structural biology studies.     

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.     

Three-color imaging using fluorescent proteins in living zebrafish embryos

The zebrafish embryo is especially valuable for cell biological studies because of its optical clarity. In this system, use of an in vivo fluorescent reporter has been limited to green fluorescent protein (GFP). We have examined other fluorescent proteins alone or in conjunction with GFP to investigate their efficacy as markers for multi-labeling purposes in live zebrafish. By injecting plasmid DNA containing fluorescent protein expression cassettes, we generated single-, double-, or triple-labeled embryos using GFP, blue fluorescent protein (BFP, a color-shifted GFP), and red fluorescent protein (DsRed, a wild-type protein structurally related to GFP). Fluorescent imaging demonstrates that GFP and DsRed are highly stable proteins, exhibiting no detectable photoinstability, and a high signal-to-noise ratio. BFP demonstrated detectable photoinstability and a lower signal-to-noise ratio than either GFP or DsRed. Using appropriate filter sets, these fluorescent proteins can be independently detected even when simultaneously expressed in the same cells. Multiple labels in individual zebrafish cells open the door to a number of biological avenues of investigation, including multiple, independent tags of transgenic fish lines, lineage studies of wild-type proteins expressed using polycistronic messages, and the detection of protein-protein interactions at the subcellular level using fluorescent protein fusions.     

Universal GFP reporter for the study of vascular development

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.     

Rapid in vivo labeling of identified zebrafish neurons

We report a simple and rapid method to label individual neurons in live zebrafish embryos and to examine their gene expression profiles. Injection of plasmid DNA encoding an alpha-tubulin promotor driving GFP expression results in mosaic embryos containing a limited number of GFP-positive neurons. Labeled neurons express GFP in their soma and axon, providing the opportunity to analyze pathfinding behaviors of identified neurons in vivo. Moreover, the presence of only a small subset of GFP tagged neurons permits the rapid anatomical identification of these neurons based on soma position and axonal trajectory. Analysis of injected embryos reveals that most, if not all, spinal cord cell types and many other neuronal cell types elsewhere in the nervous system can be GFP tagged. Finally, by combining GFP labeling of individual neurons with fluorescent in situ hybridization, we demonstrate the potential of this method to elucidate gene expression patterns at single cell resolution.     

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).     

In vivo oxygen imaging using green fluorescent protein

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs. heart; ischemia; hypoxia; molecular imaging     

Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.     

Multiview light-sheet microscope for rapid in toto imaging

We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biological specimens with subcellular resolution. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.     

Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed “nuclear injection”. To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.     

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.     

Fluorescent protein-based technologies: shedding new light on the plant endomembrane system

Without doubt, GFP and spectral derivatives have revolutionized the way biologists approach their journey toward the discovery of how plant cells function. It is fascinating that in its early days GFP was used merely for localization studies, but as time progressed researchers successfully explored new avenues to push the power of GFP technology to reach new and exciting research frontiers. This has had a profound impact on the way we can now study complex and dynamic systems such as plant endomembranes. Here we briefly describe some of the approaches where GFP has revolutionized in vivo studies of protein distribution and dynamics and focus on two emerging approaches for the application of GFP technology in plant endomembranes, namely optical tweezers and forward genetics approaches, which are based either on the light or on genetic manipulation of secretory organelles to gain insights on the factors that control their activities and integrity.     

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual‐labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N‐terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin‐C promoter. mCherry is expressed in almost all tissues analyzed including pre‐ and post‐implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals. genesis 48:723–729, 2010. © 2010 Wiley‐Liss, Inc.     

Correlative video-light–electron microscopy: development,impact and perspectives

Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the ‘history,’ or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light–electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular ‘context’ to the fluorescent dynamic structures of interest. CLEM ‘multiplies’ the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging.     

Analysis of fast dynamic processes in living cells: high-resolution and high-speed dual-color imaging combined with automated image analysis

The generation of spectral mutants of the green fluorescent protein (GFP) set the stage for multiple-color imaging in living cells. However, the use of this technique has been limited by a spectral overlap of the available GFP mutants and/or by insufficient resolution in both time and space. Using a new setup for dual-color imaging, we demonstrate here the visualization of small, fast moving vesicular structures with a high time resolution. Two GFP-fusion proteins were generated: human chromogranin B, a secretory granule matrix protein, and phogrin, a secretory granule membrane protein. They were tagged with enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), respectively. Both fusion proteins were cotransfected in Vero cells, a cell line from green monkey kidney. EYFP and ECFP were excited sequentially at high time rates using a monochromator. Charged coupled device (CCD)-based image acquisition resulted in 5-8 dual-color images per second, with a resolution sufficient to detect transport vesicles in mammalian cells. Under these conditions, a fully automated time-resolved analysis of the movement of color-coded objects was achieved. The development of specialized software permitted the analysis of the extent of colocalization between the two differentially labeled sets of cellular structures over time. This technical advance will provide an important tool to study the dynamic interactions of subcellular structures in living cells.     

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.     

Subcellular imaging in the live mouse

Fluorescent proteins are available in multiple colors and have properties such as intrinsic brightness and high quantum yield that make them optimally suited for in vivo imaging with subcellular resolution in the live mouse. In this protocol, cancer cells in live mice are labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. GFP nuclear labeling is effected by linkage of GFP to histone H2B, and a retroviral vector is used for cytoplasmic labeling with RFP. Double-labeled cells are injected by various methods. High-resolution imaging systems with microscopic optics, in combination with reversible skin flaps over various organs, enable the imaging of dual-color labeled cells at the subcellular level in live animals. The double transfection and selection procedures described here take 6-8 weeks. Cancer cell trafficking, deformation, extravasation, mitosis and cell death can be imaged with clarity.