The Surface Charge of Mesodermal and Neural Cells of Triturus Embryos

The electrical surface charge of disaggregated mesodermal and neural cells of the neurula stage of Triturus vulgaris embryos is estimated by using the apparatus described in Figure 1. The results showed a significant difference between the net charges of these two cell types: the neural cells had a clearly negative charge, whereas the mesodermal cells seemed to be more or less neutral.
The difference of the surface charges may partly explain the "sorting-out" phenomenon in the mixed reaggregates of the disaggregated cells.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development

Mouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.     

Live Imaging of Bicoid-Dependent Transcription in Drosophila Embryos

1. Download : Download high-res image (242KB)
2. Download : Download full-size image

     

Locomotion of Fundulus Deep Cells during Gastrulation

SYNOPSIS. Mechanism of locomotion of deep cells of Fundulusheteroclitus was studied in vivo during gastrulation with theaid of time lapse cinemicrography (Nomarski differential interferencecontrast optics), scanning electron microscopy of cells knownto be moving at the time of fixation, and cell culture. Theseare our findings. 1) Deep cells usually move rapidly, at about10–15 µ/min, regardless of whether they move byblebbing or spreading. Evidence suggests that this high speedis associated with weak adhesion of the trailing edge: it remainsrounded, without large retraction fibers, and it advances continuouslywith advance of the leading edge, not sporadically, as it wouldif it adhered strongly. 2) In contrast, when stationary cellsin close contact separate, they remain connected by retractionfibers, suggesting strong punctate adhesions. 3) Locomotionby shortening of a long lobopodium is really a form of spreadingmovement; the tip of a lobopodium always spreads. Also, sincespeed of shortening decreases with continuance, it may dependprimarily on elastic recoil rather than active contraction.4) Fundulus deep cells appear to move in two ways: a) protrusionof blebs, followed by much cytoplasmic flow; b) protrusion oflamellipodia, accompanied by filopodia and frequent cell shortening.5) Filopodia were not found except at the leading edge of aspreading lamellipodium and often spread themselves; perhapsfilopodia and lamellipodia are interconvertible. 6) A lamellipodialmargin may form undulations in vivo that move backward likeruffles in vitro. 7) At all times, whether stationary or moving,the surface of deep cells is smooth, raising unanswered questionsconcerning the source of surface for their rapid protrusiveactivity.     

Conserved and Divergent Roles of FGF Signaling in Mouse Epiblast Stem Cells and Human Embryonic Stem Cells

1. Download : Download high-res image (158KB)
2. Download : Download full-size image

Highlights? Nanog is a direct target of Activin and SMAD2/3 but not FGF-ERK in EpiSCs ? FGF signaling inhibits neuroectodermal commitment of EpiSCs and hESCs ? FGF inhibition relieves Klf2 repression and reverts EpiSCs to an ESC-like state ? mESCs transition to an EpiSC-like state with LIF inhibition and FGF activation     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

The Transcriptional and Functional Properties of Mouse Epiblast Stem Cells Resemble the Anterior Primitive Streak

1. Download : Download high-res image (284KB)
2. Download : Download full-size image

     

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.     

Conversion of Mouse Epiblast Stem Cells to an Earlier Pluripotency State by Small Molecules

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach, we found that blockage of the TGFβ pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However, full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1, ALK5, MEK, FGFR, and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.     

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.Since its advent 20 years ago, single-molecule fluorescence imaging has given rise to a host of exciting experiments (Ambrose and Moerner 1991). Beyond enabling fundamental investigations of the physics of emissive molecules, one main advantage of this technique is its use in biologically relevant, live-cell experiments. Optical fluorescence microscopy is an important instrument for cell biology, as light can be used to noninvasively probe a sample with relatively small perturbation of the specimen, enabling dynamical observation of the motions of internal structures in living cells. Single-molecule epifluorescence microscopy extends these capabilities by achieving nanometer-scale resolution, taking advantage of the fact that one can precisely characterize the point spread function (PSF) of a microscope, allowing the center of a distribution, and thus the exact position of an emitter, to be localized with accuracy much better than the diffraction limit itself. This localization accuracy improves beyond the diffraction limit roughly as one over the square root of the number of detected photons (Thompson et al. 2002). Detecting 100 photons from a single, isolated molecule can therefore improve the resolution of an optical measurement from the ∼250-nm diffraction limit down to 25 nm.Single-molecule imaging has been used in the investigation of a number of live-cell samples. In 2000, the lateral heterogeneity of the plasma membrane was investigated by tracing the motion of single dye-labeled lipids in native human airway smooth muscle (HASM) cells (Schütz et al. 2000), and epidermal growth factor (EGF) receptor signaling was explored with a fluorescent protein fusion and a labeled ligand (Sako et al. 2000). Single fluorophore-labeled molecules have subsequently been used in many ways (Moerner 2003), for instance to investigate the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane of Chinese hamster ovary (CHO) cells (Vrljic et al. 2002; Vrljic et al. 2005) and to explore the real-time dynamic behavior of cell-penetrating-peptide (CPP) molecular transporters on the plasma membrane of CHO cells (Lee et al. 2008). Furthermore, in 2001, Harms et al. characterized the emission of fluorescent proteins in biocompatible environments and noted that the yellow fluorescent protein EYFP was well-suited to single-molecule imaging in cells (Harms et al. 2001). Such fluorescent proteins can be genetically encoded as tags for native proteins in cells; these fusions have been used in many live-cell single-molecule experiments.More recently, single-molecule epifluorescence microscopy has been used to probe the inner workings of live bacteria. The small size of prokaryotic cells makes the optical diffraction limit particularly noticeable, which has stimulated the push toward superlocalization and superresolution to overcome this obstacle. As a result, the nascent field of bacterial structural biology has benefited greatly from single-molecule investigations of proteins in live cells. The overall shapes of such cells can be seen in a standard light microscope, but those interested in probing subcellular details, such as protein structure and localization, have typically had to resort to in vitro characterization combined with extrapolation to the cellular environment, as well as to indirect methods such as biochemical assays. Although cryo-electron microscopy can provide extremely high spatial resolution, fixation or plunge-freezing is essential, and methods for identifying specific proteins out of many are still lacking. As a consequence, bacterial cell biology is an area of study ripe for investigation with direct, noninvasive optical methods of probing position, coupling and structure, with resolution below the standard diffraction limit.Several groups have extended single-molecule imaging techniques to live bacterial samples. In 2004, single PleC proteins were visualized in Caulobacter crescentus cells (Deich et al. 2004), and the behavior of this system is described in more detail later. More recently, Xie and coauthors have used single-molecule fluorescence techniques to study DNA-binding proteins, mRNA, and membrane proteins to provide much insight into the mechanisms of bacterial gene expression; these efforts have been documented in a recent review (Xie et al. 2008). As well, Conley et al. used covalently linked Cy3-Cy5-thiol switchable fluorophores to illuminate the stalks of C. crescentus cells with high resolution (Conley et al. 2008). In this article, we focus on the application of single-molecule imaging and single-molecule-based superresolution imaging to investigate the localization, movement, and structure of three important proteins, PleC, PopZ, and MreB, in live C. crescentus cells.     

Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.     

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.     

Imaging Proteolytic Activity in Live Cells and Animal Models

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50–100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase''s was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.     

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.     

Ultrastructural Study of Changes in Cell Morphology and Extracellular Matrix in Prospective Endodermal Cells of Newt Embryos (Cynops pyrrhogaster) before and during Gastrulation

The cell morphology, cell-to-cell contact behavior and extracellular matrix (ECM) of inner cells (prospective endodermal cells) of newt ( Cynops pyrrhogaster ) embryos were examined from the morula to gastrula stage by light and electron microscopy. The inner cells showed increased cell-to-cell contact from the early blastula to early gastrula stage. The cells formed blebs (5–15 μm in diameter) during the blastula stage, and started to form filopodia and lamellipodia before gastrulation. Alcian blue and lanthanum nitrate treatment revealed ECM components on the cell surface in the early blastula stage and these components increased in amount from the late blastula to early gastrula stage. It is suggested that the increase in ECM components on the cell surface may have some relation with changes in cell-to-cell contact and formation of processes on the cell surface. Besides the cell surface ECM components, glycogen-like granules were observed in intercellular spaces. From the distribution of granules in gastrulae, it is suggested that these may be important in maintaining intercellular spaces for migration of invaginating cells.     

小鼠胚胎对人卵巢癌细胞生物学行为影响的体外研究

探讨体外共培养环境中小鼠胚胎对人卵巢癌细胞HO8910PM的影响.通过小鼠胚胎与肿瘤细胞体外共培养模型观察小鼠胚胎对肿瘤细胞的形态及生长行为的影响,Annexin V-EGFP/PI原位检测与小鼠胚胎共培养后肿瘤细胞的凋亡情况,MTT粘附实验、transwell迁移及侵袭实验研究与小鼠胚胎共培养后的人卵巢癌细胞的粘附性、迁移性及侵袭性的变化.共培养时小鼠胚胎能够侵入人卵巢癌细胞并推开肿瘤细胞形成自己的生长空间,激光共聚焦显微镜下见胚胎周围的肿瘤细胞凋亡增加,与对照组比较共培养后的HO8910PM肿瘤细胞的粘附性、迁移性及侵袭性均显著降低(P0.05、P0.01).结果表明体外共培养体系中小鼠胚胎能够侵袭肿瘤细胞,促进人卵巢癌细胞的凋亡,并使其粘附性、迁移及侵袭相关恶性行为降低.