Name: live cell imaging of microtubule cytoskeleton and micromechanical manipulation of the arabidopsis shoot apical meristem
Uploaded: 2020-05-23T14:30:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem at JoVE.com

1. Plant growth
<ol>
	<li>Sow Arabidopsis seeds expressing microtubule binding domain fused with green fluorescent protein (MBD-GFP)10 on soil and keep in long day (16 h day /8 h night), 20 &#176;C/6 &#176;C conditions for 1 week for germination.</li>
	<li>After germination, transfer seedlings to new pots with sufficient growth space to allow robust vegetative growth. Keep plants in short day (8 h day /16 h night), 20 &#176;C/16 &#176;C conditions for 3-5 weeks.</li>
	<li>Transfer plant...

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alignment+between+PIN1+polarity+and+microtubule+orientation+in+the+shoot+apical+meristem+reveals+a+tight+coupling+between+morphogenesis+and+auxin+transport.">Alignment between PIN1 polarity and microtubule orientation in the shoot apical meristem reveals a tight coupling between morphogenesis and auxin transport.</a> PLoS Biology. 8 (10), e1000516 (2010).</li><li>Louveaux, M., Rochette, S., Beauzamy, L., Boudaoud, A., Hamant, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+mechanical+compression+on+cortical+microtubules+in+Arabidopsis:+a+quantitative+pipeline.">The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline.</a> Plant Journal. 88 (2), 328-342 (2016).</li><li>Nakayama, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+auxin-mediated+growth.">Mechanical regulation of auxin-mediated growth.</a> Current Biology. 22 (16), 1468-1476 (2012).</li><li>Marc, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GFP-MAP4+reporter+gene+for+visualizing+cortical+microtubule+rearrangements+in+living+epidermal+cells.">A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells.</a> Plant Cell. 10 (11), 1927-1940 (1998).</li><li>Strauss, S., Sapala, A., Kierzkowski, D., Smith, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Plant+Growth+and+Cell+Proliferation+with+MorphoGraphX.">Quantifying Plant Growth and Cell Proliferation with MorphoGraphX.</a> Methods in Molecular Biology. 1992, 269-290 (2019).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Erguvan, O., Louveaux, M., Hamant, O., Verger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+SurfCut:+a+user-friendly+pipeline+for+high-throughput+extraction+of+cell+contours+from+3D+image+stacks.">ImageJ SurfCut: a user-friendly pipeline for high-throughput extraction of cell contours from 3D image stacks.</a> BMC Biology. 17 (1), 38 (2019).</li><li>Boudaoud, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FibrilTool,+an+ImageJ+plug-in+to+quantify+fibrillar+structures+in+raw+microscopy+images.">FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images.</a> Nature Protocols. 9 (2), 457-463 (2014).</li><li>Sampathkumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+wall+cellulose+synthase+regulates+shoot+apical+meristem+mechanics+and+growth.">Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.</a> Development. 146 (10), (2019).</li><li>Hervieux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mechanical+Feedback+Restricts+Sepal+Growth+and+Shape+in+Arabidopsis.">A Mechanical Feedback Restricts Sepal Growth and Shape in Arabidopsis.</a> Current Biology. 6 (8), P1019-P1028 (2016).</li><li>Sampathkumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+and+supracellular+mechanical+stress+prescribes+cytoskeleton+behavior+in+Arabidopsis+cotyledon+pavement+cells.">Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.</a> Elife. 3, e01967 (2014).</li><li>Sampathkumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+wall+cellulose+synthase+regulates+shoot+apical+meristem+mechanics+and+growth.">Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.</a> Development. 146 (10), dev179036 (2019).</li><li>Uyttewaal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stress+acts+via+katanin+to+amplify+differences+in+growth+rate+between+adjacent+cells+in+Arabidopsis.">Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.</a> Cell. 149 (2), 439-451 (2012).</li><li>Hamant, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+patterning+by+mechanical+signals+in+Arabidopsis.">Developmental patterning by mechanical signals in Arabidopsis.</a> Science. 322 (5908), 1650-1655 (2008).</li></ol>

The assessment of mechanical signal transduction events is crucial to identify molecular regulators involved in the mechano-perception and transduction pathways. The protocol described here provides a quantitative view of such events by using the cortical microtubule response as a readout for such a process in Arabidopsis SAMs. The procedure described here is routinely used in several studies in various tissue types16,17,18</...

Plant cells are surrounded by an extracellular matrix of polysaccharides and glycoproteins that mechanically resembles a fiber reinforced composite material capable of dynamically changing its mechanical properties1. Growth in plant cells is driven by the uptake of water into the cell, which results in a concomitant buildup of tensile forces on the cell wall. In response to such forces, modifications to the physical state of the cell wall allows for cell expansion. Cells with primary walls are capable of undergoing rapid growth compared to secondary cell wall containing cells mainly due to differences in the chemical composition of the polysaccharides within. Primary wall cells are composed of cellulose, hemicellulose, and pectin in addition to glycoproteins, and lack lignin, a component that is present in the secondary cell wall2. Cellulose, a glucose polymer linked via &#946;-1,4 bonds, is the major component of the cell walls. It is organized into fibrillar structures that are capable of withstanding high tensile forces experienced during cell growth3. In addition to withstanding tensile forces, mechanical reinforcement along a preferential direction results in turgor-driven expansion along an axis perpendicular to the net orientation of the cellulose microfibril. The organization of the cellulose microfibrils is influenced by the cortical microtubule cytoskeleton, as they guide the directional movement of the cellulose-synthesizing complexes located at the plasma membrane4. Therefore, monitoring cortical microtubule organization using a microtubule-associated protein or tubulin fused with a fluorescent molecule serves as a proxy for the observation of overlying patterns of cellulose in plant cells.
The patterning of the cortical microtubule cytoskeleton is under the control of cell and tissue morphology derived mechanical forces. Cortical microtubule organization does not have any preferential organization over time in cells located at the apex of the SAM, whereas cells in the periphery and the boundary between the SAM and the emerging organ have a stable, highly organized supracellular array of cortical microtubules5. Several approaches have been developed to physically perturb the mechanical status of the cells. Changes to osmotic status, as well as treatment with pharmacological and enzymatic compounds that influence the stiffness of the cell wall can result in subsequent changes in the tensile forces experienced by the cell6,7. The use of contraptions that allow for the gradual increase in compressive forces experienced by tissues is another alternative8. Application of centrifugal forces has also been shown to influence the mechanical forces without any physical contact with the cells9. However, the most widely used means of changing directional forces in a group of cells take advantage of the fact that all epidermal cells are under tension and physical ablation of cells will eliminate turgor pressure locally as well as disrupting cell-to-cell adhesion, thereby modifying the tensile forces experienced by the neighboring cells. This is performed either by targeting a high-powered pulsed ultraviolet laser or by means of a fine needle.
Here we elaborate on the process of imaging and assessing cortical microtubule behavior for mechanical perturbation at the SAM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FibrilTool</td>
			<td></td>
			<td></td>
			<td>Boudaoud, A. et al., Nat Protoc. 2014</td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>Schindelin, J. et al., Nat Methods. 2012</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Merck</td>
			<td>1.04201.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td>DM6000 CS</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP4-GFP</td>
			<td></td>
			<td></td>
			<td>Marc, J. et al., Plant Cell 1998</td>
		</tr>
		<tr>
			<td>micropore tape</td>
			<td>Leukopor</td>
			<td>02482-00</td>
			<td></td>
		</tr>
		<tr>
			<td>MorphographX</td>
			<td></td>
			<td></td>
			<td>Strauss, S. et al., Methods Mol Biol. 2019</td>
		</tr>
		<tr>
			<td>myo-inositol</td>
			<td>Sigma</td>
			<td>I5125</td>
			<td></td>
		</tr>
		<tr>
			<td>N6-benzyladenine</td>
			<td>Sigma</td>
			<td>B3408</td>
			<td></td>
		</tr>
		<tr>
			<td>nicotinic acid</td>
			<td>Sigma</td>
			<td>N4126</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic hinged box</td>
			<td>Electron microscopy sciences</td>
			<td>64312</td>
			<td></td>
		</tr>
		<tr>
			<td>PPM (Plant Preservative Mixture)</td>
			<td>Plant Cell Technology</td>
			<td>PPM</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>pyridoxine hydrochloride</td>
			<td>Sigma</td>
			<td>P9755</td>
			<td></td>
		</tr>
		<tr>
			<td>SURFCUT</td>
			<td></td>
			<td></td>
			<td>Erguvan, O. et al., BMC Biol. 2019</td>
		</tr>
		<tr>
			<td>thiamine hydrochloride</td>
			<td>Sigma</td>
			<td>T4625</td>
			<td></td>
		</tr>
	</tbody>
</table>

live cell imaging of microtubule cytoskeleton and micromechanical manipulation of the arabidopsis shoot apical meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Figure 1 shows typical projection images obtained from MBD-GFP lines with cells at the center of the dome containing disorganized cortical microtubules, and cells at the periphery having a circumferential distribution (Figure 1A,B), whereas the boundary domain cells contain cortical microtubules aligned parallel to the cell&#39;s long axis. These observations show differences in the spatial distribution of cortic...

Watch this Scientific Journal Video about Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem at JoVE.com

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

This protocol it helps us understand how Microtubule Cytoskeleton behavior occurs in plants. At the same time, it also helps us understand how these structural components that influence cell and organ shape, respond to changes in mechanical forces. Although, this method requires little sophistication, it provides robust, quantitative read-after, the mechanical response differences between different genotypes and conditions.Begin by growing arabidopsis seeds expressing microtubule binding domains fused with green fluorescent protein in soil, at 20 and 6 degrees Celsius under long day conditions for one week. After germination, transfer the seedlings into new pots with sufficient growth space to allow robust vegetative growth, and place the plants at 20 and 16 degrees Celsius under short day conditions. After three to five weeks, transfer the plants back to long day conditions at the same temperature until the plants bolt, allowing the inflorescence to grow up to two to five centimeters long.For Shoot Apical Meristem dissection, putt the inflorescence, and use sharp forceps to peel the flowers at the base of the peduncles, until it is difficult to see the peduncles with the naked eye to remove the older flower buds. Use the forceps to create a slit in the agros in a dissecting dish, and plant the inflorescence base into the thick Augur. Place the dish under a dissecting microscope.Starting with the oldest flower bud, push with the forceps to remove the flower bud from the plant. The shoot apical meristem is usually exposed when older flowers up to stage six to seven are removed. And use the forceps to push each flower bud down until the shoot apical meristem is visible.As soon as the shoot apical meristem is exposed, let the freshly dissected sample in growth medium in an ethanol-sterilized rectangular plastic hinged culture box with the shoot apical meristem just exposed above the medium surface. Add a few drops of sterile deionized water to the edges of the culture box and close the lid to maintain the humidity inside the box. Wrap the box with micropore tape, and place the growth box under long or continuous day conditions at 22 degrees Celsius for 12 to 24 hours.When the plant has recovered from the dissection procedure, cover the sample with sterile deionized water and check for air bubbles under the dissecting microscope. Transfer the culture box to an upright confocal microscope stage, and select the 40 or 60X water dipping lens. Lower the objective into the water and check for air bubbles on the front lens of the object.To remove bubbles, lower the stage and gently wipe the lens with an optical tissue. Then use a Pasteur pipette to add a small volume of water to the front lens of the objective before re-immersing the lens in the water. Next, use the GFP filter and epi-illumination module of the confocal microscope to adjust the XY controller to locate the sample.Adjusting the position of the oculars, position the shoot apical meristem directly under the light source, and focus along the Z-axis until the apex is located. Use a laser capable of exciting GFP to illuminate the sample, and adjust the optical zoom of the microscope, so that the entire shoot apical meristem and the stage one floral primordia are in the field of view. Then, adjust the power of the laser output and the gain settings to obtain an optimal signal to noise ratio.After allowing the sample to settle for two to five minutes, acquire confocal Z stacks of the sample at 0.25 to 0.5 micrometers Z slice intervals, adding approximately 0.3 micro meter pixel size resolution. At the end of the acquisition, immediately remove the water and transfer the culture box back to the growth chamber. For Micromechanical Shoot Apical Meristem Perturbation, acquire pre-ablation image stacks of the cortical microtubule organization as just demonstrated, and decaf the water from the culture box into a culture dish.Transfer the shoot apical meristem under a dissecting microscope, and slowly approached the shoot apical meristem with a clean 0.4 by 20 millimeter syringe needle. Briefly contact the periphery of the dome of the shoot apical meristem with the needle tip to confirm that the ablation has been completed. And refill the culture dish with sterile deionized water.Then add 10 micrograms per milliliter of propidium iodide to the dish and immediately acquire image stacks as demonstrated every two hours for six hours, returning the culture dish to the incubation between each time point. For data analysis, generate surface projections of the image stacks using an appropriate image analysis software program, and perform extraction of the cortical microtubule anisotropy using the Fibril Tool Macro in Fiji. Here, typical projection images obtained from microtubule binding domain GFP lines with cells at the center of the dome containing disorganized cortical microtubules, cells at the periphery, having a circumferential distribution and boundary domain cells containing cortical microtubules aligned parallel to the cell's long axis can be observed.Time-lapse imaging, shows cortical microtubule alignment changing from a highly disordered array to a more organized array within six hours of ablation. Tensors could be superimposed on the cortical microtubule images, and the extracted information could be represented by plotting the mean anisotropy over time. When adjusting laser output power and gain settings, we should ensure it clear observation of the filaments and try to avoid overexposure, as over saturated signals could bias the tensile direction in later analysis.Atomic force microscopy can also be performed to assess how the changes in the mechanical forces actually affect the physical status of the cell wall.

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Developmental Biology

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...